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https://f1000research.com/articles/8-666/v1	15 May 19	{ "type": "Case Report", "title": "Case Report: Investigation and molecular genetic diagnosis of familial hypomagnesaemia: a case report", "authors": [ "Jamie Willows", "Maryam Al Badi", "Chloe Richardson", "Noel Edwards", "Sarah Rice", "John A. Sayer", "Jamie Willows", "Maryam Al Badi", "Chloe Richardson", "Noel Edwards", "Sarah Rice" ], "abstract": "Genetic mutations causing familial hypomagnesaemia syndromes are well-recognised. Affected patients can present with severe symptoms of hypomagnesaemia, such as seizures or cardiac arrhythmia. We report an affected child, from a consanguineous family, who presented in the first weeks of life with seizures secondary to hypomagnesaemia, without other associated clinical features. We performed whole exome sequencing in the affected child and segregation analysis within the family, which revealed a novel homozygous missense mutation in TRPM6, which was confirmed as a heterozygous allele in both parents and two younger siblings who had transient hypomagnesaemia. Using in silico modelling, we provide evidence that the missense variant p.(K1098E) in TRPM6 is pathogenic, as it disrupts stabilising TRP domain interactions. Management of familial hypomagnesaemia relies on prompt recognition, early magnesium replacement and lifelong monitoring.", "keywords": [ "hypomagnesaemia", "with secondary hypocalcaemia", "TRPM6", "molecular genetics" ], "content": "Introduction\n\nHomeostasis of the serum magnesium level is essential for human cellular function, and levels are maintained in the normal range by tight control of magnesium reabsorption by the kidney tubules1. Hypomagnesaemia can manifest with a range of symptoms, from tremor, muscle spasms or nystagmus through to seizures, arrhythmias and cardiac arrest. Early identification of the electrolyte abnormality is vital, as treatment with magnesium replacement is efficacious and inexpensive. Common causes of hypomagnesaemia in adults include refeeding syndrome, diarrhoea, malabsorption, alcohol abuse and medications such as proton pump inhibitors2,3. Renal magnesium wasting is indicated by an inappropriately high fractional excretion of magnesium in urine despite hypomagnesaemia, and is seen in post-obstructive diuresis, the recovery phase of acute tubular necrosis, hypercalcaemia and in response to certain diuretics4. However, genetically inherited mutations that cause renal hypomagnesaemia are well-recognised, and typically present in childhood if they are secondary to autosomal recessive disorders5. Genetic forms of hypomagnesaemia should also be considered in certain clinical scenarios, such as in the presence of a positive family history of related disorders, consanguinity, or fulminant presentation.\n\nOnce a genetic cause of hypomagnesaemia is suspected, work-up can be guided by associated features and age at presentation. Though obtaining a genetic diagnosis will not alter the treatment of magnesium replacement therapy, it is vital for identifying others at risk and family counselling, and may help to guide the clinician to screen for associated phenotypic features.\n\n\nCase report\n\nWe report a child from a consanguineous family (parents were second degree cousins) from Oman, who presented with seizures and hypomagnesaemia. The affected individual, a female child, presented at 20 days of age with tonic-clonic seizures. There was no history of fever or diarrhoea, and after an uncomplicated pregnancy she had been born healthy at term, without syndromic features. Serum magnesium was severely low at 0.35 mmol/L and was associated with a mild hypocalcaemia and suppressed parathyroid hormone (PTH) (Table 1). The urinary fractional excretion of magnesium was inappropriately in the normal range given the severe degree of hypomagnesaemia present, suggesting contributory renal magnesium wasting. There were no other specific clinical or biochemical features; of note peripheral oxygen saturations and capillary blood glucose levels were within normal limits. Renal ultrasound scan was normal, with no nephrocalcinosis. She was initially treated with intravenous magnesium (20% MgCl2 0.1 mmol /kg every 6 hours p.r.n.) and calcium replacement (10% Calcium Gluconate 0.11 mmol/kg). At 4 years of age she is now supported with high-dose oral magnesium supplements (magnesium sulphate 500mg qds) alone, and remains well with no further seizures, though she maintains a low serum magnesium level between 0.4-0.6 mmol/L.\n\nNR, normal range; Fractional excretion of magnesium (%) = Urine Magnesium x Plasma Creatinine / (0.7 x Plasma Creatinine x Urine creatinine) x 100.\n\nOf note, a younger sibling of the proband, also female, presented at 18 days old with abnormal eye movements in association with a complex partial seizure. Her serum magnesium was below normal limits (0.53 mmol/L), with serum calcium and PTH within the normal range (Table 1). The fractional excretion of magnesium was inappropriately high, and again renal ultrasound scan was normal and no other clinical features were noted. She was treated with intravenous magnesium replacement (20% MgCl2 0.1 mmol /kg every 6 hours p.r.n.), followed by a period of maintenance oral magnesium replacement (magnesium sulphate 300 mg b.d.). At 2 years of age she remains well with no further seizures, and she maintains magnesium levels within the normal range without additional supplementation. A younger asymptomatic male sibling was screened with serum biochemistry tests at 1 week of age. Serum magnesium was low at 0.6 mmol/L, with normal serum calcium and PTH levels (Table 1). Supplementation was not started, and by 1 year of age serum magnesium was within the normal range.\n\n\nGenetic investigations\n\nDetailed information on the techniques described below is given in the Methods section. Following informed consent, whole exome sequencing (WES) was performed in the eldest sibling, II:1 (Figure 1). Analysis using a combination of homozygosity mapping and variant calling revealed a homozygous missense mutation c.3292A>G, p.(K1098E) in TRPM6 within a large region of homozygosity by descent (Figure 1). The missense variant was confirmed by Sanger sequencing, and cascade screening confirmed this variant was in its heterozygous state in both parents and both mildly affected siblings. In silico tools confirmed evolutionary conservation (Figure 1) as well as the rarity and predicted pathogenicity of the variant (Table 2). Using predictive modelling of the protein structure we were able to show that the lysine residue at position 1098 is predicted to form a stabilising interaction within the TRP domain, and that the missense mutation of TRPM6 K1098 to glutamate is predicted to disrupt this interaction (Figure 2).\n\n(A) Pedigree diagram showing proband (arrowed) who is homozygous for c.3292A>G p.(K1098E) missense mutation in TRPM6 and segregation of alleles from each parent. Parents were consanguineous (second degree cousins). (B) Homozygosity mapping across chromosomes 1-22. Red bars indicate regions of homozygosity. Candidate genes CLDN19 and TRPM6 (arrowed) are located within regions of homozygosity. (C) Chromatograms from proband (II:1) and sibling (II:2) showing TRMP6 variant in homozygous and heterozygous state, respectively. (D) Amino acid (AA) alignment showing conservation of the K1098 residue of TRPM6.\n\n# Reference sequence NM_017662.5\n\n(A) Sequence alignments of TRP domain residues from human TRPM1-TRPM8. Fully-conserved residues are highlighted in black and semi-conserved residues in grey. TRPM6 K1098 is outlined in red. (B) Cryo-EM structure of Ca2+- and Na+-bound TRPM2 (PDB 6CO7). One monomer of the homotetrameric channel is highlighted in orange and the co-ordinated Ca2+ and Na+ ions are shown as green and purple spheres, respectively. (C) Superposition of the TRPM2 monomer (orange) and TRPM6 homology model (green). The TRP domain region (box and inset), wherein the TRPM6 K1098E variant lies, is shown to highlight the predicted structural homology between TRPM6 and TRPM2. The Ca2+ ion (green sphere) is shown for orientation. (D) Close up view of TRPM2 Q896 and E1110 involved in co-ordination of the Ca2+ ion (green sphere) and R1114 (homologous to TRPM6 K1098, shown in (E)) predicted to form a stabilising interaction within the TRP domain. (F) Mutation of TRPM6 K1098 to glutamate (magenta) is predicted to disrupt stabilising TRP domain interactions. The relative position of the Ca2+ ion (green sphere) in TRPM2 is shown in (E) and (F) for orientation.\n\n\nDiscussion\n\nAs the second most abundant intracellular cation, magnesium is vital for normal cell function1. The majority of ingested magnesium load is absorbed in the distal small bowel via paracellular mechanisms, and the remainder is absorbed in the colon by transient receptor potential melastatin type 6 (TRPM6) ion channels in gut epithelium1. Serum magnesium levels make up a relatively tiny proportion of whole-body magnesium content, but needs to be kept within a narrow range to maintain neuronal, skeletal muscle and cardiac muscle cell stability. Serum magnesium homeostasis is therefore tightly regulated by reabsorption in the kidney; the majority is reabsorbed in the thick ascending limb of the loop of Henle via a paracellular route, and the ‘fine-tuning’ is performed in the distal convoluted tubule (DCT) via apically located TRPM6 channels.\n\nHypomagnesaemia is a common electrolyte disturbance, with a prevalence of 20% in hospitalised patients6. Causes in adults include inadequate intake, refeeding syndrome, renal losses, gastrointestinal losses in diarrhoea, gastrointestinal malabsorption, and medications such as proton pump inhibitors (PPIs)7. Serum magnesium levels may be requested as part of an extended biochemical panel if there is clinical concern about these risk factors, if symptoms or cardiac arrhythmia are present, or if other disturbances such as hypokalaemia or hypocalcaemia prompt the consideration of magnesium depletion. Measurement of urinary magnesium may help distinguish between gastrointestinal and renal losses. Urinary magnesium levels will be low if hypomagnesaemia is secondary to gastrointestinal losses, as the kidneys appropriately work to maximally reabsorb filtered magnesium, but raised or inappropriately normal despite low serum magnesium levels in renal magnesium wasting conditions. The majority of renal causes of hypomagnesaemia are not genetic, such as renal losses induced by post-obstructive diuresis, the recovery phase of acute tubular necrosis, hypercalcaemia, or drugs such as loop and thiazide diuretics, cisplatin, tacrolimus and aminoglycosides.\n\nMagnesium wasting disorders found in families have been shown to be associated with over a dozen genes5. Similar to other monogenic diseases causing renal tubule phenotypes, the study of these diseases has greatly contributed to our knowledge of the renal tubular transport proteins responsible for homeostatic and physiological functioning. Familial hypomagnesaemic renal disorders may be inherited in both autosomal dominant and recessive patterns, and the underlying genes uncovered so far all encode proteins found in the thick ascending limb of the loop of Henle or DCT. Familial hypomagnesaemias may be categorised into four groups. These include hypercalciuric hypomagnesaemias (secondary to mutations in CLCNKB (Bartter syndrome type 3), CLDN16, CLDN19, CASR); Gitelman-like hypomagnesaemias (secondary to mutations in SLC12A3 (Gitelman syndrome), BSND (Bartter syndrome type 4), KCNJ10, FYXD2, HNF1B, PCBD1); mitochondrial hypomagnesaemias; (mutations in SARS2, MT-TI and Kearns–Sayre syndrome) and other hypomagnesaemias (secondary to mutations in TRPM6, CNMM2, EGF, EGFR, KCNA1, FAM111A)5.\n\nTRPM6 is expressed in both the colon and the DCT of the kidney, and mutations here can cause the condition known as hypomagnesaemia with secondary hypocalcaemia. There have been dozens of distinct mutations in TRPM6 associated with this condition, and different variants can cause different effects on the function of the TRPM6 transporter1. In patients with TRMP6 mutations magnesium absorption from the colon is decreased (primary intestinal hypomagnesaemia), and the DCT is unable to perform the ‘fine-tuning’ of magnesium reabsorption and inappropriately wastes magnesium via the urine. Due to this dual pathology, the condition can cause the most profound electrolyte wasting of the genetic hypomagnesaemias. It typically presents in the neonatal period with severe symptoms due to hypomagnesaemia and hypocalcaemia such as seizures, which are subsequently responsive to magnesium administration8. The hypocalcaemia is thought to be secondary to hypoparathyroidism, which is induced by hypomagnesaemia9. Interestingly, the observation that treatment with PPIs is associated with hypomagnesaemia has led to a proposed mechanism of PPI-induced inhibition of TRPM6 and TRPM7 channels in the gastrointestinal tract10. TRPM6 may also be downregulated in the DCT in response to cyclosporine, resulting in renal magnesium wasting11.\n\nTreatment of all the genetic hypomagnesaemia disorders, including those caused by TRPM6 mutations, is with magnesium replacement therapy, either oral or intravenous depending on urgency and the tolerability of oral products. The major side-effect of oral magnesium replacement is diarrhoea, which can limit treatment compliance and paradoxically cause worsening of hypomagnesaemia due to increased gastrointestinal losses. Overall the prognosis of hypomagnesaemia with secondary hypocalcaemia is excellent, and serum calcium levels normalise as serum magnesium levels improve.\n\nGiven what is known about hypomagnesaemia with secondary hypocalcaemia, our first patient presented typically, with severe symptoms and the expected biochemical profile, including low PTH. WES confirmed a homozygous missense mutation in TRPM6, and clearly the family history of consanguinity was consistent with the diagnosis of an autosomal recessive disorder. In keeping with previous case reports she did not maintain magnesium concentration in the normal range, despite high dose oral replacement. Interestingly, the second child also had severe symptoms at presentation despite ultimately proving to be heterozygous for the TRPM6 mutation. However, it can be seen that her presentation was less fulminant, without the development of tonic-clonic seizures and with milder derangement of biochemical parameters. In keeping with this less severe phenotype, she now maintains normal serum magnesium levels without supplementation. Finally, the third sibling had documented transient and asymptomatic hypomagnesaemia, which corrected by 1 year of age. These two siblings provide some evidence that a heterozygous allele in infants may lead to a transient biochemical phenotype, presumably related to the immaturity of the DCT to regulate magnesium. Adults heterozygous for TRPM6 pathogenic variants have never been reported to have abnormal serum magnesium levels12. Heterozygous Trpm6 knockout mice exhibit mild hypomagnesaemia under a normal diet, suggesting that a milder phenotype may be associated with the loss of one TRPM6 allele13.\n\nThe location and predicted pathological effect of the missense mutation warrants further discussion. Previously described missense mutations in TRPM6 include p.(S141L) and p.(P1017R), which lead to either trafficking or gating impairment of the TRMP6 channel14,15. Additional missense mutations p.(I174R), p.(T354P) and p.(C707T) have also been reported16. Here we have taken advantage of recently published cryo-EM structures of TRPM217–19, TRPM420–23, TRPM724 and TRPM825, which suggest a conserved global architecture for TRPM family members, consistent with sequence conservation (see Extended data, Supplementary Figure 1)26. We therefore utilised all structures to interpret the likely pathogenic effect of the TRPM6 K1098E variant (with the exception of TRPM8, due to low resolution in the homologous region25). TRP domain sequence analysis (Figure 2A) revealed conservation of a basic residue at the homologous position to K1098 in TRPM6, indicating an important functional role for the positively charged side-chain. Studies of TRPM6-TRPM8 suggest that positively charged residues in the TRP domain may interact with the negatively charged phosphate groups in phosphatidylinositol-4,5-bisphophate (PIP2) to mediate channel activation27,28. Neutralisation of the positive charge by substitution with glutamine was shown to abolish channel activity in TRPM6 K1098Q27 and TRPM8 R1008Q28, although surprisingly no significant effect on channel activity was seen in the homologous TRPM7 K1125Q variant27. Moreover, the homologous TRPM4 variant (R1072Q) exhibited normal sensitivity to PIP2, arguing against this residue being involved directly in PIP2 binding29. Based on the available TRPM structures, we predict K1098 may mediate one of several stabilising interactions in TRPM6. In the cryo-EM structure of TRPM2 (Figure 2B), and TRPM6 homology model (Figure 2C), the TRP domain lies in close proximity to the ion conduction pathway, with mutations in this domain likely to affect channel gating. Indeed, mutation of TRP domain residues in TRPM2 (E1110) and TRPM4 (E1068) were shown to impair the binding of Ca2+ necessary for priming the channel for voltage-dependent opening19,20,30. In TRPM2, E1110 in the TRP domain stabilises Q896 in the S2 helix (Figure 2D), correctly orienting Q896 for Ca2+-binding19. Interestingly, the glutamine residues involved in co-ordination of the Ca2+ ion in TRPM2 (Q896) and TRPM4 (Q83120) are conserved in all Ca2+-dependent TRPM channels (Figure 3), but are replaced by glutamate (E889) in the Ca2+-independent TRPM6 (Figure 2E). TRPM6 modelling suggests that K1098 in the TRP domain could form stabilising interactions with E889 and E885 (equivalent to R1114 and E892 in TRPM219; Figure 2D) in the S2 helix (Figure 2E), thereby priming the channel for activation in a Ca2+-independent manner. Alternatively, TRPM6 K1098 may potentially serve to stabilise the TRP domain helix itself, either through a cation-π interaction with Y1095 (Figure 2E), equivalent to that identified in the cryo-EM structure of TRPM4 between R1072 and F106922, or via interaction with the hydroxyl side-chain of T1094 (equivalent to TRPM2 E1110; Figure 2E). This latter potential interaction is analogous to that modelled between TRPM8 R1008 and E1004, whereby agonist/antagonist binding was predicted to modulate the position and intra-protein contacts of R1008 (equivalent to TRPM6 K1098), with resultant changes in the TRP domain helix effecting channel opening/closing, respectively31. Substitution of TRPM6 K1098 with the negatively charged glutamate (K1098E; Figure 2F) is predicted to be pathogenic since this change would destabilise any of the potential interactions discussed.\n\nCryo-EM structures of (A) TRPM2 (PBD 6CO7), (B) TRPM4 (PDB 6BQV), (C) TRPM7 (PDB 6BWD) and (D) TRPM8 (PDB 6BPQ). One monomer of the respective tetrameric channel structure is highlighted. Spheres denote: Ca2+ (green) and Na+ (purple) ions in TRPM2; Ca2+ ions (green) in TRPM4; and Mg2+ ions (green) in TRPM7.\n\n\nConclusion\n\nHere we provide evidence for a novel pathogenic missense mutation p.(K1098E) in TRPM6 which leads to a severe hypomagnesaemia with secondary hypocalcaemia phenotype in an affected child. In silico modelling of homologs of the TRPM channels supports an important stabilising role for this residue.\n\n\nMethods\n\nClinical summaries were prepared and DNA samples taken from whole blood following informed and written consent. Ethical approval for this study was obtained from the National Research Ethics Service (09/H0903/36).\n\nA DNA sample from the affected proband underwent WES, performed via GATC Biotech. A DNA library was prepared using enrichment with SureSelectXT and a human All Exon Kit. Sequencing was performed using Illumina with paired end reads of 2 x150 bp with a >30X average on target coverage. Raw data was analysed via a commercial bioinformatics pipeline (GATC Eurofins), which included mapping against genomic reference sequence and detection of SNPs and InDels using GATK’s Haplotype caller32. Resulting vcf files were analysed using Qiagen Ingenuity Variant analysis software (Build 5.5.20190412) (or open access equivalent VCF-Explorer 1.0)and HomozygosityMapper.\n\nVariants in genes and segregation in other family members were confirmed using exon PCR followed by Sanger sequencing.\n\nHuman TRPM6 (UniProt accession Q9BX84) was modelled against the cryo-EM structures of TRPM2 (PDB accession 6CO7;19), TRPM4 (PDB 6BQV;20 and TRPM7 (PDB 6BWD;24 using HHPred33, Modeller (version 9.21)34 and I-TASSER35 software. TRPM structure figures were prepared using PyMOL 2.3.\n\n\nData availability\n\nWhole exome sequencing data available from BioProject, accession number PRJNA541906: https://identifiers.org/bioproject/PRJNA541906.\n\nFigshare: Alignment of human TRPM channels 1-8 amino acid sequences. https://doi.org/10.6084/m9.figshare.806353726.\n\nThis project contains the following extended data:\n\nSupplementary Figure 1. Alignment of human TRPM channels 1-8 amino acid sequences (UniProt accession codes Q7Z4N2, O94759, Q9HCF6, Q8TD43, Q9NZQ8, Q9BX84, Q96QT4 and Q7Z2W7, respectively), with sequences for the cryo-EM structures of N. vectensis (Nv) TRPM2 (UniProt and mouse (Ms) TRPM7 (UniProt Q923J1). The position of the TRP domain, TRPM6 K1098 and homologous residues are highlighted.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nWritten informed consent was obtained from the patients’ family for publication of this case report and accompanying images.", "appendix": "Author contributions\n\n\n\nThe project was conceived and directed by JAS. JW and JAS drafted the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nGrant information\n\nWe thank Northern Counties Kidney Research Fund (BH160804) who supported this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nde Baaij JH, Hoenderop JG, Bindels RJ: Magnesium in man: implications for health and disease. Physiol Rev. 2015; 95(1): 1–46. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "48551", "date": "24 Jun 2019", "name": "Karl Peter Schlingmann", "expertise": [ "Reviewer Expertise Molecular genetics of hereditary disorders of electrolyte metabolism." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear editors and authors, In their manuscript entitled \"Investigation and molecular genetic diagnosis of familial hypomagnesemia: a case report” Jamie Willows and colleagues describe a patient with the typical clinical presentation of familial hypomagnesemia with secondary hypocalcemia (HSH). Molecular genetic studies identified a homozygous mutation in the TRPM6 gene as the underlying pathology. The TRPM6 gene encodes a member of the transient receptor potential (TRP) family of ion channels that is involved in the formation of epithelial magnesium permeable ion channels in intestine and kidney. If it does so alone or in cooperation with TRPM7 has been a matter of debate.\n\nIn addition to the initial studies and two larger follow-up reports,1, 2, 3, 4 mostly small case series or case reports have been published of patients with HSH and mutations in TRPM6. HSH is thought to represent a classic autosomal-recessive disease with unaffected heterozygous parents and siblings. Most patients were found to carry non-sense mutations in TRPM6 including stop mutations, small deletions/insertions, leading to a shift in the reading frame and premature stops of translations, splice site mutations, and also deletions of larger parts of the gene. Only a small number of missense mutations have been reported of which a subset has been analyzed functionally.4, 5, 6 These almost uniformly lead to a complete loss-of-function.\n\nStudy design and results of this report are presented accurately and the appropriate literature is cited correctly. The methods used for molecular diagnosis and in-silico modeling of the identified mutant are provided. The initial presentations as well as diagnostic tests performed, treatments given and clinical outcomes of the index patient and siblings are described in sufficient detail. In the context of the published literature, this case report is unique for two reasons: to my knowledge, the discovered p.K1098E variant is the first missense mutation directly affecting the TRP domain of the TRPM6 ion channel subunit. The TRP domain is thought to play a crucial role in ion channel multimerization as well as in channel activation by PIP2 (phosphatidylinositol-4,5-bis-phosphate). Accordingly, the authors present comprehensive data analyzing the putative effects of the discovered mutant by comparing this naturally occurring mutant to engineered mutants at the identical position in TRPM6 and related TRPM channel subunits. Though the effects of engineered mutants on channel activity as well as PIP2 mediated channel activation are not consistent, the genetic data presented here together with the disease phenotype clearly argue for the pathogenetic role of the discovered mutant. It will be interesting to study this mutant functionally in an overexpression system (especially in combination with wildtype TRPM6 subunits and TRPM7, see below). The second intriguing finding presented here is that, in addition to the index patient, two siblings carrying the p.K1098E variant in heterozygous state presented in infancy with hypomagnesemia and a cerebral seizure in case of one sibling. Such a finding in heterozygous mutation carriers has not been reported before. The authors consider this finding a general feature of the disease and attribute it to a possible immaturity of the renal tubule in early life. Serum magnesium levels have not been systematically evaluated in clinically unaffected siblings so far. However, measurements of serum magnesium levels in the first weeks of life have been advocated in siblings to exclude disease before clinical presentation with cerebral seizures and before quick genetic testing became feasible. Newborns usually start with their mothers serum magnesium at birth and in case of defective TRPM6 show a continuous decline of serum levels over the following weeks. At least in single families, measurement of serum magnesium yielded normal levels and was able to exclude disease in younger siblings (unpublished data, personal observation). Therefore, the information on maternal serum magnesium is critical in this family to better classify the observed changes in the heterozygous siblings as following the mentioned approach, the measurement of serum magnesium levels would have suggested the diagnosis of HSH also in the heterozygous siblings of this family rather than excluding classic disease. Finally, it is also conceivable that the hypomagnesemia observed in the siblings of the family presented here represents a mutation specific phenomenon rather than a general finding in patients with heterozygous TRPM6 mutations which could also be discussed in the report. Are there indications of a functional effect of the p.K1098E mutant on heteromultimerization with wildtype TRPM6 or TRPM7 via TRP domain interactions? Were there additional variants in the TRPM6 gene identified by WES (especially on the unaffected alleles of siblings)? Do the two siblings share the identical unaffected allele of mother or father? Could an additional variant potentially explain the transient phenotype?\nMinor comments and suggestions:\nthe dose of daily oral magnesium should please be provided in mmol/kg/day as it allows easier comparison with published doses (500mg Mg-sulphate should be 4.06mmoles) discussion line 7: “need” instead of “needs” (serum levels) could you please add a citation for the physiological summary in the first discussion section (maybe Dai & Quamme, Phys Rev 2001?7) page 6, second paragraph: FXYD2 instead of FYXD2, CNNM2 instead of CNMM2. page 6, second paragraph: I am not aware of inherited EGFR mutations?! page 6, second paragraph: ATP1A1 could be added. page 6, second paragraph: the possibility of de-novo mutational events (in addition to recessive and dominant) should be mentioned (especially for ATP1A1 and CNNM2). please provide OMIM number and nomenclature for HSH. page 6, third paragraph: patients typically present in the neonatal period and in infancy (ranging from a couple of days to ~8 months, rarely later)\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "50062", "date": "03 Jul 2019", "name": "Lilia Romdhane", "expertise": [ "Reviewer Expertise Rare genetic diseases", "consanguinity", "NGS data analysis", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a molecular investigation of a familial hypomagnesaemia using whole exome sequencing in a consanguineous family from Oman. A novel missense mutation in the TRPM6 gene at homozygous state has been identified in the index patient. A bioinformatic analysis using a modelling approach was performed to explain the pathogenic effect of the novel mutation.\nGeneral comments: The authors have to provide the OMIM numbers of the diseases reported in the manuscript. The version of the human genome used for read alignment and annotation also has to be mentioned. The status of the identified mutation “novel or new” is only provided in the abstract and the conclusion. Is this family the first one reported with the disease from Oman? The result section drastically lacks details. In addition, details on the methods and tools used during this study have to be provided. Please divide the “Methods” section into sub-sections: Example: Whole Exome Sequencing, WES data analysis, Homozygosity mapping, TRMP6 ortholog alignment, Structural modelling …\nSpecific comments:\nQuestion: Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?\nReviwer’s comment: Clinical description of the parents are lacking as it is mandatory to explain why heterozygous siblings show a moderate phenotype and therefore, helps the interpretation of the genetic results. Are the parents also showing abnormal level of magnesium ?\nQuestion: Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?\nReviwer’s comment: Familial hypomagnesaemia (OMIM#602014) is a genetic autosomal recessive disorder caused by mutations in the TRPM6 gene in homozygous or compound heterozygous state. The authors identified a novel missense mutation at homozygous state in the index patient. The parents and the sister and brother were heterozygous. In the absence of the precise clinical description of the parents, the finding of the expression of some clinical symptoms in the brother and sister harboring the mutation at the heterozygous state is not consistent with the autosomal recessive transmission mode of the disease. Therefore, are the parents completely healthy or are they showing some hypomagnesaemia? The authors state that no adults heterozygous for TRPM6 mutations have been reported showing abnormal serum magnesium levels. Is this statement supported by any biochemical dosages in the parents of the studied family? As heteroygous carriers with moderate expression of an autosomal recessive disease have been reported in a consanguineous population (Mokni et al1) the authors have to provide further clinical details of the parents and reformulate their hypothesis. Moreover, as the authors performed a whole exome sequencing, they have access to the completed catalogue of coding variants of the patient. Is the patient harboring other variants of functional effects in the TRPM6 gene and/other candidate genes? in modifier genes? Are these variants present in the brother and sister that help explaining their phenotypes? Lainez et al2, reported a patient with a TRPM6 heterozygous mutation with familial hypomagnesaemia. They explained the phenotype by the presence of a variant, that when combined with the pathogenic mutation, drastically decreases the magnesium level. They supported they statement with functional assay. Therefore, the authors are invited to perform again the bioinformatic analysis of variant annotation and filtering. Consequently, both the result and discussion sections have to be revised.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "50544", "date": "15 Jul 2019", "name": "Abhijit Dixit", "expertise": [ "Reviewer Expertise Renal and neurological genetic disorders. Dysmorphology. Disorders of sex development." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Jamie Willows and colleagues describe the genetic diagnosis of hypomagnesemia with secondary hypocalcemia (HSH) in a consanguineous family, with identification of a novel homozygous TRPM6 missense variant in the proband. The authors have done a good job of placing this rare condition in the context of wider genetic and acquired causes of hypomagnesemia. There is a fair degree of detail in terms of the clinical course and investigations - see comments below for suggested changes.\nThe identification of a missense variant is noteworthy. The authors do not highlight the fact that the vast majority of previously reported TRPM6 mutations result in loss-of-function of the protein. Only a small number of missense mutations have been reported and still fewer have had functional assessment - still showing loss of TRPM6 channel function (Lainez et al., 20141). Functional studies are, however, not easy to undertake and the authors have provided evidence supporting pathogenicity of the novel variant in their family using protein modelling.\nThe most interesting and possibly contentious issue is the identification of neonatal hypomagnesemia in the siblings with heterozygous p.Lys1098Glu variant. My understanding from the literature is that even asymptomatic hypomagnesemia has not been documented in adult carriers of TRPM6 mutations, and therefore symptomatic presentation would be very unusual indeed. The authors attribute the hypomagnesemia in the heterozygous siblings to immaturity of the DCT. The following points, in my view, merit some extra thought:\nThe only serum magnesium value provided for the sibling II:2 is 0.53mmol/L in the context of ‘abnormal eye movements.’ The complex partial seizure is not described and no further definite seizure episode is documented. Is it possible to be certain that this episode represented a symptomatic seizure and suggest a causal relationship with modest hypomagnesemia? Were other investigations (e.g. MRI brain, blood sugar etc.) performed to identify an alternative explanation for the child's paroxysmal episode?\n\nInformation on magnesium levels in the parents would be very important and, if possible, maternal magnesium levels around the time of delivery.\n\nIt is not specified for how long the magnesium supplementation was prescribed in the two children.\n\nIt would be helpful to clarify the timeline of events further. Before exome sequencing revealed the diagnosis, what were the possible diagnostic considerations in the two older siblings?\n\nLastly, some minor changes would further improve the manuscript:\n\nThree letter amino acid nomenclature is preferable to the single letter codes now, i.e. Lys1098Glu instead of K1098E.\n\nIn Table 1, the current magnesium levels of patient II:1 has a decimal point missing. ‘0.4-0.6mmol/L’ is presumably what was intended.\n\nIt is preferable to say ‘second cousins’ rather than ‘second-degree cousins’, as family relationships in degree terms implies parents/siblings and children as first degree relatives and uncles/grandparents as second degree relatives. First cousins would be third degree relatives. Ideally, a proper pedigree should be drawn so that the familial relationship is clear.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "48695", "date": "09 Aug 2019", "name": "Na Zhu", "expertise": [ "Reviewer Expertise statistical study on human genetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors identified that a novel deleterious missense variant is causal to hypomagnesemia. They found this variant from a pediatric patient in consanguineous family. Both parents are heterozyous carriers while the patient is homozygoty carrier. They inferred the pathogenicity of this variant using in silico method. This variant is never seen in any large population datasets, it is predicted to be deleterious using various prediction tools. This variant is also indicated disrupting stabilising TRP domain from the protein structure analysis tool. The call is also verified to be true using chromatogram. Most importantly, TRPM6 has been reported to be associated with hypomagnesemia. Therefore, I agree with the authors conclusion, this variant is a causal variant to this patient.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-666
https://f1000research.com/articles/8-2056/v1	04 Dec 19	{ "type": "Systematic Review", "title": "Measurement properties of the translations of instruments evaluating the subjective effects of tobacco- and nicotine-containing products: a systematic review of the literature", "authors": [ "Catherine Acquadro", "Céline Desvignes-Gleizes", "Nelly Mainy", "Matthew Hankins", "Rolf Weitkunat", "Christelle Chrea", "Céline Desvignes-Gleizes", "Nelly Mainy", "Matthew Hankins", "Rolf Weitkunat", "Christelle Chrea" ], "abstract": "Background: Several instruments are widely used for assessing dependence, craving, withdrawal symptoms, and reinforcing effects in users of tobacco- and nicotine-containing products (TNP), including the Fagerström Test for Nicotine Dependence (FTND), Questionnaire of Smoking Urges, original (QSU) and brief (QSU-b) versions; Minnesota Nicotine Withdrawal Scale, original (MNWS) and revised (MNWS-R) versions; and Cigarette Evaluation Questionnaire, original (CEQ) and modified (mCEQ) versions. Although these instruments have been translated extensively, their translations and corresponding measurement properties have not been systematically assessed. This study aimed to (1) identify the translations of these instruments for which psychometric properties have been published, (2) describe the methods used for translation, and (3) describe the measurement properties and the context in which these translations were evaluated (e.g., target population and TNP used). Methods: Embase and MEDLINE databases were systematically searched. Results: While no information could be found for the CEQ/mCEQ, several translations were available for the remaining instruments: FTND, 25; QSU and QSU-b, 4 each; QSU (12-item version), 1; MNWS, 4; and MNWS-R, 1. Cigarette smokers represented the main target population in which the validation studies were conducted. Information about the translation process was reported for 25 translations. In most cases, the properties of the translations mirrored those of the originals. Differential item functioning was explored in only one case. Conclusions: There are few publications describing the measurement properties of the translations of the FTND, QSU/QSU-b, and MNWS/MNWS-R. None of these translations have been validated for TNPs other than cigarettes, which suggests the need for greater development and validation of instruments in this area.", "keywords": [ "Fagerström Test for Nicotine Dependence", "Questionnaire of Smoking Urges", "Minnesota Nicotine Withdrawal Scale", "Cigarette Evaluation Questionnaire", "translations", "measurement properties", "cross-cultural equivalence", "tobacco research" ], "content": "List of abbreviations\n\nABOUT: Assessment of Behavioral OUtcomes related to Tobacco and nicotine products; BAI: Beck Anxiety Inventory; BDI: Beck Depression Inventory; CDER: Center for Drug Evaluation and Research; CDS: Cigarette Dependence Scale; CEQ: Cigarette Evaluation Questionnaire; CESDS: Center for Epidemiological Studies Depression Scale; CO: carbon monoxide; COSMIN: COnsensus-based Standards for the selection of health Measurement Instruments; CTP: Center for Tobacco Products; CTT: classical test theory; DIF: differential item functioning; DSM: Diagnostic and Statistical Manual; F: factor; FDA: US Food and Drug Administration; FTND: Fagerström Test for Nicotine Dependence; IRT: item-response theory; mCEQ: Modified Cigarette Evaluation Questionnaire; MNWS: Minnesota Nicotine Withdrawal Scale; MNWS-R: revised version of the Minnesota Nicotine Withdrawal Scale; MRTP: Modified risk tobacco product; PREP: potential reduced exposure products; NDSS: Nicotine Dependence Syndrome Scale; PRO: Patient-reported outcomes; PROQOLID: Patient-Reported Outcome and Quality of Life Instruments Database; Q: question; QSU: Questionnaire of Smoking Urges; QSU-b: brief version of the Questionnaire of Smoking Urges; TDS: Tobacco Dependence Scale (TDS); TNP: tobacco- and nicotine-containing products.\n\n\nIntroduction\n\nOn June 22, 2009, the US Congress enacted a legislation (US Congress, 2009) that granted the US Food and Drug Administration (FDA) the authority to regulate tobacco products and the advertising and promotion of such products. In March 2012, the FDA Center for Tobacco Products (CTP) issued a draft guidance regulating applications for modified risk tobacco products (MRTPs) (US Department of Health and Human Services, 2012). This draft guidance mandates that applications must include scientific evidence about the effects of the products on tobacco-use behavior among current tobacco users. In particular, the guidance clearly states that submissions should present “nonclinical and/or human studies to assess the abuse liability and the potential for misuse of the product as compared to other tobacco products on the market.” In this guidance, the FDA defines abuse liability as “the likelihood that individuals will develop physical and/or psychological dependence on the tobacco product.” Physical dependence encompasses a growing tolerance to product use and/or the inception of withdrawal symptoms when product use cessation occurs. Psychological dependence is mainly characterized by craving and persistent tobacco-seeking and tobacco-use behaviors.\n\nSeveral authors (Carter et al., 2009; Hanson et al., 2009; Institute of Medicine, 2012) have extensively reviewed measures and methods for assessing dependence, craving, withdrawal symptoms, and reinforcing effects in tobacco- and nicotine-containing product (TNP) users. They have identified some measures either widely used or recommended in tobacco research for the evaluation of tobacco products in general and MRTPs in particular. The most commonly quoted are the Fagerström Test for Nicotine Dependence (FTND) (Fagerström, 1978; Fagerström, 2012; Heatherton et al., 1991), Questionnaire of Smoking Urges (QSU) (Kozlowski et al., 1996; Tiffany & Drobes, 1991), Minnesota Nicotine Withdrawal Scale (MNWS) (Cox et al., 2001; Hughes, 2017; Hughes & Hatsukami, 1986; Hughes, 1992; Hughes & Hatsukami, 1998), and Cigarette Evaluation Questionnaire (CEQ) (Cappelleri et al., 2007; Rose et al., 1998; Westman et al., 1992). In terms of tobacco dependence assessment, the US Institute of Medicine report (2012) acknowledges that the FTND appears to contribute to a more precise estimation of dependence than the “Diagnostic and Statistical Manual of Mental Disorders” criteria. Regarding withdrawal symptoms, the same report mentions the MNWS as a well-characterized measure for assessing reduction of withdrawal symptoms. In their paper describing traditional tools and methods for abuse liability assessment, Carter et al. (2009) make references to the FTND for assessing the magnitude of nicotine dependence, the MNWS for assessing nicotine withdrawal signs and symptoms, and the QSU for measuring craving. In their review on questionnaires for measuring the subjective effects of potential reduced exposure products (PREP), Hanson et al. (2009) conclude that the most widely used scale has been the MNWS or its revised version (MNWS-R), followed by the QSU. They recommend that, at a minimum, these two scales should be included in a battery of assessment tests for PREPs. In addition, the authors also mention the CEQ and its modified version (mCEQ) as being widely used.\n\nTable 1 describes these measures (FTND, QSU, MNWS, and CEQ) and their evolution over time (QSU-brief [QSU-b], MNWS-R, and mCEQ).\n\nAbbreviations: DSM: Diagnostic and Statistical Manual.\n\nThe content and measurement properties of the original versions of these four measures in different settings and populations have been documented in the literature (Buckley et al., 2005; Burling & Burling, 2003; Cappelleri et al., 2005; Cappelleri et al., 2007; Cox et al., 2001; Davies et al., 2000; Etter & Hughes, 2006; Fagerström et al., 2012; Haddock et al., 1999; Heatherton et al., 1991; Hudmon et al., 2005; Hughes, 2017; Hughes & Hatsukami, 1986; Hughes, 1992; Hughes & Hatsukami, 1998; Hughes et al., 2004; Kozlowski et al., 1994; Kozlowski et al., 1996; Okuyemi et al., 2007; Payne et al., 1994; Pomerleau et al., 1994; Radzius et al., 2003; Rose et al., 1998; Sledjeski et al., 2007; Steinberg et al., 2005; Tiffany & Drobes, 1991; Toll et al., 2006; Toll et al., 2004; Weinberger et al., 2007; West & Ussher, 2010; West et al., 2006; Westman et al., 1992). However, in the context of globalization of tobacco consumption (with an estimate of almost 972 million smokers in the world in 2012 (Ng et al., 2014) and internationalization of tobacco control (Reubi & Berridge, 2016), it is fundamental to obtain information about the measurement properties of the translations of these self-report instruments since it is crucial to ensure cross-cultural equivalence between the original versions and their translations (Petersen et al., 2003; Regnault & Herdman, 2015; US Department of Health and Human Services, 2009).\n\nThe objectives of this paper were:\n\n1. To identify translations of the FTND, QSU/QSU-b, MNWS/MNWS-R, and CEQ/mCEQ for which psychometric properties are available;\n\n2. To describe the methods used for translation;\n\n3. To describe the measurement properties and the context in which these translations were evaluated (i.e., study design, target population, and TNP used by the study population).\n\n\nMethods\n\nEmbase and MEDLINE databases were searched (in March 2018) with no limitation in timeframe, by using the following keywords and Boolean operators: (1) translation OR language OR version or cross-cultural valid* OR internal consistency OR Cronbach’s alpha OR reliability OR validation OR responsiveness OR validity, combined (AND) with (2) QSU OR Questionnaire on Smoking Urges OR Cigarette Evaluation Questionnaire OR Fagerström Test for Nicotine Dependence OR FTND OR Fagerström Test for Cigarette Dependence OR Minnesota Nicotine Withdrawing Scale OR MNWS. The combination of (1) and (2) was limited to Abstract, Human research, and English. We screened reference lists to identify supplemental pertinent studies.\n\nAbstracts retrieved through the search strategy were reviewed and excluded if they (1) did not refer to the instruments of interest; (2) referred to the original version of the instruments of interest; or (3) referred to a translation used (a) in an epidemiological or behavioral context (i.e., not reporting measurement properties) or (b) for validating another measure and not for assessing/reporting the internal consistency or structural validity of the instruments of interest. Conference abstracts were excluded.\n\nThe reference lists of the papers considered for inclusion were reviewed, and articles of interest were included if they (a) referred to a translation for which internal consistency or structural validity was assessed at minimum or (b) provided additional information on an existing translation identified through the first round of review.\n\nTwo independent reviewers performed the selection. Initial data were extracted by one reviewer and then reviewed (and complemented if needed) by another.\n\nWe used the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) categorization (Mokkink et al., 2010a; Mokkink et al., 2010b; Mokkink et al., 2010c; Mokkink et al., 2018), to classify the measurement properties as follows: reliability, validity, and responsiveness to change. A fourth category, sensitivity and specificity, was added where appropriate (e.g., when the instrument was used for screening).\n\nReliability is described as the overall consistency of a measure, i.e., the degree to which scores for subjects who have not changed are the same when the measurement is repeated over time [test–retest reliability], is done with different evaluators on the same occasion [inter-rater reliability] or with the same evaluator on different occasions [intra-rater reliability]). As for internal consistency reliability, this estimate assesses the consistency of scores across items within a measurement instrument.\n\nValidity is the degree to which an instrument measures what it is supposed to measure and includes the following:\n\nContent validity: The extent to which the content of a questionnaire is an appropriate manifestation of the construct to be assessed. The key features are whether or not the items are relevant and that not important concept is missing, i.e., that the measure is comprehensive. As this review deals with translations, this part will include a description of the translation process and whether or not, on a qualitative level, the content of some items was changed to reflect cultural aspects.\n\nConstruct validity: The extent to which the scores of a measure are in accordance with hypotheses based on the assumption that the questionnaire accurately measures the construct to be measured (Mokkink et al., 2010a). We have included the following aspects in construct validity:\n\nStructural validity: The degree to which the scores of an instrument are an adequate reflection of the dimensionality of the construct to be measured (Mokkink et al., 2010a). Factor analysis should be performed to confirm the number of subscales present in a questionnaire.\n\nHypothesis testing: The extent to which an instrument relates to other instruments in a way that is expected if it is accurately measuring the supposed construct (i.e., in accordance with predefined hypotheses about the correlation or differences between the measures). We have included the following aspects in this category:\n\n– The degree to which the instrument scores correlate with changes in instruments assessing similar constructs, connected but dissimilar constructs, or unconnected constructs.\n\n– The degree to which the instrument scores correlate with biomarkers or measures of TNP consumption (consumption patterns).\n\n– Predictive validity: The degree to which the considered instrument score is predictive of a future outcome or event.\n\nCross-cultural validity: The extent to which the items performance in a translated or culturally adapted instrument appropriately reflects the performance of the items in the original version of the instrument (Mokkink et al., 2010a). This is evaluated using multi-group factor analysis or differential item functioning (DIF) by utilizing data from populations who completed the original version of the questionnaire and its translations.\n\nResponsiveness to change (Hays & Hadorn, 1992) is the ability of an instrument to detect change over time in the construct to be measured. Responsiveness to change is considered an aspect of validity in a longitudinal context.\n\nSensitivity and specificity are used to evaluate the screening performance of a measure. Sensitivity is the proportion of true positives that are exactly identified, whereas specificity relates to the proportion of true negatives correctly identified. Generally, an optimal cutoff point for the score is selected to reduce the sum of false-positive and false-negative results.\n\n\nResults\n\nThe search retrieved 193 articles (Table 2), of which 47 were selected for data extraction. While 46 of these articles described individual investigations on the measurement properties of translated versions of the FTND, QSU/QSU-b, and MNWS/MNWS-R, one was a review of the psychometric properties of the FTND (original and translations) (Meneses-Gaya et al., 2009). No references were found on the CEQ or mCEQ. More details are presented in Figure 1 and Supplementary Material 1 (Table S1), which provides a list of references retrieved and reasons for inclusion/exclusion.\n\nAbbreviations: IC: internal consistency; Struct V: structural validity.\n\nThe search retrieved 34 FTND studies and one review. We identified 25 different FTND translations (Table 3), including two different Arabic versions for Yemeni speakers [Yemeni immigrants in the UK (Kassim, et al., 2012) and inhabitants of Yemen (Nakajima et al., 2012)], two different Chinese versions [Taiwanese (Huang et al., 2009: Huang et al., 2006) and Chinese immigrants in the US (Yamada et al., 2009)], two different Dutch versions (Breteler et al., 2004; Vink et al., 2005), two different Farsi versions (Iran) (Robabeh et al., 2017; Sarbandi et al., 2015), and two different Portuguese versions for Brazil (Carmo & Pueyo, 2002; de Meneses-Gaya et al., 2009).\n\nAbbreviations: CDS-5: Cigarette Dependence Scale (5-item version); CDS-12: Cigarette Dependence Scale (12-item version); DIF: differential item functioning; FTCD: Fagerström Test of Cigarette Dependence; FTND: Fagerström Test for Nicotine Dependence; FTQ: Fagerström Tolerance Scale; HSI: Heaviness of Smoking Index; IRT: item response theory; LCD: Lebanese Cigarette Dependence; NDSS: Nicotine Dependence Syndrome Scale; YACD: Young Adults’ Cigarette Dependence.\n\nIn case of different studies exploring the same language (e.g., Japanese versions reported in two independent studies or French versions reported in French and Swiss studies), we contacted the authors for clarification about the version used (personal communications by email). Authors confirmed using either an existing translation [Japanese (Kawada et al., 2010)] or their own version [French (Chabrol et al., 2003); Portuguese for Brazil (de Meneses-Gaya et al., 2009); Spanish for Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017); and Dutch (Vink et al., 2005)]. In cases where we did not receive any answer, we counted only one version. We did not include the study by de Leon et al. (2003), as the results were based on a combined sample of Spanish and American smokers [study reported in the review by Meneses-Gaya et al. (2009)].\n\nThe main objective of the majority of the studies was to investigate the psychometric properties of the FTND in a specific target language and population. In some instances, the studies aimed to develop other dependence scales by using the FTND as a reference instrument [Spanish (Becoña et al., 2010); French (Etter, 2005); Italian (Grassi et al., 2014); Japanese (Kawada et al., 2010); and Arabic (Salameh et al., 2013; Salameh & Khayat, 2014)] while also exploring the properties of the FTND (Table 3).\n\nSupplementary Material 2 describes the sociodemographic/design characteristics and targeted country/language of each study retrieved (Table S2.1), translation process (steps and people involved if mentioned) as described in the paper (Table S2.2), and measurement properties of the translations (Table S2.3). Combustible cigarettes were the TNP evaluated in all studies. Studies in India had evaluated bidis (cigarettes made locally by wrapping coarse tobacco in dried temburni leaf) [Malayalam (Jayakrishnan et al., 2012) and Hindi (Jhanjee & Sethi, 2010)].\n\nA wide range of populations was investigated (Table S2.1). Participants were recruited from the general population in 24% of the studies (Becoña & Vázquez, 1998; Etter, 2005; John et al., 2004; Klinsophon et al., 2017; Nakajima et al., 2012; Salameh & Khayat, 2014; Stavem et al., 2008; Yamada et al., 2009). While 12% of the studies investigated youth samples, such as students (de Meneses-Gaya et al., 2009; Etter et al., 1997; Nakajima et al., 2012; Salameh & Jomaa, 2014), 20% studied patients with comorbidities or drug dependence (Becoña et al., 2010; de Meneses-Gaya et al., 2009; Osório Fde et al., 2013; Jhanjee & Sethi, 2010; Mikami et al., 1999; Park et al., 2004; Robabeh et al., 2017). The sex ratio was balanced, except in some languages or countries with predominantly male samples [e.g., Iran (Robabeh et al., 2017; Sarbandi et al., 2015), India (Jayakrishnan et al., 2012; Jhanjee & Sethi, 2010), Japan (Kawada et al., 2010; Mikami et al., 1999), Korea (Park et al., 2004), Malaysia (Yee et al., 2011), Taiwan (Huang et al., 2006, Huang et al., 2009), Thailand (Klinsophon et al., 2017), and the Yemeni population in the U.K. (Kassim et al., 2012)]. With regard to cigarette consumption, participants in most countries were light to moderate smokers, except in India (Jhanjee & Sethi, 2010), Italy (Ferketich et al., 2008; Grassi et al., 2014; Svicher et al., 2018), Japan (Kawada et al., 2010; Mikami et al., 1999), Spain (Becoña & Vázquez, 1998), Taiwan (Huang et al., 2006, Huang et al., 2009), and Turkey (Uysal et al., 2004; Uysal et al., 2015), where the samples included a mix of moderate to heavy smokers. The original version of the FTND (Heatherton et al., 1991) was developed with a sample of 254 adult visitors (male, 111; female, 143) at the Ontario Science Centre, who ranged in age from 17 to 77 years (mean age, 33.5 ± 12.7 years) and smoked an average of 20.7 cigarettes per day.\n\nTranslation process. Of the translations, 60% (15 out of 25) were documented with a description of the translation process used to develop each of them (Table S2.2). Out of these 15 translations, only three (Kassim et al., 2012; Uysal et al., 2004; Yee et al., 2011) were presented with a brief report of the difficulties encountered and solutions found. References to guidelines or recommendations were given for six translations (Becoña & Vázquez, 1998; Kassim et al., 2012; Klinsophon et al., 2017; Sarbandi et al., 2015; Yamada et al., 2009; Yee et al., 2011). Descriptions of the translation process were either minimal, with only a mention of the steps performed (Becoña & Vázquez, 1998; Jayakrishnan et al., 2012; Jhanjee & Sethi, 2010; Mikami et al., 1999; Nakajima et al., 2012; Robabeh et al., 2017), or more detailed, with information about the people involved in the process (Etter et al., 1999; Kassim et al., 2012; Klinsophon et al., 2017; Park et al., 2004; Salameh et al., 2013; Salameh & Khayat, 2014; Sarbandi et al., 2015; Uysal et al., 2004; Yamada et al., 2009; Yee et al., 2011). Except for one translation, i.e., French for Switzerland (Etter et al., 1999), all teams included in their process a backward translation step (i.e., the translation of the target language version back to the source language, English).\n\nMeasurement properties All translations were assessed using the classical test theory (CTT), except for the Chinese version developed for immigrants to the US (Yamada et al., 2009), which was assessed only by an item response theory (IRT)-based approach. The Dutch (Breteler et al., 2004) and Italian versions (Svicher et al., 2018) were assessed by IRT supplemented with CTT. Table S2.3 provides detailed information on all properties.\n\nMeasurement equivalence was explored for only one translation—Chinese for immigrants to the US (Yamada et al., 2009)—for which DIF was examined by using IRT. Question (Q) 2 (difficult to refrain) showed a significantly substantial DIF, indicating that users of the Chinese version were more likely to support this item and to report more difficulty in refraining from smoking at several public places even after controlling for the nicotine-dependence level. As this DIF item in the Chinese version contributed minimally at the aggregate level, the authors concluded that its impact was negligible on scale scores. Neither unidimensional nor multidimensional results showed DIF for Q1 (time to first cigarette) or Q3 (cigarette hated most to give up), indicating that these two items are DIF-free. Authors concluded that these two items should be retained in the FTND to enable comparison between Chinese- and English-speaking smokers.\n\nStructural validity was documented for 72% of the translations (18 of 25): Arabic for Lebanon (Salameh et al., 2013; Salameh & Khayat, 2014); Arabic for Yemenites living in the UK (Kassim et al., 2012) and Yemen (Nakajima et al., 2012); Chinese for Taiwan (Huang et al., 2006) and the US (Yamada et al., 2009); Dutch (Breteler et al., 2004); both Farsi versions (Robabeh et al., 2017; Sarbandi et al., 2015); French for France (Chabrol et al., 2003) and Switzerland (Etter, 2005; Etter et al., 1999); German (John et al., 2004); Hindi (Jhanjee & Sethi, 2010); Italian (Grassi et al., 2014; Svicher et al., 2018); Korean (Park et al., 2004); Malay (Yee et al., 2011); Portuguese for Brazil (de Meneses-Gaya et al., 2009); Spanish for Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017); and Turkish (Uysal et al., 2004; Uysal et al., 2015). Six of the translations had a monofactorial structure, similar to their original versions (Heatherton et al., 1991): Arabic (Lebanon) (Salameh et al., 2013); Farsi (Sarbandi et al., 2015); French for France (Chabrol et al., 2003) and Switzerland (Etter, 2005; Etter et al., 1999)]; Italian (Svicher et al., 2018); and Spanish for Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017). The bifactorial structure, described by Haddock et al. (1999) and Radzius et al. (2003) (i.e., one factor labeled “Smoking Pattern” with Q1, Q2, Q4, and Q6 and the other factor labeled “Morning Pattern” with Q3 and Q5, without and with cross-loading of Q1), was replicated in six studies: Chinese for Taiwan (Huang et al., 2006) and the US (Yamada et al., 2009); Farsi (Robabeh et al., 2017); German (John et al., 2004); Korean (Park et al., 2004); and Portuguese for Brazil (de Meneses-Gaya et al., 2009). For the Arabic version for Lebanon (different from the original version), Salameh & Khayat (2014) reported a bifactorial structure in a general population sample but a monofactorial structure in a student sample (Salameh et al., 2013).\n\nInternal consistency was explored for all translations. In monofactorial structures, Cronbach’s alpha ranged from 0.52 (Thai version, Klinsophon et al., 2017) to 0.82 (Portuguese for Brazil, Osório Fde et al., 2013). For the French version (France, Chabrol et al., 2003), Cronbach’s alpha was 0.86 after deletion of Q3, which led the authors to propose a revised FTND without Q3. Removing Q3 improved the Cronbach’s alpha of the French for Switzerland (Etter et al., 1999), Portuguese for Brazil (Carmo & Pueyo, 2002) (slightly), and Turkish (Uysal et al., 2004) versions as well. In the original version (Heatherton et al., 1991), the alpha value was low (0.61), and Q3 loaded less than 0.30 (i.e., 0.23).\n\nTest–retest reliability was assessed for 40% of the translations (10 of 25)—Dutch (Vink et al., 2005); French (Switzerland) (Etter et al., 1999); Farsi (Sarbandi et al., 2015); Japanese (Mikami et al., 1999); Malay (Yee et al., 2011); Malayalam (Jayakrishnan et al., 2012); Norwegian (Stavem et al., 2008); Portuguese for Brazil (Carmo & Pueyo, 2002; de Meneses-Gaya et al., 2009; Osório Fde et al., 2013); Thai (Klinsophon et al., 2017); and Turkish (Uysal et al., 2004)—with coefficients ranging from 0.50 (Yee et al., 2011) to 0.92 (de Meneses-Gaya et al., 2009) and intervals from 1 week (Moreno-Coutiño & Villalobos-Gallegos, 2017; Yee et al., 2011) to 1.8 years (Vink et al., 2005). In comparison, test–retest reliability analyses conducted on the original version showed coefficients ranging from 0.65 (smokers with schizophrenia, interval not specified) (Weinberger et al., 2007) to 0.87 (a sample of young smokers during military training, 6-week interval) (Haddock et al., 1999).\n\nInter-rater reliability was investigated for only one translation—Portuguese for Brazil (de Meneses-Gaya et al., 2009)—with the authors reporting an intraclass coefficient of 0.99 (95% confidence interval [CI]: 0.98–1.0) for two raters (sample size, 40).\n\nCorrelations with biomarkers and consumption patterns were documented for 52% of the translations (13 of 25): Arabic (Lebanon) (Salameh et al., 2013; Salameh & Khayat, 2014); Chinese (Taiwan) (Huang et al., 2006; Huang et al., 2009); Dutch (Vink et al., 2005); Farsi (Sarbandi et al., 2015); French (Switzerland) (Etter et al., 1999); Hindi (Jhanjee & Sethi, 2010); Italian (Ferketich et al., 2008; Grassi et al., 2014); Japanese (Kawada et al., 2010); Korean (Park et al., 2004); Malay (Yee et al., 2011); Malayalam (Jayakrishnan et al., 2012); Norwegian (Stavem et al., 2008); and Spanish [Spain (Becoña & Vázquez, 1998) and Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017)]. Correlations with biomarkers such as exhaled carbon monoxide and salivary and urinary cotinine levels were explored, as were correlations with duration of smoking (years), packs/year, packs/day, age of regular smoking, age of first cigarette, and willingness to pay for a cigarette after a day of abstinence. Correlations with biomarkers ranged from weak to moderate [e.g., CO: r = 0.288 (Salameh & Khayat, 2014) to 0.535 (Salameh et al., 2013)], in agreement with the correlations reported in studies that had used the original FTND [CO: r = 0.210 (Steinberg et al., 2005), 0.40 (Buckley et al., 2005), and 0.59 (Burling & Burling, 2003)].\n\nCorrelations with self-reported instruments investigating similar or dissimilar constructs was explored for 24% of the translations (6 of 25): Farsi (Robabeh et al., 2017); French for Switzerland (Etter et al., 1999); Japanese (Kawada et al., 2010; Mikami et al., 1999); Norwegian (Stavem et al., 2008); and Spanish [Spain (Becoña et al., 2010) and Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017)]. Correlations with the following scales were investigated: Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI) (r = 0.091 and 0.116, respectively) (Moreno-Coutiño & Villalobos-Gallegos, 2017); Cigarette Dependence Scale (CDS) 12 and 5 (r = 0.60 and 0.72, respectively) (Stavem et al., 2008); Diagnostic and Statistical Manual (DSM) diagnostic criteria for nicotine dependence (DSM II-R; r = 0.70) (Mikami et al., 1999); DSM-V nicotine dependence (no correlation) (Robabeh et al., 2017); Nicotine Dependence Syndrome Scale (NDSS) (r = 0.58) (Becoña et al., 2010); Structured Clinical Interview for DSM-IV (r = 0.38) (Becoña et al., 2010); Tobacco Dependence Scale (TDS) (r = 0.352) (Kawada et al., 2010); and withdrawal symptoms (irritability, sensation of relief, and embarrassment: relative validity 93, 100, and 100, respectively) (Etter et al., 1999). For the original version, Pomerleau et al. (1994) showed a correlation with the Classification of Smoking by Motives addictive factor (r = 0.53). No relationship was detected with depression measured by using the Center for Epidemiological Studies Depression Scale (CESDS) (r = -0.24).\n\nPredictive validity was investigated for 12% of the translations (3 of 25): French for Switzerland (Etter, 2005); Italian (Ferketich et al., 2008); and Spanish for Mexico (Moreno-Coutiño & Villalobos-Gallegos, 2017). The FTND predicted abstinence only at 7 weeks (Ferketich et al., 2008) and not in the long term (Etter, 2005; Moreno-Coutiño & Villalobos-Gallegos, 2017). In contrast, Kozlowski et al. (1994) showed that the FTND could predict smoking cessation to a small degree (Study 2: 16-month follow-up, r = -0.11).\n\nSensitivity/specificity (Se/Sp) was explored for 28% of the translations (7 of 25): Chinese for Taiwan (Huang et al., 2009); Italian (Svicher et al., 2018); Japanese (Mikami et al., 1999); Malay (Yee et al., 2011); Portuguese for Brazil (de Meneses-Gaya et al., 2009; Osório Fde et al., 2013); and Spanish for Spain (Becoña et al., 2010). Measures of reference varied among the studies: biomarkers (Huang et al., 2009: salivary cotinine – cutoff score = +4, Se = 76.2%, Sp = 67.5%); status (Svicher et al., 2018); measures of dependence (Becoña et al., 2010: score under the curve, 0.69; Osório Fde et al., 2013: cutoff score = +2, Se = 76%, Sp = 91%; de Meneses-Gaya et al., 2009: cutoff score = +4, Se = 80%, Sp = 74%; Mikami et al., 1999: cutoff score = +5/6, Se = 75%, Sp = 80%); and others (Yee et al., 2011).\n\nResponsiveness to change has never been assessed.\n\nWe retrieved four translations for the QSU, four for the QSU-b, and one for the QSU-12 (Table 4).\n\nAbbreviations: MNWS: Minnesota Nicotine Withdrawal Scale.\n\nTable 5 presents the general characteristics of each study. Combustible cigarettes were the TNP evaluated in all studies. Nearly all studies involved moderate smoker samples, on average. In comparison, the original QSU (Tiffany & Drobes, 1991) and QSU-b (Cox et al., 2001) were developed with heavy smoker samples. Most studies had recruited samples from the general population, excepting studies in Spain [QSU (Cepeda-Benito et al., 2004) and QSU-b (Cepeda-Benito & Reig-Ferrer, 2004)] and the Netherlands [QSU-b (Littel et al., 2011)], which had recruited students. The sex ratio was balanced in all countries except China [QSU-b (Yu et al., 2010)] and Malaysia [QSU-b (Blebil et al., 2015)], where men were predominant (98% and 99%, respectively) and Spain [QSU (Cepeda-Benito et al., 2004)], where the sample comprised a majority of women (81.5%).\n\nAbbreviations: F: female; M: male; NA: not available in this paper (reference to another publication); NS: not specified in this paper; SD: standard deviation; S1: Sample 1; S2: Sample 2.\n\nIn comparison, the original version of the QSU (Tiffany & Drobes, 1991) was developed in a sample of 230 daily cigarette smokers (141 men and 89 women) assigned to one of three levels of deprivation (0, 1, or 6 hours). The mean participant age in each subgroup was 20.91, 20.64, and 22.73 years (SD not shown), and the consumption rate was 23.3, 21.28, and 22.36 cig/day, respectively (SD not shown). The QSU-b (Cox et al., 2001) was developed in two populations: Study 1 included 221 continuing smokers (111 men and 110 women; mean age, 30.23 ± 10.27 years; consumption rate, 26.95 ± 10.68 cig/day), while Study 2 included 112 smokers who were contemplating quitting (49 men and 63 women; mean age, 43.15 ± 11.60 years; consumption rate, 27.82 ± 12.57 cig/day).\n\nTranslation process. The translation process (Table 6) was not described for three translations: German QSU (Müller et al., 2001); French QSU-12 (Dethier et al., 2014); and Dutch QSU-b (Littel et al., 2011). References to guidelines were provided for three translations: Brazilian (Araujo et al., 2006); Chinese (Yu et al., 2010); and Malay (Blebil et al., 2015). The description for the Chinese QSU-b was minimal. Only the Brazilian team had provided some insight into the problems that arose during translation and the solutions they found.\n\nAbbreviations: BT: backward translation; FT: forward translation.\n\n* ?: Not clearly specified in the paper, but recommended in quoted guidelines; *Translation Process referred to as Linguistic Validation Process.\n\nMeasurement properties. Table 7 reports the measurement properties explored for each translation. The results of the Spanish QSU-b (Cepeda-Benito & Reig-Ferrer, 2004) are not included, as the version developed by the authors is not a translation of the QSU-b but a new version with completely different content derived from the QSU, with items being positively keyed. It, therefore, represents a new version not comparable to the original US version of the QSU-b.\n\nAbbreviations: BAI: Beck Anxiety Inventory; Beck Depression Inventory; Cig.: cigarettes; CFA: confirmatory factorial analysis; CO: carbon monoxide; EFA: exploratory factorial analysis; F1: factor 1; F2: factor 2; FTND: Fagerström Test for Nicotine Dependence; FTQ: Fagerström Tolerance Questionnaire; ITC: item-to-total correlation; ns: not significant; PANAS: Positive Affect Negative Affect Scales; PANAS-NA: PANAS Negative Affect; PANAS-PA: PANAS Positive Affect; SHAPS: Snaith–Hamilton Pleasure Scale; VAS: visual analog scale.\n\nMeasurement equivalence using DIF was never assessed. Structural validity was explored for all translations:\n\nQSU: The Brazilian (Araujo et al., 2006) and French (Guillin et al., 2000) versions of the QSU have a similar bifactorial structure as the original (Tiffany & Drobes, 1991) (i.e., with factor 1 [F1] representing a desire and intention to smoke, with smoking anticipated as pleasurable [15 items of which 10 are negatively keyed], and factor 2 [F2] representing an anticipation of relief from negative affect and nicotine withdrawal, with an urgent desire to smoke [11 items positively keyed]). However, the French and Brazilian translations show a difference in the order of factor extraction: Craving (urgent desire to smoke) was extracted first. According to the French authors, the duration of smoking abstinence of the French sample at the time of evaluation (1.5 to 3 hours) might explain the inversion of order of the two factors. Two-thirds of the subjects in the study of Tiffany & Drobes (1991) had been abstinent for 1 hour or less, which might have resulted in lesser craving in the US sample. The Spanish authors (Cepeda-Benito et al., 2004) compared the factorial structures of the original US and translated Spanish versions and found that: (1) a better fit was found with the four-factor and two-factor models than with the one-factor model, and (2) the two-factor model provided a better fit than the four-factor model in both samples. In addition, their data suggested that the presence of mostly negatively worded items in F1 contributed largely to the two-factor structure of the QSU. Analysis with only negative items in F1 greatly improved the model fit in both data sets. According to the authors, these findings question the original interpretation of the nature of the dimensions measured by the two factors of the QSU.\n\nQSU-b: The authors of the Dutch and Malay versions reported differences from the original QSU-b (Cox et al., 2001), which, when used to derive a global measure of craving, showed high internal consistency across settings and provided reliable assessment of the desire to smoke. In contrast, factor analyses generated two instances of verbal report of craving. F1 represented a strong desire and intention to smoke, with smoking perceived as satisfying for active smokers, when an anticipation of relief from negative affect and an urgent desire to smoke was reflected by F2.\n\nThe first factor (F1) of the Dutch version (Littel et al., 2011) corresponded with the second factor (F2) of the English QSU-b (items 2, 4, 5, 8, and 9). F2 comprised items 1, 3, 6, 7, and 10. Items 2 and 5 loaded strongly on F1, whereas they had originally cross-loaded. The authors attributed this discrepancy to language differences. Items 2 and 5 (i.e., “nothing would be better than smoking a cigarette right now” and “all I want right now is a cigarette”) communicate quite extreme statements, especially when literally translated into Dutch. F2 corresponded with the first factor of the original QSU-b, although, in the Dutch study, items 1 and 6 loaded on two factors. Again, Dutch language might be an explanation for these items loading on both factors. Items 1 and 6 include the words “desire” and “urge.” Although phrases such as “I have a strong desire or urge for a cigarette,” might be used in Dutch, it is far more common to use less potent expressions (e.g., “I would like/fancy a cigarette”). Nevertheless, items 1 and 6 are less extreme than the items assigned to F1. The authors did not add “anticipation of pleasure from smoking” to the name of this factor, because the subscale was not significantly correlated with either positive or negative affect.\n\nIn the Malay version (Blebil et al., 2015), factors 1 and 2 corresponded with those in the original version, with items 2 and 5 strongly loading on F2. The authors attributed this cross loading to the phrase “strong urge” conveying extreme utterances when literally translated into Malay.\n\nInternal consistency was explored for all translations of the QSU. The alpha values for QSU F1 and F2 ranged from 0.89 (Guillin et al., 2000) to 0.96 (Araujo et al., 2006) and 0.87 (Cepeda-Benito et al., 2004) to 0.92 (Araujo et al., 2006), respectively, in line with the values of the original version, in which scores representing these two factors demonstrated strong internal consistency (Cronbach’s alpha = 0.95 and 0.93, respectively). The Cronbach’s alpha values of the QSU-b translations in Malay (Blebil et al., 2015), Dutch (Littel et al., 2011), and Chinese (Yu et al., 2010) were 0.81, 0.83, and 0.92, respectively. When scored as a 10-item scale, the original QSU-b demonstrated high reliability as a measure of global craving in both initial and follow-up sessions (alpha = 0.89 and 0.87, respectively).\n\nTest–retest reliability was never assessed.\n\nCorrelations with biomarkers and consumption patterns were explored for all translations (QSU/QSU-b) except the German (Müller et al., 2001) and Spanish (Cepeda-Benito et al., 2004) QSU versions, with the latter focusing only on factor analysis.\n\nQSU: As in the original development, correlations with biomarkers were not explored in the translations. The correlation with number of cigarettes per day was weak to moderate [Brazilian version: F1 and F2 QSU, r = 0.197 and 0.182, respectively (Araujo et al., 2006); French QSU, r = 0.54 (Guillin et al., 2000) (not explored in the original)].\n\nQSU-b: Correlation with exhaled CO was explored for the Malay version (r = 0.0024; not explored in the original development) (Blebil et al., 2015). The correlation with number of cigarettes per day was weak [Dutch QSU-b, r = 0.25 (Littel et al., 2011); Malay QSU-b, r = 0.30 (Blebil et al., 2015) (not explored in the original)].\n\nCorrelations with self-reported measures exploring similar or dissimilar constructs were not studied in the French (Guillin et al., 2000) or Spanish versions of the QSU (Cepeda-Benito et al., 2004) but were investigated in other translations (QSU/QSU-b):\n\nQSU: Correlations with the Craving Visual Analog Scale (VAS) German (Müller et al., 2001) and Brazilian (Araujo et al., 2006) versions, Fagerström Tolerance Questionnaire (FTQ) (Müller et al., 2001), FTND (Araujo et al., 2006), BAI, and BDI (Araujo et al., 2006) were explored. Weak correlations were found with the FTQ (F2, r = 0.16), FTND (F1, r = 0.244; F2, r = 0.163), BAI (F1, r = 0.249), and BDI (F1 r = 0.249). For the original QSU, correlations with the Withdrawal Symptoms Checklist (WSC) and Mood Form were assessed. F1 showed a strong correlation with the craving subscale of the WSC.\n\nQSU-b: Correlations with the FTND Malay and Dutch versions (Blebil et al., 2015; Littel et al., 2011), craving scales Dutch and Chinese versions (Littel et al., 2011; Yu et al., 2010), desire and urge VAS, the Positive Affect Negative Affect Scale (PANAS), and the Snaith–Hamilton Pleasure Scale (SHAPS) Dutch version (Littel et al., 2011) were explored. Weak correlations were found with the FTND (Malay: r = 0.24; Dutch: r = 0.14), and strong correlations were found with the craving scales (Chinese: r = 0.75; Dutch: r = 0.80). Only a weak correlation with the Negative Affect scale of the PANAS was found for F1 of the Dutch version (r = 0.25). Weak correlations were found with the SHAPS (F1 and F2 of the Dutch version: r = 0.23 and 0.22, respectively). Correlations with the Mood Form were assessed in the original version.\n\nResponsiveness to change, predictive validity, sensitivity, and specificity were not assessed.\n\nFour studies were retrieved for the MNWS (Table 8), corresponding to three translations of the MNWS (nine-item version) into Chinese (China) (Yu et al., 2010), Korean (Kim et al., 2007), and Malay (Blebil et al., 2014) and one translation of the MNWS-R and MNWS (eight-item version) into Italian (same paper) (Svicher et al., 2017).\n\nIn the MNWS, there were variations in the items included in the original versions that were used as a basis for translation. The Chinese version was based on the MNWS developed by Cappelleri et al. (2005), while the Italian version was based on that by Hughes (1992). The Malay and Korean versions included “impatience” and were based on the MNWS developed by Jorenby et al. (1996) (Table 9).\n\n Based on symptoms listed in Jorenby et al., 1996.\n\nTable 10 presents the general characteristics of each study. Combustible cigarettes were the TNP evaluated in all studies. Most subjects reported previous attempts to quit, except in the Malay sample (Blebil et al., 2014), where 77% of the subjects had not attempted to quit previously.\n\nAbbreviations: F: female; M: male; MNWS: Minnesota Nicotine Withdrawal Scale; MNWS-R: Minnesota Nicotine Withdrawal Scale revised version; NS: not specified in this paper; SD: standard deviation.\n\nAll studies were run with moderate smoker samples, on average, except for the study involving Koreans living in the US, which had recruited light smokers (Kim et al., 2007). In comparison, the original MNWS was developed with heavy smokers (Hughes & Hatsukami, 1986). Mean participant age ranged from 34 to 47.7 years. Men were predominant in all studies except in that in Italy, where women were slightly preponderant (59%) (Svicher et al., 2017).\n\nTranslation process. All four papers provided a description of the translation process used to develop each translation (Table 11). Only the Korean version (Kim et al., 2007) presented a brief report of the difficulties encountered and solutions found. References to guidelines or recommendations were given for all translations except for the Italian version (Svicher et al., 2017). Descriptions of the translation process were detailed for all translations except the Chinese version (Yu et al., 2010).\n\nAbbreviations: BT: backward translation; FT: forward translation; NS: not specified.\n\n?: Not clearly specified in the paper, but recommended in quoted guidelines; *Translation Process referred to as Linguistic Validation Process.\n\nMeasurement properties. Table 12 reports the measurement properties explored for each translation. All translations were assessed for structural validity, with a one-factor structure reported for the Italian MNWS eight-item version (Svicher et al., 2017) and the Malay nine-item version (Blebil et al., 2014).\n\nAbbreviations: ASI-3: Anxiety Sensitivity Index-3; AUDIT: Alcohol Use Disorder Identification Test; CI: confidence interval; cig.: cigarettes; CO: carbon monoxide; Coeff.: coefficient; CTT: classical test theory; FTCD: Fagerström Test for Cigarette Dependence; F1: factor 1; F2: factor 2; FTND-M: Malay version of the Fagerström Test for Nicotine Dependence; ICC: intraclass coefficient; IRT: item-response theory; ITC: item-to-total correlation; ns: not significant; PANAS: Positive and Negative Affect Schedule; SCS: Smoker Complaint Scale; SERS: Self-Efficacy in Resisting Smoking Scale\n\nA two-factor structure was reported for the Chinese version of the MNWS nine-item version (Yu et al., 2010) and the Korean nine-item version (Kim et al., 2007). The structure of the Chinese version was identical to the two-factor structure of the original version reported by Cappelleri et al. (2005): negative effect (F1, four items: depressed mood; irritability, frustration, or anger; anxiety; and difficulty concentrating), insomnia (F2, two items: difficulty going to sleep and difficulty staying asleep), and three single items (craving, restlessness, and increased appetite). A review of the items showed a slight discrepancy in those used as originals, with impatience listed in the Korean version but not in the Chinese, where insomnia represents two items (difficulty going to sleep and difficulty staying asleep). For the Korean version, F1 represented early-occurring disorders in mental functioning, and F2 represented disorders in physiological functioning and late-occurring disorders in mental functioning (i.e., increased appetite, disturbed sleep, depression, and impatience), explaining 66% of the variance. A two-factor structure was also reported in the Italian MNWS-R.\n\nInternal consistency was explored for all translations, with Cronbach’s alpha values of 0.85 (Italian MNWS eight-item version) and 0.91 (Malay nine-item version) reported for the monofactorial structure. In comparison, the Cronbach’s alpha values of the original eight-item MNWS explored by Toll et al. (2007) were 0.80 (abstinence study), 0.83 (framing study), and 0.82 (naltrexone + patch study) at the initial time point after quitting. Internal consistency was not evaluated by Jorenby et al. (1996). With regard to the bifactorial structure, only a global alpha value was provided for the Chinese version (0.90). Cappelleri et al. (2005) reported alpha values ranging from 0.76 to 0.87 for the negative domain and 0.71 to 0.83 for the insomnia domain in the studies and time assessed.\n\nMeasurement equivalence using DIF was never assessed.\n\nIn total, 75% percent of the translations (3 of 4; Italian, Korean, and Malay) were assessed for test–retest reliability, with coefficients ranging from 0.51 (Kim et al., 2007) to 0.88 (Blebil et al., 2014) and intervals from 1 (Blebil et al., 2014; Kim et al., 2007) to 3 (Svicher et al., 2017) months.\n\nCorrelations with consumption patterns and biomarkers were reported for three translations: Chinese, Italian, and Malay. Correlations with self-reported measures of dependence, craving, and anxiety were explored for all translations (Table 12).\n\nResponsiveness to change, predictive validity, sensitivity, and specificity were not assessed.\n\n\nDiscussion\n\nGiven the globalization of tobacco research and control, we expected to retrieve more than 25, 9, 4, and 1 translations—documented with measurement properties—of the FTND, QSU/QSU-b, MNWS, and MNWS-R, respectively. A search on the Patient-Reported Outcome Quality Of Life Instrument Database (PROQOLID™; https://eprovide.mapi-trust.org/) reveals that there are 19 translations available for the QSU-b and 12 for the MNWS-R. No information was retrieved for the FTND, as it is not listed or documented on PROQOLID™.\n\nAmong the 19 QSU-b translations listed on PROQOLID™ and indicated as translated by Mapi, we found two versions overlapping with our research (i.e., the Dutch and Spanish versions for Spain), indicating that there are at least two versions of the QSU-b in those languages. PROQOLID™ does not mention whether those 19 translations have undergone any evaluation of their measurement properties. Our review found three translations of the QSU-b (Chinese, Dutch, and Malay) and one Spanish version derived from the Spanish QSU, with content different from the original QSU-b.\n\nA review of the information on the Vermont University website reveals the existence of seven translations of the MNWS (nine-item versions: Chinese, Czech, Dutch, Japanese, and Korean; 11-item version: Arabic; 14-item version: Portuguese) and five of the MNWS-R (Bosnian, German, Italian, Russian, and Spanish), all listed under the acronym of MNWS. A comparison of the information on PROQOLID™ and the Vermont University website reveals no overlap for the MNWS-R translations, raising the number to 17 translations available. Our review found only one MNWS-R translation (Italian) with documented measurement properties.\n\nOverall, our review showed that the process used to elaborate the translations of the FTND, QSU/QSU-b, and MNWS/MNWS-R is not standardized and is not always documented. This could prove to be a challenge if the US FDA CTP aligns any future guidance with the 2009 patient-reported outcome (PRO) guidance published by the US FDA Center for Drug Evaluation and Research (CDER) (US Department of Health and Human Services, 2009). Appendix VIII of this PRO guidance outlines that all translation documents should be provided for CDER review. This includes a report on the process(es) used and challenges encountered during the translation process, especially during testing of the translation on the target population.\n\nThere is a great heterogeneity in the populations recruited for each study, in terms of sample characteristics (e.g., sex [samples with mixed sexes or a majority of male subjects], age, or level of cigarette consumption [light to heavy smokers]). In addition, depending on the objectives of the research teams, not all properties are explored for each language. Our review showed that most of the translations have measurement properties similar to their original versions.\n\nResults concerning the MNWS might be found to be problematic as the number of items used are different across the languages (e.g., eight for Italian vs. nine for Chinese, Korean, and Malay), and there is variation in which items are included, making it impossible to compare scores across studies. Translations of the FTND revealed the same concerns about the structural validity of the original version (mono vs. bifactorial), low internal consistency [except for some versions (de Meneses-Gaya et al., 2009; Osório Fde et al., 2013], and validity of Q3 (hated the most to give up). Those translation measurement outcomes questioning the validity of the original instrument may raise questions about the need to modify the content of the original. In this context, there is a well-known precedent: The International Quality of Life Assessment Project (Aaronson et al., 1992) is a notorious example of the development of translations of a PRO measure (i.e., the Short Form-36 [SF-36] Health Survey, which led the developer to change the original US instrument). The development and validation of the translated versions contributed to improvements in item wording and response categories and to the creation of the SF-36v2 Health Survey (Ware, 2007).\n\nOur review showed that cross-cultural validity is rarely explored. Measurement equivalence using an IRT-based approach for examining DIF is almost never applied. This is a concern, as it might make it difficult to know if the scores obtained with the translations of these measures are comparable across languages and cultures and whether or not it is relevant to aggregate data from studies conducted in different countries. Based on their extensive experience in cross-cultural evaluation, researchers from the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Group have suggested that DIF should be part of the validation of questionnaire translations (Petersen et al., 2003; Scott et al., 2006; Scott et al., 2009). In their research, DIF analyses were conducted to identify items answered differently by language administration, reflecting either linguistic issues (e.g., imperfect translation) or cultural differences. Overall, they showed that, although most of the EORTC QLQ-C30 items seemed to have good linguistic equivalence, several scales presented highly conflicting results for some translations. They implied that some of these effects might be substantial enough to affect the outcomes of clinical studies, as translation differences in an item could result in clinically important differences at the scale score level.\n\nFinally, our review showed that none of the translations has been validated with candidate MRTPs, indicating that more research is needed to comply with regulatory recommendations on the development of self-reported measures for use in labeling claims (US Department of Health and Human Services, 2009).\n\nThe main limitation of our research lies in its descriptive design. We did not provide insights on the quality of the translated versions (i.e., ratings on the translation process and the quality of the measurement properties) (Schellingerhout et al., 2011; Thoomes-de Graaf et al., 2016). Further research is needed to critically appraise the quality of the translations and guide researchers in their search for the best translation for their studies.\n\nThese results showing (1) discrepancies between the number of translations available, with and without documented information about their measurement properties, (2) heterogeneity in the scope of measurement properties explored and in the characteristics of the samples recruited, and (3) lack of validation with TNPs other than conventional cigarettes raise the need for generating a new initiative with two main goals (i.e., information and development).\n\nFirst, implementation of a centralized repository for measurement instruments (original version and translations) with a licensing structure (endorsed by the developers of the originals) would enable researchers to have access to the most up-to-date information about measures (i.e., development story and psychometric properties). By identifying existing translations and documenting them, this implementation might also help prevent the development of multiple translations for the same language and avoid concerns about which translation to use (Anfray et al., 2009). Furthermore, engaging the developers of the original versions in this process might help protect the integrity of each measurement instrument included (Anfray et al., 2018).\n\nSecond, if the original versions and translations of these measures are not appropriate for candidate MRTPs, fit-for-purpose measurement instruments (i.e., concept-driven instruments providing interpretable outcomes for the intended purpose) should be developed to enable comparison of combustible and noncombustible products on the same risk continuum. A similar initiative was launched several years ago, which led to the development of the ABOUT™ Toolbox (Assessment of Behavioral OUtcomes related to Tobacco and nicotine products) (Chrea et al., 2018). The measurement instruments included in this Toolbox are at different degrees of development. With their dissemination on ePROVIDE™ , researchers will be able to use instruments that are (1) developed and validated with state-of-the-art scientific methods to be psychometrically sound, straightforward to implement in clinical and population-based studies, and easy to interpret; (2) created to be relevant and applicable across the whole spectrum of TNPs and across various populations; and (3) designed to enhance standardization and comparison of data on perception and behaviors toward MRTPs across academic, industry, and public health research communities.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework. Measurement properties of the translations of instruments evaluating the subjective effects of tobacco- and nicotine-containing products: a systematic review of the literature. https://doi.org/10.17605/OSF.IO/3Z2EV (Acquadro, 2019).\n\nThis project contains the following extended data:\n\nSupplementary file 1: List of the 193 references retrieved during the literature search.\n\nSupplementary file 2: Tables presenting detailed information on the FTND translations.\n\n– Table S2.1. Sociodemographic/design characteristics and targeted country/language of studies evaluating the measurement properties of the translations.\n\n– Table S2.2. Description of translation processes used (steps and people involved if mentioned) for the FTND translations.\n\n– Table S2.3. Measurement properties of the translations of the FTND.\n\nOpen Science Framework: PRISMA checklist for: “Measurement properties of the translations of instruments evaluating the subjective effects of tobacco- and nicotine-containing products: a systematic review of the literature” https://doi.org/10.17605/OSF.IO/3Z2EV (Acquadro, 2019).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Author contributions\n\n\n\nCA, CC and NM conceived the idea of the manuscript. CDG performed the literature search. CA and CDG reviewed the abstracts and selected the articles for review. CA developed and drafted the manuscript. CC, CDG, MH, NM, and RW reviewed critically the manuscript. All authors read and approved the final version of the manuscript.\n\n\nReferences\n\nAaronson NK, Acquadro C, Alonso J, et al.: International Quality of Life Assessment (IQOLA) Project. 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PubMed Abstract \| Publisher Full Text\n\nSarbandi F, Niknami S, Hidarnia A, et al.: Psychometric properties of the Iranian version of the Fagerström Test for Nicotine Dependence and of Heaviness of Smoking Index. J Res Health. 2015; 5: 96–103. Reference Source\n\nSchellingerhout JM, Heymans MW, Verhagen AP, et al.: Measurement properties of translated versions of neck-specific questionnaires: a systematic review. BMC Med Res Methodol. 2011; 11: 87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott NW, Fayers PM, Aaronson NK, et al.: The practical impact of differential item functioning analyses in a health-related quality of life instrument. Qual Life Res. 2009; 18(18): 1125–1130. PubMed Abstract \| Publisher Full Text\n\nScott NW, Fayers PM, Bottomley A, et al.: Comparing translations of the EORTC QLQ-C30 using differential item functioning analyses. Qual Life Res. 2006; 15(6): 1103–1115. PubMed Abstract \| Publisher Full Text\n\nSledjeski EM, Dierker LC, Costello D, et al.: Predictive validity of four nicotine dependence measures in a college sample. Drug Alcohol Depend. 2007; 87(1): 10–19. PubMed Abstract \| Publisher Full Text\n\nStavem K, Rogeberg OJ, Olsen JA, et al.: Properties of the Cigarette Dependence Scale and the Fagerström Test of Nicotine Dependence in a representative sample of smokers in Norway. Addiction. 2008; 103(9): 1441–1449. PubMed Abstract \| Publisher Full Text\n\nSteinberg ML, Williams JM, Steinberg HR, et al.: Applicability of the Fagerström Test for Nicotine Dependence in smokers with schizophrenia. Addict Behav. 2005; 30(1): 49–59. PubMed Abstract \| Publisher Full Text\n\nSvicher A, Beghè A, Mangiaracina G, et al.: Factor Analysis and Psychometric Properties of the Minnesota Nicotine Withdrawal Scale and the Minnesota Nicotine Withdrawal Scale-Revised: Italian Version. Eur Addict Res. 2017; 23(3): 157–162. PubMed Abstract \| Publisher Full Text\n\nSvicher A, Cosci F, Giannini M, et al.: Item Response Theory analysis of Fagerström Test for Cigarette Dependence. Addict Behav. 2018; 77: 38–46. PubMed Abstract \| Publisher Full Text\n\nThoomes-de Graaf M, Scholten-Peeters GG, Schellingerhout JM, et al.: Evaluation of measurement properties of self-administered PROMs aimed at patients with non-specific shoulder pain and \"activity limitations\": a systematic review. Qual Life Res. 2016; 25(9): 2141–2160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTiffany ST, Drobes DJ: The development and initial validation of a questionnaire on smoking urges. Br J Addict. 1991; 86(11): 1467–1476. PubMed Abstract \| Publisher Full Text\n\nToll BA, Katulak NA, McKee SA: Investigating the factor structure of the Questionnaire on Smoking Urges-Brief (QSU-Brief). Addict Behav. 2006; 31(7): 1231–1239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToll BA, McKee SA, Krishnan-Sarin S, et al.: Revisiting the factor structure of the questionnaire on smoking urges. Psychol Assess. 2004; 16(4): 391–395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToll BA, O’Malley SS, McKee SA, et al.: Confirmatory factor analysis of the Minnesota Nicotine Withdrawal Scale. Psychol Addict Behav. 2007; 21(2): 216–225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nU.S. Congress: Family Smoking Prevention and Tobacco Control Act of 2009, Public Law 111-31, 123 Stat. 1776 (June 22, 2009). 2009. Reference Source\n\nU.S. Department of Health and Human Services, Food and Drug Administration, Center for Tobacco Products: Guidance for industry - Modified risk tobacco product applications - Draft Guidance. 2012. Reference Source\n\nU.S. Department of Health and Human Services, Food and Drug Administration: Guidance for Industry. Patient-reported outcome measures: use in medical product development to support labeling claims. 2009. Federal Register, 74, 65132–65133. Reference Source\n\nUysal MA, Kadakal F, Karşidağ C, et al.: Fagerstrom test for nicotine dependence: reliability in a Turkish sample and factor analysis. Tuberk Toraks. 2004; 52(2): 115–121. PubMed Abstract\n\nUysal MA, Öztuna D, Bahadir A, et al.: Psychometric properties of the Turkish version of the Fagerström test for nicotine dependence. Tuberk Toraks. 2015; 63(4): 250–256. PubMed Abstract \| Publisher Full Text\n\nVink JM, Willemsen G, Beem AL, et al.: The Fagerström Test for Nicotine Dependence in a Dutch sample of daily smokers and ex-smokers. Addict Behav. 2005; 30(3): 575–579. PubMed Abstract \| Publisher Full Text\n\nWare J: User’s Manual for the SF-36v2 Health Survey. Second ed. Lincoln, RI QualityMetric Incorporated. 2007.\n\nWeinberger AH, Reutenauer EL, Allen TM, et al.: Reliability of the Fagerström Test for Nicotine Dependence, Minnesota Nicotine Withdrawal Scale, and Tiffany Questionnaire for Smoking Urges in smokers with and without schizophrenia. Drug Alcohol Depend. 2007; 86(2–3): 278–282. PubMed Abstract \| Publisher Full Text\n\nWest R, Ussher M: Is the ten-item Questionnaire of Smoking Urges (QSU-brief) more sensitive to abstinence than shorter craving measures? Psychopharmacology (Berl). 2010; 208(3): 427–432. PubMed Abstract \| Publisher Full Text\n\nWest R, Ussher M, Evans M, et al.: Assessing DSM-IV nicotine withdrawal symptoms: A comparison and evaluation of five different scales. Psychopharmacology (Berl). 2006; 184(3–4): 619–627. PubMed Abstract \| Publisher Full Text\n\nWestman EC, Levin ED, Rose JE: Smoking while wearing the nicotine patch: Is smoking satisfying or harmful? Clinical Research. 1992; 40: 871A. Reference Source\n\nWild D, Grove A, Martin M, et al.: Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: report of the ISPOR Task Force for Translation and Cultural Adaptation. Value Health. 2005; 8(2): 94–104. PubMed Abstract \| Publisher Full Text\n\nYamada H, Acton GS, Tsoh JY: Differential item functioning of the English and Chinese versions of the Fagerstrom Test for Nicotine Dependence. Addict Behav. 2009; 34(2): 125–133. PubMed Abstract \| Publisher Full Text\n\nYee A, Ng CG, Rusdi AR: Validation of the Malay version of Fagerström Test for Nicotine Dependence (FTND-M) among a group of male staffs in a university hospital. Malaysian Journal of Psychiatry. 2011; 20: 1. Reference Source\n\nYu X, Xiao D, Li B, et al.: Evaluation of the Chinese versions of the Minnesota nicotine withdrawal scale and the questionnaire on smoking urges-brief. Nicotine Tob Res. 2010; 12(6): 630–634. PubMed Abstract \| Publisher Full Text" }	[ { "id": "77538", "date": "23 Mar 2021", "name": "Jennifer Rose", "expertise": [ "Reviewer Expertise Nicotine dependence", "statistics", "data analysis", "psychometric assessment." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reports on a comprehensive review of the psychometric properties of the translated versions of some frequently used nicotine dependence (ND) assessments. The range of psychometric properties used to test equivalence of the translated ND measures was as rigorous as possible, given that there was considerable variability in the translation process and reporting of psychometric properties across the studies.\nThe review found a surprising lack of rigorous assessment of measurement invariance (differential item functioning) for translated versions compared to the original English language versions. This is mentioned in the Discussion, but it seems important to more strongly emphasize that the psychometric properties examined in this study can evaluate measurement equivalence, but not measurement invariance. Although factor structure, reliability, and validity appeared similar for most of the translations, it is possible that the meaning of the ND measure was different for the translated versions compared to the English version. This potential lack of measurement invariance might be a reason for the psychometric differences that were found. Without testing for measurement invariance, the possibility of unaccounted noninvariance remains a potential confounder in subsequent statistical analyses using the translated ND measures, and may be an important factor in frequent failure to replicate findings across studies using the same measures.\nIn the Introduction, the authors indicate that it is crucial to ensure “cross-cultural equivalence between the original versions and their translations”. This suggests that observed translation differences in the ND measures are related to cultural differences. However, the reasons for the differences are indeterminate. The differences may be attributed to cultural differences, but also they could be due to changes in the wording in the translated measures, study design, or a myriad number of other factors. This should be clarified.\nThe drawback of such a comprehensive review is that it is very difficult to keep track of all the psychometric differences, and there is no clear way to determine when the identified differences may be considered significant or meaningful and when they may be insignificant or inconsequential. In addition, the supplemental tables provide more information, but they were not easy to locate. Perhaps there could be more detailed instructions for accessing those tables.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "136674", "date": "30 May 2022", "name": "Maria Rosaria Galanti", "expertise": [ "Reviewer Expertise Population and clinical studies of tobacco use and cessation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors did a considerable effort in tracing and summarizing scientific articles related to the use of instruments for the assessment of nicotine dependence in languages different from the one in which they were originally created.\nDespite this effort, I find the review quite difficult to follow because it is lacking a definite focus, ultimately resulting in point-by point description of what was or was not done in different studies. Indeed, the discussion of the results leads to rather general statements, some of which could be anticipated: not standardized processes translation/adaptation; heterogeneity of populations assessed; deficit of analysis of differential item functioning (DIF); lack of cross-cultural validation.\nI suggest that the authors re-organize and summarize their valuable material along one (or both) of the following themes:\nCompare the procedures and the analyses done for the several language-specific versions of the scales with pre-defined gold-standards (as described in the methods section), for instance how should a sound translation be conducted and reported; how should content, construct validity, predictive validity, etc. be minimally investigated. As a result of this summary the reader will be informed on how many of the x-language versions of the y-instrument meet these criteria.\n\nCompare the psychometric properties of the different language-specific translations of a given instrument with the original one(s) analyzing the possible reasons for discrepancies, above all the characteristics of the involved samples. This work would be of crucial importance, since smoking populations are increasingly diversifying across different countries, with the emergence of both low-frequency smokers and heavy smokers with psychiatric co-morbidity. Suggested comparisons: population vs. clinical samples; heavy smokers vs light/moderate smokers.\n\nApart from these overarching considerations I have two additional points:\nTitle: would be better to rephrase it so that it would be clear that “subjective effects” refer to symptoms of dependence (even dizziness after smoking is a subjective effect).\n\nI wonder why the Cigarette Dependence Scale (Etter et al., 20031) was not included in the search/results. I am aware of at least one study (Rydell et al., 20162) dealing with both translation of the original instrument and its adaptation to smokeless tobacco use.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2056
https://f1000research.com/articles/8-1459/v1	19 Aug 19	{ "type": "Software Tool Article", "title": "HDCytoData: Collection of high-dimensional cytometry benchmark datasets in Bioconductor object formats", "authors": [ "Lukas M. Weber", "Charlotte Soneson" ], "abstract": "Benchmarking is a crucial step during computational analysis and method development. Recently, a number of new methods have been developed for analyzing high-dimensional cytometry data. However, it can be difficult for analysts and developers to find and access well-characterized benchmark datasets. Here, we present HDCytoData, a Bioconductor package providing streamlined access to several publicly available high-dimensional cytometry benchmark datasets. The package is designed to be extensible, allowing new datasets to be contributed by ourselves or other researchers in the future. Currently, the package includes a set of experimental and semi-simulated datasets, which have been used in our previous work to evaluate methods for clustering and differential analyses. Datasets are formatted into standard SummarizedExperiment and flowSet Bioconductor object formats, which include complete metadata within the objects. Access is provided through Bioconductor's ExperimentHub interface. The package is freely available from http://bioconductor.org/packages/HDCytoData.", "keywords": [ "benchmarking", "high-dimensional cytometry", "Bioconductor", "ExperimentHub", "clustering", "differential analyses" ], "content": "Introduction\n\nBenchmarking analyses are frequently used to evaluate and compare the performance of computational methods, for example by users interested in selecting a suitable method, or by developers to demonstrate performance improvements of a newly developed method. A critical part of any benchmark is the selection of appropriate benchmark datasets1,2. In some cases, suitable publicly available datasets may be found in the literature. Alternatively, new experimental or simulated datasets containing a known ground truth may be created by the authors of the benchmark1,2.\n\nHigh-dimensional cytometry refers to a set of recently developed technologies that enable measurement of expression levels of up to dozens of proteins in hundreds to thousands of cells per second, using targeted antibodies labeled with various types of reporter tags. This includes multi-color flow cytometry, mass cytometry (or CyTOF), and sequence-based cytometry (or genomic cytometry). Due to the large size and high dimensionality of the resulting data, numerous computational methods have been developed for analyzing these datasets3. Many of these methods are based on the fundamental concept of analyzing cells in terms of cell populations, for example using clustering to define cell populations, or detecting differential cell populations between conditions.\n\nIn our previous work, we have collected a number of benchmark datasets to evaluate methods for clustering4 and differential analyses5 in high-dimensional cytometry data. This includes publicly available datasets previously published by other groups or our experimental collaborators, as well as new semi-simulated datasets that we generated. In these previous publications, we recorded links to original data sources and made all data available via FlowRepository6. FlowRepository is a widely used resource in the cytometry community, which has also been used by other authors to distribute benchmark datasets (e.g., 7,8). However, downloading and loading the data from these sources for further analysis in R requires customized code and matching of metadata (e.g., sample information), which can hinder accessibility and reproducibility.\n\nHere, we introduce the HDCytoData package, which provides a resource for re-distributing high-dimensional cytometry benchmark datasets through Bioconductor’s ExperimentHub9, in order to improve accessibility. ExperimentHub provides a flexible platform for hosting datasets in the form of R/Bioconductor objects, which can be directly loaded within an R session. HDCytoData provides datasets in the form of standard SummarizedExperiment and flowSet Bioconductor object formats10–12, which include all required metadata within the objects and facilitate interoperability with R/Bioconductor-based workflows. We envisage that these datasets will be useful for future benchmarking studies, as well as other activities such as teaching, examples, and tutorials. The package is extensible, allowing new datasets to be contributed by ourselves or other researchers in the future. The package is freely available from http://bioconductor.org/packages/HDCytoData.\n\n\nMethods\n\nThe benchmark datasets currently included in the HDCytoData package consist of experimental and semi-simulated data, and can be grouped into datasets useful for benchmarking algorithms for (i) clustering and (ii) differential analyses. Table 1 and Table 2 provide an overview of the datasets.\n\nFor more details on these datasets, see Table 2 in 4, or the HDCytoData help files.\n\nFor more details on these datasets, see Supplementary Note 1 in 5, or the HDCytoData help files.\n\nThe raw datasets were collected from various sources (Table 1 and Table 2), and have been extensively reformatted and documented for inclusion in the HDCytoData package. Each dataset is stored in both SummarizedExperiment and flowSet formats, since these are the most commonly used R/Bioconductor data structures for high-dimensional cytometry data. The objects each contain one or more tables of expression values, as well as all required metadata. Following standard conventions used for cytometry data13, rows contain cells, and columns contain protein markers. Row metadata includes sample IDs, group IDs, patient IDs, reference cell population labels (where available), and labels identifying ‘spiked in’ cells (where available). Column metadata includes channel names, protein marker names, and protein marker classes (cell type or cell state). Note that raw expression values should be transformed prior to performing any downstream analyses. Standard transformations include the inverse hyperbolic sine (asinh) with cofactor parameter equal to 5 for mass cytometry or 150 for flow cytometry data (14, Supplementary Figure S2); several other alternatives also exist15.\n\nMost of these datasets include a known ground truth, enabling the calculation of statistical performance metrics. The ground truth information consists of reference cell population labels for the clustering datasets, and labels identifying computationally ‘spiked in’ cells for the differential analysis datasets. The datasets without a ground truth instead consist of experimental datasets that contain a known biological signal, which can be used to evaluate methods in qualitative terms; i.e., whether methods can reproduce the known biological result.\n\nExtensive documentation is available via the help files for each dataset — including descriptions of the datasets, details on accessor functions required to access the expression tables and metadata, and links to original sources. In addition, reproducible R scripts demonstrating how the formatted SummarizedExperiment and flowSet objects were generated from the original raw data files are included within the source code of the package. New datasets may be contributed by ourselves or other authors by providing (i) formatted SummarizedExperiment and flowSet objects containing the data as well as all necessary metadata, (ii) reproducible R scripts showing how the formatted objects were generated from the original raw data files, and (iii) comprehensive documentation.\n\nThe HDCytoData package can be installed by following standard Bioconductor package installation procedures. All datasets listed in Table 1 and Table 2 are available in Bioconductor version 3.10 and above. Minimum system requirements include a recent version of R (3.6 or later; this paper was prepared using R version 3.6.1), on a Mac, Windows, or Linux system. Example installation code is shown below.\n\n\n\nOnce the HDCytoData package is installed, the datasets can be downloaded from ExperimentHub and loaded directly into an R session using only a few lines of R code. This can be done by either (i) referring to named functions for each dataset, or (ii) creating an ExperimentHub instance and referring to the dataset IDs. Example code for each option for one of the datasets is shown below. Note that each dataset is available in both SummarizedExperiment and flowSet formats. After an object has been downloaded, the ExperimentHub client stores it in a local cache for faster retrieval. For more details on accessing ExperimentHub resources, refer to the ExperimentHub vignette available from Bioconductor.\n\n\n\nOnce the datasets have been downloaded and loaded, they are available to the user as R objects within the R session. They can then be inspected and manipulated using standard accessor and subsetting functions (for either the SummarizedExperiment or flowSet object class). Example code to inspect a SummarizedExperiment is displayed below. For more details on how to load and inspect datasets, including the expected output from each function shown here, refer to the HDCytoData vignette available from Bioconductor.\n\n\n\nDocumentation describing each dataset is available in the help files for the objects, which can be accessed using the standard R help interface, as shown below.\n\n\n\n\nUse cases\n\nThe datasets currently included in the HDCytoData package (Table 1 and Table 2) can be used to benchmark methods for either (i) clustering or (ii) differential analyses. In addition, these datasets may be useful for other activities such as teaching, examples, and tutorials (e.g., demonstrating how to use a new computational tool).\n\nFor benchmarks using the clustering datasets (Table 1), performance can be evaluated by calculating metrics such as the mean F1 score or adjusted Rand index, which measure the similarity between two sets of cell labels (i.e., the cluster labels and the ground truth reference cell population labels)1. For examples (including reproducible R code), see the evaluations in our previous study4. An additional visual example is displayed in Figure 1, which compares the performance of three different dimensionality reduction algorithms (principal component analysis [PCA], t-distributed stochastic neighbor embedding [tSNE]22,23, and uniform manifold approximation and projection [UMAP]24,25) in visually separating the known cell populations in the Levine_32dim dataset (see Table 1). R code to reproduce Figure 1 using data downloaded from the HDCytoData package is available at http://github.com/lmweber/HDCytoData-example.\n\nThis example compares performance (in visual terms) of three dimensionality reduction algorithms — principal component analysis (PCA), t-distributed stochastic neighbor embedding (tSNE), and uniform manifold approximation and projection (UMAP) — for representing known cell populations in the Levine_32dim dataset (Table 1).\n\nFor benchmarks using the differential analysis datasets (Table 2), methods can be evaluated by their ability to recover the known differential signals, either in quantitative terms using the ground truth spike-in cell labels (for the semi-simulated datasets), or in qualitative terms (for the experimental datasets). The differential signals consist of either differential abundance of cell populations, or differential states within cell populations (i.e., differential expression of additional functional markers within cell populations), providing conceptually distinct differential analysis tasks. For examples (including reproducible R code), see the evaluations in our previous study5.\n\n\nSummary\n\nThe HDCytoData package is an extensible resource providing streamlined access to a number of publicly available benchmark datasets used in our previous work on high-dimensional cytometry data analysis. Datasets are provided in standard Bioconductor object formats, and are hosted on Bioconductor’s ExperimentHub platform. By facilitating access to these datasets, we hope they will be useful for other researchers interested in designing rigorous benchmarks for method development or other computational analyses, as well as other activities such as teaching, examples, and tutorials.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: http://bioconductor.org/packages/HDCytoData\n\nSource code available from: https://github.com/lmweber/HDCytoData\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.336284726\n\nLicence: MIT License", "appendix": "Grant information\n\nLMW was supported by a Forschungskredit (Candoc) grant from the University of Zurich [FK-17-100].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors thank Mark D. Robinson (University of Zurich, Switzerland) for supervising the projects where these datasets were previously used for benchmarking, and feedback on the manuscript; and Lori Shepherd (Bioconductor Core Team and Roswell Park Cancer Institute, Buffalo, NY, USA) for assistance in making the datasets available through Bioconductor’s ExperimentHub.\n\n\nReferences\n\nWeber LM, Saelens W, Cannoodt R, et al.: Essential guidelines for computational method benchmarking. Genome Biol. 2019; 20(1): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMangul S, Martin LS, Hill BL, et al.: Systematic benchmarking of omics computational tools. Nat Commun. 2019; 10(1): 1393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaeys Y, Van Gassen S, Lambrecht BN: Computational flow cytometry: helping to make sense of high-dimensional immunology data. Nat Rev Immunol. 2016; 16(7): 449–462. 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PubMed Abstract \| Publisher Full Text\n\nBodenmiller B, Zunder ER, Finck R, et al.: Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat Biotechnol. 2012; 30(9): 858–867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Maaten L, Hinton G: Visualizing data using t-SNE. J Mach Learn Res. 2008; 9: 2579–2605. Reference Source\n\nvan der Maaten L: Accelerating t-SNE using tree-based algorithms. J Mach Learn Res. 2014; 15: 3221–3245. Reference Source\n\nMcInnes L, Healy J, Melville J: UMAP: Uniform manifold approximation and projection for dimension reduction. arXiv, 1802.03426 (v2). 2018. Reference Source\n\nBecht E, McInnes L, Healy J, et al.: Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol. 2019; 37(1): 38–44. PubMed Abstract \| Publisher Full Text\n\nWeber LM, Soneson C: lmweber/HDCytoData: Version from paper (Weber and Soneson, 2019) (Version v1.5.12). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3362847" }	[ { "id": "52681", "date": "28 Aug 2019", "name": "Shila Ghazanfar", "expertise": [ "Reviewer Expertise statistics", "high throughput genomics", "transcriptomics", "R software", "high-dimensional data analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWeber and Soneson have written a software article presenting HDCytoData (currently version 1.4.0), a Bioconductor package aimed at making multiple high-dimensional cytometry (HDC) datasets available in a consistent R friendly format as either SummarizedExperiment or flowSet objects. The authors have framed this as an aid for facilitating benchmarking studies and for use for examples or tutorials in future. HDCytoData provides links to these datasets through ExperimentHub and includes some helpful commands for downloading such data. Currently eight datasets are included in the package which are accessed with easy-to-use function calls in the workspace.\nWith this in mind, I have some comments & questions below that could improve the manuscript and useability of the HDCytoData package.\nThis approach effectively duplicates the data from flowRepository and into the Bioconductor ExperimentHub ecosystem, is it more worthwhile to provide the functions to extract and process the data from the original flowRepository source? This manuscript could contain more motivation for hosting the processed data versus providing functions to download + process the data from flowRepository.\n\nIs it extensible to other additional characteristics? e.g. data arising from imaging mass cytometry with further measured features, or similar. The authors could discuss the breadth of experimental data types they imagine HDCytoData to encompass or accept from contributors.\n\nThe authors should discuss the continued curation of the data within the HDCytoData package and mention how it behaves in case of changes or updates to the 'original' data in flowRepository. Describe further how one can contribute their dataset(s)?\n\nI'm confused as to the framing of this package as principally for benchmarking studies. Whilst it's an important aspect of understanding and improving methodology, users of this package may be more interested in a convenient and consistent way of loading the flow cytometry data altogether, especially so for some integrative analysis of multiple HDC datasets.\n\nIt's unclear how large the data files are that are being downloaded into the local cache, ideally the user would want to know this information before going ahead and downloading it.\n\nIt's not clear in this manuscript how one would remove the data once it's no longer needed, or how to clear the cache. It appears that it's assumed users are also fairly familiar with the ExperimentHub interface. The authors should make it more clear what level of experience they imagine package users should have, i.e. who are they aiming the software towards?\n\nIt would be useful to have a bulk download to cache, or possibly a bulk load to workspace option for these datasets.\n\nIs there a functionality to switch from SummarizedExperiment object to flowSort format? If this exists in another package it should be pointed to.\n\nFor the Bodenmiller data, I was surprised to find that the help file for Bodenmiller_BCR_XL_SE() says there are measurements for 24 proteins but the exprs assay has 35 columns, looking at the colData, features are classed into \"type\" and \"state\" with the remainder having a class of \"none\". Some of these columns do not appear to measure specifically protein abundance but rather cell-specific (as opposed to sample-specific) features, for instance \"Cell_length\". Have the authors anticipated this type of extra information and how it would fit into the SummarizedExperiment or flowSort object? The slot name \"exprs\" suggests that the data within this slot should be some molecular quantities, should these other features go into the rowData() slot instead? Or furthermore, whether ideally for downstream analysis (such as differential expression) these extra columns should be discarded? (Note these extra columns than what is listed in the function help descriptions appears for multiple datasets.)\n\nHow do you ensure that there is enough information available here to be able to accurately normalise/standardise the data, especially so for flow cytometry data, given the particular combinations of fluorescent markers associated with the proteins, and potential overlap of the fluorescence for these markers?\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5052", "date": "04 Dec 2019", "name": "Lukas Weber", "role": "Author Response", "response": "Thank you for your comments and suggestions. We have updated the text, vignettes, and help files to clarify each of the issues raised above. Below are also responses to the specific questions: (1) Code to process the raw .fcs files from FlowRepository into the SummarizedExperiment and flowSet formats is provided in the ‘make-data’ scripts saved in the ‘inst/scripts’ directory in the source code of the HDCytoData package. Here we have followed the standard setup for ExperimentHub packages – i.e. processed data objects that are ready to load into R, together with reproducible scripts saved in ‘inst/scripts’ – as described in the ExperimentHub vignettes. We believe this is a useful setup for these datasets. FlowRepository is primarily intended as a permanent public repository for .fcs files associated with peer-reviewed publications, which cannot be updated. FlowRepository is also primarily accessed via the web interface, so it would be much less user-friendly to only provide scripts that re-format the .fcs files after downloading. Providing these datasets as pre-formatted SummarizedExperiment and flowSet objects makes them much more easily accessible for users. (2) In principle, any data types that can be formatted into SummarizedExperiment and flowSet formats could be added to the package. However, we believe it makes sense to keep the scope of the package relatively limited, to facilitate modularity and maintainability. For now, we plan to include only the current set of technologies, although in the future it may make sense to develop similar packages for other data types (e.g. imaging mass cytometry) (see Summary). (3) According to the policies of FlowRepository, original .fcs files stored in FlowRepository cannot be updated after publication of the associated peer-reviewed paper. Similarly, data objects stored in ExperimentHub can only be updated manually by contacting the ExperimentHub maintainers. Therefore, we do not expect any major updates to the datasets currently stored in the HDCytoData package (except possibly minor bug fixes). We have included some additional text explaining this. We have also included a new vignette on “Contribution guidelines” (see comments for Reviewer 2). (4) While users could indeed use the HDCytoData package to load these datasets in a consistent way for other purposes, we believe the main use cases for these particular datasets are for benchmarking and teaching / examples / tutorials. These datasets are well-characterized and have been studied in a number of previous publications, making them ideal for benchmarking. Formatting the datasets into consistent SummarizedExperiment and flowSet formats requires significant effort, so we expect this will mainly be worthwhile for datasets that can be re-used a number of times, e.g. for benchmarking. (5-7) We have updated the text, main vignette, and help files to mention the size of the data files. The datasets range in size from 2.4 MB to 194.5 MB. We have also explained how to clear the local download cache, and updated the text to mention the expected level of experience with Bioconductor. We are not aware of a bulk download option in the ExperimentHub interface, so we have not included this. (If this functionality were added in the future, we believe it would better belong in the ExperimentHub package than in HDCytoData.) (8) There is no simple way to convert between the SummarizedExperiment and flowSet formats. This is one of the major contributions of this package – we have pre-processed the datasets into both of these formats (with reproducible code saved in the ‘inst/scripts’ directory), so that users do not need to do this manually. We have included additional text to mention this. (9) The additional columns of raw data (which are labeled as “none” in the “marker_class” column) contain additional information from the raw .fcs files from the mass cytometry machine, including barcodes for sample deconvolution, and event length and DNA content to identify live single cells. These columns are usually stored in the expression matrices in the original raw .fcs files, so we have also left them in the objects, e.g. for users who wish to check the pre-processing steps. We labeled these columns as “marker_class = none” to make them easier to identify, especially for users who are not already familiar with mass cytometry data. We have updated the help files to clarify that these columns are not needed for downstream analyses. (10) Compensation for fluorescence spillover has already been performed by the original authors of the flow cytometry datasets, so users of these datasets do not need to perform this. However, users still need to apply a transformation (e.g. arcsinh), which we have described in the vignettes and help files." } ] }, { "id": "52679", "date": "02 Sep 2019", "name": "Laurent Gatto", "expertise": [ "Reviewer Expertise Computational biology", "method development", "research software engineering." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWeber and Soneson present HDCytoData, a Bioconductor data package providing pre-formatted high-dimensional cytometry data. The preparation of the datasets as SummarizedExperiment and flowSet objects makes these amendable for benchmarking, a crucial step when developing new methods.\nMy main comment centres around the contribution of new data. While the curated/formatted data in the package have already been useful to the authors in their previous work, the ambition is to make it possible for others to benefit from them and, to enable this in the longer term, to expand the package with additional data. These contributions are anticipated to come from the original authors and, ideally, also by new contributors.\nThe contribution procedure, while crucial, (1) isn't described very clearly and, at least in its current form, (2) only applies to seasoned R users/programmers. These two points constitute a serious barrier to external contributions.\nIndeed, the only information that is provided are a list of three required artefacts (objects, scripts and documentation), without details as to how to produce these, nor how to provide them. I would suggest to add a 'How to contribute' vignette to the package, describing all these aspects, including an example for one of the existing data. I would also suggest to include a contribution code of conduct, given that external contributions are explicitly advertised.\nI would suggest asking new contributors to send a pull request (PR) on Github, with possible alternative methods for those that aren't familiar with GitHub. The use a PR provides traceability (as opposed to an email, for instance) and publicly recognises the external contribution, as PRs are publicly recorded on GitHub. I would also suggest to explicitly define how external contributions are to be acknowledged in the contribution guide (for example addition as a 'contributor' in the DESCRIPTION file).\nThese additions will clarify what is expected for a contribution to be considered, how it will be managed by the authors, and how it will be acknowledged, thus hopefully facilitating the process.\nMinor suggestions:\nHow can a potential user find out if/when new data have been added to the package? While `?HDCytoData` gives a list of dataset, a function returning a vector or dataframe with dataset names and possibly some annotation would be useful for programmatic access (given here that `data(package = \"HDCytoData\")` doesn't work for data on ExperimentHub).\n\nIt could be useful to expand the 'Use cases' section with (1) example calculations of the F1 scores and Rand indices for the clustering example and (2) adding a similar short example for the differential analysis use case.\n\nI am curious as to why the content of the lmweber/HDCytoData-example isn't included as a vignette in the HDCytoData package (and thus lacking the usual control and documentation that comes with R packages).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5053", "date": "04 Dec 2019", "name": "Lukas Weber", "role": "Author Response", "response": "Thank you for your comments and suggestions. As suggested, we have provided significant additional material on the procedure for contributing new datasets. We have expanded the section in the text on external contributions, and added an additional Bioconductor vignette titled “Contribution guidelines”. This vignette describes the required files (data objects, scripts, documentation, metadata), as well as the submission procedure. We have requested that contributions be submitted via GitHub issues and pull requests, clarified the acknowledgment procedure, and added a code of conduct. Regarding the minor suggestions, we have also (i) updated the main vignette and package help file to show how to programmatically retrieve a data frame of all available datasets, and (ii) added a new vignette titled “Examples and use cases”, which includes the example from the previous repository (https://github.com/lmweber/HDCytoData-example), as well as new examples showing how to use the datasets in the HDCytoData package to evaluate clustering performance (e.g. adjusted Rand index) and perform differential analyses." } ] } ]	1	https://f1000research.com/articles/8-1459
https://f1000research.com/articles/8-264/v1	07 Mar 19	{ "type": "Research Article", "title": "Investigating gateway effects using the PATH study", "authors": [ "Peter N Lee", "John S Fry", "John S Fry" ], "abstract": "Background: A recent meta-analysis of nine cohort studies in youths reported that baseline ever e-cigarette use strongly predicted cigarette smoking initiation in the next 6-18 months, with an adjusted odds ratio of 3.62 (95% confidence interval 2.42-5.41). A recent review of e-cigarettes agreed there was substantial evidence for this “gateway effect”. However, the number of confounders considered in the studies was limited, so we investigated whether the effect might have resulted from inadequate adjustment, using Waves 1 and 2 of the Population Assessment of Tobacco and Health study. Methods: Our main analyses considered Wave 1 never cigarette smokers who, at Wave 2, had information available on smoking initiation. We constructed a propensity score for ever e-cigarette use from Wave 1 variables, using this to predict ever cigarette smoking. Sensitivity analyses accounted for use of other tobacco products, linked current e-cigarette use to subsequent current smoking, or used propensity scores for ever smoking or ever tobacco product use as predictors. We also considered predictors using data from both waves to attempt to control for residual confounding from misclassified responses. Results: Adjustment for propensity dramatically reduced the unadjusted odds ratio (OR) of 5.70 (4.33-7.50) to 2.48 (1.85-3.31), 2.47 (1.79-3.42) or 1.85 (1.35-2.53), whether adjustment was made as quintiles, as a continuous variable or for the individual variables. Additional adjustment for other tobacco products reduced this last OR to 1.59 (1.14-2.20). Sensitivity analyses confirmed adjustment removed most of the gateway effect. Control for residual confounding also reduced the association. Conclusions: We found that confounding is a major factor, explaining most of the observed gateway effect. However, our analyses are limited by small numbers of new smokers considered and the possibility of over-adjustment if taking up e-cigarettes affects some predictor variables. Further analyses are intended using Wave 3 data which should avoid these problems.", "keywords": [ "Cigarettes", "Confounding", "E-cigarettes", "Gateway effects", "Modelling", "Propensity score" ], "content": "Introduction\n\nIn youths, use of e-cigarettes (“vaping”) and cigarette smoking are strongly associated, as shown in, e.g. Canada (Aleyan et al., 2018), France (Dautzenberg et al., 2016), Great Britain (Eastwood et al., 2015), Korea (Lee et al., 2014) and Poland (Goniewicz et al., 2014), as well as the U.S. (e.g. Cooper et al., 2016; Dutra & Glantz, 2014; Wills et al., 2017). Since vaping significantly reduces exposure to harmful constituents compared to smoking (National Academies of Sciences Engineering and Medicine, 2018), one might expect risks from vaping to be substantially lower (Nutt et al., 2014). While the benefits of introducing e-cigarettes would seem clear for smokers switching to e-cigarettes who would have continued smoking otherwise, for established smokers who are helped to quit, and for individuals who would otherwise have started smoking who start vaping instead, there are possible downsides. While risk increases may be modest for smokers intending to quit who vape instead, and for smokers who vape but retain their usual cigarette consumption, there is concern vaping may encourage individuals to start smoking who would otherwise not have done so.\n\nThis so-called “gateway effect” is our focus, following recent analyses (Soneji et al., 2017) based on nine U.S. cohort studies in young people linking previous vaping to subsequent initiation of smoking. This publication reported that among baseline never-smokers, ever vaping at baseline strongly predicted initiation in the next six to 18 months, with an odds ratio (OR) of 3.62 (95% confidence interval (CI) 2.42-5.41) after adjustment for various predictors of initiation. Similarly baseline past 30-day vaping also predicted subsequent 30-day cigarette use (OR 4.25, 95% CI 2.52-7.37).\n\nA recent review of e-cigarettes (National Academies of Sciences Engineering and Medicine, 2018) considered there was “substantial evidence” of a gateway effect, noting the “wide range of covariates” the relevant studies had considered, and thought it “unlikely” that confounding entirely accounts for the association, as reductions in the association following adjustment were not consistently observed.\n\nWhile some studies (Barrington-Trimis et al., 2016; Primack et al., 2015; Primack et al., 2016) do report that the association increases following adjustment, many more (Best et al., 2018; Conner et al., 2018; Hammond et al., 2017; Hornik et al., 2016; Leventhal et al., 2015; Loukas et al., 2018; Lozano et al., 2017; Miech et al., 2017; Spindle et al., 2017; Unger et al., 2016; Watkins et al., 2018; Wills et al., 2017) report a decrease. Furthermore, though adjusted associations are usually statistically significant, adjustment is often limited. Factors never considered include, for example, school performance, parental smoking and peer attitudes to smoking. Information from a large cohort study which collected data on many factors would therefore clearly be useful, as would gaining some insight into the extent of bias resulting from misclassification of such variables.\n\nHere we report detailed analyses of the gateway effect based on Wave 1 (2013–2014) and Wave 2 (2014–2015) of the Population Assessment of Tobacco and Health (PATH) study (Berry et al., 2019b; Hyland et al., 2017), a longitudinal cohort study in the U.S. supported by federal funds. The databases provide extensive information on tobacco product use and on many other factors possibly linked to smoking initiation. At each Wave, data are separately collected for youths aged 12–17 and adults aged 18+, and our analyses, which concern smoking initiation by youths, use the youth data of each Wave, plus Wave 2 data for adults previously youths at Wave 1.\n\n\nMethods\n\nThe main analysis considers those who had never smoked cigarettes by Wave 1 and who, at Wave 2, had information available on whether initiation of cigarette smoking had occurred. Use of other tobacco products is not considered. For a youth to be considered, data should be available on each of five demographics (age, sex, Hispanic origin, race, region) and on vaping.\n\nThe analyses, which relate ever having vaped by Wave 1 to initiation of cigarette smoking by Wave 2, after adjustment for factors linked to e-cigarette use recorded at Wave 1, was conducted in two steps.\n\nStep 1. In step 1, Wave 1 data were used to develop a propensity score for e-cigarette use based on the five demographic variables and on 60 smoking predictor variables selected from a much longer list. As described more fully in the Extended Data, we ignored questions only asked in a population subset, only really relevant to smokers, or of dubious relevance. Also, where many related questions were asked, attention was limited to those seemingly more likely to be relevant.\n\nWe used a logistic regression model where the propensity for vaping (Pi) for a youth i (i = 1 to n) was linked to various smoking predictors xij (j = 1 to m) by\n\n\n\nFor a given set of predictors, we refer to the value of the term on the left as the propensity score.\n\nAll logistic regression analyses were weighted by the person-level weights provided on the PATH database, with the weights normalized to sum to 1.\n\nIntroducing all 65 variables simultaneously into the model would have involved two problems. First, as the analysis required individuals with complete data on all variables, substantial information may be lost. Second, analyses including very many variables sometimes fail to solve. We therefore introduced variables in stages, using groups of conceptually-related variables, with missing values likely to be on the same individuals.\n\nAt stage 1 the variables were divided into groups numbered 1–11. In each group, an analysis was carried out for each variable individually, followed by a forward stepwise approach with the most significant variable introduced first, then the next, until no further variables in the group significant at p<0.01 could be introduced. At stage 2 significant variables from the first stage were divided into three groups (A, B and C), and a forward stepwise approach again used to identify significant variables.\n\nFinally, at stage 3, the stepwise approach was applied to the stage 2 significant variables to generate a final list of variables for the propensity score, which was then re-calculated based on youths with complete data on all these variables.\n\nAt each stage, the analyses involved all participants with complete data for each variable considered in the group being analysed.\n\nStep 2. Step 2 involved the outcome of interest, initiation of cigarette smoking between Wave 1 and Wave 2. The first analysis was a weighted logistic regression analysis to determine the unadjusted OR and 95% CI for the association of ever vaping at Wave 1 with subsequent initiation of cigarette smoking. The second analysis was similar but adjusted for propensity by dividing the youths into five quintiles of the propensity score, the separate OR (95% CI) values for each stratum being then combined to form an overall propensity-adjusted estimate. Propensity-adjusted analyses were also conducted using the score as a continuous variable, and also using the variables making up the score individually rather than combined.\n\nFive sets of sensitivity analyses were conducted linking vaping to initiation of cigarette smoking, along the lines of the main analysis. For each set, ORs (95% CIs) were again calculated with no adjustment for propensity, adjustment as quintiles, adjustment as a continuous variable, and adjustment for the variables making up the score.\n\nSensitivity analysis 1 Youths who, by Wave 1, had ever used any other tobacco product (i.e. than cigarettes or e-cigarettes) were excluded. As there were considerably fewer significant predictor variables from the stage 1 analyses, the stage 2 analyses were omitted.\n\nSensitivity analysis 2 Here ever users of other tobacco products by Wave 1 were not excluded but use of other products was included as an extra predictor. The stage 1 and 2 analyses were not repeated. Rather the final model used was one that included the same final set of variables plus that for ever using other tobacco products.\n\nSensitivity analysis 3 Whereas the main analysis and sensitivity analyses 1 and 2 linked a propensity score for ever vaping by Wave 1 to ever cigarette smoking by Wave 2, sensitivity analysis 3 linked a propensity score for current e-cigarette use at Wave 1 to current cigarette smoking at Wave 2, last 30-day use being considered current. Again, as few significant variables emanated from the stage 1 analyses, the stage 2 analyses were omitted.\n\nSensitivity analysis 4 Whereas all the analyses described above relate to a propensity score for vaping, sensitivity analysis 4 was essentially the same as for the main analysis but based on a propensity score for ever cigarette smoking.\n\nSensitivity analysis 5 Sensitivity analysis 5 was also like the main analysis, but the propensity score was based on ever use of any tobacco product.\n\nFor sensitivity analyses 4 and 5, the full three-stage process described in Step 1 was used to determine the variables included in the propensity score.\n\nIn the main analysis, the propensity score for e-cigarettes used data provided by youths at Wave 1. Although there was no gold standard to validate reported answers, it seemed possible that more accurate predictors could be based on data from Wave 1 and 2 combined. For those variables forming the propensity score we investigated whether there was further useful information available in Wave 2 and, if so, created a revised variable. How this was done is detailed in the Results section, the procedure depending on which variables were selected for the propensity score. Once the revised predictor variables were created, the main analyses were rerun using a modified propensity score.\n\nRelevant data were transferred for analysis to a ROELEE database, and analysed using the ROELEE program (Release 59, Build 49). SAS version 9.4 (SAS Institute Inc, 2017) was also used to check some results from ROELEE and generate results when ROELEE failed to converge. The GLM Package and the Step function from the R Program (https://www.r-project.org) could be used to run all the analyses.\n\n\nResults\n\nThe propensity score for ever vaping by Wave 1 was developed using five demographic variables and 60 other predictors (Table 1). Each variable, except for variable 52, depended on a question involving a few possible answers (see Table 1 footnotes) with the regression analyses estimating a coefficient per level. Exceptionally, variables 10–13, where numbers of youths were very small for some levels, were treated as continuous variables in analysis.\n\nFor each of the 65 Wave 1 predictor variables considered, Table 1 gives details of their type (graded or continuous) and of the stage at which they were eliminated from consideration for the final propensity score.\n\naFor graded variables, the number of levels is shown in the table, and the levels they represent is shown below. Continuous variables are indicated by the letter C. Exceptionally variables 10–13 were treated as a continuous variable with levels 1 to 4.\n\nb Those not eliminated at stage 1 went into stage 2 analyses A, B or C as indicated.\n\ncThose variables not eliminated at stage 2 went into stage 3 (final = F) analysis.\n\ndThose variables not eliminated at stage 3 were included in the propensity score (P).\n\nStage 1 in developing the propensity score involved separate regression analyses within each of the 11 groups. As Table 1 shows, 38 variables were eliminated from consideration at that stage, with 27 retained for stage 2, 8 considered in group A, 10 in group B and 9 in group C. Following eliminating 9 more variables at stage 2, 18 variables entered stage 3 with 6 more eliminated. After rerunning the regression analysis based on 10,671 youths with data on all 12 predictors, rather than 10,361 with data on 18 predictors, the final model was as shown in Table 2.\n\nThe table shows the final model used for the propensity score. It is based on those 12 of the 18 predictor variables which entered stage 3 and is based on data from those 10,671 youth with data on all the 12 predictors.\n\naThe variables are shown in order of their inclusion into the model\n\nbOdds ratio are per unit of the graded variable which represents decreasing curiosity\n\ncOdds ratio are per unit of the graded variable which represents decreasing likelihood\n\nEver e-cigarette use was independently associated with older age, male sex, use of alcohol and prescription drugs, social networking, and preferring exciting and unpredictable friends. It was also associated with cohabitants using tobacco, parents or guardians not being very upset if they found the youth using tobacco, agreeing that some tobacco products are safer than others, the youth being curious about smoking, and the youth thinking they will smoke a cigarette in the next year. Note that, for these last two variables, the grading system ascribed lower scores for greater curiosity or greater likelihood to smoke cigarettes in the next year so the fitted ORs were <1. The results for the variable regarding enjoying using tobacco was less straightforward to interpret as very few youths strongly agreed they would enjoy it. However, those who strongly disagreed that they thought that they would enjoy using tobacco had a clearly lower odds of ever e-cigarette use than those who simply agreed or disagreed.\n\nAs Table 3 shows, the unadjusted OR for the association of vaping by Wave 1 with cigarette smoking initiation by Wave 2 was 5.702 (95% CI 4.334-7.502). The OR was markedly reduced by adjustment for the propensity score, whether as quintiles (2.476, 1.852-3.310), as a continuous variable (2.474, 1.791-3.419), or for the 12 variables making up the score (1.847, 1.347-2.533). Table 3 also shows the effects of introducing the variables successively. With one minor exception, introducing each variable reduced the OR, the largest reductions relating to the first four variables considered, which in combination already reduced the OR to 2.185 (1.608-2.969).\n\nTable 3 first shows the unadjusted OR, and then the OR adjusted for the propensity score in two ways. Finally, it shows the results of a stepwise regression based on successively including the most significant adjustment variables.\n\nTable 4 summarizes the sensitivity analysis results, comparing them with those from the main analysis. While the number of significant variables included varies between analyses, all show that adjustment markedly reduces the unadjusted association, reducing ORs of over 5 to less than 3. The effect of adjustment was always greater when made for each of the individual variables making up the score. Relating ever vaping by Wave 1 to initiation of cigarette smoking by Wave 2 (main analysis, sensitivity analyses 1 and 2), the lowest adjusted ratio of 1.586 (1.194-2.198) is seen in sensitivity analysis 2, where adjustment is made for 13 predictor variables, including smoking of other products. Here, the adjustment explains 87.5% of the unadjusted association (as estimated from the ratio of the excess ORs, i.e. OR – 1). Sensitivity analysis 3, which concerns current (last 30 day) rather than ever use of both products also produced similar results, though the estimates are more variable due to the very few new cigarette smokers among e-cigarette users. Sensitivity analysis 4, where the score was based on variables linked to Wave 1 cigarette smoking rather than vaping also gave similar results, as did sensitivity analysis 5, where the score was based on variables linked to use of any tobacco product.\n\nFor the main analysis Table 4 shows the number of new smokers considered, as well as the OR (95% CI) for the relationship between ever e-cigarette use at Wave 1 and ever smoking initiation by Wave 2 with no adjustment for propensity to smoke, or with adjustment in three ways. Similar results are also shown for the five sensitivity analyses.\n\na12 variables shown in Table 2\n\nb10 variables (variables 1, 10, 11, 23, 32, 44, 48, 57, 59 and 62 from Table 1)\n\ncEstimated using SAS version 9.4 as failed to converge using ROELEE\n\nd12 variables shown in Table 2 plus ever smoked other products\n\ne7 variables (variables 1, 10, 14, 19, 30 and 33 from Table 1 plus ever smoked other products)\n\nf18 variables (variables 1, 6, 17, 21, 23, 24, 32, 33, 41, 42, 45, 48, 50, 51, 52, 59 and 62 from Table 1 plus ever smoked other products)\n\ng16 variables (variables 1, 10, 11, 21, 24, 30, 32, 39, 41, 44, 48, 51, 59, 60, 62 and 64 from Table 1)\n\nThe propensity score used in the main analyses was revised using modified versions of the 12 predictor variables. The age range of 12–14 or 15–17 at Wave 1 was modified to be 12–13, 14, 15–16, 17 depending on whether 12–14 year-olds at Wave 1 were 15 at Wave 2, and whether 15–17 year-olds at Wave 1 were adults at Wave 2. Gender was unchanged, being consistent between waves. For three ever use variables (alcohol; prescription drugs; social networking) non-users at Wave 1 were now considered users if use was reported at Wave 2. For four variables where questions were asked at both waves (reaction if parent or guardian found using tobacco; think would enjoy tobacco; relative safety of tobacco products; cohabitant uses tobacco) the level most associated with vaping use was used (i.e. maximum for reaction and minimum for the other three). These questions were only asked of youths, so if the participant became an adult at Wave 2, the Wave 1 response was used. For the other three variables (curiosity about smoking; think will smoke a cigarette; prefer exciting and unpredictable friends) a corresponding question was not asked at Wave 2, so the Wave 1 value was used.\n\nAs the subjects included in this analysis were as for the main analysis, the unadjusted OR remained 5.702 (95% CI 4.334-7.502). After adjusting for propensity score as quintiles, the OR value reduced to 2.390 (1.791-3.188), somewhat lower than the 2.476 (1.852-3.310) in the main analysis, with no misclassification adjustment. After adjusting as a continuous variable, the OR reduced to 2.262 (1.625-3.150), again somewhat lower than the 2.474 (1.791-3.519) in the main analysis. Adjusting for the 12 variables making up the score reduced the OR to 1.772 (1.264-2.484), somewhat lower than the 1.847 (1.347-2.533) in the main analysis.\n\n\nDiscussion\n\nWe have described analyses aimed at gaining insight into how much the claimed “gateway effect” of e-cigarettes leading to cigarette smoking may be overestimated by limited control for factors linked to initiation. We used a propensity score approach, intended to remove confounding in the analysis of outcomes by balancing exposure groups on potential confounders, the score being developed prior to, so independently of, the analysis of outcomes. This approach attempts to transpose observational data into what would have been obtained from a randomized trial, using groups balanced on baseline covariates. The main analysis, which aims to balance potential confounders across vapers and non-vapers at Wave 1 (the comparison groups in the analysis where cigarette smoking is the outcome) is strictly designed for this approach. An alternative approach addressed using sensitivity analysis 5, views the propensity outcome more broadly, considering use of any nicotine-containing product as indicative of an inclination to initiate cigarette smoking. The difficulty in the propensity score approach, as with use of observational data generally, is to ensure that all relevant variables are considered in the score, and to account for possible inaccuracies in the variables included.\n\nThe main and five sensitivity analyses summarized in Table 4 all show that adjustment for propensity determined at Wave 1 markedly weakens the gateway effect, the association between vaping by Wave 1 and subsequent initiation of smoking. This was true whether propensity was based on variables associated with vaping (the main analyses), cigarette smoking (sensitivity analysis 4) or any tobacco product use (sensitivity analysis 5). Sensitivity analyses 1–3 also demonstrated this marked reduction, whether users of other products at Wave 1 were excluded or included, whether or not adjustment was made for such use, or whether analyses were based on ever or current use. There was no consistent difference between results adjusted for propensity as quintiles or as a continuous variable, but adjustment for the individual variables making up the score produced lower adjusted ORs. The proportion of the unadjusted excess OR (i.e. OR – 1) explained by adjustment for the individual variables was at least 75.4% in the main and sensitivity analyses, with a maximum of 87.5% for sensitivity analysis 2, where other product use was adjusted for, as well as the 12 main analysis predictor variables.\n\nOur analyses have limitations. One is the small numbers of new smokers considered, never exceeding 71 and as low as six in sensitivity analysis 3. We plan future work intending to obtain more precise answers using the recently available Wave 3 data.\n\nA second issue is the possibility of over-adjustment. One can argue that vaping by Wave 1 may have affected some answers given then. For example, taking up e-cigarettes may make youths more curious about cigarettes, or more likely to think they will smoke or enjoy them. Using Wave 3 data, we also plan further analyses relating initiation of cigarette smoking at Wave 3 to vaping at Wave 2, restricting attention to those who, at Wave 1, had never vaped, and using propensity indicators recorded at Wave 1.\n\nIt is possible that more complete propensity adjustment might have explained more of the “gateway effect”. Our analyses only included variables showing an effect significant at p<0.01. Weakening significance to p<0.05 or p≤0.1 might have included more variables in the propensity score and explained even more of the association.\n\nAs is well documented, inaccurately determining confounding variables may limit the ability to fully adjust. Thus, many years ago (Tzonou et al., 1986), it was demonstrated that “even misclassification rates as low as 10% can prevent adequate control of confounding” and other publications highlight the residual confounding problem (Ahlbom & Steineck, 1992; Fewell et al., 2007; Greenland, 1980; Phillips & Davey Smith, 1994; Savitz & Baron, 1989). Proper adjustment for residual confounding requires a gold standard to compare reported answers with, but such data were unavailable in the PATH study. However, some insight was given by using predictor variables derived from answers given at both Wave 1 and Wave 2. Thus, if a youth reported alcohol use at one wave and not the other, it is possible this was not reported at one wave, and a predictor based on ever reported use may better predict smoking initiation. Such analyses usually weakened the adjusted association of prior vaping with subsequent smoking initiation, but only slightly. This may, however, reflect methodological limitations rather than lack of serious residual confounding.\n\nWhile our analyses do not demonstrate there is no gateway effect, it seems clear that most of the claimed relationship results from confounding. As noted above, more reliable information may emerge from the further analyses we plan using Wave 3.\n\nAnother gateway analysis based on PATH Waves 1 and 2 has recently been published (Watkins et al., 2018). This differed from ours in that their “unadjusted” model already included all non-cigarette tobacco products as indicators of cigarette initiation, and their “adjusted” models added only a restricted list of predefined variables, rather than using models to include more relevant variables. While their adjusted models did include some established determinants of cigarette use (sensation seeking; alcohol use; living with a tobacco user; and variables regarding health warnings and advertising), their adjusted ORs were higher than ours. Thus, whereas the variables we included in our main analysis reduced the OR from 5.702 to 1.847, their similar analysis reduced it only from 3.50 to 2.53. Consequently, although they recognized uncontrolled confounding may exist, they dubiously considered vaping was “independently associated with cigarette smoking one year later”.\n\nIn considering whether a true important gateway effect exists, one should note the lack of any increase in the US in cigarette smoking prevalence following the rise in vaping (Levy et al., 2018), and the fact that, in the PATH study, considerably more (279 vs. 79) Wave 1 cigarette only smokers took up e-cigarettes by Wave 2, than Wave 1 e-cigarette only users who took up smoking. Despite any possible gateway effect, introducing e-cigarettes may have reduced overall youth smoking prevalence.\n\n\nConclusions\n\nThe results presented, based on Waves 1 and 2, strongly suggest that reported estimates of the gateway effect (Soneji et al., 2017) are much too high. Indeed, it is unclear whether prior vaping actually increases uptake of cigarette smoking, if potential confounding effects were to be fully accounted for.\n\n\nAddendum\n\nAt the time this paper was being finalised, an analysis was published (Berry et al., 2019a) investigating gateway effects based on data from Waves 1, 2 and 3 of the PATH study. The authors reported that prior e-cigarette use was associated with increases in the odds of ever and current cigarette use by, respectively, 4.09 (95% CI 2.97-5.63) and 2.75 (1.60-4.73). Though noting that they could not rule out the possibility of residual confounding, they concluded that their findings supported a gateway effect. We intend to examine this claim in detail when we conduct our own planned analyses using the data from all three Waves.\n\n\nData availability\n\nNational Addiction & HIV Data Archive Program: Population Assessment of Tobacco and Health (PATH) Study [United States] Public-Use Files (ICPSR 36498). https://doi.org/10.3886/ICPSR36498.v8 (United States Department of Health and Human Services, 2018).\n\nData are available under the Terms of Use as set out by ICPSR, which can be accessed when users start the process of downloading the data.\n\nOpen Science Framework: Investigating gateway effects using the PATH study https://doi.org/10.17605/OSF.IO/QCFZR (Lee, 2019).\n\nThis project contains the following extended data files:\n\nGateway PATH_F1000 Research_Supplementary file.docx\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nFinancial support was provided by Philip Morris Products SA, through Project Agreement No. 19 with P N Lee Statistics and Computing Ltd.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Mrs Y Cooper and Mrs D Morris for typing the various drafts of this report, and Philip Morris for financial support.\n\n\nReferences\n\nAhlbom A, Steineck G: Aspects of misclassification of confounding factors. 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PubMed Abstract \| Publisher Full Text\n\nSoneji S, Barrington-Trimis JL, Wills TA, et al.: Association Between Initial Use of e-Cigarettes and Subsequent Cigarette Smoking Among Adolescents and Young Adults: A Systematic Review and Meta-analysis. JAMA Pediatr. 2017; 171(8): 788–797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpindle TR, Hiler MM, Cooke ME, et al.: Electronic cigarette use and uptake of cigarette smoking: A longitudinal examination of U.S. college students. Addict Behav. 2017; 67: 66–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTzonou A, Kaldor J, Smith PG, et al.: Misclassification in case-control studies with two dichotomous risk factors. Rev Epidemiol Sante Publique. 1986; 34(1): 10–17. PubMed Abstract\n\nUnger JB, Soto DW, Leventhal A: E-cigarette use and subsequent cigarette and marijuana use among Hispanic young adults. Drug Alcohol Depend. 2016; 163: 261–4. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "48433", "date": "12 Jun 2019", "name": "Raymond Niaura", "expertise": [ "Reviewer Expertise Tobacco regulatory science." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aimed to evaluate the “gateway effect” of baseline e-cigarette use predicting cigarette smoking initiation among youths, addressing the inadequate adjustment of confounders considered in the previous published studies. The first two waves of PATH data from the youth sample were used for this purpose.\n\nThe strength of this study is that the authors used multiple approaches to evaluate this gateway effect, and performed five sets of sensitivity analysis. Propensity scores for e-cigarette use at wave 1 were created. Propensity score based approaches are preferred to statistically control for numerous confounding variables; however, this approach was not completely adequate as it appears in this study for several reasons. A few major concerns are below.\nFirst, due to the availability of data at the time of analyses, the confounding variables were measured concurrently with e-cigarette exposure or afterwards (i.e., analyses investigating residual confounding). Considering the Wave 3 PATH data have been available since fall 2018, the authors are encouraged to revise the manuscript using data from the first 3 waves. They state they will do so but they could probably access the data at this point.\nSecond, the authors aimed to address the inadequate adjustment of confounding in previous studies, however, this limitation still exists in the current study. As a result of Step 1, only a set of 13 covariates were included of the main analysis, sensitivity analyses, and residual confounding analysis. Moreover, it is unclear whether the balancing of covariates was achieved between the (e-cigarette) exposure and non-exposure groups.\n\nA few details regarding each approach need to be provided. Specifically, it was unclear what the authors meant by saying “the first analysis was a weighted logistic regression analysis.” How was a weighting variable created? Was this what the authors called “no adjustment of propensity” afterwards (in the first paragraph of the Sensitivity Analysis section, and in Table 4)? The results from the “adjustment as quintiles” and “adjustment as a continuous variable” approaches typically agree, and the effect of e-cigarette exposure would be biased if only a subset of confounding variables were considered in analyses. Moreover, the “using the variables making up the score individually” approach was actually a traditional covariate approach, which assumed a linear association between the outcome and each covariate. Authors need to justify whether this assumption was satisfied or not before they concluded the findings of the study.\nThird, in analyses investigating residual confounding, authors used some of the covariates from Wave 2, which might be influenced by the exposure of “e-cigarette” at Wave 1. In this case, the partial coefficient of e-cigarette use on outcome is actually the controlled direct effect in a mediation analysis. This coefficient only showed the partial effect of e-cigarette use on outcome, which was an underestimated total effect of exposure. In this set of analysis, the confounding was over-adjusted.\nFourth, all analyses only involved participants with complete data for each variable considered in the group being analyzed. Missing complete at random was actually assumed in all analyses. Authors need to report the sample sizes of the original data and the working data, and justify whether/why this assumption was valid in this study.\nIn addition, a few minor issues are below.\nMethods: Some information in the last paragraph of the Introduction section need to be relocated in the Methods section, under the title of “sample and data collection”. Software: what is the ROELEE program? Please briefly introduce this software. Under what circumstance did ROELEE fail to converge (and SAS did not)? Supplementary document: the very long list of variables was not helpful to understand how confounding variables were selected. It seems like the authors have already selected (without details on criteria) 60 smoking predictor variables out of 200+. Please provide sample questions as needed. Sensitivity analysis: report sample size of each sensitivity analysis in Table 4.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4710", "date": "28 Jun 2019", "name": "Peter Lee", "role": "Author Response", "response": "Response by the first author, Peter Lee.In his first major point, Dr Niaura expressed concern about the possibility of confounding arising if vaping by Wave 1 had affected some answers given then. We noted this in our discussion saying that further analyses are planned relating initiation of cigarette smoking at Wave 3 to vaping at Wave 2, restricting attention to those who, at Wave 1, had never vaped, and using propensity indicators recorded at Wave 1. We have had access to the Wave 3 data for some time, and in due course will publish a further paper. We do not propose to revise the current manuscript.As regards to the second concern, our final analyses were based on adjustment for a limited number of confounding variables. However, this list was based on a much longer list, with no further variables adding significantly to the final model, the much longer list (shown in Supplementary File 1) being itself derived from a review of the literature. The fact that the model enormously reduced the association between vaping and subsequent initiation of cigarette smoking was the main point we wished to make. It is possible other models may explain more of the unadjusted association but that will be addressed in the paper currently being prepared.Dr. Niuara refers to reporting whether the balancing of covariates was achieved between the e-cigarette exposed and non-exposed groups. Presumably, he is referring to balancing after propensity adjustment. We intend to report on this in the planned new paper. Dr. Niaura asked how the weighting variable was created. This variable is supplied directly to users of the PATH study and allows the user to generate results representative of the U.S. population. It is stated that “the effect of e-cigarette exposure would be biased if only a subset of confounding variables were considered in analyses”. As noted above we did take into account a very large number of potential confounders in our analyses. It would be expected that adjustment for some variables would not in fact have a material effect when other variables were already included in the analyses, i.e. they would not be true confounders. As regards linear associations it should be noted that most of the variables were yes/no variables. We will consider non-linear relationships in our planned analyses using Wave 3.It is well known that adjustment for inaccurately measured confounding variables may be incomplete. In the absence of a gold standard we attempted to gain insight into the problem of looking at variation in answers to corresponding questions at different Waves. It is true that this may cause problems if the answers are affected by e-cigarette use, but this could only be addressed by analyses involving more than three Waves, relating e-cigarette use at Wave 3 to initiation of smoking by Wave 4, with adjustment for questions asked at Waves 1 and 2 in non-tobacco users.We will report fuller details regarding missing data in our planned analyses based on Waves 1 to 3.As regards the other points we do not propose to modify the current paper, preferring to take all the points into account in the paper on Waves 1 to 3. For information, the ROELEE program is one my company started to develop over 30 years ago, initially to get output in a user-friendly form not available using programs like SAS or SPSS. It has undergone numerous updates over the years and is fully tested. It is available from ROELEE Statistics Ltd. While, for logistic regression analyses, it gives the same results as SAS, but in a more convenient form, the version of ROELEE we used at the time sometimes failed to converge when there are large numbers of predictor variables, whereas SAS did not. We are currently modifying the code of ROELEE to avoid this non-convergence problem.As regards the sample sizes used in the 6 sets of analyses in Table 4, these were as follows:Main, S2 and S5 9423; S1 8900; S3 9907; S4 9354." } ] }, { "id": "55826", "date": "08 Nov 2019", "name": "Reiner Hanewinkel", "expertise": [ "Reviewer Expertise I am trained as Psychologist" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n“On 19 December 2018, Altria announced an offer of US$12.8billion for a 35% share of Juul Labs. The deal combines Altria (formerly Philip Morris), the company with the largest share of the US cigarette market, with the large and rapidly growing vaping product (aka e-cigarette) company, Juul Labs. The acquisition price was based on a US$38billion valuation, which was more than twice Juul Labs’ August valuation, and a surprise to many investors on Wall Street.” 1\nWe have to have this information in mind, because the manuscript submitted to F1000Research by Peter Lee and John Fry is financed by Philip Morris. My personal opinion is that Philip Morris has an interest to show that the so called gateway effect is not existent.\nI am not an expert in the propensity score approach, but what I understand is, that the authors tried everything to show, that the gateway effect does not exist. It seems trivial to me, that adjustments reduce an OR. Even after adjustment for a broad range of confounders, there is still a significant and clinical meaningful association between e-cigarette use at baseline and smoking initiation in the observational period.\nMy main point is that I do not agree with the conclusion and discussion of the results. Instead of saying that confounding is a major factor explaining most of the observed gateway effect, the authors should emphasize that even after controlling for a large number of confounders, the gateway effect is still present.\nI am counting nearly two dozens of cohort studies, which investigated the gateway effect. The authors only cite a portion of these studies. In detail, young people have been recruited in the US and Canada 2-15, UK 16-18, Finland 19, Mexico 20, the Netherlands 21, Romania 22, Taiwan 23, and Germany 24. More than 100,000 young people have been recruited for these studies with observational periods of up to 24 months. Outcome variables range from one cigarette to daily smoking. All these studies found a significant association between e-cigarette use at baseline and uptake of smoking in the observational period. Applying the well-known Bradford-Hill criteria, it seems to me that the following criteria are fulfilled: (1) Strength (effect size), (2) Consistency (reproducibility), (3) Temporality, and (4) Plausibility.\nIn the Introduction the authors should describe the two opponent explanations for the observed association between e-cigarette use and the later uptake of smoking: the so called “common liability” theory vs. the “gateway” theory 25. Another important theoretical paper should also be discussed 26.\nIf anything, the results reported are in line with the gateway theory and not with the common liability theory.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5055", "date": "04 Dec 2019", "name": "Peter Lee", "role": "Author Response", "response": "We thank Dr. Hanewinkel for his comments. However, it seems unfortunate that he suggests our interpretation may have been affected by the funding source. As experienced medical statisticians/epidemiologists our interest is in unbiased assessment of data, not in trying to reach conclusions favourable to any sponsor. The objective of our paper was to provide insight into how much the observed gateway effect could be explained by adjustment for confounding factors, in order to provide insight into the possible magnitude of any true effect. That adjustment could reduce an unadjusted OR from 5.70 to 1.59 should be interpreted as providing doubt as to whether there is any real effect, despite the 1.59 being statistically significant. Our discussion, and also our companion paper in F1000Research[1], already highlight the potential problems of interpretation due to residual confounding.Dr. Hanewinkel states that “it seems trivial” to him “that adjustments reduce an OR”. However, theoretically adjustment can increase or decrease an OR. He cites numerous cohort studies that reported a significant gateway effect after adjustment, and we have cited all but the most recently published ones in our present paper or in its companion[1]. We note that many of these papers have also shown a large reduction in the OR after allowing for confounding variables. The existence of a significant association following adjustment does not necessarily mean that the adjusted association is real, for the reasons we have already discussed. We have now extended the introduction as suggested to describe the “common liability” and the “gateway” theory, making it clear that both can contribute to the observed association between e-cigarette use and subsequent initiation of smoking.Our abstract already made clear that a significant association remained after adjustment, and that its interpretation is not straightforward. We have now modified the conclusion in the abstract to point out that further work is needed to clarify the extent of any true gateway effect that may exist.We have also extended the discussion section of the paper. We feel that our results adequately support our conclusion that “confounding is a major factor, explaining most [though not all] of the observed gateway effect”. Further analysis of later waves of the PATH study could well help to establish whether the residual effect is or is not real. Reference1 Lee PN, Coombs KJ and Afolalu EF. Considerations related to vaping as a possible gateway into cigarette smoking: an analytical review [version 3; peer review: 2 approved]. F1000Research 2018; 7: (https://doi.org/10.12688/f1000research.16928.3) [DOI: doi.org/10.12688/f1000research.16928.3]" } ] } ]	1	https://f1000research.com/articles/8-264
https://f1000research.com/articles/8-1774/v1	18 Oct 19	{ "type": "Software Tool Article", "title": "RCy3: Network biology using Cytoscape from within R", "authors": [ "Julia A. Gustavsen", "Shraddha Pai", "Ruth Isserlin", "Barry Demchak", "Alexander R. Pico", "Julia A. Gustavsen", "Shraddha Pai", "Ruth Isserlin", "Barry Demchak" ], "abstract": "RCy3 is an R package in Bioconductor that communicates with Cytoscape via its REST API, providing access to the full feature set of Cytoscape from within the R programming environment. RCy3 has been redesigned to streamline its usage and future development as part of a broader Cytoscape Automation effort. Over 100 new functions have been added, including dozens of helper functions specifically for intuitive data overlay operations. Over 40 Cytoscape apps have implemented automation support so far, making hundreds of additional operations accessible via RCy3. Two-way conversion with networks from \\textit{igraph} and \\textit{graph} ensures interoperability with existing network biology workflows and dozens of other Bioconductor packages. These capabilities are demonstrated in a series of use cases involving public databases, enrichment analysis pipelines, shortest path algorithms and more. With RCy3, bioinformaticians will be able to quickly deliver reproducible network biology workflows as integrations of Cytoscape functions, complex custom analyses and other R packages.", "keywords": [ "Bioconductor", "Cytoscape", "Automation", "Scripting", "Network biology" ], "content": "Introduction\n\nIn the domain of biology, network models serve as useful representations of interactions, whether social, neural or molecular. Since 2003, Cytoscape has provided a free, open-source software platform for network analysis and visualization that has been widely adopted in biological and biomedical research fields1. The Cytoscape platform supports community-developed extensions, called apps, that can access third-party databases, offer new layouts, add analytical algorithms, support additional data types, and much more2,3.\n\nIn 2011, the CytoscapeRPC app was created to enable R-based workflows to exercise Cytoscape v2 functionality via functions in the corresponding RCytoscape R package over XML-RPC communications protocols4. In 2015, the CyREST app was created to enable R-based workflows to exercise Cytoscape v3 functionality. This was achieved by the first version of the RCy3 R package, which re-implemented much of RCytoscape’s organization, data structures and syntax over REST communications protocols3,5.\n\nHere, we describe version 2.0 of the RCy3 package, which is better aligned with Cytoscape’s CyREST API. We have rewritten every function, deprecated 43 functions and added over 100 new functions. This work provides a more intuitive and productive experience for Cytoscape users learning about the RCy3 package, and it positions RCy3 to take advantage of future Cytoscape Automation6 development and evolution. The goal of this paper is to describe the implementation and operation of the updated RCy3 package and to provide detailed use cases relevant to network biology applications in common and advanced bioinformatics workflows.\n\n\nMethods\n\nRCy3 is a component of Cytoscape Automation. At the core of Cytoscape Automation is CyREST, which implements an interface to the Cytoscape Java application via the REST protocol6. A collection of GET, POST, PUT and DELETE operations practically cover the complete feature set of the Cytoscape desktop software. Additional features, including those provided by user-installed Cytoscape apps, are covered by a separate command-line interface called Commands (Figure 1). For version 2.0, we redesigned the RCy3 package to parallel CyREST and Commands APIs to standardize the syntax and organization of its functions and streamline its future development. RCy3 functions are grouped into categories to aid the parallel development with Cytoscape Automation APIs and to facilitate navigation and comprehension of the overall package (Table 1).\n\nThe Java desktop version of Cytoscape (bottom) supports Commands (red) and CyREST (green) interfaces for scripting and automation either directly (blue) or via R and Python harmonization libraries (purple).\n\nThe categories correspond to separate R files in the package. Brief descriptions and example functions are listed for each category.\n\nAll RCy3 functions are ultimately implemented as calls to a small set of utility functions that execute the CyREST or Commands REST protocol (e.g., cyrestGET and commandsPOST). The internals of these functions handle the composition of operations and parameters to be sent via httr functions to CyREST, as well as the processing of results from JSON to appropriate R objects using RJSONIO.\n\nIn most RCy3 functions there is an optional argument for base.url. This is the URL used by RCy3 to connect to the Cytoscape desktop application via CyREST, and it defaults to \"http://localhost:1234/v1\". The default CyREST port is 1234, and it can be changed in Cytoscape through Edit/Preferences/Properties or by command-line (see CyREST setup guide). If you change the CyREST port, you should reflect the change in the base.url argument per function call or change each function’s default value using the default package.\n\nThe second most common argument in RCy3 functions is network. If left as NULL (default), the currently selected network in the Cytoscape application is referenced. Network name or SUID (session unique identifier) can thus be explicitly specified or inferred from the current state of the application. The current network can also be controlled and retrieved by setCurrentNetwork and getCurrentNetwork. Given a base.url and network (when needed), the majority of RCy3 functions simply validate parameters and construct arguments in order to call one of the cyrest* or commands* functions.\n\nThe commandsRun function is a special RCy3 function that allows users to directly issue commands via Cytoscape’s command-line syntax (e.g., \"node list network=current\"), including commands implemented by Cytoscape app developers (see Use cases). This single function can perform hundreds of operations made available by both Cytoscape and automation-enabled apps. Over 40 of these RCy3-supported apps are currently registered in the Cytoscape App Store2. The cyrestAPI and commandsAPI open interactive Swagger documentation for the CyREST and Commands programmatic interfaces. Cytoscape Automation can be performed via these Swagger web pages. The same operations and parameters are supported by the cyrest* and commands* functions in RCy3. Command-line syntax can also be run from the Automation panel in Cytoscape, at manual.cytoscape.org/en/stable/Command_Tool.html.\n\nThe primary goal of RCy3 is to provide wrappers for every feature made available by CyREST and Commands. However, we also have a secondary goal of providing useful and intuitive functions for common workflows in R. So, in addition to the generic functions implemented to parallel the CyREST and Commands APIs, we have also implemented sets of specific helper functions.\n\nAs an example, consider the common Cytoscape operation of mapping network data values to visual style properties. CyREST has a POST endpoint for /styles/{style name}/mappings that takes a JSON data structure defining the mapping. We implemented updateStyleMapping which takes a style.name and mapping arguments and sends them out via cyrestPOST. We also implemented mapVisualProperty to help construct the mapping argument. With these generic functions one can perform any of the hundreds of visual style mappings supported by Cytoscape, including new ones added in the future. However, these functions are not simple to use, requiring knowledge of specific property names, like \"NODE_FILL_COLOR\", and mapping data structures. To simplify usage for common situations, we therefore also implemented specific functions for over a dozen of the most commonly used mappings (e.g., setNodeColorMapping). With autocomplete in tools like RStudio, after just typing setNode... a script author is presented with a series of intuitively named functions with obvious arguments.\n\nNetworks are a popular visualization option in R often implemented as graph models by igraph and Biocondutor’s graph (i.e., graphNEL). RCy3 can create networks in Cytoscape from either igraph, graphNEL or dataframe objects (createNetworkFrom). Likewise, igraph and graphNEL objects can be created from networks (createFromNetwork), and dataframes from node and edge tables in Cytoscape (getTableColumns).\n\nIn the case of createNetworkFromDataFrames, two dataframes are accepted as arguments, one for nodes and one for edges. The nodes dataframe must include a column named \"id\", and the edges dataframe must include \"source\" and \"target\" columns. Additional columns are imported as node and edge attributes into Cytoscape. The function can also work with just one dataframe. If a dataframe of only edges is passed to createNetworkFromDataFrames, then a connected network will be created with all of the nodes. If a dataframe of only nodes is passed, then a network with no connections, only nodes, will be created.\n\nRCy3 can also import network file formats supported by Cytoscape natively (e.g., SIF, xGMML and CX7) and via user-installed apps (e.g., GPML8 and adjacency matrices). With these functions RCy3 can interoperate with any other Bioconductor packages that deal with networks in a standardized manner, providing advanced network visualization options and advanced network analytics from the Cytoscape ecosystem (see Table 2).\n\nRCy3 can provide and consume network models to and from these packages (checkmarks). Asterisks indicate where a Cytoscape app is required (* aMatReader, ** WikiPathways).\n\nIn order to work with RCy3 you must have Cytoscape v3.7 or later installed and running. Cytoscape can be installed from cytoscape.org. The RCy3 package can be installed from Bioconductor:\n\n\n\nLaunch Cytoscape and keep it running whenever using RCy3. Confirm that you have everything installed and that RCy3 is communicating with Cytoscape via CyREST:\n\n\n\nAs with any R package, one can access the documentation and browse over a dozen vignettes included in the RCy3 package:\n\n\n\n\nUse cases\n\nThe following sections demonstrate a variety of common and advanced network biology use cases as runnable R code snippets. The code for these use cases is also available as an online Rmd notebook and Rmd file in the Cytoscape Automation repository (see Data availability). The first set focuses on fundamental Cytoscape operations that are common to most use cases:\n\nLoading networks (from R objects, Cytoscape files and public databases)\n\nVisualizing network data\n\nFiltering by node degree or data\n\nSaving and exporting networks\n\nAdditionally, there are examples that demonstrate analytical workflows, relying not only on Cytoscape, but also on Cytoscape apps and other R packages:\n\nBuilding maps of enrichment analysis results using EnrichmentMap and AutoAnnotate\n\nVisualizing integrated network analysis using BioNet\n\nPerforming advanced graph analytics using RBGL\n\nLoading Networks. Networks come in all shapes and sizes, in multiple formats from multiple sources. The following code snippets demonstrate just a few of the myriad ways to load networks into Cytoscape using RCy3.\n\nFrom R objects. . .\n\n\n\nFrom Cytoscape-supported file formats. . .\n\n\n\nFrom public databases via Cytoscape apps. . .\n\n\n\nVisualizing data on networks. Cytoscape excels at generating publication-quality network visualization with data overlays. This vignette demonstrates just one of the hundreds of visual style mapping options using RCy3.\n\n\n\nFiltering networks by degree and by data. Network topology and associated node or edge data can be used to make selections in Cytoscape that enable filtering and subnetworking. The filters are added to the Select tab in the Control Panel of Cytoscape’s GUI and saved in session files.\n\n\n\nSaving and exporting networks. There are local and cloud-hosted options for saving and sharing network models and images. The Cytoscape session file (CYS) includes all networks, collections, tables and styles. It retains every aspect of your session, including the size of the application window. Network and image exports include only the currently active network. Export to NDEx requires account information you can obtain from ndexbio.org. Files are saved to the current working directory by default, unless a full path is provided.\n\n\n\nBuilding maps of enrichment analysis results. This workflow illustrates how to plot an annotated map of enrichment results using the EnrichmentMap Pipeline Collection of apps in Cytoscape9. An enrichment map is a network visualization of related genesets in which nodes are gene sets (or pathways) and edge weight indicates the overlap in member genes10. Following the construction of the enrichment map, AutoAnnotate clusters redundant gene sets and uses WordCloud11 to label the resulting cluster (Figure 2). The code uses the Commands interface to invoke EnrichmentMap and AutoAnnotate apps. After installing apps, run commandsAPI() to open the live Swagger documentation to browse and execute command-line syntax.\n\n\n\nA few of the largest clusters of pathways from WikiPathways are displayed as a network with WordCloud-based labels annotating groups (yellow areas). The size of the labels correspond to the size of the groups.\n\nVisualizing integrated network analysis using BioNet. The BioNet package implements analytical methods to perform integrated network analysis, for example, of gene expression data and clinical survival data in the context of protein-protein interaction networks. Partnered with RCy3, the analytical results from BioNet can be visualized in Cytoscape with vastly more options for customization. Starting with the \"Quick Start\" tutorial from BioNet, we pass the results directly to Cytoscape for visualization:\n\n\n\nPerforming advanced graph analytics using RBGL. As an interface to the BOOST library, the RBGL Bioconductor package offers an impressive array of analytical functions for graphs. Here we will start with a network in Cytoscape, load it into R as a graph object, perform shortest path calculation using RBGL and then visualize the results back in Cytoscape (Figure 3).\n\n\n\nIn Cytoscape, the start and finish nodes were colored green and magenta, respectively (left panel). The shortest path calculated by RBGL was then selected and nodes along the path were highlighted with thick borders (middle panel). Finally, a subnetwork was created from the selected path (right panel). Note that while common gene names are displayed as node labels, official yeast identifiers are the actual node names that are referenced in the script.\n\n\nDiscussion\n\nEvery operation exposed by Cytoscape’s REST API has now been implemented as a function in RCy3 2.0. Furthermore, RCy3 provides dozens of higher-level helper functions in support of common usage, such as setNodeColorMapping, to make script writing more intuitive and efficient. The issue trackers for CyREST and RCy3 are linked for functions pending implementation, such as mergeNetworks, as well as for bug fixes. Thus, RCy3 is expected to keep pace with future development of Cytoscape Automation. More broadly, RCy3 is an integral part of the Cytoscape ecosystem, which includes the network repository NDEx7, Cytoscape apps and services, and web components like cytoscape.js12. The ecosystem has also defined interfaces and standard formats to interoperate with interaction databases and annotation services. Adopting RCy3 for network analysis will establish a connection to the Cytoscape ecosystem and enable Cytoscape Automation workflows6. As the sharing and publishing of analysis scripts and workflows become more routine (if not mandated), software tools designed to work in an open and accessible ecosystem are becoming essential.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nRCy3 vignettes: https://bioconductor.org/packages/release/bioc/html/RCy3.html\n\nRCy3 Rmd notebooks: https://cytoscape.org/cytoscape-automation/for-scripters/R/notebooks/\n\nRCy3 workshop presentations: http://tutorials.cytoscape.org/#automation\n\nVideo demonstrations of RCy3: https://www.youtube.com/playlist\n\nCytoscape Automation training: http://automation.cytoscape.org/\n\nCytoscape Automation code repository: https://github.com/cytoscape/cytoscape-automation\n\n\nSoftware availability\n\nRCy3 is available from Bioconductor: https://bioconductor.org/packages/release/bioc/html/RCy3.html\n\nSource code available from: https://github.com/cytoscape/RCy3\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3473421\n\nIssue tracker: https://github.com/cytoscape/RCy3/issues\n\nLicense: MIT License.", "appendix": "Author contributions\n\n\n\nAP, SP and RI redesigned and implemented version 2 of RCy3. AP, JAG, SP and BD drafted the manuscript. AP, JAG, SP and RI contributed use cases.\n\n\nAcknowledgments\n\nWe would like to acknowledge contributions by other developers on the original implementation of RCy3 by Paul Shannon, Tanja Muetze and Georgi Kolishkovski. We also greatly appreciate the input from Mark Grimes (testing), Martin Morgan (Bioconductor), and the excellent work on CyREST and Cytoscape Commands by David Otasek and John “Scooter” Morris.\n\n\nReferences\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLotia S, Montojo J, Dong Y, et al.: Cytoscape app store. Bioinformatics. 2013; 29(10): 1350–1351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDemchak B, Otasek D, Pico AR, et al.: The Cytoscape Automation app article collection [version 1; peer review: not peer reviewed]. F1000Res. 2018; 7: 800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShannon PT, Grimes M, Kutlu B, et al.: RCytoscape: tools for exploratory network analysis. BMC Bioinformatics. 2013; 14: 217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOno K, Muetze T, Kolishovski G, et al.: CyREST: Turbocharging Cytoscape Access for External Tools via a RESTful API [version 1; peer review: 2 approved]. F1000Res. 2015; 4: 478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOtasek D, Morris JH, Bouças J, et al.: Cytoscape Automation: empowering workflow-based network analysis. Genome Biol. 2019; 20(1): 185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPratt D, Chen J, Pillich R, et al.: NDEx 2.0: A Clearinghouse for Research on Cancer Pathways. Cancer Res. 2017; 77(21): e58–e61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Iersel MP, Kelder T, Pico AR, et al.: Presenting and exploring biological pathways with PathVisio. BMC Bioinformatics. 2008; 9: 399. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReimand J, Isserlin R, Voisin V, et al.: Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc. 2019; 14(2): 482–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerico D, Isserlin R, Stueker O, et al.: Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One. 2010; 5(11): e13984. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOesper L, Merico D, Isserlin R, et al.: WordCloud: a Cytoscape plugin to create a visual semantic summary of networks. Source Code Biol Med. 2011; 6: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranz M, Lopes CT, Huck G, et al.: Cytoscape.js: a graph theory library for visualisation and analysis. Bioinformatics. 2016; 32(2): 309–311. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55403", "date": "31 Oct 2019", "name": "James Denvir", "expertise": [ "Reviewer Expertise Bioinformatics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents an R package, RCy3, designed to control and automate the network visualization software application Cytoscape. The article describes both a low-level (\"generic\") and a high-level (\"specific\") API and presents use-cases with some code samples.\nOverall this appears to be an API with great potential utility for the bioinformatics community, and the article presents the package clearly and with several useful and informative examples.\n\nI have a couple of minor comments and suggestions, which I hope will serve to improve the article.\nThe abstract mentions the potential for RCy3 to deliver reproducible workflows. In my opinion, reproducibility is among the most important benefits of code/script-based workflows over workflows performed using \"point and click\" GUI-based applications. However, the article does not elaborate on this outside of the abstract. A couple of sentences in the discussion describing the potential for RCy3 to enhance reproducibility would be worthwhile.\nThe article mentions that the Cytoscape App store currently lists over 40 \"RCy3-supported apps\". A quick note on what is needed for a third party app to be \"RCy3-supported\" might be pertinent here. This may be already sufficiently covered in existing publications (e.g. reference 6), in which case a simple note to that effect would suffice.\nThe code block for the section \"Building maps of enrichment analysis results\" is not quite as clear as the other code blocks in the article. A comment indicating that commandsAPI() opens a web browser page (externally; at least in my environment) and comments after the print() statements showing the expected output, as in the other blocks of code, would be helpful. The uses of file.path and paste(... sep='/') are a little confusing here (to be honest, I was surprised to find these actually worked on Windows systems); brief comments added to the code might aid readability here.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5040", "date": "27 Nov 2019", "name": "Alexander Pico", "role": "Author Response", "response": "Thank you for your careful review and suggestions. We share your opinion regarding the importance of reproducibility and will add a sentence accordingly to the Discussion section. The support of automation by Cytoscape apps is indeed a major topic of reference 6; we will note that in the text as well. Regarding the code block, we will likely remove the commandsAPI() call to not surprise/distract users with a browser window opening. The use of file.path is simply to avoid a really long string, i.e., longer than the column width of this publication. The use of forward slash is in paths is a generic solution to avoid platform-specific conventions and avoid having to escape backslashes. Neither of these last two “tricks” are at all specific to RCy3, so we might just leave those as-is." } ] }, { "id": "55401", "date": "21 Nov 2019", "name": "Krithika Bhuvaneshwar", "expertise": [ "Reviewer Expertise Bioinformatics", "Genomic Data Science", "Translational and Precision Health Informatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a review of the manuscript titled \"RCy3: Network biology using Cytoscape from within R\". RCy3 is a Bioconductor package that communicates with Cytoscape via its REST API to build and visualize networks. This manuscript is about a re-designed/updated RCy3 package and its function and implementation.\nMy comments are below:\nThe authors say \"We have re-written every function\". Is it backward compatible? Should existing users of the older version of the package have to make changes in their code?\n\nThe authors say \"Over 100 new functions have been added, including dozens of helper functions specifically for intuitive data overlay operations\". This is a great achievement. In addition to this statement, perhaps it would be helpful for users to summarize/compare what the main improvements are in this newer version?\n\nDoes this newer version include more or better (or the same) interoperability with other major packages that use RCy3? For example, exporting and importing to/from Ndex. From the example code listed in the paper, it seems there is no change in the way the interoperability works?\nMinor comment: It is helpful to call the package RCy3 2.0 or something to note the version change? Would keeping the same name be confusing for users?\nOverall great work! RCy3 is one of the major packages that work with Cytoscape so very important for the R/BioC Community. Not all authors of Bioconductor and R packages take effort to update their packages, so I commend this effort. I have used RCy3 in the past and am excited to try out this newest version of the package.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5041", "date": "27 Nov 2019", "name": "Alexander Pico", "role": "Author Response", "response": "Thank you for your careful review and questions. The package is not backward compatible. We in fact initially submitted it as a completely new package, but were encouraged by Bioconductor reviewers to coordinate with the original RCy3 authors and submit it as a new version of the same package, which we did. The version number is kept separate from the package name per Bioconductor and semantic versioning conventions, so we maintained the original package name. There is a vignette dedicated to upgrading scripts from the original to version 2: (https://bioconductor.org/packages/release/bioc/vignettes/RCy3/inst/doc/Upgrading-existing-scripts.html), which highlights the major differences and improvements. The original version focused on graphNEL objects, so interoperability with other graphNEL-based packages perhaps hasn’t changed. But I just now double checked the old code base and I can’t find any NDEx, igraph, data frame or adjacency matrix related functions for network interoperability in the original version." } ] } ]	1	https://f1000research.com/articles/8-1774
https://f1000research.com/articles/8-2051/v1	03 Dec 19	{ "type": "Case Report", "title": "Case Report: Acute pancreatitis as a complication for the use of furosemide during transurethral resection of the prostate", "authors": [ "J. Antonio Grandez-Urbina", "Anggie Santillan-Romero", "Mariela Corrales-Acosta", "J. Eduardo Tejeda-Mariaca", "J. Luis Fernandez-Luque", "Anggie Santillan-Romero", "Mariela Corrales-Acosta", "J. Eduardo Tejeda-Mariaca", "J. Luis Fernandez-Luque" ], "abstract": "Background: Pancreatitis caused after transurethral resection of the prostate (TURP) is an uncommon complication, with few reports found in the literature. Here we present the case of a middle-aged patient without comorbidities with this pathology. Clinical case: A 66-year-old male patient with no prior medical history underwent TURP surgery due to benign prostatic hyperplasia. The evolution was unfavourable, leading to post TURP syndrome. On the fifth day after surgery, the patient was admitted for emergency dialysis. During his emergency stay, the patient was unstable; he was diagnosed with pancreatitis, moving to an intensive care unit for management The patient progressed favourably from renal failure and pancreatitis, and on the 5th day the patient was discharged, tolerating oral feeding and stabilized renal failure. Conclusions: Pancreatitis caused by TURP is very rare. This complication has been reported in the literature infrequently and should be suspected when there is pain in the epigastrium. If this complication is suspected, imaging tests and pancreatic enzyme levels should be immediately requested, as it can have an unfavourable evolution until death.", "keywords": [ "Furosemide", "pancreatitis", "Transurethral Resection of Prostate", "Drug-Related Side Effects and Adverse Reactions" ], "content": "Introduction\n\nAcute pancreatitis caused after transurethral resection of the prostate (TURP) is an uncommon complication, with few reports found in the literature. It usually occurs in those who develop TURP syndrome1.\n\nThe prognosis depends on suspicion of this complication in a timely manner because it lead to multi-organ failure and even death1.\n\nThe case presented herein describes the case of a middle-aged patient who develops pancreatitis after TURP. The report describes how this was managed, the treatment used, and a review of the literature to identify similar cases.\n\n\nClinical case\n\nA timeline of the patient’s case can be seen in Figure 1.\n\nA 66-year-old male with a prior history of high blood pressure (treatment, Valsartan 80 mg bid), without any prior history of alcoholism, previous pancreatitis episodes, or gallbladder disease underwent TURP surgery after diagnosis with obstructive benign prostatic hyperplasia (BPH). This was detected with an approximate weight of 45 gr by ultrasound and a verum-neck distance of 3 cm in cystoscopy. The patient had no family history of hypertriglyceridemia or gallbladder stones.\n\nMonopolar TURP surgery was performed using 13 liters of distilled water for continuous bladder irrigation. During surgery, 80mg of furosemide EV was used to reduce the risk of dilutional hyponatremia. The operative time was 55 minutes, removing 17gr of prostate tissue; with no intraoperative complications. No lesion of the capsule or any significant bleeding was reported.\n\nAfter surgery, a physical abdominal and genitourinary exploration was carried out. There was evidence of oral intolerance, abdominal distention and increased abdominal resistance, with pain at the epigastrium that radiated to the lumbar region. Permeable Foley catheter with clear urine was reported.\n\nAt 24 hours post-surgery, the patient presented elevation of azoles (Cr = 3.7mg/dl; Urea = 92 mg/dl) and oliguria, therefore furosemide 20mg was administered twice a day. After 72 hours of follow-up, the condition persisted, and it was decided to increase treatment with furosemide to 20 mg EV three times a day; despite this, it evolves in an unfavourable manner. On the fifth day after surgery, elevation azoles continued (Cr = 12.43 mg/dl; Urea = 235mg/dl), and there was also an increase in pancreatic enzymes (Amylase = 329U/L(Normal value <105); Lipase = 304U/L(Normal value: <50) ; leukocytes = 20880ml/mm3 ( Normal value = 4500 ml/mm3 to 10500 ml/mm3). The aggregate infectious process was improved. An abdominal-pelvic CT scan was performed, finding a slight increase in the density of peripancreatic mesenteric fat with the presence of low laminar free fluid (Figure 2). The patient was transferred to the Intensive Care Unit (ICU) for emergency dialysis and stabilization.\n\nPancreas is not increased in size, no pancreatic necrosis, no peripancreatic collections. Slight increase in the density of mesenteric fat. Presence of low laminar free liquid.\n\nThree sessions of haemodialysis and treatment of pancreatitis were required to stabilize the condition. On the fourth day of admission to the ICU, the patient progressed favourably from renal failure and pancreatitis by reduction in levels of pancreatic enzymes and creatinine, and on the 5th day the patient was discharged, tolerating oral feeding and stabilized renal failure.\n\nAmoxicillin/clavulanic acid 500mg three times a day was prescribed for five days. The Foley catheter was removed on the fifth day after surgery as it was not necessary to monitor urine volume. Renal impairment was progressively restored, and the level of creatinine three months after surgery was 0.98mg/dl. No urethral stricture or any other urinary complications were reported.\n\n\nDiscussion\n\nAcute pancreatitis is an inflammatory disease of the pancreas that can also involve neighbouring tissues or organs, with a highly variable clinical presentation. In some cases, it has significant morbidity and mortality1,2; 20% of patients adopt a severe evolutionary course, with persistent organ failure leading to mortality in 25%. The reported age range is between 50 and 75 years, as the incidence of acute pancreatitis increases with age1,2.\n\nApproximately 80–90% of cases are due to alcohol consumption and gallbladder lithiasis, while 10–20% have variable aetiology, including idiopathic, hyperlipaemias, viral infections, impaired pancreatic perfusion, ductal obstructions, drugs, and hypercalcemia3,4.\n\nDrug-associated pancreatitis is an underreported entity with an incidence of 0.1 to 2%5. Drugs that have been associated with causing pancreatitis that have been reported in the literature include antihypertensive agents, proton pump inhibitors, anticonvulsants, and chemotherapeutic agents5.\n\nLesions in the pancreatic tissue is caused by aggressor factors (drugs, infection, or metabolic disorder) and by the secondary activation of digestive zymogens within acinar cells, which trigger a subsequent inflammatory response3. The mechanisms by which these complications develop are not fully understood, but intestinal endotoxins and inflammatory mediators play an important role1.\n\nThe diagnosis of drug-associated pancreatitis is by exclusion, so it is recommended to use the “Naranjo Scale of drug adverse reaction probability” in order to calculate the probability of adverse drug effect (Table 1). In 1981 Naranjo et al. validated this scale for cases of adverse drug reactions. In our case, the total score of our patient was 5 (answered affirmatively in questions 1, 2, 3 and 10), classified as probable in relation to furosemide6.\n\nTotal score is the sum of the subcategories. The relationship is categorized as Definitive if the score is> 8; likely if it is between 5 – 8; possible if it is between 1 – 4; and doubtful if the score is 0\n\nDrugs such as furosemide have been described that could damage pancreatic perfusion by decreasing intravascular volume, affecting blood flow, producing ischemia and the subsequent development of acute pancreatitis. It may also stimulate the exocrine pancreas producing a hypersensitivity reaction1,6,7.\n\nIn our patient, Valsartan was suspected of having contributed to the presentation. However, due to the prolonged exposure time, it was dismissed as a possible aetiology.\n\nIn the literature, risk factors associated with drug pancreatitis have been described, of which our patient presented advanced age and vascular risk factors. As a result of these factors an increase in pancreatic toxicity may have occurred, due to atherosclerosis of mesenteric vessels, resulting in furosemide making the perfusion worse by diuresis and intravascular volume depletion8. However, the exact mechanism is not yet known. However, some hypotheses include that furosemide could stimulate exocrine secretion of the pancreas, or mechanisms of hypersensitivity or immune response against a drug-protein. As evidenced in multiple reports, there are many confounding factors, but they only increase pancreatic susceptibility. In our case, the precipitating event is the administration of furosemide.\n\nThe present patient did not present capsule perforation or post-surgery bleeding. Berber-Deseusaa et al. present the case of a patient who presents capsule perforation during the intraoperative period1. In our case, pancreatitis was mild, presenting improvement on the eighth post-surgery day after dialysis. However, the case reported by Berber-Deseusaa et al. presented severe necrotizing pancreatitis, leading to the death of the patient1.\n\nAcute pancreatitis is an infrequent complication reported after TURP1,9–11; it can delay diagnosis if it is not considered as a possibility1. In our patient, the surgery was without complication, unlike previous cases presented in the literature where capsule perforation was observed1. The present surgical time was 55 minutes.\n\nOn the other hand, the evolution of our patient was favourable in contrast to what was presented by Berber et al., where the two patients presented unfavourable evolution with acute renal failure, ventilatory-circulatory deterioration, and death1.\n\n\nConclusions\n\nThe clinical features of pancreatitis following TURP surgery has been reported previously in the literature but infrequently. This condition should be suspected when dealing with pain in the epigastrium post TURP. If this complication is suspected, imaging tests and pancreatic enzyme levels should be immediately requested, as it can have an unfavourable evolution until death.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nBerber-Deseusaa A, Rosas-Navaa JE, de los Santos-Gonzáleza J, et al.: Pancreatitis posterior a resección transuretral de próstata. Publisher Full Text\n\nSánchez AC, Aranda G, Alberto J: Pancreatitis aguda. Bol Méd Hosp Infant México. 2012; 69(1): 3–10. Reference Source\n\nBreijo Puentes A: Pancreatitis aguda. Artículo de revisión. IntraMed J [Internet]. 2014 [citado 1 de abril de 2018]; 3(2). Reference Source\n\nVege SS, Gardner TB, Chari ST, et al.: Low mortality and high morbidity in severe acute pancreatitis without organ failure: a case for revising the Atlanta classification to include \"moderately severe acute pancreatitis\". Am J Gastroenterol. 2009; 104(3): 710–5. PubMed Abstract \| Publisher Full Text\n\nBadalov N, Baradarian R, Iswara K, et al.: Drug-induced acute pancreatitis: an evidence-based review. Clin Gastroenterol Hepatol. 2007; 5(6): 648–61; quiz 644. PubMed Abstract \| Publisher Full Text\n\nNaranjo CA, Busto U, Sellers EM, et al.: A method for estimating the probability of adverse drug reactions. Clin Pharmacol Ther. 1981; 30(2): 239–45. PubMed Abstract \| Publisher Full Text\n\nKaurich T: Drug-induced acute pancreatitis. Proc Bayl Univ Med Cent. 2008; 21(1): 77–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChao C-T, Chao J-Y: Case report: furosemide and pancreatitis: Importance of dose and latency period before reaction. Can Fam Physician. 2013; 59(1): 43–5. PubMed Abstract \| Free Full Text\n\nLevine SR, Gambill EE, Greene LF: Acute pancreatitis following transurethral prostatic resection: report of six cases. J Urol. 1962; 88: 657–63. PubMed Abstract \| Publisher Full Text\n\nNicholls AJ, Catto GR: Acute pancreatitis and acute tubular necrosis following transurethral resection of the prostate. Br J Clin Pract. 1981; 35(6): 235–7. PubMed Abstract\n\nLee MH, Chen KK, Lin AT, et al.: Acute pancreatitis following transurethral resection of prostate. Eur Urol. 1993; 23(3): 419–22. PubMed Abstract \| Publisher Full Text" }	[ { "id": "78702", "date": "15 Feb 2021", "name": "Hemendra N Shah", "expertise": [ "Reviewer Expertise endourological management of kidney stone and enlarged prostate" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a case report of mild pancreatitis that was diagnosed 5 days after TURP. Valuable information about the indication for TURP, patients PSA level, the trial of medical management for BPH, associated medical conditions is missing. There is no description of the possible cause of postoperative Acute kidney injury after TURP. What were serum electrolytes levels? What was the ABG report? Why was frusemide given? A detailed account of postoperative labs including CBC, BMP, and culture if performed will be useful. Was there any hydronephrosis? Any element of resection close to ureteric orifice causing a transient bilateral ureteric obstruction? It will be beneficial to the reader to have a chart of pre and post-operative biochemical investigations. Was the patient on any nephrotoxic medications? Acute pancreatitis can occur in post-operative cases and due to any metabolic disorders. (PONKA JL, LANDRUM SE, CHAIKOF L. Acute Pancreatitis in the Postoperative Patient. Arch Surg. 1961;83(3):475–490.)1 How can authors be so sure that frusemide is the potential cause? It appears multifactorial in the present case.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "78703", "date": "05 Mar 2021", "name": "Hrishi Joshi", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce a case of acute pancreatitis requiring intensive care support. The initial causation is argued to be the surgical procedure (transurethral resection of the prostate), however, it becomes conflicted with the arbitrary use of furosemide peri-procedure without presented rationale. The clinical diagnosis of pancreatitis isn’t wholly convincing with borderline enzyme levels which in other countries of the world is only just diagnostic. There is no information regarding prior patient details regarding BPE and trials of medical management. Procedure details are also not consistent with normal global practice including the use of distilled water as the irrigation fluid and use of loop diuretic both peri-procedure and post-procedure. The leap to conclude that the operation is the cause of the pancreatitis isn’t explained with any substantial supporting evidence and there are a plethora of alternative diagnoses equally as likely. There is little relevance to the practicing urologist from this case.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/8-2051
https://f1000research.com/articles/8-2045/v1	03 Dec 19	{ "type": "Research Article", "title": "Determining the lx and estimating the force of mortality for children under five in Bangladesh", "authors": [ "Md. Rafiqul Islam", "Md. Sohel Rana", "Md. Mosharaf Hossain", "Aziza Sultana Rosy Sarkar", "Ruhani Mat Min", "Md. Rafiqul Islam", "Md. Sohel Rana", "Aziza Sultana Rosy Sarkar", "Ruhani Mat Min" ], "abstract": "Background: Under-five mortality is a health indicator in population health and the health sciences. Force of death is a more accurate measure of mortality than others, which indicates the situation of mortality when time tends to zero. The purpose of this research is to construct a simulation for lx (the number of people living at exact age x) for under-five mortality in Bangladesh as a whole, and separately for rural and urban areas, and then estimating the mortality rate in regard to these matched models. Methods: Secondary data were collected from Bangladesh Demographic and Health Survey 2007. A polynomial model was selected to match the lx values. To prove the accuracy of the model, the rule of cross-validity was applied. Results: It has been shown that fourth degree polynomial models can be adjusted to lx values with explanation of more than 94% variation. It was noted that the mortality rate shows a rapidly decreasing pattern for people aged 0-20 months, a monotonically increasing pattern for those aged 20-53.5 months and then it begins to decrease. It is found that the mortality rate in rural areas is higher than in urban areas of all ages. Conclusions: It has been shown that the mortality rate in rural areas is higher than for urban areas of all ages, except for those aged 53.5 months. The health situation should therefore be improved to reduce mortality in rural Bangladesh.", "keywords": [ "Children under five years", "Force of mortality", "Urban and rural mortality", "F-test", "Bangladesh" ], "content": "Abbreviations\n\nANOVA, Analysis of variance; BDHS, Bangladesh Demographic and Health Survey; BBS, Bangladesh Bureau of Statistics; CVPP, cross validity prediction power; IMR, infant mortality rates; EA, enumeration area; MDG 4: Millennium Development Goal 4; NGO, non-governmental organization\n\n\nIntroduction\n\nThe mortality rate of infants is a reflection of the well-being and nutritional status of children, and also indicates the socio-economic progress in a given country or region. In truth, it reflects the well-being of mothers, the availability and standard of care for well-being, and how easy it is to receive support in the community and/or country. Child mortality rates and, in particular, infant mortality rates (IMRs) dropped sharply in developed countries, but over the past five decades it has been difficult to lower rates in developing countries such as Bangladesh. It has been found that 29,000 children under the age of five die every day in Bangladesh1.\n\nFive years of previous mortality studies of children under five (2010–2014) is 46 deaths per 1000 live births. Bangladesh achieved its Millennium Development Goal 4 (MDG 4) target for deaths under the age of five, decreasing the rate of child mortality to 48 per 1000 births by 2015. The current infant mortality rate is 38 deaths per 1000 live births, and the mortality rate for children is 8 per 1000 children. In childhood, the risk of death in the first month of life (28 deaths per 1000 live births) is almost three times higher than in the next 11 months (10 deaths per 1000 live births). It has also been found that mortality in the neonatal stage constitutes around 61% of all deaths under the age of five2.\n\nIt was noted that in 2007, around 9.2 million live births resulted in death of the child before their fifth birthday3, but in the next twelve months this number fell to 8.8 million4,5, with 41% of these deaths occurring in newborns5. The IMRs are 44.5 and 76 per 1,000 live births universal for more developed and less developed countries, respectively, indicating that infant death is still high in some vulnerable groups and areas, despite declining trends6. The highest under-five death rates are still observed in sub-Saharan Africa, while South Asia has the second highest rates in the world5,7. Many countries have not reached MDG 45, but Bangladesh is currently one of the few countries in the world, particularly in South Asia and sub-Saharan Africa, to have reached this goal. Bangladesh decreased death rates from 151 deaths per 1,000 live births in 1990 to 50 deaths per 1,000 live births in 20158. Current studies have revealed that the mortality risk index is 45 per 1,000 live births6 and that under-five mortality is between 719 and 6510 per 1,000 live births. Although the recent downward trend in infant mortality is notable11, it remains high in Bangladesh due to the high incidence of malnutrition and childhood diseases. One child dies every three or four minutes, resulting in 14 child deaths every hour in Bangladesh12. Deaths usually occur during the neonatal period13.\n\nFor this reason, a number of studies have been carried out on the mortality of infants and children and under five; however, in Bangladesh, there have been few studies investigating the mortality rate of infants and children under the age of five. The polynomial model can be used to approximate a complex nonlinear relationship, which is related to the polynomial degree, which depends on the data pattern. In addition, the response variable is estimated at any time within the domain of the matched model in terms of the explanatory variable, i.e. the respondent's age. Furthermore, mortality is important because it is considered a mortal situation when time tends to zero, but age-specific mortality is the mortality rate within a specific age group. Therefore, the main purpose of this article is to match some models to lx values for children under five in Bangladesh as a whole, as well as in rural and urban areas separately.\n\n\nMethods\n\nTo achieve the above-mentioned goals of this study, secondary data on the lx value for age (in months), i.e. the group data for children under five in Bangladesh as a whole and separately for rural and urban areas, are from Bangladesh Demographic and Health Survey (BDHS) 200714. The 2007 BDHS was representative at national level and included the entire population that did not live in institutional housing units in the country. The survey was used as a sampling frame for the list of enumeration areas (EAs) prepared for the Population and Housing Census 2007, provided by Bangladesh Statistics Office (BBS). The BDHS study was conducted using a two-stage stratified sample of households. A total of 600 areas were initially selected, with 207 clusters in urban areas and 393 in rural areas. A complete list of households was then compiled in all selected areas to provide a sampling framework for the second phase of household selection. In the second phase of the sampling, a systematic sample of an average of 30 households per area was selected to provide statistically reliable estimates of the most important demographic and health variables for the country as a whole, for urban and rural areas individually, and for each of the seven divisions (Dhaka, Rajshahi, Rangpur, Chittagong, Khulna, Barisal and Sylhet). The total sample size is 8,721, of which 5,840 come from rural areas and 2,881 from urban areas during the ten years preceding the survey, in which the sample consists of eligible women, i.e. women who have ever been married and who are aged between 10 and 49 with at least one child. Their data are used as the raw data for this study.\n\nTo better adjust the model, the age (in months) associated with the lx values in Bangladesh and rural and urban areas was smoothed separately using the 4253H smoothing method twice15. The smoothing method was done using Minitab 12.1. It should be noted that the smoothing method is performed to remove age as well as reporting errors before model fitting for better performance as well as better prediction. There are various methods of smoothing, such as the free-hand curve method, the moving average method and 4253H, the double method. The free-hand curve method does not have a mathematical background and the moving average method loses some observations. For this reason, the 4253H method is used, a double method for better efficacy that does not lose any observations.\n\nThe lx values for under-five mortality showed a non-linear distribution. Therefore, an n degree polynomial model is identified in this case and the model is given as:\n\n\n\nin which x represents the age in months that is used as explanatory variable in the model, y represents the lx value, a0 is a constant, ai is the coefficient of the xi (i = 1, 2, 3, ..., n) and u is the error term of the model16. Here, a suitable n is chosen for which the sum of the squares of the error is the lowest. These models were built using SPSS 22.\n\nCross-validation is a technique used in model selection to better estimate the test error of a predictive model. The idea behind cross-validation is to create a better assessment of a model’s predictive performance. For this, to check the legitimacy of these models over the population, the cross validity prediction power (CVPP), ρcv2, is employed at this juncture. The mathematical formula for the CVPP is specified below:\n\n\n\nwhere n is the quantity of classes, k is the number of explanatory variables in the fitted model and R is the correlation between the observed and the fitted values of the response variable. The shrinkage coefficient of the fitted model is the absolute value of (ρcv2−R2); where ρcv2 is the CVPP and R2 is the proportion of variation of this model. Furthermore, the stability situation of R2 of this model is (1 – shrinkage). The estimated CVPP corresponding to R2 and the results for the model fittings are summarized in Table 1. It has been shown that the CVPP can also be used as a model validation technique17–26.\n\nCVPP, cross validity prediction power; df, degrees of freedom.\n\nTo identify the overall significance level of the formulated model and the significance of R2 of the model, the F-test is used in this paper. The formula for the F-test is given as:\n\n\n\nwhere p is the number of parameters of the fitted model, n is the number of cases and R2 is the coefficient of determination of the fitted model27.\n\nThe instantaneous force of mortality at age x is defined as the ratio of the instantaneous rate of decrease in lx to the value of lx. It is denoted by μx and, in the limiting case, it is given using differential calculus by:\n\nμx=Ltt→0lx+t−lxt.lx=−1lxddx(lx)=−ddx(lnlx). [28, 29]\n\nIn brief, it is also called the force of mortality. In fact, the force of mortality is a more accurate measure in mortality studies than age-specific death rates. It measures the mortality situation when time tends to zero. This is an instantaneous measurement, rather than an interval measurement.\n\n\nResults\n\nThe polynomial model is used to fit the age associated with the lx values for children under five in Bangladesh in rural and urban areas, and these fitted models are presented (Figure 1, Figure 2, Figure 3) below:\n\ni) For Bangladesh, y=950.1666-39.3148x+2.1053x2-0.0449x3+0.0003x4; t-value: (42.33616) (-5.84934) (3.96269) (-3.12604) (2.64699)\n\nii) For rural areas, y=966.3290-26.1108x+1.3809x2-0.0294x3+0.0002x4; t-value: (63.16859) (-5.69949) (3.81335) (-3.00112) (2.54014)\n\niii) For urban areas, y=984.0909-13.2389x+0.7251x2-0.0155x3+0.0001 x4; t-value: (138.4389) (-6.2189) (4.3088) (-3.4093) (2.8858)\n\nX axis represents age group and Y axis represents lx.\n\nX axis represents age group and Y axis represents lx.\n\nX axis represents age group and Y axis represents lx.\n\nThe results of the fitted models and the CVPP corresponding to the R2 of these fitted models of lx values for children under five in Bangladesh and in the rural and urban areas are presented in Table 2. It seems that the constructed models (i) to (iii) are cross-validated, and their shrinkages are also shown in Table 2. These imply that the fitted models (i) to (iii) are more than 81% stable, and this is demonstrated in Table 2.\n\nIn addition, it can be seen that the parameters of these models are significant, with more than 94% of the explained variance, which is also shown in Table 3. The stability position for R2 of these matched models is over 87%. The calculated values for the F-test, i.e. the analysis of variance (ANOVA) for these matched models with (5, 7) df, are 26.08, 27.09 and 24.548 and are displayed in Table 2, while the corresponding tabular values are 9.15 for models (i) to (iii) at a significance level of 1%. Consequently, on the basis of these test statistics, it is concluded that the F-test is highly significant, and therefore, that these models are highly significant. That is why the fitting of the models is good. Hence, forecasts based on these models are shown in Table 1.\n\nIt should be mentioned here that other more typical models, such as the linear model (R2 = 0.53), exponential (R2 = 0.55), square (R2 = 0.73) and the cubic model (R2 = 0.85), were also used for the Bangladesh data set, but they did not show a better fit due to the low coefficient of determination.\n\nThe ages associated with force of mortality for children aged under five in Bangladesh, and in rural and urban areas, are estimated from the fitted model of the lx values that are presented in Table 3 and shown in Figure 4 to Figure 6.\n\nX axis represents age in months and Y axis represents force of mortality.\n\nX axis represents age in months and Y axis represents force of mortality.\n\nX axis represents age in months and Y axis represents force of mortality.\n\nIn Table 3, as well as in the numbers described above, it can be seen that the mortality of children under five in Bangladesh, as well as in rural and urban areas, shows a rapidly decreasing pattern at 0–20 months, followed by a monotonically increasing pattern for age from 20 to 53.5 months and then it begins to decrease. In addition, it has been demonstrated that the mortality rate in rural areas is higher than for urban areas of all ages excluding those aged 53.5 months. All three cases show a similar pattern.\n\n\nDiscussion\n\nExplanation of the empirical age patterns of death using these models is one of the most important topics in population studies, especially in demographics. In this study, the fourth degree polynomial models are well-suited to lx values for children ages under five, with an explanation for the huge proportion of variation. The mortality of infants and children in Bangladesh is significantly reduced, but the future increase in life expectancy requires an additional reduction in mortality30. This study estimates how age is related to the mortality of children under the age of five in Bangladesh as a whole and in rural and urban areas separately. We expect these recent mortality findings to convince the government, non-governmental organizations and those responsible for policy planning of the need for a planned increase in socio-demographic development and health care facilities. For this reason, the survival (lx) function taken from the data is selected as the raw material in this study. The mortality force is the ratio between the instantaneous rate of decrease in lx and the value of lx, which is obtained from the functional dependence between lx and age x. Mortality rate should be treated as the probability of death at a given moment, taking into account survival until that time. It was reported that the lx values for the Bangladeshi male population are in line with the four-parameter cubic polynomial model30 and it has been observed that the lx values for the Bangladeshi population are consistent with the third degree polynomial model31. However, in this study, efforts were made to focus on which mathematical model best suits the relationship between age and lx values for minors in Bangladesh as a whole, and in rural and urban areas separately. For this purpose, the polynomial model is chosen. It should be mentioned that a number of studies were carried out using the polynomial model32–35. One of the limitations of the survey is that, except for a few questions, almost all information collected in BDHS surveys is subject to reporting errors.\n\n\nConclusions\n\nIn this study, it was found that the relationship between age and lx values for children under five in Bangladesh results from a four-level polynomial model containing five parameters. The mortality rate shows a rapidly decreasing pattern at 0–20 months and a monotonically increasing pattern between 20 and 53.5 months, and then begins to decrease in all cases. This research result has fundamental strategic implications, especially for educational and awareness program requirements for a real decrease in child mortality and compliance with public health interventions. The researcher recommends additional research, taking into account these unobserved issues that are probably associated with the death of infants and children, to better understand the relationship between family and public factors and the death of a child in Bangladesh. Intervention programs and policies should focus on this research to improve the health consequences for children, in order to achieve future improvement of the situation in Bangladesh as well as in other areas such as sub-Saharan Africa.\n\n\nEthical approval\n\nEthical approval for this study was not applicable, since ethical approval for the collection of data was previously approved for BDHS.\n\n\nData availability\n\nThe data from BDHS 2007 are free to access (https://dhsprogram.com/data/dataset/Bangladesh_Standard-DHS_2007.cfm?flag=0); however, before you can download data, users must register as a DHS data user for reasons laid out on the DHS website (https://www.dhsprogram.com/data/Registration-Rationale.cfm). Dataset access is only granted for legitimate research purposes (https://dhsprogram.com/data/new-user-registration.cfm).", "appendix": "Acknowledgements\n\nWe are heartily grateful and indebted to the Global Research Forum in Rajshahi University, Bangladesh for giving me the opportunity to present this article in a seminar and providing valuable suggestions and constructive criticism that has boosted the quality of the manuscript.\n\n\nReferences\n\nTadesse H, Deribew A, Woldie M: Predictors of defaulting from completion of child immunization in south Ethiopia, May 2008: a case control study. BMC Public Health. 2009; 9: 150–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitra SN and Associates: Bangladesh demographic and health survey 2014. National Institute of Population Research and Training (NIPORT), Dhaka, Bangladesh. Reference Source\n\nUNICEF: Millennium Development Goals, United Nations Children’s Emergency Fund, 2009. Dhaka, Bangladesh. 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Publisher Full Text\n\nMontgomery DC, Peck EA: Introduction to linear regression analysis. John Wiley and Sons, New York. 1982. Reference Source\n\nIslam MR: Mathematical modelling of age and of income distribution associated with female marriage migration in Rajshahi, Bangladesh. Res J App Sci Eng Technol. 2012; 4(17): 3125–29. Reference Source\n\nIslam MR: Modelling and projecting population for Muslim of urban area in Bangladesh. International Journal of Probability and Statistics. 2012; 1(1): 4–10. Publisher Full Text\n\nIslam MR: Modelling age structure and ASDRs for human population of both sexes in Bangladesh. International Journal of Anthropology. 2013; 28(1): 47–53. Reference Source\n\nIslam MR, Hossain MS: Mathematical modelling of age specific adult literacy rates of rural area in Bangladesh. American Open Demography Journal. 2013; 1(1): 1–12.\n\nIslam MR, Hossain MS: Mathematical modelling of age specific participation rates in Bangladesh. Int J Sci Innov Math Res. 2013; 1(2): 150–59. Reference Source\n\nIslam MR, Ali MK, Islam MN: Construction of life table and some mathematical models for male population of Bangladesh. American Journal of Computational and Applied Mathematics. 2013; 3(6): 269–76. Reference Source\n\nHossain MK, Islam MR, Khan MN, et al.: Contribution of socio-demographic factors on antenatal care in Bangladesh: Modelling approach. Public Health Research. 2015; 5(4): 95–102. Reference Source\n\nHossain MS, Islam MR: Age specific participation rates of Curacao in 2011: Modelling approach. American Open Computational and Applied Mathematics Journal. 2013; 1(2): 08–21. Reference Source\n\nIslam MR, Hossain S: Some standard physical characteristics of students in Seoul: Modelling approach. American Journal of Mathematics and Statistics. 2015; 5(5): 230–37. Reference Source\n\nIslam MR, Hoque MN: Mathematical modelling and projecting population of Bangladesh by age and sex from 2002 to 2031. Emerging Techniques in Applied Demography. 2014; 4: 53–60. Publisher Full Text\n\nGujarati DN: Basic econometrics. Third Edition, McGraw Hill, Inc., New York. 1998.\n\nBiswas S: Stochastic processes in demography and applications. Wiley Eastern Limited, New Delhi. 1988. Reference Source\n\nKeyfitz N: Introduction to the mathematics of population. Addison Wesley Publishing Company, Reading, Massachusetts. 1968. Reference Source\n\nIslam MR: Construction of abridged life tables and indirect estimation of some mortality measures of Bangladesh in 2005. Journal of Population Indonesia. 2007; 11(2): 117–30.\n\nIslam MR: Estimation of some mortality measures, modelling of lx and age associated with force of mortality of Bangladesh in 2007. Middle East Journal of Age and Ageing. 2007; 4(6): 32–38.\n\nIslam MR, Hossain MS, Faroque O: U-Shaped pattern of employees’ job satisfaction: Polynomial model approach. Int J Ecosyst. 2014; 4(4): 170–75. 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Reference Source" }	[ { "id": "65215", "date": "03 Jul 2020", "name": "Duah Dwomoh", "expertise": [ "Reviewer Expertise Statistical Methods and its application to health data", "Mathematical Modeling of Infectious diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract:\nThe authors indicated in the results section that mortality rate was monotonically increasing for those aged 20-53.5 months but in the conclusion it is restricted to only those aged 53.5 months. Authors should revise that to reflect the findings as stated in the results\n\nIntroduction:\nThe fact Bangladesh achieved the millennium development goal 4 has been repeated in the first and second paragraphs of the manuscript.\n\nPlease provide a reference to this statement ‘’ … it remains high in Bangladesh due to the high incidence of malnutrition and childhood diseases’’\n\nMethods:\nThe statement ‘’ i.e. the group data for children under five in Bangladesh as a whole and separately for rural and urban areas, are from Bangladesh Demographic and Health Survey (BDHS) 2007’’. I think there is a word missing there. Perhaps are ‘’obtained’’ from the Bangladesh Demographic and Health Survey (BDHS) 2007.\n\nThe authors fitted a polynomial model explaining the number of people living at exact age x. The number of people living at the exact age group x is influenced by factors associated with their survival rate throughout the neonatal period. There are more critical factors such as multiple births, birth spacing, facility delivery, etc. that could influence the outcome measure of interest apart from age in a month. Authors should explain why these critical factors were omitted from the model. Can the addition of these key predictors improve the predictive power of the model?\n\nAuthors conducted internal validation of the model using cross-validation which is good but they could have applied the developed model to a more recent Bangladesh DHS data to validate the model. This could have provided a more rigorous assessment of the predictive performance of the model. In addition, the could have estimated Root Mean Square Percentage Error (RMSPE), Brier Score, and Mean Absolute Percentage Error to assess the predictive performance of the model especially when we have a typical count outcome.\n\nResults:\nThere should be a legend on the graph explaining which graph represents smoothed and predicted for readers who are not statistically inclined. This should go for all the graphs. However, for policy decisions, it could have been interesting to investigate the pattern of the outcome among the wealth quintile as well. I mean conduct similar analysis among various socioeconomic groups.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "66568", "date": "03 Aug 2020", "name": "Mahmudur Rahman", "expertise": [ "Reviewer Expertise I am a Medical Epidemiologist" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDetermining the lx and estimating the force of mortality for children under five in Bangladesh\n\nReview Comments:\n\nIntroduction:\nIntroductory paragraphs focus on IMR whereas this analysis is on Child mortality. The citations are also mostly on IMR than under 5 child mortality.\n\nIt has also been mentioned that “Child mortality rates and, in particular, infant mortality rates (IMRs) dropped sharply in developed countries, but over the past five decades it has been difficult to lower rates in developing countries such as Bangladesh” but the IMR and under 5 Child mortality rate trend of Bangladesh does not support this statement.\n(Ref: https://www.macrotrends.net/countries/BGD/bangladesh/infant-mortality-rate#:~:text=The%20infant%20mortality%20rate%20for,a%204.49%25%20decline%20from%202017) (Ref: https://knoema.com/atlas/Bangladesh/Child-mortality-rate).\n\nThe statistics given regarding under five child mortality (“29,000 children under the age of five die every day in Bangladesh”) is from 2009. However, updated data on IMR and <5yrs mortality could have been cited. Generally, speaking this article referred to data in which most of the cases are old although, recent data were available.\n\nMethod:\nThe analysis of this study was based on the BDHS data of 2007 whereas there were reports/data of 2014, 2017-18 on which this analysis could have been done.\n\nThe observed smoothed, and predicted survival function (lx values) for children under five in Bangladesh:\nThe model explained about 94%, which is a strength of this analysis Table 2: Smoothed and predicted survival function could have been shown for ages in months. At least at 6 months 12 months 18 months…… would be more practical.\n\nIt seems that potential confounding variables were not considered in the best fit models.\n\nSome modifiable risk factors that could have been considered is the analysis of this study.\n\nResults:\nDecreasing and increasing pattern have been shown at specific ages (in months) and also according to residential status. It would be better to show the results either for every month up to the age of 5yrs or at least at some specific ages like 6 months, 12 months, 18 months, so on... This type of presentation of the results might help policy makers to take policy decisions and the researchers to further undertake research to explore the reasons for such findings. As regards the rural/urban distribution, urban slum people are the most vulnerable therefore, breakdown analysis for the slum population would be more practical. However, if secondary data does not allow to do so then it should be stated as a limitation of the study under the discussion section.\n\nDiscussion:\nDiscussion centered on the appropriateness of the modelling, however, some probable explanation(s) of the findings could have been discussed e.g., why a decreasing pattern was observed in ages 0-20 months and increasing trend in ages 20-53.5 months. Nothing has been mentioned about the methodological limitations of this study and the reason for using 2007 BDHS data where data od 2017-18 are available.\n\nConclusion: Concluded on the relationship of age and force of child mortality but did not take into consideration of the rural/urban relationship though it was one of the objectives of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2045
https://f1000research.com/articles/8-2039/v1	02 Dec 19	{ "type": "Clinical Practice Article", "title": "Nonsurgical endodontic management of dens invaginatus: a report of two cases", "authors": [ "Amjad Abu Hasna", "Daniela Maria de Toledo Ungaro", "Allana Agnes Pereira de Melo", "Karen Cristina Kazue Yui", "Eduardo Galera da Silva", "Frederico Canato Martinho", "Ana Paula Martins Gomes", "Daniela Maria de Toledo Ungaro", "Allana Agnes Pereira de Melo", "Karen Cristina Kazue Yui", "Eduardo Galera da Silva", "Frederico Canato Martinho", "Ana Paula Martins Gomes" ], "abstract": "Dens invaginatus is a malformation affecting mainly the superior lateral incisors. It is defined as an infolding of the crown hard tissues, including the enamel and dentin, and can extend up to the root apex. Root canal treatment of this abnormality is considered difficult due to the complex anatomy presented by these teeth. This case series presents nonsurgical endodontic treatment in two cases of dens invaginatus (type II and III) in maxillary lateral incisors. This nonsurgical or conventional endodontic treatment results in healing of the periapical lesions associated with both cases, with no need for extra intervention e.g. surgical or invasive management. The manual instrumentation associated with sodium hypochlorite and calcium hydroxide were able to completely heal the lesions. Radiographic exams were carried out to control and asses the healing. Nonsurgical treatment was successful in both cases with adequate repair after a 6-year follow-up with radiographic and tomographic assessments.", "keywords": [ "Dens invaginatus", "Follow-up", "Root canal treatment." ], "content": "Introduction\n\nDens invaginatus is a growth deformity mainly affecting the superior lateral incisors. It is considered a developmental anomaly and its main characteristic is crown invagination prior to the occurrence of tooth calcification (Rajendran & Sivapathasundharam, 2009). As it is a developmental anomaly, its pathogenetic mechanism is not yet well understood; however, the deformation is through to be due to excessive pressure during dental arch formation, internal enamel epithelium growth failure, rapid and aggressive proliferation, alteration of the enamel, tooth germ accidental fusion or infection process, or trauma injuries (Hülsmann, 1997). In the literature, its incidence ranges from 0.04% to 10% (Hovland & Block, 1977; Rotstein et al., 1987) and the maxillary lateral incisors are considered the most affected (Marwah et al., 2009).\n\nDens invaginatus is grouped into three categories according to the degree of invagination. Type I characterizes an enamel invagination well delimited to the tooth crown; type II is a delimited type of enamel that surrounds the root and can interconnect with the pulp; type III is a severe condition in which invagination exceeds the limit of the cementoenamel junction generating a second foramen (Oehlers, 1957).\n\nThe root canal system of dens invaginatus is characterized by morphologic and anatomic complexity, thus, the diagnoses and treatment planning of such cases is considered difficult (Wayama et al., 2014). Treatment approaches include the sealing of the invagination, surgical or non-surgical endodontic treatment, one-step apexification technique or tooth extraction (Demartis et al., 2009; Wayama et al., 2014). This case series presents nonsurgical endodontic treatment in two cases of dens invaginatus (type II and III) in maxillary lateral incisors.\n\n\nCase 1\n\nA 10-year-old male was indicated to treat the right maxillary lateral incisor. Upon palpation, the patient related positive responses to percussion and palpation in the apical region. Intra-oral examination showed a small change in shape on the crown, without presence of caries or color alteration. Radiographic examination revealed dens invaginatus type III associated with periradicular lesion (Figure 1A).\n\n(A) Preoperative radiograph of right maxillary lateral incisor showing dens invaginatus with periapical area of radiolucency. (B) Radiography after endodontic root canals filling. (C) 18 months follow-up showing new bone formation in the periapical area.\n\nThe tooth had primary and secondary canals, the secondary canal with incomplete apex and associated radiolucent area. The patient had a good general health, without anterior dental trauma history. Conventional root canal treatment was indicated.\n\nThe patient received local anesthesia (Citanest 3% - Dentsply, York, Pennsylvania, USA), absolute isolation took place, and access cavity was carried out, with two root canals being located separately without communication. Both canals presented necrotic pulp.\n\nThe canals were negotiated using size 35 K-flexofile (Dentsply-Maillefer, Ballaigues, Switzerland) and 2.5% sodium hypochlorite NaOCl (Biodinâmica, Ibiporã, PR, Brasil). The working length was measured by using Root ZX apex locator (J. Morita, Kyoto, Japan). The primary canal was instrumented through step-back technique using manual instrument, size 60 K-file (Dentsply-Maillefer, Ballaigues, Switzerland), and then the cervical third of the root canal was widened using Gates-Glidden drills. The secondary canal (invagination) was prepared with a size 90 K-file with abundant irrigation with 2.5% NaOCl.\n\nThe final irrigation of both canals was carried out with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3 minutes activated by ultrasonic stream (Jet Sonic Gnatus Medical and Dental Equipment, Brazil), 5 mL of sterile saline solution to rinse out the EDTA, and sterile paper points used to dry the canals. The intra canal medication used was calcium hydroxide Ca(OH)2 paste (Calen, SSWhite, Rio de Janeiro, RJ, Brazil) for 14 days, the paste was spatulated over a sterile glass plate with a sterile saline solution in a proportion of 1:1 (powder: saline solution), obtaining a tooth paste-like texture\n\nThen the patient returned for a second visit, and the tooth was asymptomatic. Clinical examination revealed no positive responses for vertical percussion or digital palpation and the soft tissues were healthy; Ca(OH)2 was removed. The root canals were obturated by means of thermo-mechanical compactors size 35 (Dentsply-Maillefer, Ballaigues, Switzerland) and lateral condensation technique was performed using gutta-percha cones (Tanari, Tanariman Industrial Ltda, Amazonas, Brazil) and Sealapex sealer cement (SybronEndo Kerr, Romulus Michigan, USA) (Figure 1B). The access cavity was restored with a light cured composite resin (Z100 – 3M ESPE, Minnesota, USA).\n\nClinical and radiographic controls sessions were carried out at 6, 12 and 18 months after treatment (Figure 1C). During the follow-up period, no signs and symptoms related to the respected tooth were reported by the patient. Bone neoformation was noticed in the periapical area. Figure 2 shows cone beam computed tomography 6-years after root canal treatment.\n\n\nCase 2\n\nA 11-year-old female patient was referred for endodontic treatment, indicating sensitivity to palpation in tooth 12 and recurrent sinus tract in the vestibular area of the tooth. Endodontic treatment had been begun 8 months previously by a general practitioner, in which only one canal was located. After several unsuccessful intracanal medication changes, the practitioner referred the patient to treatment with an endodontic specialist.\n\nRadiographic examination showed the presence of dens invaginatus type II associated with periradicular lesion (Figure 3A). A negative response to pulp sensitivity test occurred in this area.\n\n(A) Maxillary lateral incisor showing dens invaginatus and periapical lesion. (B) Radiography image after root canal treatment. (C) 18 months follow-up radiograph showing complete bone repair.\n\nAt the first visit, after intra- and extra-oral clinical examination, the sinus tract was screened and the tooth anesthetized (Citanest 3% - Dentsply, York, Pennsylvania, USA). A rubber dam was applied, the access cavity was modified, and a second canal (invagination) was located. The root canals were negotiated with 15 K-flexofile (Dentsply-Maillefer, Ballaigues, Switzerland) and irrigated with 2.5% NaOCl.\n\nThe working length was measured using Root ZX apex locator. The primary canal was instrumented through step-back technique using manual instrument, a size 50 K-file (Dentsply-Maillefer, Ballaigues, Switzerland), and then the cervical third of the root canal was widened using Gates-Glidden drills. The second canal was prepared to a size 40 K-file using 2.5% NaOCl without use of Gates-Glidden drills.\n\nDuring the instrumentation, the canals were irrigated with 2.5% NaOCl, finally rinsing with 5 mL of 17% EDTA for 3 minutes activated by ultrasonic stream. The EDTA was rinsed out using 5 mL of sterile saline solution. After drying with paper points, the canals were filled with Ca(OH)2 paste.\n\nFourteen days later, the patient related no signs and symptoms of the treated tooth and the sinus tract had cured. At this second visit, the canals were obturated with gutta-percha cones and AH Plus cement (Dentsply DeTrey GmbH, Germany) (Figure 3B) using lateral condensation technique and the tooth was sealed with composite resin in the same session. Clinical and radiographic control of the case was performed at 6, 12 and 18 months, showing complete periapical repair (Figure 3C). The patient underwent orthodontic treatment after the end of root canal treatment. The patient was lost to follow-up and did not come back for cone beam tomography after 6-years of the root treatment.\n\n\nDiscussion\n\nSuccessful endodontics treatment requires debridement and disinfection of the entire root canal system; however, complex root canal morphologic variations make the management of dens invaginatus a challenge (Narayana et al., 2012). Conventional endodontic treatment of dens invaginatus is commonly difficult and complicated, particularly when large periapical lesions are associated (Lichota et al., 2008). Nevertheless with periradicular lesion dimensions, nonsurgical endodontic treatment must be considered before any surgical treatment (Pai et al., 2004; Wayama et al., 2014).\n\nDens invaginatus requires as early as possible diagnosis planning and treatment execution, and generally is found during radiographic investigation (Er et al., 2007). Patients often look for a professional due to acute pain or sinus tract presence. During endodontic treatment, the mechanical instrumentation of the invaginated canal might presents technical difficulties (Lichota et al., 2008).\n\nThe management of such cases when presented with necrotic pulp can be either be treatment with conventional endodontic treatment, surgical treatment, combined endodontic treatment and periapical surgery, intentional reimplantation, or tooth removal (Beltes, 1997; Chaniotis et al., 2008; Wayama et al., 2014). The patient’s age and physical condition, and the case complication and variability are factors that can affect the treatment decision (Beltes, 1997). However, type II or III dens invaginatus cases with periapical lesion, could be managed successfully through conventional endodontic treatment, and considered successful as it results in lesion regression (Lichota et al., 2008; Pai et al., 2004).\n\nIn previous cases (Alessandro et al., 2018; Gound & Maixner, 2004; Nallapati, 2004; Soares et al., 2017), an upper lateral incisor with type III dens invaginatus associated with periradicular lesion and vital pulp in a separate root canal have been managed non-surgically or surgically. The maintenance of pulp vitality of the main canal is possible once the invagination presents no connection with the root canal system (de Sousa & Bramante, 1998). When one of the canals is vital but there is contact between the two canals, a pulpotomy (Kunert et al., 2017) or pulpectomy in the vital canal can be performed. In the literature, intervention in the main canal may not be necessary, especially in cases where the main canal has vital pulp or when there is no anatomical communication with the invaginated canal (Gonçalves et al., 2002).\n\nRoot canals are instrumented with manual files (Goel et al., 2017; Kumar et al., 2014; Rodekirchen et al., 2006; Soares et al., 2017). In the present cases, the widening of the main canal with hand K-files was possible and safe but required more attention and prolonged sessions. The use of microscopy can help to define the access of the invagination and contribute in following preparations. The invagination can be cleaned and shaped with manual or rotary instruments, even if the use of rotary instrumentation within the lesion of type II dens invaginatus is not endorsed due to the fact that the surface is predominantly enclosed by enamel and has varying shapes, which may increase the possibility of endodontic instrument fracture (Bishop & Alani, 2008).\n\nIn many cases, after the instrumentation with manual files and irrigation with NaOCl, Ca(OH)2 paste was primarily selected as an intracanal medication (Gound & Maixner, 2004; Nallapati, 2004; Rodekirchen et al., 2006; Soares et al., 2017; Zhang & Wei, 2017). Ca(OH)2 is the first choice intracanal medication due to its antibacterial action (Gound & Maixner, 2004) and dissolution of organic tissue capacity (Hasselgren et al., 1988). Ca(OH)2 accompanied with various vehicles can also prevent microbial growth (Vianna et al., 2005) and detoxifies residual lipopolysaccharide (Tanomaru et al., 2003). Antibiotic paste when used as an intracanal medication shows an additional effect in serious infections (Goel et al., 2017). This requires changing the paste at various times, with intervals of one month for each one and at least for 3 months (Zhang & Wei, 2017).\n\nCase 1 presented here was classified as dens invaginatus type III. This type of invagination allows the entrance of irritations into the periradicular area, and thus results in periapical pathology (Gonçalves et al., 2002; Jung, 2004).\n\nRecently, pulp revascularization was introduced as a method to treat immature teeth with open apex (Yang et al., 2013). This treatment shows that apexification and total healing of the periradicular lesion occurred, showing the relevance of conservative treatment; however, surgical management may be performed in cases in which the traditional conservative management is not preferred (Wayama et al., 2014).\n\nComputed tomographic scanning is indicated for anatomically complex cases and difficult diagnosis making (Demartis et al., 2009; Wayama et al., 2014). Even more, cone beam computed tomography aids in treatment planning and execution (Kaneko et al., 2011; Patel, 2010).\n\nDens invaginatus associated with periapical lesion can be managed with nonsurgical endodontic treatment, which can result in acceptable periradicular curing (Er et al., 2007). The two cases presented were followed up clinically and radiographically for 18 months, indicating that healing had occurred through the bone neoformation. At 6-year follow-up, cone-beam computed tomography was performed for case 1. Both patients did not report signs, symptoms or problems with the respected teeth at follow-up visits.\n\nThese cases are part of a study assessing nonsurgical endodontic management of dens invaginatus. This study was approved by the Ethics Committee of the Institute of Science and Technology of São Paulo State University (approval number 3.711.314). Written informed consent for participation in the study was obtained from the guardians of the two patients.\n\nWritten informed consent for publication of the patients’ clinical details and clinical images was obtained from the guardians of the two patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAlessandro L, Fabrizio DF, Gennaro DM, et al.: Dens invaginatus with necrotic pulp in a right maxillary lateral incisor with preserved vitality. J Conserv Dent. 2018; 21(1): 109–113. PubMed Abstract \| Free Full Text\n\nBeltes P: Endodontic treatment in three cases of dens invaginatus. J Endod. 1997; 23(6): 399–402. PubMed Abstract \| Publisher Full Text\n\nBishop K, Alani A: Dens invaginatus. Part 2: clinical, radiographic features and management options. Int Endod J. 2008; 41(12): 1137–1154. PubMed Abstract \| Publisher Full Text\n\nChaniotis AM, Tzanetakis GN, Kontakiotis EG, et al.: Combined endodontic and surgical management of a mandibular lateral incisor with a rare type of dens invaginatus. J Endod. 2008; 34(10): 1255–1260. PubMed Abstract \| Publisher Full Text\n\nde Sousa SM, Bramante CM: Dens invaginatus: treatment choices. Endod Dent Traumatol. 1998; 14(4): 152–158. PubMed Abstract \| Publisher Full Text\n\nDemartis P, Dessì C, Cotti M, et al.: Endodontic treatment and hypotheses on an unusual case of dens invaginatus. J Endod. 2009; 35(3): 417–421. PubMed Abstract \| Publisher Full Text\n\nEr K, Kuştarci A, Ozan U, et al.: Nonsurgical endodontic treatment of dens invaginatus in a mandibular premolar with large periradicular lesion: a case report. J Endod. 2007; 33(3): 322–324. PubMed Abstract \| Publisher Full Text\n\nGoel S, Nawal RR, Talwar S: Management of Dens Invaginatus Type II Associated with Immature Apex and Large Periradicular Lesion Using Platelet-rich Fibrin and Biodentine. J Endod. 2017; 43(10): 1750–1755. PubMed Abstract \| Publisher Full Text\n\nGonçalves A, Gonçalves M, Oliveira DP, et al.: Dens invaginatus type III: report of a case and 10-year radiographic follow-up. Int Endod J. 2002; 35(10): 873–879. PubMed Abstract \| Publisher Full Text\n\nGound TG, Maixner D: Nonsurgical management of a dilacerated maxillary lateral incisor with type III dens invaginatus: a case report. J Endod. 2004; 30(6): 448–451. PubMed Abstract \| Publisher Full Text\n\nHasselgren G, Olsson B, Cvek M: Effects of calcium hydroxide and sodium hypochlorite on the dissolution of necrotic porcine muscle tissue. J Endod. 1988; 14(3): 125–127. PubMed Abstract \| Publisher Full Text\n\nHovland EJ, Block RM: Nonrecognition and subsequent endodontic treatment of dens invaginatus. J Endod. 1977; 3(9): 360–362. PubMed Abstract \| Publisher Full Text\n\nHülsmann M: Dens invaginatus: aetiology, classification, prevalence, diagnosis, and treatment considerations. Int Endod J. 1997; 30(2): 79–90. PubMed Abstract\n\nJung M: Endodontic treatment of dens invaginatus type III with three root canals and open apical foramen. Int Endod J. 2004; 37(3): 205–213. PubMed Abstract \| Publisher Full Text\n\nKaneko T, Sakaue H, Okiji T, et al.: Clinical management of dens invaginatus in a maxillary lateral incisor with the aid of cone-beam computed tomography--a case report. Dent Traumatol. 2011; 27(6): 478–483. PubMed Abstract \| Publisher Full Text\n\nKumar H, Al-Ali M, Parashos P, et al.: Management of 2 teeth diagnosed with dens invaginatus with regenerative endodontics and apexification in the same patient: a case report and review. J Endod. 2014; 40(5): 725–731. PubMed Abstract \| Publisher Full Text\n\nKunert GG, Kunert IR, de Figueiredo JA, et al.: Nonconventional Therapeutic Protocol for Type III Dens Invaginatus. J Contemp Dent Pract. 2017; 18(3): 257–260. PubMed Abstract \| Publisher Full Text\n\nLichota D, Lipski M, Woźniak K, et al.: Endodontic treatment of a maxillary canine with type 3 dens invaginatus and large periradicular lesion: a case report. J Endod. 2008; 34(6): 756–758. PubMed Abstract \| Publisher Full Text\n\nMarwah N, Goenka P, Nigam AG, et al.: Combined surgical and nonsurgical endodontic therapy in the treatment of dens invaginatus type 3: a case report. Int J Clin Pediatr Dent. 2009; 2(3): 43–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNallapati S: Clinical management of a maxillary lateral incisor with vital pulp and type 3 dens invaginatus: a case report. J Endod. 2004; 30(10): 726–731. PubMed Abstract \| Publisher Full Text\n\nNarayana P, Hartwell GR, Wallace R, et al.: Endodontic clinical management of a dens invaginatus case by using a unique treatment approach: a case report. J Endod. 2012; 38(8): 1145–1148. PubMed Abstract \| Publisher Full Text\n\nOehlers FA: Dens invaginatus (dilated composite odontome). I. Variations of the invagination process and associated anterior crown forms. Oral Surg Oral Med Oral Patholy. 1957; 10(11): 1204–18 contd. PubMed Abstract \| Publisher Full Text\n\nPai SF, Yang SF, Lin LM: Nonsurgical endodontic treatment of dens invaginatus with large periradicular lesion: a case report. J Endod. 2004; 30(8): 597–600. PubMed Abstract \| Publisher Full Text\n\nPatel S: The use of cone beam computed tomography in the conservative management of dens invaginatus: a case report. Int Endod J. 2010; 43(8): 707–713. PubMed Abstract \| Publisher Full Text\n\nRajendran A, Sivapathasundharam B: Shafer’s Textbook of Oral Pathology. 6/e. 6th Edition. 2009. Reference Source\n\nRodekirchen H, Jung M, Ansari F: Dens invaginatus type II: case report with 2-year radiographic follow-up. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 102(4): e121–5. PubMed Abstract \| Publisher Full Text\n\nRotstein I, Stabholz A, Heling I, et al.: Clinical considerations in the treatment of dens invaginatus. Endod Dent Traumatol. 1987; 3(5): 249–254. PubMed Abstract \| Publisher Full Text\n\nSoares TRC, Silva LPD, Andrade Risso P, et al.: Management of a Permanent Maxillary Lateral Incisor with Vital Pulp and Necrotic Dens Invaginatus Type III. J Dent Child (Chic). 2017; 84(3): 149–151. PubMed Abstract\n\nTanomaru JM, Leonardo MR, Tanomaru Filho M, et al.: Effect of different irrigation solutions and calcium hydroxide on bacterial LPS. Int Endod J. 2003; 36(11): 733–739. PubMed Abstract \| Publisher Full Text\n\nVianna ME, Gomes BP, Sena NT, et al.: In vitro evaluation of the susceptibility of endodontic pathogens to calcium hydroxide combined with different vehicles. Braz Dent J. 2005; 16(3): 175–180. PubMed Abstract \| Publisher Full Text\n\nWayama MT, Valentim D, Gomes-Filho JE, et al.: 18-year follow-up of dens invaginatus: retrograde endodontic treatment. J Endod. 2014; 40(10): 1688–1690. PubMed Abstract \| Publisher Full Text\n\nYang J, Zhao Y, Qin M, et al.: Pulp revascularization of immature dens invaginatus with periapical periodontitis. J Endod. 2013; 39(2): 288–292. PubMed Abstract \| Publisher Full Text\n\nZhang P, Wei X: Combined Therapy for a Rare Case of Type III Dens Invaginatus in a Mandibular Central Incisor with a Periapical Lesion: A Case Report. J Endod. 2017; 43(8): 1378–1382. PubMed Abstract \| Publisher Full Text" }	[ { "id": "57373", "date": "13 Dec 2019", "name": "Carla Renata Sipert", "expertise": [ "Reviewer Expertise Endodontics", "cell and mollecular biology", "innate immunity." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHasna and coworkers aimed to demonstrate the success of an endodontic treatment protocol for dens invaginatus committed by pulpal necrosis and apical periodontitis. The authors showed a 6-year control by using cone beam computer tomography confirming the treatment success. First described by Ploquet in 1794 in a whale's tooth1, this malformation was once referred to as a \"tooth within a tooth\". Interestingly, due to the nature of the malformation, the root canal anatomy becomes unique and unpredictable. Therefore, the clinical report here presented using conventional endodontic treatment might still be considered as the gold standard protocol to control the pulpal cavity and invagination lumen contamination.\nOverall, the manuscript is very well written. A suggestion for improving the content, the authors could include data regarding the bilateral occurrence of this anomaly at upper lateral incisors (43%2) in the Introduction’s first paragraph. The structure named by the authors as “secondary canal” could be instead be named as the “invagination lumen” since structurally they are distinctly developed. The invagination lumen is not filled by pulpal tissue such as the tooth canal. Also at the sentence “Both canals presented necrotic pulp”, I would suggest to replace the term “pulp” for “necrotic tissue” as the invagination lumen is not expected to be found with pulpal tissue either vital or necrotic. Finally, when discussing the effects of calcium hydroxide, mentioning “detoxifies residual lipopolysaccharide”, please consider to cite also the study of Safavi & Nichols (1993)3 who unequivocally demonstrated the LPS lipid A hydrolysis by calcium hydroxide.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "57371", "date": "16 Dec 2019", "name": "Vera Lúcia Schmitt", "expertise": [ "Reviewer Expertise Restorative Dentistry", "Tooth Adhesion", "Dental Bleaching and Dental Materials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well written and can help the clinician to perform a better diagnosis and treatment plans in such cases. The case series seems to be well-executed and respected the scientific evidence in the literature.\nThe abstract was concise and explained well the purpose and results obtained in the case series.\nIt can be suggested the use of more recent references (2015-2019).\nIn the 5th paragraph of the discussion, the authors discussed the instrumentation methods, comparing manual x rotary. It could be useful to discuss more about different points of view.\n\nIn the 7th paragraph of the discussion, the authors discussed new methods such as revascularization and surgical therapy. Why revascularization not used in this case? And in which cases is the traditional conservative management not preferred? This information could help the clinicians to perform better treatment planning.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2039
https://f1000research.com/articles/8-1429/v1	14 Aug 19	{ "type": "Research Article", "title": "Peak expiratory flow rate and chronic respiratory symptoms among restaurant workers: a cross-sectional study from Thailand", "authors": [ "Chudchawal Juntarawijit" ], "abstract": "Background: Cooking smoke is a major source of indoor air pollution affecting millions of people worldwide. To date, there has been no epidemiological study to show the variation in health effects resulting from work at different kinds of restaurants in Thailand. This study determines lung function and chronic respiratory symptoms of workers in four types of eateries commonly found in Thailand. Methods: This is a cross-sectional study of 321 people working in four common types of restaurants in Thailand: ‘tamsang’ restaurants (from the Thai word ร้านอาหารตามสั่ง, a restaurant that makes a variety of foods to order) (170 people), papaya salad restaurants (51 people), noodle restaurants (50 people), and barbecue stalls (50 people). The restaurant workers’ demographic data as well as information on their working conditions was collected using a questionnaire administered in a face to face interview. Each worker’s peak expiratory flow rate was measured using a portable peak flow meter. Results: This study found that working in a ‘tamsang’ restaurant is associated with a higher risk of poor lung function (OR = 2.59, 95% CI 1.33–5.06) and a higher prevalence of moderate dyspnea symptoms (OR = 3.79, 95% CI 1.63–8.79) compared to working in a papaya salad restaurant. The study also found that each of the following were associated with poor lung function and/or chronic respiratory symptoms: cooking with palm oil, having irritated teary eyes while cooking, cooking without a ventilation hood, long past experience working at restaurants, and working in a small cooking area (1–6 m2). Conclusions: Work in different kinds of restaurants with variations in cooking methods and work conditions produces diverse effects on airway and lung function. Regulatory organizations should pay careful attention to protecting the health of restaurant workers, especially those working in ‘tamsang’ restaurants.", "keywords": [ "Indoor air pollution", "cooking smoke", "restaurant workers", "peak expiratory flow rate", "chronic respiratory symptoms", "lung function" ], "content": "Introduction\n\nRecently cooking smoke has received increased public attention as an indoor and outdoor source of air pollution. The World Health Organization estimated that in 2018 inefficient cooking using solid fuels (biomass, kerosene and coal) caused premature death of about 4 million people worldwide1. Previous studies have clearly established that cooking oil fumes (COFs) commonly contain fine particulate matter and many other toxic compounds, including volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), aldehydes, alkanoic acids, nitrogen dioxide (NO2), and carbon monoxide (CO)2–4. The exact composition and concentration of COFs depend on several factors, including cooking temperature, cooking methods, and cooking oil/fuel type5. Other research has reported that the cooking method releasing the most particles, especially in the ultrafine size range, is deep frying, followed by regular frying, stir frying, boiling, and steaming6,7. Concentrations of volatile organic compounds can vary from 257.5 to 3,494.0 µg/m3, depending on cooking style3. A study of two Chinese cooking styles found concentrations of fine particles, mostly fatty acids, to be 1,406.3 ± 293.4 µg/m3 for Hunan cooking and 672.0 ± 295.8 µg/m3 for Cantonese cooking4. It has been estimated that cooking with natural gas could add 21–30% to weekly average indoor concentrations of CO and 25–39% to those of NO2, depending on ventilation and season of the year8.\n\nPeople working in a kitchen, whether at home or in a restaurant setting, are at risk of exposure to cooking smoke and related health consequences. A study of people using natural gas to cook at home without a ventilation hood predicted that they would be exposed to NO2, CO, and formaldehyde at 62%, 9%, and 53% above established safety limits, respectively8. Lai et al.9 reported a close correlation between levels of toxic chemicals in restaurant air and levels of those same chemicals in the urine of chefs. Exposure to cooking smoke can result in various kinds of respiratory problems. A recent study from Taiwan reported an increased risk of lung cancer among chefs cooking Chinese food10. Studies from various parts of the world have reported an elevated prevalence of both acute and chronic respiratory symptoms and diseases among people involved in cooking11–17.\n\nLung function is another effect that has been associated with cooking smoke. A large study of elementary school children found reductions of peak expiratory flow up to 3.4% among children in families cooking with gas18. A study of Chinese restaurants in Hong Kong reported that workers cooking with gas had poorer lung function than those cooking with electricity12. A similar result was also observed in a study from India2. In Nigeria, workers exposed to wood smoke and oil fumes from a local style of grilled meat called “mai suya” have lower forced expiratory volume in one second (FEV1) and lower forced vital capacity (FVC) compared to an unexposed control group17.\n\nIn Thailand, four common types of eateries are: ‘tamsang’ restaurants (a kind of Thai restaurant that makes a variety of foods to order, from the Thai word ร้านอาหารตามสั่ง), noodle restaurants, papaya salad restaurants, and roadside barbecue stalls19 The kinds of food available at these four sorts of eateries are not the same. Of the four, ‘tamsang’ restaurants sell the widest variety of food, mainly popular Thai dishes, including red curry, basil stir fry, fried rice and numerous other fried dishes. Most of the food from ‘tamsang’ restaurants is cooked at high temperatures, creating a lot of oily steam and pungent vapors from frying chili peppers and other spicy ingredients. In noodle restaurants, much of the food is boiled but some noodle restaurants also make fried dishes, like phat Thai and other fried noodle dishes. At roadside barbecue stalls, various kinds of meat (beef, pork, chicken, and seafood) are grilled, usually over charcoal, producing abundant smoky vapors. In papaya salad restaurants, the salad is often served with some side dishes, such as sour soup, spicy meat salad, and grilled chicken. Therefore, papaya salad restaurants do employ a variety of cooking methods, including boiling, frying, grilling, but there is relatively less frying than at a ‘tamsang’ restaurant and less grilling than at a barbecue stall19.\n\nTo date, there has been no epidemiological study to show the variation in health effects resulting from work at different kinds of restaurants in Thailand. A previous study did find a higher occurrence of respiratory symptoms among restaurant workers in general, but for statistical reasons the study was unable to demonstrate a significant difference in health effects between different types of restaurants16. The current study investigates peak expiratory flow rate (PEFR) and chronic respiratory symptoms among workers at different types of restaurants in Thailand. The study results are useful for the prevention and control of occupational health problems among restaurant workers.\n\n1. To measure peak expiratory flow rate (PEFR) and to investigate the prevalence of chronic respiratory symptoms of workers in different types of restaurants\n\n2. To determine the associations between restaurant types/other risk factors and PEFR/ chronic respiratory symptoms\n\n\nMethods\n\nThis study is a cross-sectional study.\n\nPhitsanulok is a medium size province in northern Thailand, about 400 km from Bangkok, with a population of 865,368 in 2017, and its largest city is Phitsanulok City. In 2017, Phitsanulok City reported that there were 2,511 restaurants within the city limits (personal communication, December 15, 2017).\n\nIn this study, a cluster sampling technique was employed by first divided restaurant types into four groups (‘tamsang’restaurant, noodle restaurant, papaya salad restaurant, and barbecue stall), and then, a consecutive sampling technique was applied for choosing the individual restaurants. In each restaurant one worker, at least 20 years old, who were free from certain chronic diseases (heart disease; cancer; or respiratory diseases such as asthma, chronic obstructive pulmonary disease, and emphysema) employees were interviewed. If possible we aimed to interview a chef. A total of 321 participants (170 from ‘tamsang’ restaurants, 50 from noodle restaurants, 51 from papaya salad restaurants, and 50 from barbecue stalls) were selected, this proportion roughly representing the frequency with which each restaurant type is encountered in the city\n\nThe sample size calculation for a proportion or descriptive study was used to calculate the optimal sample size which found to be 322 using OpenEpi (version 3.0). The sample size calculation employed the following assumptions: population size = 2,500 (based on data from Phitsanulok City Hall), proportion of population with outcome = 0.40 (based on the previous study of the author, which found a prevalence of respiratory symptoms to be about 12% to 54%16, confidence interval = 95%, and standard error = 0.05.\n\nA questionnaire administered by interviewers was used to collect data in face-to-face interviews with restaurant workers (provided as extended data in English and Thai20). The interviews took place in a restaurant. The data was collected during the period of January–March 2018, when the weather is dry and mild in Thailand and therefore is unlikely to affect the respiratory health of the participants. The questionnaire was identical to that in a previous study by the current author on respiratory symptoms among restaurant workers16. The questionnaire was not validated but before use, it was tested for question sequencing and understanding. In addition to demographic data, the study also collected information on cooking fuel, cooking oil, kitchen size (approximated by interviewer) and types, use of ventilation hood, and frequency of tears while cooking (TWC) caused by eye irritation.\n\nThe questions asked on chronic respiratory symptoms were developed based on British Medical Research Council (Medical Research Council Questionnaire, MRCQ)21 and American Thoracic Association (ATS-DLD-78) questionnaires22. Information on respiratory symptoms that were collected in this study included: chronic cough, chronic phlegm, wheeze, moderate dyspnea, and severe dyspnea. People with ‘chronic cough’ refers to those who cough with or without phlegm at least 4 to 6 times a day for 4 or more days out of the week. ‘Chronic phlegm’ refers to people who have sputum for at least twice a day for 4 or more days per week. ‘Wheeze’ refers to those who breathe with a whistling sound whether or not they have a cold. ‘Moderate dyspnea’ refers to people with shortness of breath when walking briskly or exercising. ‘Severe dyspnea’ refers to people with shortness of breath even when undertaking ordinary daily activities.\n\nPeak expiratory flow rate (PEFR) was measured using a portable peak flow meter from MicroPeak (MPE8200EU), CareFusion Company, United Kingdom. The meter can measure flow rates in the range of 60–900 L/min, with an accuracy of ±5% (10 L/min). The PEFR of each study participant was measured three consecutive times, and the highest reading was selected to be their PEFR record. This figure was then used to compare with a standard PEFR of Thai people, and study participants with a PEFR of less than 80% of the standard are considered to have abnormal lung function23.\n\nThe study data was collected by two graduate students from Naresuan University’s Environmental Sciences master degree program.\n\nData was analyzed using IBM SPSS (version 19) software. Demographic and frequency variables were analyzed using descriptive statistics. The associations between restaurant types and PEFR/ respiratory symptoms were analyzed using multiple logistic regression. All analyses were two-sided, with a 95% confidence interval, and a p-value of <0.05 was considered to be statistically significant.\n\nWritten informed consent was obtained from each study participant before the interviewing process. At that time, study participants were informed of the study’s purposes, the data collection procedure, and their right to refuse participation in the study. This study was approved in advance by the Naresuan University Board of Ethics (Certificate of Approval (COA) number: 033/2018).\n\n\nResults\n\nMost of the study participants were female cooks with a similar mean age across all four types of restaurants studied. Most of the participants had been working for more than one year. Less than 20% of the participants were current cigarette smokers. Additional information on the demographic data is shown in Table 1 and underlying data24.\n\nSignificant with p<0.05 (2-sided)\n\nChi-square test for category data, and ANOVA for continuous variable\n\nThe prevalence of abnormal peak expiratory flow rate (PEFR) and chronic respiratory symptoms in the different types of restaurants (‘tamsang’ restaurant, noodle restaurant, papaya salad restaurant, barbecue stall) are shown in Table 2. Results of this study reveal that participants working in ‘tamsang’ restaurants have a significantly lower mean PEFR (p<0.001) as well as higher rates of abnormal PEFR than participants at other restaurant types. These workers also had a higher prevalence of moderate dyspnea.\n\nSignificant with p <0.05 (2-sided), ANOVA test\n\n*Significant with p <0.05 (2-sided), Chi-square test\n\nFurther analysis using multiple logistic regression showed that those who worked in ‘tamsang’ restaurants are at a significantly higher risk (p <0.05) of having poor PEFR (OR = 2.59, 95% CI 1.33–5.06), and moderate dyspnea (OR = 3.79, 95% CI 1.63–8.79) compared to papaya salad restaurant workers (Table 3). A higher risk of poor PEFR and/ or chronic respiratory symptoms were also significantly associated (p <0.05) with the use of palm oil for cooking, with having frequent TWC, use of a ventilation hood, and with working in 1–6 m2 kitchen. All analyses were carried out using the following covariates: age, gender, BMI, cigarette smoking, and cooking at home.\n\naAdjusted for: Gender (male vs. female), age (continuous), body masss index (BMI) (continuous), tobacco use (current smoker, ex-smoker, never smoked), cooking at home (almost always, sometimes, rarely)\n\nbAnalysis using only data from ‘tamsang’ restaurant workers\n\n Statistically significant\n\n\nDiscussion\n\nThis study revealed that a large proportion of restaurant workers have poor PEFR, with the highest frequency of 64.7% found in ‘tamsang’ restaurants and the lowest rate of 41.2% in papaya salad restaurants (Table 2). Compared to workers in papaya salad restaurants, workers in ‘tamsang’ restaurants are more likely to have a low PEFR (OR = 2.59, 95% CI 1.33–5.06) and moderate dyspnea symptoms (OR = 3.79, 95% CI 1.63–8.79) (Table 3). These results are consistent with previous literature, which has reported that cooking smoke contains pollutants, such as fine particulate matter, acrolein, formaldehyde, and NO2, that can cause airway irritation2,25,26. These pollutants are found more often in high temperature frying than in boiling or steaming3,27, which poses a real hazard to workers at ‘tamsang’ restaurants, since, as mentioned earlier, ‘tamsang’ restaurants do a lot of frying. More pollutants can also be expected from barbecue stalls, which usually grill meat with charcoal. However, this type of eatery is usually located outdoors in the open air, therefore exposure can be greatly reduced by the wind. Also somewhat surprising in this study, ‘tamsang’ restaurant workers were found to have a lower risk of severe dyspnea (OR = 0.45, 95% CI 0.20–0.99). This might be the result of the so called ‘healthy worker effect’ (HWE), in which people who have severely illness are excluded from the job28. Similar findings were also observed in the association between working as a cook and prevalence of severe dyspnea (OR = 0.33, 95% CI 0.11–0.98) (Table 3).\n\nThis study surprisingly found that cooking with palm oil is associated with 5.38 (95% CI 1.43–20.31) times the risk of having low PEFR compared to cooking with lard (Table 3). This finding is contrary to information that palm oil contains mostly saturated and monounsaturated fats, similar to lard, and that both of them are resistant to oxidation and create relatively few harmful pollutants when cooked27,29. However, a study by Lee et al.30 also found a negative effect from palm olein in the form of higher levels of aldehyde when frying chicken with palm olein compared to other cooking oils, despite similar amounts of fatty acids. At present, data on health effects of palm oil is limited. Further research is therefore needed to investigate this issue.\n\nThe current study also found that low PEFR is correlated with tears while cooking (TWC) in a dose-response fashion, with an OR of 3.07 (95% CI 1.09–8.63) for those who often had TWC and an OR of 2.80 (95% CI 1.29–6.06) for those who only occasionally had TWC (Table 3). TWC was also significantly correlated with many chronic symptoms, including cough, wheezing, and moderate dyspnea. This result confirms that TWC is a good marker for cooking smoke. In a previous study of the current author, TWC was also found to be correlated with respiratory symptoms16.\n\nThis study also showed that not using a ventilation hood is significantly associated with an increased risk of chronic cough (OR = 2.25, 95% CI 1.03–4.94) and severe dyspnea (OR = 2.58, 95% CI 1.14–5.86) (Table 3). This finding was consistent with general thinking and previous research that has found that ventilation hoods, even when operating inefficiently, can significantly reduce cooking smoke exposure8,31. On the other hand, regarding kitchen location, there were no significant associations found. A study in Ghana similarly found that concentrations of PM2.5 and black carbon did not significantly differ across outdoor, enclosed, and semi-enclosed kitchens5.\n\nInside ‘tamsang’ restaurants, people working in a smaller kitchen area of 1–6 m2 have an increased risk of abnormal PEFR (OR = 2.22; 95% CI 1.13–4.36), compared to those working in a larger kitchen area of ≥7 m2 (Table 3). One possible explanation is that restaurants with a larger kitchen area tend to have and use ventilation hoods more often than restaurants with a smaller kitchen area. Another possible explanation is that because a large kitchen area has more space, there is a greater volume of air to dilute the pollutants. Further analysis revealed that, in fact, large kitchens use ventilations hoods less frequently than small kitchens (27.5% vs. 44.3%, respectively). Thus, the first idea can be rejected.\n\nThis study employs a cross-sectional design, in which data on both cooking smoke exposure and health outcome were collected at the same time. Thus, a causal association between the two cannot be drawn. Further study using an alternate design would be useful. Study results might also be adversely affected by small sample size and non-probabilistic sampling technique. This study used data of restaurants located in only one municipality area which may not represent all the restaurants in the whole country. However, all communities in each province of Thailand have similar types of restaurants and cooking style.\n\nAlthough the questionnaire was developed with reliable questions from respected medical associations, there is a chance of information bias regarding respiratory symptoms, which were reported by study participants rather than with confirmation from medical doctors.\n\nApart from cooking smoke, restaurant workers might also be exposed to roadside air pollution, and this could skew results. However, based on data from the Pollution Control Department of Thailand, all six criteria pollutants (PM10, Pb, CO, NOx, SO2, O3) in the Phitsanulok area were within safe air standards.\n\n\nConclusion\n\nWorking in a Thai restaurant increases risk of abnormal lung function and chronic respiratory symptoms. Compared to working in a papaya salad restaurant, working in a ‘tamsang’ restaurant is associated with a lower PEFR, and higher rates of moderate dyspnea. Use of palm oil, having frequent tears while cooking, not using a ventilation hood, and working in a small kitchen area are significant predictors of poor lung function and/ or chronic respiratory symptoms. Relevant regulatory organizations need to pay attention to and address the danger in order to protect the health of restaurant workers, particularly those in ‘tamsang’ restaurants.\n\n\nData availability\n\nFigshare: PEFR and chronic respiratory symptoms. https://doi.org/10.6084/m9.figshare.8980592.v224\n\nThis project contains the following underlying data:\n\n- PEFR_Chronic_symptoms.sav (Collected demographic and respiratory data)\n\n- Data Dictionary.docx (Word document containing dictionary for study dataset)\n\nFigshare: Questionnaire-PEFR-chronic respiratory symptoms. https://doi.org/10.6084/m9.figshare.9114503.v120\n\nThis project contains the following extended data:\n\n- Questionnaire-English.docx (Study questionnaire, English)\n\n- Questionnaire-Thai.docx (Study questionnaire, Thai)", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe author is grateful to all the restaurant workers who took time to participate in this study and provided their valuable information. Thank you very much to Ms. Jintana Peangkhamrak and Ms. Waraporn Uraisri, graduate students in the Environmental Science program, for collecting so much data. Thank you also to Mr. Paul Freund of Naresuan University’s Writing Clinic (DIALD) for editing assistance.\n\n\nReferences\n\nWorld Health Organization: Household air pollution and health. 2018. Reference Source\n\nSingh A, Kesavachandran CN, Kamal R, et al.: Indoor air pollution and its association with poor lung function, microalbuminuria and variations in blood pressure among kitchen workers in India: a cross-sectional study. Environ Health. 2017; 16(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng S, Wang G, Lang J, et al.: Characterization of volatile organic compounds from different cooking emissions. Atmos Environ. 2016; 145: 299–307. Publisher Full Text\n\nHe LY, Hu M, Huang XF, et al.: Measurement of emissions of fine particulate organic matter from Chinese cooking. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLai CH, Jaakkola JJ, Chuang CY, et al.: Exposure to cooking oil fumes and oxidative damages: a longitudinal study in Chinese military cooks. J Expo Sci Environ Epidemiol. 2013; 23(1): 94–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin PC, Peng CY, Pan CH, et al.: Gender differences and lung cancer risk in occupational chefs: analyzing more than 350,000 chefs in Taiwan, 1984–2011. Int Arch Occup Environ Health. 2019; 92(1): 101–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSvendsen K, Sjaastad AK, Sivertsen I: Respiratory symptoms in kitchen workers. Am J Ind Med. 2003; 43(4): 436–9. PubMed Abstract \| Publisher Full Text\n\nWong TW, Wong AH, Lee FS, et al.: Respiratory health and lung function in Chinese restaurant kitchen workers. Occup Environ Med. 2011; 68(10): 746–52. PubMed Abstract \| Publisher Full Text\n\nDuflo E, Greenstone M, Hanna R: Cooking Stoves, Indoor Air Pollution, and Respiratory Health in India \| The Abdul Latif Jameel Poverty Action Lab. J-PAL. 2010. Reference Source\n\nKurmi OP, Semple S, Simkhada P, et al.: COPD and chronic bronchitis risk of indoor air pollution from solid fuel: a systematic review and meta-analysis. Thorax. 2010; 65(3): 221–8. PubMed Abstract \| Publisher Full Text\n\nKilabuko JH, Nakai S: Effects of cooking fuels on acute respiratory infections in children in Tanzania. Int J Environ Res Public Health. 2007; 4(4): 283–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuntarawijit C, Juntarawijit Y: Cooking smoke and respiratory symptoms of restaurant workers in Thailand. BMC Pulm Med. 2017; 17(1): 41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdewole OO, Desalu OO, Nwogu KC, et al.: Respiratory symptoms and lung function patterns in workers exposed to wood smoke and cooking oil fumes (mai suya) in Nigeria. Ann Med Health Sci Res. 2013; 3(1): 38–42. PubMed Abstract \| Free Full Text\n\nMoshammer H, Hutter HP, Neuberger M: Gas cooking and reduced lung function in school children. Atmos Environ. 2006; 40(18): 3349–54. Publisher Full Text\n\nIt’s better in Thailand: Thai food guide to different restaurant types - It’s better in Thailand. 2019. Retrieved July 26, 2019. Reference Source\n\njuntarawijit c: Questionnaire-PEFR-chronic respiratory symptoms. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9114503.v1\n\nCotes JE, Chinn DJ: MRC questionnaire (MRCQ) on respiratory symptoms. Occup Med (Chic Ill). 2007; 57(5): 388. Publisher Full Text\n\nTennant S, Szuster F: Nationwide monitoring and surveillance question development: Asthma. Working Paper Series No. 2. 2003; (2). Reference Source\n\nDejsomritrutai W, Nana A, Maranetra KN, et al.: Reference spirometric values for healthy lifetime nonsmokers in Thailand. J Med Assoc Thai. 2000; 83(5): 457–66. PubMed Abstract\n\njuntarawijit c: PEFR and chronic respiratory symptoms. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8980592.v2\n\nAmaral AF, Ramasamy A, Castro-Giner F, et al.: Interaction between gas cooking and GSTM1 null genotype in bronchial responsiveness: results from the European Community Respiratory Health Survey. Thorax. 2014; 69(6): 558–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulin L, Hansel N: Particulate air pollution and impaired lung function [version 1; peer review: 3 approved]. F1000Res. 2016; 5: pii: F1000 Faculty Rev-201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng CY, Lan CH, Lin PC, et al.: Effects of cooking method, cooking oil, and food type on aldehyde emissions in cooking oil fumes. J Hazard Mater. 2017; 324(Pt B): 160–7. PubMed Abstract \| Publisher Full Text\n\nShah D: Healthy worker effect phenomenon. Indian J Occup Environ Med. 2009; 13(2): 77–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarwar A, Vunguturi S, Ferdose A: A Study on Smoke Point and Peroxide Values of different widely used Edible Oils. 2016. Reference Source\n\nLee CY, Zhou WB, Bansal G: Determination of the Aldehyde Levels in Frying Oil Using p-Anisidine Test. Glass. 2007. Reference Source\n\nDelp WW, Singer BC: Performance assessment of U.S. residential cooking exhaust hoods. Environ Sci Technol. 2012; 46(11): 6167–73. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53449", "date": "16 Sep 2019", "name": "Satoshi Nakai", "expertise": [ "Reviewer Expertise Environmental Epidemiology", "Exposure Sciences", "Health Risk assessment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author discussed the health effects of cooking smoke at restaurants to workers. It has interesting views and it is valuable to index the manuscript. It should be useful to evaluate and control the indoor air environment of cooking places in restaurants, even though the study information may be limited to Thailand.\nHowever, several things shown in the following comments should be revised:\nSome sentences are incomplete. Please check the manuscript again.\n\nThe reviewer considers that papaya salad restaurants are referenced, so the author had better set the salad restaurants at the end of the list. Readers can understand which type of restaurants the reference is.\n\nDoes the author have some quantitative information about the concentration inside restaurants in Thailand? The author mentioned the other countries’ situation, but no one can image the environment of Thailand. It is very useful for the readers. If not, the author must discuss the environment of the restaurant, and the similarity and difference between Thailand and other countries.\n\nAre interviewers and examiners of lung function the same persons? Are the interviews and PEFR test conducted at the same time?\n\nSecond line of “Data analysis”: What are “frequency variables”? Unclear.\n\nThe author uses “abnormal lung function”, “poor PEFR”, “low PEFR” in this manuscript. The reviewer considers these are the same. Please consider the usage of these terms.\n\nThe author shows the covariates in the analysis at “Results”. These should be shown in “Methods”.\n\nThe author writes “cooking at home” in the covariate list, but no data related to cooking at home are shown. Why? The author discussed the effects other than cooking smoke in restaurants. The above information should be important.\n\nWhy did the author not include “years of working” in this analysis? The reviewer considers it is related to exposure duration and important as an exposure index.\n\nThe author mentioned the healthy worker effect for severe dyspnea. Why not the other outcomes related to HWE? Please add some comments.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5044", "date": "02 Dec 2019", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "1. Some sentences are incomplete. Please check the manuscript again.The manuscript was rechecked and the errors were corrected.2. The reviewer considers that papaya salad restaurants are referenced, so the author had better set the salad restaurants at the end of the list. Readers can understand which type of restaurants the reference is.The list was rearranged and papaya salad restaurants were presented last.3. Does the author have some quantitative information about the concentration inside restaurants in Thailand? The author mentioned the other countries’ situation, but no one can image the environment of Thailand. It is very useful for the readers.If not, the author must discuss the environment of the restaurant, and the similarity and difference between Thailand and other countries.Unfortunately no specific information about concentration of smoke pollutants inside restaurants in Thailand appears to exist. However, discussion about the environment of the restaurants was added to the discussion. 4. Are interviewers and examiners of lung function the same persons? Are the interviews and PEFR test conducted at the same time?Yes, the interviewers and examiners of lung function are the same persons, and the interviews and PEFR test was conducted at the same time.This information was added to the method section.5. Second line of “Data analysis”: What are “frequency variables”? Unclear.The “Data analysis” was duly revised and the term no longer needed to be used.6. The author uses “abnormal lung function”, “poor PEFR”, “low PEFR” in this manuscript. The reviewer considers these are the same. Please consider the usage of these terms.The inconsistency issue was corrected by using only one term, “poor PEFR”.7. The author shows the covariates in the analysis at “Results”. These should be shown in “Methods”.Thank you for the reminder. The statement was added to “Methods”8. The author writes “cooking at home” in the covariate list, but no data related to cooking at home are shown. Why? The author discussed the effects other than cooking smoke in restaurants. The above information should be important.Data on “cooking at home” was added to Table 1.9. Why did the author not include “years of working” in this analysis? The reviewer considers it is related to exposure duration and important as an exposure index.Actually, “years of working” is included in the analysis (Table 3), and it was found to have significant association only to wheezing symptoms.10. The author mentioned the healthy worker effect for severe dyspnea. Why not the other outcomes related to HWE? Please add some comments.That is a good point. The reason may be that severe dyspnea is a relatively obvious and serious symptom, comparing with the other symptoms. So, severe dyspnea is more likely to affect their job performance.This comment was added to the discussion." } ] }, { "id": "54898", "date": "11 Nov 2019", "name": "Bjørn Hilt", "expertise": [ "Reviewer Expertise Occupational medicine and epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWith certain limitations, this is an interesting and well-written manuscript focusing on the health of cooks in different types of kitchen in Thailand. The main limitation is of course the cross-sectional design, the description of the selection of participants, and to some extent also the local approach (see below).\nThe introduction is very good and covers most of the adequate knowledge in the field. If the author want to seem entirely up to date, they could also have included this very recent reference: Sindre Rabben Svedahl, Bjørn Hilt, Kristin Svendsen. Work environment factors and respiratory complaints in Norwegian Cooks. Int Arch Occup Environ Health. 2019 Sep 17. doi: 10.1007/s00420-019-01473-w. [Epub ahead of print].\nAs is common, the author use operational criteria in the description of the objectives of the study. The research question is not whether cooks differ in PEFR, but if they have impaired lung function related to work-exposures. A better and a more conceptual way to state the objectives would in my view be: “To investigate if cooks working in different types of kitchen have impaired lung function and an increased occurrence of respiratory symptoms”. Then to use PEFR as a measure for lung function is legitimate and purely operational.\nThe description of the selection of study participants is not absolutely crystal-clear (last para on p3 and first on p4). Somehow, it does not quite make sense. In the very first sentence there seems to be a misprint in “… by first diveded…” which in my eyes should be “…by first dividing…”. Please forgive my ignorance, but what is a “consecutive sampling technique”? Then the reader (at least this one) gets confused about who was selected from which restaurants. Was it one worker from each restaurant fulfilling certain criteria employees was interviewed? And, it should preferably be a chef. This does not quite make sense to me. Does it mean that they visited 321 restaurants and interviewed one person in each? This needs to be made clearer so that even this reader can understand it easily.\nAnother thing with the selection of subjects is that it might have led to a selection of respiratory healthy cooks free from asthma, COPD and emphysema which again has strengthened the so called “healthy worker effect” that the author come back to in the discussion.\nSome other (mostly minor) points as they occur:\nIntroduction last para: Was it for statistical reasons that the previous study failed to demonstrate a significant difference? I would rather say that it was due to small numbers that the study failed to unveil a statistically significant difference.\nP4, 2nd para: When we do power assessments we do not calculate the “optimal” sample size, but the “necessary” sample size. What does the last sentence of the para mean? Please clarify.\nIn the introduction the author use both “cooking oil fumes (COF)” and “cooking smoke”. Is there a difference between them? For my part I am used to the term “cooking fume”.\nWere the PEFR measurements done at random times of the day? I mean, if all measurements from one type of restaurants were done in the morning and the measurement from another in the evening, it could possibly have biased the results due to circadian differences in lung function.\nDo the author have a literature reference for the expected lung function values for the Thai people? Instead of a dichotomized (>< 80 %) variable for PEFR they could also have given us the actual percentages. In table 2 they give the actual mean values without any adjustments. For those figures with three main figures, I think it is unnecessary to put it with two decimals.\nFor table 1 and 2 the reader needs to know the meaning of the column with p-values. For what comparision?\nTable text for table 2 needs to be more comprehensive and explanatory.\nSince table 3 is divided in two, it needs to be stated that it will be continued at the bottom of the first part. For table 3 it is not necessary to explain in a footnote for the reader that a 95% confidence interval that not includes the value 1 is statistically significant on a 5% level.\nI see tears while cooking more at another effect that a real predictor/determinant of the other effects. In the same time it is of course correct to say that if the exposure is so strong that you have the symptom TWC, you can also have other, and worse effects.\nA better reference and explanation of the tables should be given in the text.\nIn the first para of the discussion the so called healthy worker effect is noting but selection bias and no phenomenon on its own. Selection bias may well have been introduced since the participants apparently should be free from respiratory diseases.\nThe paras on study limitations are insightful and well written. The fact that the study is quite local and that the external validity (generalizability) is limited could also have been included. Even so, it is worthwhile to report on deleterious health effects from professional and domestic cooking from different parts of the world with different cooking methods.\nFor the conclusion I believe that the term Thai restaurants is a term for all the four types of restaurants included, while it was in “tamsang” restaurants that the effects were found. Perhaps the first sentence of the conclusion could be omitted and that they only start with “This study shows that employees in “tamsang” restaurants in Thailand have…..”\nThe consequences of the results are not worth the attention for “regulatory organizations” only, but for everyone concerned with the health and welfare of restaurant workers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5045", "date": "02 Dec 2019", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "Comment: The introduction is very good and covers most of the adequate knowledge in the field. If the author want to seem entirely up to date, they could also have included this very recent reference: Sindre Rabben Svedahl, Bjørn Hilt, Kristin Svendsen. Work environment factors and respiratory complaints in Norwegian Cooks. Int Arch Occup Environ Health. 2019 Sep 17. doi: 10.1007/s00420-019-01473-w. [Epub ahead of print].Response: Thank you. The reference was added to the manuscript.Comment: As is common, the author use operational criteria in the description of the objectives of the study. The research question is not whether cooks differ in PEFR, but if they have impaired lung function related to work-exposures. A better and a more conceptual way to state the objectives would in my view be: “To investigate if cooks working in different types of kitchen have impaired lung function and an increased occurrence of respiratory symptoms”. Then to use PEFR as a measure for lung function is legitimate and purely operational.Response: Thank you for this helpful insight. The study objective was restated as suggested. Comment:The description of the selection of study participants is not absolutely crystal-clear (last para on p3 and first on p4). Somehow, it does not quite make sense. In the very first sentence there seems to be a misprint in “… by first diveded…” which in my eyes should be “…by first dividing…”. Please forgive my ignorance, but what is a “consecutive sampling technique”? Then the reader (at least this one) gets confused about who was selected from which restaurants. Was it one worker from each restaurant fulfilling certain criteria employees was interviewed? And, it should preferably be a chef. This does not quite make sense to me. Does it mean that they visited 321 restaurants and interviewed one person in each? This needs to be made clearer so that even this reader can understand it easily.Response:The description of participant selection was completely revised. The term “by first divided” was change to “by first dividing”. Yes, all 321 restaurants were visited and one person was interviewed at each shop. Actually, the study wanted to interview every worker in a restaurant, but most restaurants refused to do that way. After negotiation, only one participant, preferring to be a chef, was interviewed.Most of restaurants in Thailand are small size and they usually have only one chef, the rest is waitperson and chef assistance.Another thing with the selection of subjects is that it might have led to a selection of respiratory healthy cooks free from asthma, COPD and emphysema which again has strengthened the so called “healthy worker effect” that the author come back to in the discussion.I agree. From the field experience, we found no workers with those chronic diseases and thus, to be excluded from this study. Therefore, we decide to delete the criteria to avoid confusion.Comment:Introduction last para: Was it for statistical reasons that the previous study failed to demonstrate a significant difference? I would rather say that it was due to small numbers that the study failed to unveil a statistically significant difference.Response: The sentence has been revised as suggested.Comment: P4, 2nd para: When we do power assessments we do not calculate the “optimal” sample size, but the “necessary” sample size. What does the last sentence of the para mean? Please clarify.Response: Thank you for the reminding. The word “optimal” has been replaced with “necessary”.Comment:In the introduction the author use both “cooking oil fumes (COF)” and “cooking smoke”. Is there a difference between them? For my part I am used to the term “cooking fume”.Response:You are right. The term “cooking smoke” and cooking oil fume (COFs) has been replaced with “cooking fumes”.Comment:Were the PEFR measurements done at random times of the day? I mean, if all measurements from one type of restaurants were done in the morning and the measurement from another in the evening, it could possibly have biased the results due to circadian differences in lung function.Response:Yes, the PEFR measurements of each type of restaurants were done at random times of the day, and the bias should be minimal.Comment:Do the author have a literature reference for the expected lung function values for the Thai people? Instead of a dichotomized (>< 80 %) variable for PEFR they could also have given us the actual percentages. In table 2 they give the actual mean values without any adjustments. For those figures with three main figures, I think it is unnecessary to put it with two decimals.Response:Yes, we do have a reference for expected lung function of Thai people. To measure PEFR percentage, participants have to perform lung function test and the measured PEFR was then compared with PEFR standard which depend on gender, age, and height of the participant. We agree that more data should be presented. So, we decide to add data on average standard PEFR, and data on distribution of PEFR percent. The decimals was deleted.Comment:For table 1 and 2 the reader needs to know the meaning of the column with p-values. For what comparision?Response:More information on p-values was added to make sure that the readers known what the comparisons are.Table text for table 2 needs to be more comprehensive and explanatory.The table text was completely revised.Comment:Since table 3 is divided in two, it needs to be stated that it will be continued at the bottom of the first part. For table 3 it is not necessary to explain in a footnote for the reader that a 95% confidence interval that not includes the value 1 is statistically significant on a 5% level.Response: All the suggestions were taken, and the footnote was deleted.Comment: I see tears while cooking more at another effect that a real predictor/determinant of the other effects. In the same time it is of course correct to say that if the exposure is so strong that you have the symptom TWC, you can also have other, and worse effects.Response: I agree that TWC can be either effect of its own or a predictive factor. However, in this study, TWC was used as a marker of smoke exposure.Comment: A better reference and explanation of the tables should be given in the text.Response: Thank you for the suggestion. The paper was revised in many places, e.g. table 2 text.Comment:In the first para of the discussion the so called healthy worker effect is noting but selection bias and no phenomenon on its own. Selection bias may well have been introduced since the participants apparently should be free from respiratory diseases.Response:I agree that by using the selection criteria the bias was introduced. However, healthy worker effect is difficult to avoid unless control group with similar health was used as a comparison group. As in this study, health of workers in different types of restaurant was compared to each other, thus HWE will have not much an effect to the study result.Comment:The paras on study limitations are insightful and well written. The fact that the study is quite local and that the external validity (generalizability) is limited could also have been included. Even so, it is worthwhile to report on deleterious health effects from professional and domestic cooking from different parts of the world with different cooking methods.Response: Thank you for your thoughtful and generous notion. The idea was added to the limitation statement.Comment: For the conclusion I believe that the term Thai restaurants is a term for all the four types of restaurants included, while it was in “tamsang” restaurants that the effects were found. Perhaps the first sentence of the conclusion could be omitted and that they only start with “This study shows that employees in “tamsang” restaurants in Thailand have…..”Response: It may not true that the effects were found only in “tamsang” restaurant. The study results showed that 41.2% to 64.7% of the workers have poor PEFR. However, the problem was more pronounce in “tamsang” restaurant.Comment: The consequences of the results are not worth the attention for “regulatory organizations” only, but for everyone concerned with the health and welfare of restaurant workers.Response: Thank you for the insightful suggestion. The idea was added to the conclusion." } ] }, { "id": "54980", "date": "12 Nov 2019", "name": "Jan G.C. van Amsterdam", "expertise": [ "Reviewer Expertise Pharmaco-toxicology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study of Juntarawijit describes adverse health effects among employees working in four common types of restaurants in Thailand. Main results are that employees of ‘tamsang’ restaurants are more affected by cooking smoke than those working in papaya salad restaurants. This is a nice study which suffers from some methodological errors.\n\nMain points The selection method was poorly described. Did all invited participants agree to participate? Part of the participants was no cook. What was their function? Were they also exposed to cooking smoke? What proportion of the participants were allergic? E.g. mites or pollen? Why were for example waiters (not exposed to cooking smoke not used as comparison group? The group ‘Other job’ (Table 1) should be deleted from data analysis, because they are ill characterized and group size is too small.\n\nMinor points Abstract: Please describe that much less adverse effects were seen in the participants in the other three groups. 3rd line: replace ‘health effects’ by ‘adverse health effects’.\nIntroduction: Please distinguish the effects of cooking smoke vs. methods of heating (coal, gas). Give in paragraph 3 some indication of effect sizes observed.\nMethods: Study participants, Line 5-6: please specify ‘each restaurant worker’ and use singular in this sentence. Why was only one worker per restaurant included? Any data of homogeneity in effects per restaurant? Data from the Pollution Control Department showed that pollutants in the Phitsanulok area were within safe air standards. However, did they vary during the study period? To which extent was traffic related air pollution involved in the outcome? Was any restaurant situated near a busy road (probably it was!)?\nResults: Define TWC when first time used. Was TWC related to allergy? Was TWC due to irritating smoke or rather an allergic reaction? Please present the results of the combination: ‘Use of ventilation’ and ‘enclosed location’. There may be an adding effect as both affect the indoor air quality.\nDiscussion: Probably palm oil and lard give a different heating temperature (frying temperature without burning; ‘boiling point’). Suggest to include this difference to explain the differences found in adverse effects. Similar for soy bean oil.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5046", "date": "02 Dec 2019", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "Comment:Main points: The selection method was poorly described. Did all invited participants agree to participate? Part of the participants was no cook. What was their function? Were they also exposed to cooking smoke? What proportion of the participants were allergic? E.g. mites or pollen? Why were for example waiters (not exposed to cooking smoke not used as comparison group? The group ‘Other job’ (Table 1) should be deleted from data analysis, because they are ill characterized and group size is too small.Response:Participant selection method was completely revised. Yes, all invited participants agree to participate. Nearly all of the participants are chefs and the rest are waitperson or chef assistant. Sorry, we have no data on allergies among participants. The research data showed that all workers in a restaurant are exposed to cooking smoke.The number of waitperson is not also enough to be a comparison group. At first, we plan to collect data from everyone working in a restaurant but the idea reject by the restaurants’ owner. After a negotiation, they allow us to collect data from one worker. Yes, data on ‘other job’ is too small. However, it is an actual field data and we prefer to present it.Comment:Minor points:Abstract: Please describe that much less adverse effects were seen in the participants in the other three groups. 3rd line: replace ‘health effects’ by ‘adverse health effects’.Response: Abstract was amended as suggestion and the term was revised.Comment:Introduction:Please distinguish the effects of cooking smoke vs. methods of heating (coal, gas).Give in paragraph 3 some indication of effect sizes observed.Response:To avoid confusion, the term “cooking fumes” has been used instead of ‘cooking smoke’ and ‘cooking oil fume’. Cooking fumes are usually from both burning fuel (heating by using coal, gas), and cooking oil fume (COFs), which caused by overheating cooking oil. However, fumes from these two sources cannot be distinguished in field study.Comment:Methods:Study participants, Line 5-6: please specify ‘each restaurant worker’ and use singular in this sentence. Why was only one worker per restaurant included? Any data of homogeneity in effects per restaurant?Data from the Pollution Control Department showed that pollutants in the Phitsanulok area were within safe air standards. However, did they vary during the study period? To which extent was traffic related air pollution involved in the outcome? Was any restaurant situated near a busy road (probably it was!)?Response:The mistakes were corrected and the whole section has been revised. At the beginning, we planned to collect data from everyone working in a restaurant but the idea was rejected by restaurants’ owners. After a negotiation, they allowed us to collect data from only one worker. We believe that ambient air and traffic pollution will not much affect the health outcomes even though most of the restaurants situated in a main road. Besides, the fact that level of pollution is still within standard limit, the main purpose of this study was to compare health of workers working in between types of restaurants, which located in the same area.Comment:Results:Define TWC when first time used. Was TWC related to allergy? Was TWC due to irritating smoke or rather an allergic reaction?Please present the results of the combination: ‘Use of ventilation’ and ‘enclosed location’. There may be an adding effect as both affect the indoor air quality.Response:The term TWC has been defined as suggested. In this study, TWC refers to an acute reaction caused by smoke exposure rather than allergic reaction. Results of the combination effects of ‘use of ventilation’ and ‘enclosed location’ has been analyzed as suggested. However, it was not found to be significantly associated with PEFR and chronic symptoms. The data was added to Table 3.Comment:Discussion: Probably palm oil and lard give a different heating temperature (frying temperature without burning; ‘boiling point’). Suggest to include this difference to explain the differences found in adverse effects. Similar for soy bean oil.Response:Actually, palm oil and lard did not have a different boiling point. Both have high boiling point as compare to soybean oil. Therefore, it was expected to see similar low health impacts from cooking with palm oil and lard, but more from soybean. However, issue is interesting, and the discussion has been amended to clarify these points." } ] } ]	1	https://f1000research.com/articles/8-1429
https://f1000research.com/articles/8-2032/v1	29 Nov 19	{ "type": "Study Protocol", "title": "Protocol for the Effectiveness of an Anesthesiology Control Tower System in Improving Perioperative Quality Metrics and Clinical Outcomes: the TECTONICS randomized, pragmatic trial", "authors": [ "Christopher R. King", "Joanna Abraham", "Thomas G. Kannampallil", "Bradley A. Fritz", "Arbi Ben Abdallah", "Yixin Chen", "Bernadette Henrichs", "Mary Politi", "Brian A. Torres", "Angela Mickle", "Thaddeus P. Budelier", "Sherry McKinnon", "Stephen Gregory", "Sachin Kheterpal", "Troy Wildes", "Michael S. Avidan", "TECTONICS Research Group", "Christopher R. King", "Joanna Abraham", "Thomas G. Kannampallil", "Bradley A. Fritz", "Arbi Ben Abdallah", "Yixin Chen", "Bernadette Henrichs", "Mary Politi", "Brian A. Torres", "Angela Mickle", "Thaddeus P. Budelier", "Sherry McKinnon", "Stephen Gregory", "Sachin Kheterpal", "Troy Wildes" ], "abstract": "Introduction: Perioperative morbidity is a public health priority, and surgical volume is increasing rapidly. With advances in technology, there is an opportunity to research the utility of a telemedicine-based control center for anesthesia clinicians that assess risk, diagnoses negative patient trajectories, and implements evidence-based practices. Objectives: The primary objective of this trial is to determine whether an anesthesiology control tower (ACT) prevents clinically relevant adverse postoperative outcomes including 30-day mortality, delirium, respiratory failure, and acute kidney injury. Secondary objectives are to determine whether the ACT improves perioperative quality of care metrics including management of temperature, mean arterial pressure, mean airway pressure with mechanical ventilation, blood glucose, anesthetic concentration, antibiotic redosing, and efficient fresh gas flow. Methods and analysis: We are conducting a single center, randomized, controlled, phase 3 pragmatic clinical trial. A total of 58 operating rooms are randomized daily to receive support from the ACT or not. All adults (eighteen years and older) undergoing surgical procedures in these operating rooms are included and followed until 30 days after their surgery. Clinicians in operating rooms randomized to ACT support receive decision support from clinicians in the ACT. In operating rooms randomized to no intervention, the current standard of anesthesia care is delivered. The intention-to-treat principle will be followed for all analyses. Differences between groups will be presented with 99% confidence intervals; p-values <0.005 will be reported as providing compelling evidence, and p-values between 0.05 and 0.005 will be reported as providing suggestive evidence. Registration: TECTONICS is registered on ClinicalTrials.gov, NCT03923699; registered on 23 April 2019.", "keywords": [ "Telemedicine", "Artificial Intelligence", "Machine Learning", "Forecasting Algorithms", "Decision Support", "Randomized Controlled Trial", "Anesthesiology" ], "content": "List of Abbreviations\n\nACT Anesthesiology Control Tower\n\nACTFAST Anesthesiology Control Tower—Feedback Alerts to Supplement Treatments\n\nAKI Acute Kidney Injury\n\nBJH Barnes-Jewish Hospital\n\nEHR Electronic Health Record\n\nML Machine Learning\n\nNINR National Institute of Nursing Research\n\nPHI Protected Health Information\n\nPI Principal Investigator\n\nSMC Safety Monitoring Committee\n\nOR Operating Room\n\nRCT Randomized Controlled Trial\n\nTECTONICS Telemedicine Control Tower for the OR: Navigating Information Care and Safety Trial\n\n\nIntroduction\n\nPerioperative complications collectively contribute to numerous deaths around the world1. Following inpatient surgeries, the estimated 30-day mortality is between 1% and 5%2–10, and between 5% and 10% of surgical patients will die in the following year4,5,8,11. Furthermore, 10% to 20% of surgical patients experience major complications such as heart attacks, chronic pain12, infections and blood clots following their procedures6,9,10,13. Although some complications are unavoidable, based on the nature of the particular surgical procedure or non-modifiable patient characteristics9,14,15, others may be preventable through early identification of patient risk factors and the use of tailored treatments.\n\nSeveral factors, besides technical aspects of surgeries, contribute towards preventable perioperative complications. Clinicians16–19, including anesthesia care teams (typically comprising anesthesiologists and certified registered nurse anesthetists [CRNAs]), sometimes fail to implement and adhere to evidence-based standards of care. Many clinicians believe that they are in compliance with guidelines, when in reality they are not20. In the United States, differing backgrounds and perspectives of anesthesiologists and CRNAs can undermine effective collaboration among team members, potentially compromising patient care21,22. Clinicians also experience cognitive overload in the operating room (OR), and limitations in cognitive capacities impair information processing23 and decision making abilities24–26. In addition, the rapidly evolving OR environment and complex patient responses to surgery and anesthesia make it challenging for clinicians to accurately assess patients’ shifting risks.\n\nTelemedicine and integrated machine learning (ML) are two promising approaches for addressing cognitive and information overload, dynamically focusing resources where needed, and reducing accidental variations in care quality. However, there is no prospective evidence on telemedicine or ML in the OR context. There is an urgent need for rigorous research investigating the utility of a telemedicine-based control center to dynamically assess risk, diagnose negative patient trajectories, implement evidence-based practices, and improve outcomes for surgical patients. A collaborative telemedicine solution for the OR, through the early and accurate identification of potential risks, could facilitate the development of tailored plans for patient care risk mitigation and management27–29. It could also enhance meaningful teamwork between CRNAs and anesthesiologists, act as a complementary support for anesthesia care teams in the OR, help to decrease cognitive overload and bias, and facilitate evidence-based care.\n\nTo address this deficit, our interdisciplinary team, including academic and clinical leaders, has developed a prototype anesthesiology control tower (ACT)30,31. Our previous pilot work has demonstrated the usefulness and usability of the ACT30,32. We have also developed ML algorithms for real time decision-support instruments33–36, developed the institutional infrastructure to maximize OR integration of the ACT, and evaluated the feasibility of conducting a large scale randomized control trial using the ACT31. In this protocol we outline the Telemedicine Control Tower for the OR: Navigating Information, Care and Safety (TECTONICS) trial, which is a large-scale randomized controlled trial (RCT) to empirically evaluate the effectiveness of the ACT on preventing clinically relevant adverse perioperative outcomes and improving perioperative care.\n\nWe hypothesize that the integrated ACT system in the TECTONICS trial will improve evidence-based quality of perioperative care metrics and prevent clinically relevant adverse perioperative outcomes (postoperative delirium, renal failure, respiratory failure, and 30-day mortality).\n\n\nParticipants and setting\n\nThe study is a single center, randomized, controlled, phase 3, pragmatic superiority trial at Barnes Jewish Hospital (BJH) in St. Louis, MO. All adults (18 years and older) undergoing surgical procedures in these operating rooms will be included. Children are excluded in this study. Labor and operative delivery is conducted in a separate administrative area and is also excluded unless it occurs in the main surgical ORs. There are no other exclusion criteria related to procedure type, comorbid illnesses, or planned disposition other than the requirement that some anesthesia clinician be requested (excluding e.g. organ procurement and minor procedures performed without anesthesiology services) and the requirement for the procedure to take place in an operating room (excluding sedation-based procedure suites such as the cardiac diagnostic laboratory).\n\nAn estimated 10,000 patients will be enrolled annually, and enrollment will be over four years for approximately 40,000 total patients enrolled (Figure 1). Cases started during the hours of operation of the ACT will be included regardless of the stop time.\n\nThis study has been approved by the Human Research Protection Office at Washington University (St. Louis, MO # 201903026) for enrollment with a waiver of consent. Participant data is collected from the electronic health record (EHR) of Barnes-Jewish and its affiliated hospital and clinic databases until 30-days after their surgery.\n\n\nInterventions\n\nThe participant groups are intraoperative telemedicine support from the ACT (“intervention”) and usual care.\n\nThe ACT has been established in a location remote from the OR with sophisticated hardware and software. It is staffed by at least two clinicians from the research team (an anesthesiologist and one or more other clinicians). Intraoperative data streams used by the ACT clinicians include real-time access to the hospital’s EHR, web-based visualization of vital signs and waveforms, treatment guidelines, protocols for care, as well as AlertWatch® (Ann Arbor, MI) (Figure 2, Figure 3). AlertWatch® is a Food and Drug Administration-approved patient monitoring and alerting system that displays integrated patient information. AlertWatch® was primarily conceptualized for use in individual ORs; we have modified the AlertWatch® software based on stakeholder engagement and feedback for use in the ACT, creating a customized dashboard and interface specifically designed for the telemedicine setting32. The ACT AlertWatch® version that we developed in our preliminary studies is a customized research product with innovative information technology and communication components and is distinct from the current commercial product in several important respects. Real-time forecasting (from the machine-learning algorithms discussed below) of adverse outcomes for individual patients is provided to ACT clinicians. BJH is currently installing high definition cameras in the ORs; video feeds will be incorporated in the ACT if these are available during the trial. Video or audio will not be stored.\n\nThe team in the ACT receives data form the electronic health record, web-interfaced monitors in the operating room (OR), video cameras in the OR, multipath convolutional neural network machine learning algorithms, and alerting software has been customized to provide maximum utility in an ACT. The team weaves together disparate data strands, and collaboratively formulates a plan to address the patient’s risk and optimize outcomes. The plan is discussed collegially with OR clinicians, who exercise judgement in delivering the best individualized perioperative management to each surgical patient. Dynamic data from OR patient monitors. (The photo was taken in our prototype ACT). CRNA, certified registered nurse anesthetist.\n\nIn ORs randomized to intervention, ACT clinicians contact OR clinicians in two phases. First, the ACT messages the OR clinician an individualized risk assessment and considerations / recommendations based on the preoperative evaluation and real time information from the monitors in the OR. The recommendations are geared towards (1) preventing major complications (Table 1) based on patients’ specific risk profiles (e.g. history of stroke, hypertension, type 1 diabetes, coronary artery disease, valvular heart disease, pulmonary disease) and (2) adhering to general quality of care indicators (Table 2). The recommendations are based on the best currently available evidence (e.g. intensive insulin management in type 1 diabetes). OR clinicians are encouraged to reply to the message with specific concerns they would like to discuss, risk assessments they believe to be erroneous, and additional monitoring that they would like the ACT to perform. The second phase occurs during procedures. The ACT monitors physiologic and process alerts generated by AlertWatch® and ML algorithms, filters these alerts for those believed relevant and actionable, and contacts the anesthesia team where deemed appropriate. OR clinicians receiving notification from the ACT may choose to carry out whatever course of action they deem clinically appropriate. Anesthesia clinicians in the OR have access to the institution’s “clinical” AlertWatch software, but do not have access to the “research” view.\n\nIn ORs randomized to usual care, the ACT monitors patients, but the ACT clinicians does not contact the OR clinicians unless the ACT clinicians believe it to be clinically necessary for patient safety purposes (e.g. neuromuscular blockade without evidence of hypnotic agent administered). Concomitant care is provided in the usual perioperative setting with no modifications based on the trial. Anesthesia clinicians in the OR have access to the institution’s “clinical” AlertWatch software, but do not have access to the “research” view.\n\nUsing data from over 110,000 patients, we developed calibrated ML algorithms to predict adverse postoperative outcomes. The details of the dataset and algorithm development are published elsewhere34–36. Briefly, we implemented deep neural network models for 30-day mortality, acute kidney injury, and postoperative ventilatory failure among other anesthesiology-relevant outcomes based on routinely collected clinical data. We tested numerous other prediction methods, and found that deep neural networks processing extensive patient information (i.e., demographic characteristics, surgical risk, co-morbidities) as well as time series physiological data (e.g., blood pressure, temperature, heart rate) predict outcomes such as death with high area under receiver operator characteristics curve (0.880), acceptable sensitivity (~50%) and excellent specificity (~95%). We adapted models to improve their interpretability and incorporated advanced post-processing methods to uncover the data which drives individual predictions. The training of these deep neural network models jointly estimates data filtering and imputation steps with prediction33. We implemented appropriate data validation and quality filtering steps for the live environment and display forecasts of mortality updated every 5 minutes. In contrast to standard forecasting models, we have previously demonstrated that ML and data mining approaches for patients in ICUs are markedly superior in predicting clinical outcomes such as mortality42. The feasibility of this integration is supported by a previous successful trial, where members of our investigative team, using live data from the EHR, implemented ML algorithms to guide a rapid response team in medical wards43–46.\n\nOver the course of the TECTONICS study, with ongoing acquisition of high-resolution data and outcomes on thousands of surgical patients, our algorithms will undergo regular evaluation and refinement. Periodically updating the model will be necessary, which we plan to do at 6-month intervals with newly collected data in the control arm. We will also use a human expert to review the face validity of the model’s predictions and most important input features. Such a dynamic feedback loop will continuously improve and adjust the model. We will test for the overall percentage of correct forecasts, the percent of correctly forecasted events, and accuracy when data include noise and missing values on several adverse perioperative outcomes. One key metric that we will evaluate and compare will be the sensitivity at 95% specificity, since it is important to maintain a high specificity (i.e., low false alarm rate) for meaningful decision support. Validation techniques will include cross-validation47 and systematically different hold-out samples (e.g. distinct time periods or OR locations).\n\n\nOutcome measures and data acquisition\n\nThe primary objective is to determine whether the ACT system is effective in preventing clinically relevant adverse perioperative outcomes including thirty-day postoperative mortality, postoperative delirium, postoperative respiratory failure and postoperative acute kidney injury. Secondary objectives are to determine whether the ACT system is effective at improving perioperative quality of care metrics. The study outcomes will be assessed according to established criteria (Table 1 and Table 2).\n\nMultiple sources are used for standardized data collection. Data on patient outcomes and perioperative care metrics is extracted from the EHR. Preoperative patient characteristics, comorbidities, surgical and clinical history, as well as perioperative and immediate post-operative information are pulled from the EPIC EHR (Verona, WI, USA). Additional postoperative patient outcomes data (for sub-studies) will be obtained from clinical registries (American College of Surgeons National Surgical Quality Improvement Program48, Society of Thoracic Surgeons49,) as well as the EHR. BJH determines vital status through multiple mechanisms including follow-up contact and state death databases. Non-mortality outcomes are not tracked after discharge from hospital. Although the development of incident serious acute kidney injury (AKI) or delirium post-discharge is possible, we anticipate that these will be uncommon enough to not warrant large-scale surveillance.\n\nData on clinician responses to individual alerts, generated from the AlertWatch® Control Tower platform, is logged by ACT staff to a database for qualitative studies and internal quality improvement.\n\n\nAssignment of treatments\n\nThe 58 ORs (all the ORs at BJH excluding “remote” locations, procedure suites, and labor and delivery) are 1:1 randomized daily to receive intraoperative support from the ACT or usual care without any form of stratification. In other words, participants are randomized in clusters whose size randomly depends on the number of cases assigned to a room. The randomization script for the TECTONICS trial has been incorporated into the AlertWatch® ACT infrastructure, and runs automatically early every morning.\n\nDue to the nature of the intervention it is not possible to blind OR clinicians since feedback alerts from the ACT inform them that they are in the intervention group. However, patients and those evaluating outcomes are blinded to group assignment.\n\n\nSample size calculation and recruitment\n\nIn recent years, more than 20,000 surgeries have been performed annually in the Barnes Jewish Hospital main ORs. However, the ACT is only staffed during work-week hours and when the assigned attending anesthesiologist is available. During our pilot, we made efforts to ensure staffing during all appropriate times; however, under half of cases took place during ACT staffed times. We therefore conservatively estimate that 10,000 patients will participate per year, but we anticipate that more rapid accrual is likely based on more complete recent staffing. The TECTONICS trial will be adequately powered to answer with precision whether the ACT system has a meaningful impact on clinically relevant outcomes (primary outcomes, see Table 3) and quality of care metrics (secondary outcomes, see Table 4). Despite the fact that we are evaluating multiple surrogate outcomes, the very large sample size will provide adequate statistical power to determine whether or not there is improvement with the ACT system. Individuals with multiple surgeries within 30 days will be analyzed with the assignment of their index surgery. Individuals with multiple independent encounters (>30 days separation) will be treated as distinct observations. Surgeries which take place outside BJH will not be accounted for.\n\nThe adjusted power was calculated assuming a cluster-randomized design allowing for an intracluster correlation between 0.005 and 0.01 and varying number of patients per OR. The current incidence estimates on which these power analyses are based are consistent with findings in our previous studies4,5,12,55,56,57,58,50,51 as well as from the ACTFAST2 pilot study, where we have approximations of these complications from ~110,000 (mostly inpatient) surgical patients at our institution over 5 years.\n\nThe adjusted power was calculated assuming a cluster-randomized design allowing for an intracluster correlation between 0.01 and 0.03 and varying number of patients per OR for a total N=40,000.\n\n\nAssessing Hawthorne and contamination effects\n\nWe anticipate a possible contamination (or learning) effect over time in the usual care group. Clinicians are included in both intervention and control ORs (possibly on the same day) and may become sensitized to the standards of practice and the surrogate outcome measures being tracked, leading to “overlapping” improvements in these measures (as well as clinical outcomes) in both groups over the course of the study. This learning effect might manifest most strongly among clinicians who spend time in the ACT. Furthermore, with the knowledge that clinical behaviors are being observed, there is a high likelihood of a Hawthorne effect59,60. In the reverse of the usual Hawthorne problem, the effect of being observed in the intervention arm represents part of the actual effect of the intervention; knowing that they are being observed, clinicians may feel more accountable to following perioperative best practices, and this is a meaningful effect. However, the non-contact rooms are also aware of the trial and enjoy the same improvement, falsely decreasing the estimated effect size. The data we have obtained prior to instituting the ACT will be useful in assessing the extent of contamination. Specifically, we will assess the intensity/frequency of contamination by comparing the outcomes of the control group patients to those of matched patients who had similar surgeries, demographics, and health conditions during the immediate period prior to the ACT implementation. We will also analyze control arm results in 3-month time bins (with baseline data for reference) to evaluate secular tends which may represent contamination, as well as the magnitude of clustering design effects.\n\n\nAnalysis of primary and secondary outcomes\n\nComparisons between groups during the randomized study will be with parametric and non-parametric statistical tests, according to the distributions of the measures of interest. Fisher’s exact or χ2 test will be used to assess differences between proportions (the majority of assessments). Contingency statistical tests will be used to compare occurrence of hypotension, hyperglycemia, hypoglycemia, and hypothermia between groups; and unpaired t-test or Mann-Whitney U-test, as appropriate, will be used to compare the durations of these occurrences between groups. All comparisons will use the intention-to-treat principle.\n\nResults in statistical tests with a p value <0.005 will be viewed as providing compelling evidence, whereas results with a p value between 0.05 and 0.005 will be interpreted as providing suggestive evidence61. We will report p-values adjusted for multiple hypothesis tests (within the primary and secondary outcome blocks) using permutation methods that account for the correlation across outcomes62. Within the secondary outcomes, false-discovery-rate control will be reported63.\n\n\nHandling of missing data\n\nWe anticipate that the prospectively collected data will be high quality with few missing outcomes. AKI is informatively missing in patients who are judged as low risk by the surgical team and do not have assessments of postoperative creatinine or urine output. Delirium is similarly infrequently assessed at our institution in patients who do not require intensive care unit admission. A screening bias in both outcomes is possible where patients in the treatment arm are more accurately identified as elevated risk and checked for complications. Ventilatory failure is unlikely to occur in the 48-hour time window among discharged patents. We will report the number of patients without assessments for each outcome. The primary analysis will treat patients discharged without measures of AKI or delirium as negative. Patients who are informatively censored by death will be treated as positive for other outcomes. All outcomes will be required to be incident. Individuals without preoperative measures of renal status or delirium will be assumed to have normal values.\n\n\nAdverse event and safety monitoring\n\nThis study will have a Safety Monitoring Committee (SMC) according to the National Institute of Nursing (NINR) Research Data Safety Monitoring policy. The SMC will be comprised of a small group of experts with at least two members independent of the study team. They will be responsible for reviewing all adverse events, compliance with the IRB requirements, investigator compliance, minimizing risks and protecting the confidentiality of participant data. The SMC will review study data every six months.\n\nThe study team will prepare reports for the SMC, NINR and the IRB. In the event a serious adverse event occurs that is deemed to be reasonably associated with the conduct of the study by an external SMC, the study may be halted. Local regulatory agents in agreement with the study principle investigator may decide to stop the study at any time.\n\n\nStrengths, limitations and alternative strategies\n\nThe TECTONICS trial has important strengths. It is a pragmatic RCT conducted in a high volume, real-world clinical setting that incorporates telemedicine for the OR. The adverse outcomes under study are serious and meaningful to patients. The TECTONICS trial can be conducted efficiently as many components of the proposed study are incorporated into existing infrastructures and processes at Washington University: 1) with no known risk associated with the support offered by the ACT, participants are included with a waiver of informed consent; 2) the members of the care team (anesthesiologists, CRNAs, residents and student registered nurse anesthetists) participate in the trial in the course of their routine clinical work; and 3) most of the surrogate and clinical outcomes data are obtained from existing IT resources or from established and ongoing registries64. Randomization of ORs is implemented easily, and the process for providing feedback alerts from the ACT does not require any lead-in time or advanced preparation. The study includes all adult surgical-patients at BJH, including both men and women, and those who are recognized to be vulnerable and understudied in clinical research. The feasibility of the trial is enhanced by participation of a highly committed cadre of CRNAs and attending anesthesiologists, student registered nurse anesthetists and residents in the Anesthesiology Department, as well as an experienced team of CRNA and anesthesiology investigators that has established a track record of scientific collaboration and completion of major trials4,5,55–57,65–67.\n\nThere are also relevant limitations. The TECTONICS study will be vulnerable both to Hawthorne and contamination effects. Although we do not think that these effects can be eliminated, we have considered how best to account for them in the analyses. In addition, it will not be possible to ensure blinding68 of clinicians. However, surgical patients and those evaluating outcomes will be blinded to group assignment. Another major constraint relates to both accuracy and completeness of outcome measures. Outcomes routinely tracked in the EHR are often well represented. However, we know from previous experience that EHR, registry, and patient reported outcomes data are occasionally inaccurate58. Missing and inaccurate outcomes data will be partially mitigated by the large number of patients included in the trial, and are expected to be randomly distributed across groups.\n\n\nEthics/protection of human subjects\n\nThis study has been approved by the Human Research Protection Office at Washington University (St. Louis, MO # 201903026). It satisfies the criteria for a waiver of informed consent (there is minimal risk with the intervention, the research could not practically be conducted without a waiver, and the rights and welfare of patients are not adversely affected by their involvement in the study, and there is no deception requiring additional disclosure) and is being conducted accordingly. This protocol was written in compliance with the SPIRIT Checklist Guidelines for Interventional Trials. Only the minimum necessary private patient information will be collected for the purposes of the study. Any protected health data is kept in a secure digital environment that is digitally encrypted, password protected and limited to research team only. De-identified data may be kept and used in future studies not pre-specified in the above protocol. The investigators are responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data collected.\n\n\nData management\n\nThe ACT will use clinical applications available to all clinicians to monitor ongoing surgical procedures. These applications can only be accessed over the secure hospital network or by virtual private network logins. This arrangement meets and/or exceeds Health Insurance Portability and Accountability Act standards for Patient Health Information (PHI) security. AlertWatch® is an approved clinical application at Barnes-Jewish Hospital, and therefore maintains these same levels of protection. Access to the data collected in this study will be restricted to approved personnel. It is a strict policy that PHI content cannot be reviewed outside of this protected environment. Prior to data analysis, information will be de-identified. When possible, extracts from this project will avoid the use of PHI. De-identified patients can be identified using a special, non-PHI primary key, which we have used previously.\n\nThe research material obtained in this proposed trial will consist of the already established infrastructure and resources of the SATISFY-SOS (NCT02032030), NSQIP and STS registry studies, Anesthesiology Control Tower—Feedback Alerts to Supplement Treatments (ACTFAST) 1, 2 and 3 studies, the intra-operative electronic medical record and the AlertWatch® evidence-based alerting system. Patient demographic information and preoperative characteristics are collected and entered into the electronic health record as part of routine clinical care at the Center for Preoperative Assessment and Planning. Perioperative and intraoperative drug administration and vital signs are also charted in the electronic health record.\n\nData will be maintained for at least 5 years after the end of the grant funding period. Further study record retention will be at the discretion of the study investigator. Data may be used for future studies not mentioned in the protocol.\n\n\nPublication/data sharing policy\n\nThe investigative team is comprised of a range of stakeholders, including scientists, clinical investigators, and relevant end users. We will each disseminate in our respective networks through presentations to relevant stakeholder groups, through peer-reviewed publications, and by providing brief summaries for hospital administrators and policy makers. We will also utilize the BJC Collaborative, which aligns multiple health networks in the region (including rural settings), as a vehicle for dissemination.\n\nData from the TECTONICS trial will be made available for analysis in compliance with the recommendations of the International Committee of Medical Journal Editors69. For this study, individual participant data that underlie the results of the trial will be made available after appropriate de-identification, along with the study protocol and statistical analysis plan. We plan to make this information accessible to researchers who provide a methodologically appropriate proposal for the purpose of achieving the aims of that proposal. Data will be available beginning 9 months and ending 36 months following trial publication at a third-party website. Data requestors will need to sign a data access agreement to gain access to trial data. Proposals should be directed to avidanm@wustl.edu. TECTONICS is registered on clinicaltrials.gov, NCT03923699.\n\n\nAuthorship eligibility and contributorship\n\nAuthorship for this study will be given to key personnel involved in study design, data collection, and data analysis. There are no publication restrictions and no professional writers will be involved in the generation of the manuscript. M. Avidan, A. Ben Abdallah, J. Abraham, Y. Chen, B. Fritz, C. King, B. Henrichs, T. Kannampallil, M. Politi, A. Sharma, B. Torres, S. Kheterpal, T. Wildes are responsible for conceptualizing study design.\n\nAll authors, including King, Avidan, Ben Abdallah, Abraham, Chen, Fritz, Henrichs, Kannampallil, Politi, Sharma, Torres, Mickle, Budelier, McKinnon, Gregory, Wildes, have critically revised the TECTONICS protocol and approved the final version. All authors agree to be accountable for the accuracy and integrity of all aspects of the TECTONICS trial. No paid writers will be used.\n\n\nProtocol amendments\n\nProtocol amendments will be approved by the steering, operations, safety, and data management committees, communicated to the IRB and NINR, and posted to Clinicaltrials.gov. This protocol corresponds to version 1.1 and was agreed on October 1 2019.\n\n\nConclusion\n\nWhile ML and telemedicine have been extensively studied over the past decade, telemedicine has often been implemented without a strong research foundation. When it has been studied, this has often been done in the context of observational before and after cohort trials. ML algorithms have often been studied for risk calculations and predictions, but there has been limited investigation of their application to improving patient outcomes. In contrast, TECTONICS is designed as a pragmatic, randomized clinical trial including telemedicine and ML. The over-arching strength of TECTONICS is that it combines and leverages a telemedicine initiative with advanced machine-learning algorithms. The net innovation is a fully integrated clinical decision support system, comprised of remote surveillance of patient risks in real-time, human expert judgment, and computer-generated rules. This realization of the ACT concept provides an empowering and unobtrusive socio-technical telemedicine infrastructure and decision support solution for OR teams. The ACT also provides a practical and innovative solution to the challenge of implementing evidence-based guidelines in the OR. Although the ACT system requires an initial modest financial investment, if it proves to be effective in promoting and enhancing evidence-based perioperative care, it is possible that it could lead to decreased costs via improvements in surgical patient outcomes.\n\nThe proposed study can have a major impact on healthcare if it demonstrates that the ACT system enhances OR care quality and patient safety while simultaneously increasing teamwork. Following the TECTONICS study, the ACT system will be further refined, and its implementation will be expanded. The logical next step will be to conduct a larger multisite trial focusing on expanded clinical and patient-reported outcomes. We are well positioned to track such outcomes, based on electronic access to ICD codes58, our experience in building a patient reported outcomes registry50,58,70, as well as our collaborations with NSQIP and the Society for Thoracic Surgeons. Importantly, one of the TECTONICS contributors (Kheterpal) is the principal investigator of the Multi-Center Perioperative Outcomes Group (MPOG), which includes data from >4 million patients, and has an established and sophisticated international IT infrastructure. We envisage that future dissemination and implementation of the ACT system could occur efficiently using the MPOG infrastructure.\n\nThroughout the study, we will collect data from ACT users on reach (percent of clinicians and staff eligible who use and engage with the ACT), adoption (user confidence that they will continue to use the ACT after the study ends), implementation (user confidence that the ACT can be consistently delivered as intended), and maintenance (user confidence that the ACT will produce lasting benefits beyond the study)71. When planning for wider-scale implementation, we will use established guidelines set-out by the Expert Recommendations in Implementing Change (ERIC) team72.\n\n\nData availability\n\nNo data are associated with this article.\n\nRepository: SPIRIT checklist for ‘Protocol for the Effectiveness of an Anesthesiology Control Tower System in Improving Perioperative Quality Metrics and Clinical Outcomes: the TECTONICS randomized, pragmatic trial’. https://doi.org/10.6084/m9.figshare.1025572473.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe TECTONICS Research Group is made up of Michael Avidan2, Joanna Abraham1, Amrita Aranake-Chrisinger2, Anchal Bansal2, Kara Battig2, Arbi Ben Abdallah2, George Benzinger2, Mara Bollini2, Walter Boyle2, Thaddeus P. Budelier2, Kathryn Cass2, Laura Cavallone2, Yixin Chen3, Zhicheng Cui3, Daniel Emmert2, Mitchell Fingerman2, Bradley Fritz2, Jason Gillihan2,Marie Goez2, Shreya Goswami2, Thomas Graetz2, Stephen Gregory2, Ryan Guffey2, Katharine Gurba2, Charles Hantler2, Yujie He3, Daniel Helsten2, Bernadette Henrichs2, Erin Herrera2, Omokhaye Higo2, Robert Hovis2, Gary Hubbard2, Rocco Hueneke2, Mark Ingram2, Ivan Kangrga2, Thomas Kannampallil1, Menelaos Karanikolas2, Christopher R. King2, Sachin Kheterpal5, Justin Knittel2, Helga Komen2, Alexander Kronzer2, Anand Lakshminarasimhachar2, Alyssa McClellan2, Jacob McDowell2, Sherry McKinnon2, Angela Mickle2, Alexander Mohrmann2, David Monks2, Jordan Oberhaus2, Hilary Odorizzi2, Daniel Park2, Sarah Perez2, Mary Politi4, Debra Pulley2, Elizabeth Quillen2, Rashmi Rathor2, Andrea Reidy2, Cameron Ritter2, Evan Roller2, Elizabeth Schappe2, Alexandra Schatz2, Anshuman Sharma2, Tracey Stevens2, Brian Tolly2, Brian Torres2, Swarup Varaday2, William Varnum2, Troy Wildes2, William Wise2, Rachel Wolfe2, Jinghan Yang3, Muhan Zhang3\n\n1Department of Anesthesiology and Institute for Informatics (I2), Washington University School of Medicine, St. Louis, MO\n\n2Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO\n\n3Department of Computer Science and Engineering, Washington University, St. Louis, MO\n\n4Department of Surgery, Washington University School of Medicine, St. Louis, MO\n\n5Department of Anesthesiology, University of Michigan, Ann Arbor, MI\n\n\nReferences\n\nNepogodiev D, Martin J, Biccard B, et al.: Global burden of postoperative death. 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PubMed Abstract \| Publisher Full Text\n\nKronzer VL, Tang RD, Schelble AP, et al.: Preoperative Falls and Their Association with Functional Dependence and Quality of Life. Anesthesiology. 2016; 125(2): 322–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRe-AIM Planning Tool. (Accessed September 29, 2017). Reference Source\n\nPowell BJ, Waltz TJ, Chinman MJ, et al.: A refined compilation of implementation strategies: results from the Expert Recommendations for Implementing Change (ERIC) project. Implement Sci. 2015; 10: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKing C: SPIRIT_Fillable-checklist-15-Aug-2013.doc. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10255724.v1" }	[ { "id": "58762", "date": "20 Jan 2020", "name": "Ben Gibbison", "expertise": [ "Reviewer Expertise Endocrine", "machine learning", "clinical trials of periooperative care", "cardiac surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is a protocol and therefore this review affects that.\n\nThe authors plan to implement a combined ML based patient parameter monitoring / alert system and human advisory system to aid in decision support. Operating rooms are individually randomised by day to the intervention or simply monitoring of the physiological parameters (\"control\" group). This will take place in a large University Hospital system in the USA.\nOutcomes assessed include: thirty-day postoperative mortality, postoperative delirium, postoperative respiratory failure and postoperative acute kidney injury.\nThe manuscript is well written and will be a fascinating study if and when they complete it.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "60684", "date": "16 Mar 2020", "name": "Leon du Toit", "expertise": [ "Reviewer Expertise All three reviewers are clinical anaesthesia providers. Our research expertise includes perioperative outcomes research", "pragmatic trial design", "feasibility studies", "process evaluation", "meta-analysis", "airway management research and device innovation", "perioperative thermoregulation", "extreme environment physiology", "and obstetric anaesthesia research." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol originates from a cross-disciplinary group of researchers including perioperative clinicians and data scientists. The project is based at a single academic hospital in North America. The protocol describes a pragmatic clinical trial of a complex systems intervention using machine learning guided remote decision support in anaesthesia care. The primary outcome is a composite of 30-day mortality, postoperative delirium, respiratory failure and acute kidney injury after surgery. The proposed trial is pragmatic in design.\nPopulation: The population includes all adult surgical patients taking place in the main theatre facilities of the trial site. Obstetric patients and other patients undergoing surgery in locations remote to the main theatre facilities are excluded by the sampling method. The study population is restricted to a single academic hospital in a high-income setting.\n\nIntervention: The intervention is complex in design and implementation, but all the components of the intervention have already been piloted and introduced into the system and the technology supporting the trial are not new to the trial site. It is stated that the intervention will change over the duration of the trial as guidelines and updated and machine learning algorithms improve. Will the effect of an evolving intervention be evaluated? The complexity of the intervention should be considered when interpreting the findings of this trial.\n\nOutcome: The outcome is clinically important to patients and healthcare providers. The components of the composite outcome are measured as part of routine care (data extracted from electronic medical records). The mortality component of the composite outcome is robust, but the event rate of the other components will likely be underestimated by this method of data collection. This will not affect validity of the estimate of effect of the intervention. The reviewers wonder what reasoning determined the components of the composite outcome. Why, for example, are cardiovascular events and surgical site infections not included?\n\nDesign: The unit of randomisation is the operating room, and randomisation is updated daily. Theoretically, randomisation to a complex intervention within a single institution risks contamination across the arms of the study. The investigators address this concern appropriately in the protocol. The concern of bias introduced by the Hawthorne effect is also addressed in the analysis plan. With a projected sample size of 40000 surgical cases, the proposed trial is sufficiently powered to answer the patient relevant question.\n\nBurden: Despite the complexity of the trial, it does not pose additional burden to healthcare providers. Data collection is part of routine care, provision of the intervention does not rely on the usual healthcare providers, and the institutional review board approved a waiver of consent.\nThe reviewers wonder which ethical considerations were discussed for observing healthcare providers. Is there an agreement for participation process for clinicians who are being observed in this study?\nWhat will be considered an adverse event in this trial? Can examples be given? Will the project use specific criteria to identify adverse events?\nMonitoring and evaluation of the program would add value. What is the explicit logical framework connecting the intervention to the outcomes being measured? When reading the result of this trial the reviewers would conceivably be interested in knowing how the intervention lead to a change in management? What factors determined translation of recommendation from the ACT to practice change in theatre? Does the person sitting in the ACT cause variability in the way the intervention is delivered and received? Will the trial measure compliance or adherence to the ACT recommended intervention?\n\nThe capital investment and running cost of this intervention must be significant. Future work, investigating the cost of benefit will facilitate considerations around generalisability of this intervention.\nThis protocol represents an exciting frontier in the field of anaesthesia research. The proposed trial incorporates precision care into routine care, assessed in a pragmatic manner. The reviewers look forward to the results and report of this trial, and future projects that build on this one.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/8-2032
https://f1000research.com/articles/8-1031/v1	09 Jul 19	{ "type": "Research Article", "title": "Does fMRI neurofeedback in the context of stress influence mood and arousal? A randomised controlled trial with parallel group design", "authors": [ "Angelo Belardi", "Jong-Hwan Lee", "Hyun-Chul Kim", "Esther Stalujanis", "Eun Kyung Jung", "Minkyung Oh", "Seung-Schik Yoo", "Jens C. Pruessner", "Marion Tegethoff", "Gunther Meinlschmidt", "Angelo Belardi", "Jong-Hwan Lee", "Hyun-Chul Kim", "Esther Stalujanis", "Eun Kyung Jung", "Minkyung Oh", "Seung-Schik Yoo", "Jens C. Pruessner" ], "abstract": "Background: Stress-related mental and physical health issues burden modern societies. New treatment opportunities could help to lessen long-term detrimental consequences of stress. Objective: To investigate whether real-time functional magnetic resonance imaging neurofeedback (rtfMRInf), aimed at modulating brain activity associated with a stressor, affects subjective mood and arousal. Methods: In total, 30 males participated in a randomised controlled trial with parallel-group design. rtfMRInf was the intervention, sham-neurofeedback the control condition, and the Stroop task the stressor. We instructed participants to modulate their stress response to the Stroop task via feedback from their anterior cingulate cortex and their insular cortex, concomitantly applying mental strategies. We assessed mood with the Multidimensional Mood State Questionnaire (dimensions: good/bad, GB; awake/tired, AT; and calm/nervous, CN), and subjective arousal with Self-Assessment Manikins (SAM). Results: We found significantly higher subjective arousal after neurofeedback phases in the experimental condition as compared to the control condition [t(26.6) = −2.216, 95%CI [−2.188,−0.083], p = 0.035; t(27.9) = −3.252, 95%CI [−2.685,−0.609], p = 0.003], but no significant differences between the conditions regarding mood [GB: b = 0.4, 95%CI [−0.67, 1.47], p = 0.467; AT: b = 0.769, 95%CI [−0.319, 1.857], p = 0.177; CN: b = 0.5, 95%CI [−0.53, 1.53], p = 0.352]. In both conditions, there was significantly worse and more tired mood after the fMRI session as compared to before [GB:b = −0.77, 95% CI [−1.31, 0.23], p = 0.009; AT: b = −0.652, 95%CI [−1.116,−0.187], p = 0.01]. Conclusions: Findings indicate that rtfMRInf led to higher arousal, which may counteract the aim to reduce stress responses. Whether the multitasking situation has triggered this neurofeedback-related arousal – and how to circumvent it – asks for further study. Trial registration: NCT01921088, ClinicalTrials.gov, 13th August 2013.", "keywords": [ "arousal", "dual task", "multitasking", "functional magnetic resonance imaging", "mood", "neurofeedback", "psychological stress" ], "content": "Introduction\n\nStress is ubiquitous in life. Getting rid of it is neither realistic nor desirable, as Hans Selye pointed out: “complete freedom from stress is death!”1, p. 137. However, accepting stress as part of our lives does not mean we are all heading towards long-term detrimental consequences of this inevitability. We can influence how a stressor affects us in the long run. For this, let us begin with how we react to a stressor: The stress response is manifested in physiological and psychological aspects and leads to specific behavior. Physiologically, stress for example increases blood pressure, heart rate, and specific brain activity, and triggers a cascade of endocrine activity which ends in glucocorticoid release2,3. Psychologically, stress leads to focused attention and increased arousal and alertness, and shows up in behavioral measures such as self-reported questionnaires2.\n\nThis response ensures survival in the presence of a life-threatening stressor, or at least increases the chance of survival. In everyday life in a modern society, however, the response might overshoot, given the nature of the stressors we are facing. And accumulated or chronic stress can lead to dire mental and physical health issues: Stress-related mental disorders like depression and anxiety, which are globally a main source of adult disability4 for example, or hypertension5, which is the top modifiable risk factor for mortality6.\n\nThe effects of stress and related disorders burden industrialized countries increasingly. Associated costs have been estimated to 20 billion euros per year, in the European Union alone7. It is also a topic which affects most of us eventually: 49% of people in a survey in the United States had a major stressful event in a one year time-window8. When asked how often they experience stress in their daily life, 44% of respondents said “frequently” and 35% “sometimes” to a Gallup poll conducted in the US in 20179.\n\nFinding ways to better deal with stressors in the short term might spare us of these long-term consequences and lessen the burden on the individual and society. In the definition of psychological stress by Lazarus and Folkman, our own appraisal of a situation and our coping abilities take a major role: “a relationship with the environment that the person appraises as significant for his or her well-being and in which the demands tax or exceed available coping resources”10, p. 63.\n\nCognitive and behavioral techniques used in occupation-specific stress management programs are also known to psychotherapeutic practice. Intervention programs for stress management often include education about and practice of time-management and coping skills, psychoeducation, relaxation techniques (e.g. Jacobson’s progressive muscle relaxation, controlled breathing, hypnosis), mindfulness-based stress reduction, exercise, leveraging social support, or training in specific job-related skills to prevent or prepare for common stressors11–14. Yoga and meditation-based therapies have both also been associated with mood changes in people with the stress-related disorders depression and anxiety15. The mechanism behind these changes might act via the biological stress system16.\n\nHowever, while there is support for classical stress management interventions17, as with any treatment it is likely that a proportion of affected people do not respond to a given intervention. In such cases, innovative neuroscientifically-informed interventions might help. Such interventions are based on neuroscientific knowledge about the stress response and can be coupled with personal neural activity in reaction to a stressor. Due to that, they are likely to help participants work deliberately on their individual stress response.\n\nGaining deliberate control over specific and individual brain activity (and thus indirectly over mental processes) is a key aim of neurofeedback, an approach that has over the last decades been applied to modulate a wide set of mental processes and to improve symptoms to specific mental disorders18. Neurofeedback describes the paradigm to feed back a signal reflecting a person’s own brain activity so that the person can use information contained in the signal to better modulate their brain activity18. An advantage of fMRI is that it allows to work with a spatially circumscribed region of the brain. Thus, one can use this approach to target various brain areas associated with different mental processes, in real-time functional magnetic resonance imaging neurofeedback (rtfMRInf). rtfMRInf has been applied to modulate diverse mental processes, including pain19, anxiety20, mood21, and many others22. Whether the procedure could also be used to modulate the central and peripheral stress response has to the best of our knowledge not yet been investigated.\n\nWithin a larger rtfMRInf study, we assessed self-reported mood and arousal measures and tested whether these differed between the participants who had received real feedback from their own brain’s activity, as compared to those who received sham feedback (the recorded brain activity of another participant). Participants thereby used one out of four different mental strategies to help them reduce their stress response. The use of these strategies has shown to improve mood when trained once a day for 13 consecutive days, using smartphone-based instructions23. Here, we aimed to elucidate whether rtfMRInf aiming at modulating the central and peripheral stress response is related to changes in mood and subjective arousal. While we addressed the primary and secondary outcomes of the study, namely physiological components of stress (brain activity and blood pressure) and adverse events, in another manuscript (Belardi, Lee, Kim, Stalujanis, Jung, Oh, Yoo, Pruessner, Tegethoff, Meinlschmidt; unpublished study), here we focus on additional outcomes, namely self-reported psychological measures of mood and arousal.\n\nWe assumed that effects of rtfMRInf on mood and arousal may show up in one of two potential directions: Neurofeedback may lead to i) improved mood and lower arousal (in line with its aim to reduce the stress response); or ii) worse mood and higher arousal (in line with increased mental workload based on the multitasking situation going along with rtfMRInf). The second direction might be due to our experiment requiring the participants to multitask: at the same time monitoring the feedback signal; applying a specific mental strategy; and conducting the Stroop task. Current research in the field of multitasking generally reports lower task performance when the task is performed in a multitasking setting, as compared to when it is done as a single task24 and higher arousal for complex and multitasking situations25,26.\n\n\nMethods\n\nWe recruited 31 subjects and analyzed data of 30 of these (with mood and arousal data lacking from one subject, due to no-show for the main experiment). They were all male students at Korea University, Seoul, Republic of Korea. We allocated 17 of them to the experimental condition and 13 to the control condition, based on a predefined block-randomized scheme (blocks of 8, 10, and 12 with random order). The mean age was 24.6 years (SD=2.1) and 24.5 years (SD=2.4) in the experimental and the control condition, respectively with a mean of education of 14.9 years (SD=1.4) and 15.3 years (1.3), respectively. There were no significant differences between the conditions for these baseline characteristics [age: t(28) = -0.0585, p = 0.954; education years: t(28) = 0.718, p = 0.479].\n\nWe based the sample size on previous studies which had shown large effect sizes for rtfMRInf19,27. Using power analysis, we estimated that with 14 subjects in each group we could detect effects of d = 1.0 with sufficient power (1 − β > .80; given α = 0.05, one-sided). Recruitment was stopped after the intended 30 subjects had participated in the experiment.\n\nA researcher in Switzerland who was not directly involved in conducting the experiment and who had no contact to the participants, generated the randomized allocation sequence. MATLAB was used for the randomization, whose underlying random number generator uses the Mersenne Twister algorithm by default28. This researcher ensured that the allocation sequence was concealed from those who recruited and assigned the participants. Participants were assigned to a condition according to the allocation sequence in the order in which they were included in the study and only after a final decision about inclusion was made, to support concealment. Researchers at Korea University did the enrollment and then assigned participants to the conditions.\n\nWe recruited participants via ads on the university’s website and a bulletin board on campus. Using the following inclusion and exclusion criteria, we checked all interested students and decided about their eligibility for the study. Inclusion criteria were being i) male, ii) between 18 and 65 years of age, iii) right-handed, iv) familiar with using a smartphone, to take part in the ambulatory training, v) having sufficient English language skills to follow the written instructions in the experiment, vi) no indication of color-blindness, vii) no history of cardiovascular or neurological diseases, and viii) no history of a severe mental disorder. After finishing the whole study procedure, we paid each participant 60,000 KRW to compensate for time and effort related to study participation.\n\nThe institutional board of Korea University approved the study and all participants gave written informed consent (approval number: KU-IRB-10-38-A-2(E-A-1)(E-A-1)(E-A-3)).\n\nWe used established tools to assess mood and arousal, applied a well-known cognitive task as a stressor in the fMRI experiment, and instructed our participants in four mental strategies, aimed at reducing their stress response during the experiment.\n\nTo assess mood, we used the English version of the established multidimensional mood state questionnaire (MDMQ) (original in German “Mehrdimensionaler Befindlichkeitsfragebogen (MDBF)”), which has good psychometric properties29,30. The questionnaire measures current mood on three dimensions: good to bad, awake to tired, and calm to nervous. Individual values on each dimension range from 4 to 24 and higher values represent more positive affect, feeling more awake, and calmer, respectively.\n\nWe assessed subjective arousal with a non-verbal pictorial rating scale to assess valence, arousal, and dominance, on a 9-point Likert Scale, called Self-Assessment Manikin (SAM)31. The arousal rating was labeled with “At the moment, I’m feeling...” and went from “very calm” to “very aroused” in addition to the original pictures. The SAM is an established tool which is used extensively in research. We were only interested in the arousal dimension, because it is clearly linked to a psychological stress response and, thus, have not analyzed the other dimensions of the SAM.\n\nDuring the rtfMRInf experiment, we induced acute stress using a cognitive task which had previously been used for this purpose and shown to elicit a cardiovascular and neural stress response: the Stroop color-word interference task32, adapted for the use in fMRI experiments and to be more challenging due to implemented adaptive time constraints33,34. We instructed the participants in four mental strategies: Body attention, contemplative repetition (mantra), emotional imagery, and facial expression (make different emotional faces). More details about these strategies and the exact instructions (text and video clips) were published elsewhere23.\n\nWe laid out the whole study as a randomised parallel-group study with rtfMRI neurofeedback as the experimental condition and sham-feedback as the control condition. Participants were blinded about their allocation, while experimenters and those analyzing the data were not. We registered the study before starting recruitment (ClinicalTrials.gov, identifier NCT01921088). Over the course of the study, participants visited the laboratory three times and conducted 13 days of smartphone-based ambulatory mental training between the two main experimental visits. We conducted the study at the Korea University, Seoul, Republic of Korea (RRID:SCR_004095) between August and October of 2013.\n\nInitially, we screened all interested students in a short telephone interview to check for their history of diseases and mental disorders described in the exclusion criteria above. On a first visit to the laboratory, we then further checked their eligibility based on all additional inclusion and exclusion criteria with a set of questionnaires. There, we also instructed participants about the whole study and the four mental strategies.\n\nAfter deciding upon the final inclusion in the study, participants then visited the laboratory two more times for the main rtfMRInf experiments. These two visits were 14 days apart and during the 13 days in-between, participants took part in a smartphone-based ambulatory mental training, where they applied the mental strategies they had already used during the experiment in short daily sessions. They were guided through the training with video clips and questionnaires on their smartphones.\n\nWe here report data from the first laboratory visit (screening day) and the first experiment day and will, thus, refer to the latter day simply as “experiment day”. More details on the procedures, especially regarding the ambulatory training, have been reported elsewhere23.\n\nWith regard to the first experiment day, the experimental procedure contained the following phases: Structural scans, where the previously defined broad regions of interest for the neurofeedback training were localized; Functional localizer phase, where participants did the Stroop task and the individual regions of interest could be pinpointed, to ensure participants get a feedback signal from areas active during the task; resting phase; Neurofeedback-only (i.e., without Stroop) phase, where participants first had to apply the four learned mental strategies in turn, and then continue using the strategy which worked best for them, and also do neurofeedback; resting phase; phase with additional structural scans; Neurofeedback with Stroop, where subjects used both, the mental strategies and the neurofeedback signal to actively modulate their brain activity associated with the stressor; resting phase; Stroop-only phase, where subjects only used the mental strategies to reduce their stress response; resting phase.\n\nThe Stroop task runs were made up of 8 blocks each; congruent and incongruent trials were alternated. During the neurofeedback phases, we presented the feedback signal continuously on one side of the screen and (if applicable) the Stroop task on the other side. We assessed current mood (MDMQ questionnaire) once before and once after the whole fMRI experiment and arousal (with the SAMs) after each individual phase of the experiment.\n\nWe defined a set of regions of interest (ROIs) encompassing the left and right anterior cingulate cortex (ACC) and insular cortex (IC), based on previously found brain activity associated with the Stroop task33,35. Within this set, a more precise individual ROI was localized during the functional localizer phase for each participant. The recorded and processed brain activity of the individual ROIs was fed back to the participants in near real-time. Participants saw the feedback signal abstracted as a white, moving thermometer-like bar on a black background, which went up and down depending on the signal strength, indicating the divergence from the baseline activity level. We instructed them to modulate the activity of the ROIs using this information, by applying the mental strategies they had learned and the information from the feedback signal. Sham-feedback for the control condition was the recording of the feedback signal from another participant.\n\nWe acquired the MRI data with a 3T Siemens Tim Trio scanner with a 12-channel head coil (Erlangen, Germany). To measure the BOLD signal, we applied a standard gradient-echo EPI pulse sequence36 using the following specifications for rtfMRInf: repetition time (TR) = 1500 ms, echo time = 25 ms, field of view 240240 mm, matrix size 6464, voxel size = 3.753.755 mm, flip angle 90, and 30 interleaved slices with 5mm thickness at approximately 30 oblique to the AC-PC line without a gap37,38.\n\nWe calculated individual ROIs for each participant during the functional localizer phase as follows: EPI preprocessing (head motion correction for six parameters, spatial smoothing with an 8 mm full-width at half maximum Gaussian kernel); estimation of beta-value maps for each incongruent and congruent Stroop trial via general linear model (GLM) implemented in SPM to get a contrast map for “incongruent > congruent Stroop trials”; calculating the neurofeedback signal then from the intersection map between ROIs from the GLM and the predefined set of ROIs.\n\nTo calculate the neurofeedback signal, we first removed possible artifacts from the raw BOLD signal of the individual ROIs, applying a bandpass-filter (0.008 - 0.1 Hz) using a third-order elliptic digital filter to avoid low-frequency linear drift36. Next, we linearly detrended the median BOLD signals within each of the ROIs as well as the whole-brain area. We then averaged the values between the 10th and 30th percentile during the cross-fixation period, using this as the baseline BOLD intensity (for ROI and whole-brain area). Percentage signal change (PSC) of the ROI relative to the whole-brain area were then estimated voxel-wise, by subtracting the estimated whole-brain PSC from the ROI PSC. This PSC difference was used as the neurofeedback signal. Finally, we averaged the signal over the last three TR periods in order to reduce potential high-frequency fluctuations occurring due to cardiac-and respiratory-related activity.\n\nAll offline data analysis was conducted using the software package R (version 3.5.1 and later; RRID:SCR_001905)39 and specific further packages for R as follows: “lme4” (RRID:SCR_015654) and “lmerTest” (RRID:SCR_015656)40, to conduct the mixed effects models, “dplyr” (RRID:SCR_016708) and “tidyr” (RRID:SCR_017102)41 for data preparation, and “ggplot2” (RRID:SCR_014601)42 and “ggpubr”43 to create and export data visualizations. For online fMRI data preparation, analysis, feedback signal calculation, and neurofeedback presentation as described above, we used MATLAB (The MathWorks, Inc., Natick, MA, USA; RRID:SCR_001622) with SPM8 (RRID:SCR_007037).\n\nTo take into account the longitudinal nature of the mood data (two measurements, before and after the fMRI session), we used linear mixed effects models. Our models included the following factors: fixed effects Time (prescan, postscan), Condition (experimental, control), and the interaction TimeCondition, and random intercept for each participant. We estimated three models, one for each of the mood dimensions as dependent variable. Together with beta values, we report 95% confidence intervals of two-sided tests using an alpha-level of 0.05 to determine statistical significance.\n\n\nResults\n\nThe flow of participants, from enrollment to allocation and analysis, is given in Figure 1 in a flow diagram consistent with the Consolidated Standards of Reporting Trials (CONSORT).\n\nMood scores were assessed with the MDMQ once before and once after the fMRI session. Results of individual mixed models for the three mood dimensions are presented in Table 1, and descriptive statistics can be found in the interaction plots in Figure 2. In the mood dimensions good/bad and awake/tired, we saw lower values after the fMRI session than before in both conditions, leading to a significant main effect of time. In the calm/nervous dimension, a slight drop in values was present only in the experimental condition, but neither the time effect nor the interaction with condition was significant in this model. None of these models, to determine effects on mood, showed a significant main effect for condition or an interaction between time and condition.\n\nNote. σ2 = within-group variance, τ00 = between-group variance, ICC = intraclass correlation coefficient, CI = confidence interval. Factor predictors were coded using effect/deviant coding to increase interpretability of the fixed effects. Comparison from the mean intercept of the factor to the level names in parentheses for each factor. In the good/bad and calm/nervous models, there was one missing value each.\n\nLighter empty symbols in the background are individual data points (jittered on the x-axis to avoid overplotting), while filled symbols with error bars are condition group values (means and standard errors).\n\nWe calculated Welch two sample t-tests for unequal variances to assess differences in SAM arousal values between the experimental and control conditions. To account for heteroscedasticity, these tests model different variances for both levels of the factor condition. Descriptive values for subjective arousal can be found in Figure 3. In the “Neurofeedback-only” phase, we found SAM arousal to be significantly higher for participants in the experimental condition [t(26.6)=-2.216, 95%CI[-2.188, -0.083], p=0.035, (two-tailed test)], as compared to those in the control condition. In the phase “Neurofeedback with Stroop”, SAM arousal was also significantly higher in the experimental condition [t(27.9)=-3.252, 95%CI[-2.685, -0.609], p=0.003, (two-tailed test)]. This difference was not present in the “Functional localizer” phase, before the neurofeedback intervention started [t(24.1)=-1.429, 95%CI[-1.869, 0.339], p=0.166, (two-tailed test)].\n\nCombination of dot plot with individual data points and a boxplot for each condition group. The boxplot’s lower and upper hinges mark the 25th and 75th percentiles. Whiskers extend from the hinges to the largest/smallest value up to 1.5 the inter-quartile range. Black X shapes mark the condition group means. There was one missing value in the experimental condition during the functional localizer phase.\n\n\nDiscussion\n\nSubjective arousal was higher after neurofeedback training as compared to the sham-feedback control. This was true when the stressor task was present and when not. In our mood data, we could not observe changes specifically related to real neurofeedback, but participants in both conditions reported worse mood and being more tired after the fMRI session as compared to before.\n\nThese findings pose several questions: First, why did subjective arousal rise, contrary to our goal to reduce stress with our intervention? Arousal rose for experimental condition participants but not for those in the control condition, even when neurofeedback was practiced without a stressor. This finding is in contrast to the assumption that with neurofeedback, subjects reduce their stress response going along with reduced subjective arousal. The finding is, however, in line with the assumption that the cognitive demand on subjects in the experimental condition was higher as compared to subjects in the control condition. Let us recapitulate what participants did in this phase of the experiment: They applied previously learned mental strategies and used the feedback signal from their ACC and IC, trying to reduce the activity in these brain regions. This was the same for experimental and control condition participants. The only difference was the kind of feedback signal they saw (real or sham).\n\nOne possible explanation supporting the idea of increased cognitive demand in the experimental condition is the multitasking situation, present in the experiment. Participants had to do several tasks simultaneously. This might have lead to increased mental load in subjects who got real feedback compared to those who got sham-feedback, because those receiving sham-feedback might have (consciously or unconsciously) realized that the shown signal was not contingent with their brain activity. They might then have given less attention to this signal and the neurofeedback training and could focus better on other task(s) (applying mental strategies and solving the Stroop task). Arousal levels might, thus, here be an indicator for multitasking and increased mental load instead of an effect of the Stroop-induced stress. In this sense, the multitasking aspect of the experiment may have itself become a stressor, because it increased the cognitive demand of the participant.\n\nThe second set of questions concerning our results is: Why could we not observe any statistically significant mood changes related to the intervention and what could explain the increased tiredness and worse mood after the fMRI session?\n\nWe observed no statistically significant mood changes associated with neurofeedback, even though we would have expected better mood after the training in accordance with the study aim to reduce the physiological and psychological stress response with the help of neurofeedback. This could be linked to one limitation in the experimental design, namely that we measured mood only completely before and after the fMRI session. The observed mood effects can, thus, primarily be interpreted in relation to the participant’s experience over the whole session and unfortunately they can not be matched to putative mood changes related to single experiment phases. Interestingly, subjective arousal, which was measured directly after each experiment phase during the fMRI session, did show differences between the conditions. We can, thus, assume that the sampling rate of mood might not have been fine-grained enough to pick up differences between the conditions and was only able to represent the overall experiment effects on all participants.\n\nRegarding tiredness, fatigue due to the experiment and cognitive demand is expected. A one hour fMRI session is tiring and such overall effects might overshadow the miniscule differences between conditions due to the manipulation (real vs. sham feedback). Especially also since the neurofeedback manipulation was only present in some phases of the experiment. The lower awake/tired mood values after the experiment are, thus, not surprising. Participants may become tired after a demanding experiment in an fMRI scanner where they have to repeatedly solve a monotonous cognitive task. Furthermore, we expected our participants to relax and calm down. Thus, their indication of being more tired can be interpreted in line with what they actually did.\n\nTo explain the decrease in the good/bad mood dimension, we can look at the individual items in the questionnaire that made up this dimension: Subjects rated to what degree they felt uncomfortable, content, discontent, good, bad, happy, unhappy, great, superb, and wonderful. For example, it is unlikely to feel more comfortable and content after the experiment, given that lying in an fMRI scanner can be somewhat uncomfortable, and considering that participants were challenged with a cognitive task with adaptive difficulty, ensuring that they did not perform too well. Even if participants could modulate their immediate stress response, the overall mood change from before to after the experiment towards a worse mood might, thus, be explainable.\n\nWe also did not target to specifically change mood with our intervention. In comparison to another rtfMRI study, which did exactly that21, we used different target brain regions. Where these researchers targeted brain regions that most highly reflected activity differences in response to positive vs. neutral images, we focused on regions associated with our stressor task. Our modulated regions were thus less likely to be directly involved in supporting positive mood and we could only expect a potential side-effect in the mood due to the down-regulation of the stress response.\n\nFuture studies should aim to overcome the above-mentioned limitations, including limited time resolution in mood assessment: They could profit from using a shorter mood assessment instrument that can be applied in higher frequency. One example is a visual analog scale (VAS) to rate perceived stress, which could be implemented during an fMRI experiment and which allows to sample rapidly and repeatedly, yet with lower precision as compared to the MDMQ. Longer questionnaires like the applied MDMQ interrupt the experiment for a longer time and are thus not ideal to be applied in higher frequency.\n\nBroader implications for future research include a notion that the multitasking situation during an experiment can itself influence the measured values. While it is important to make the best of experimental time for economic reasons and to avoid prolonging an experiment unnecessarily, overloading an experiment might result in unexpected and intertwined effects. In our case, the multitasking present during the experiment might have led to our finding of increased arousal connected to the neurofeedback intervention. Even though challenging, future rtfMRInf studies on stress should try to prevent multitasking situations as good as possible. One could also more explicitly look at this multitasking aspect and conduct experiments to elucidate this component in the context of rtfMRInf research.\n\nWe had set out asking whether rtfMRInf to modulate the stress response would influence participants’ subjective perception of mood and arousal. The mood effects reflected the overall experimental experience due to sampling only before and after the fMRI session, and probably reflected rather general fatigue due to the cognitively demanding experiment than specific neurofeedback effects. To the best of our knowledge, we are the first to report a phenomenon of neurofeedback-related arousal: With regard to arousal, our findings are in line with the assumption that the multitasking nature of conducting neurofeedback during a stress task may have increased acute stress perceived by our participants, being in contrast to short-term neurofeedback effects on reduced subjective indicators of stress. Future studies should take into account multitasking situations in the experimental design, and further elucidate the neurofeedback-related arousal phenomenon, especially in the context of stress.\n\n\nData availability\n\nFull underlying (non-aggregated) data cannot be made publicly available since the ethics approval of this study does not cover openly publishing non-aggregated data.\n\nIn order to access this data, it must be requested from the corresponding author. Data requestors will have to provide: i) written description and legally binding confirmation that their data use is within the scope of the study; ii) detailed written description and legally binding confirmation of their actions to be taken to protect the data (e.g., with regard to transfer, storage, back-up, destruction, misuse, and use by other parties), as legally required and to current national and international standards (data protection concept); and iii) legally binding and written confirmation and description that their use of this data is in line with all applicable national and international laws (e.g., the General Data Protection Regulation of the EU).\n\nOpen Science Framework: CONSORT checklist for “Does fMRI neurofeedback in the context of stress influence mood and arousal? A randomised controlled trial with parallel group design”. https://doi.org/10.17605/OSF.IO/XFQHZ44.\n\nThe completed CONSORT checklist is available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was funded by the Swiss National Science Foundation, SNSF (project no. PZ00P1_137023 to MT). Further, GM, JL, and MT received funding from the National Research Foundation of Korea (NRF) within the Global Research Network Program (under project no. 2013S1A2A2035364). JL received funding in form of a NRF grant, MSIP of Korea (NRF-2016M3C7A1914450), and in part in form of a National Research Council of Science Technology (NST) grant by the Korean government (MSIT) [No. CAP-18-01-KIST]. GM received funding from the Stanley Thomas Johnson Stiftung & Gottfried und Julia Bangerter-Rhyner-Stiftung under projects no. PC_28/17 and PC_05/18, from the Swiss Cancer League (Krebsliga Schweiz) under project no. KLS-4304-08-2017, from the Research Foundation of the International Psychoanalytic University (IPU) Berlin, from Gesundheitsförderung Schweiz under project no. 18.191, and from the Swiss National Science Foundation (SNSF) under project no. 100014_135328.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Peter J. Gianaros, Ph.D. from the Department of Psychology, University of Pittsburgh, Pittsburgh, PA, USA, for his valuable contributions to this study by supporting us with the paradigm central to our experiment and by sharing his insights during the design phase.\n\n\nReferences\n\nSelye H: Stress without distress. In: George Serban, editor, Psychopathology of Human Adaptation. Springer US, Boston, MA. 1976; 137–146. Publisher Full Text\n\nChrousos GP: Stressors, stress, and neuroendocrine integration of the adaptive response. The 1997 Hans Selye Memorial Lecture. 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Publisher Full Text\n\nKassambara A: ggpubr: ’ggplot2’ based publication ready plots. 2018. Reference Source\n\nBelardi A: Consort checklist accompanying the manuscript “does fmri neurofeedback in the context of stress influence mood and arousal? a randomised controlled trial with parallel group design” (belardi et al.). 2019. Reference Source" }	[ { "id": "50963", "date": "26 Jul 2019", "name": "Ryuichiro Hashimoto", "expertise": [ "Reviewer Expertise neuroimaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-done study of fMRI-neurofeedback, which is gaining considerable attention as a potential new method for clinical intervention and increasing mental functions. The study is commended for its rigorous sampling and other procedures for the randomized controlled trial. The reviewer is particularly impressed by rigid applications of statistical tests and all the information necessary for assessing the effect of neurofeedback was clearly presented, including effect size and 95%CI. Overall, the study provides significant information for researchers who are interested in the application of fMRI-neurofeedback for various purposes.\n\nThe reviewer would like to raise some concerns in the hope of improving the clarity of the study even more as follows: Although other parts of the study designs are well written, I feel that the procedures of neurofeedback are less satisfactory. Particularly, the reviewer would like to ask the following points:\n\nThe authors seemed to have identified ROI in the individual brain using a contrast map of incongruent > congruent. How was the statistical threshold determined? How different were identified ROIs between individuals? Probably mean and SD of the cluster sizes should be described. How were ACC and IC combined?\n\nSubjects were instructed to apply one of the 4 mental strategies that were given to them before the study. What were they actually? The author stated that each subject selected the best one that worked for him/herself (page 5 left column), how was the \"best\" one determined? The reviewer would like to know whether and how the 4 mental strategies are expected to change ACC and IC activation.\n\nThe reviewer could not locate the description of the length of each fMRI run, including functional localizer, NF only, resting-state, NF+Stroop, and Stroop-only.\n\nIn page 5, it says that \"We instructed them to modulate the activity of the ROIs ...\" . Does \"modulate\" include both increase and decrease activation, or only decrease activation? In page 9 (left column), it says \"trying to reduce the activity in these brain regions\".\n\nSham condition. In page 5, it says \"sham feedback for the control condition was the recording of the feedback signal from another participant\". How was \"another participant\" selected? Was it selected from the control group or from experiment group or both?\n\nWere subjects in the experiment group able to increase scores significantly at the end of the day 1.\n\nWas there any relationship between changes in NF scores and psychological scores between individuals?\n\nProbably related to (5) and (6), were the degrees of score changes controlled between the experiment and control groups? Can the authors exclude the possibility that the psychological scores related to stress responses are associated with the NF scores?\nMinor points\nThe reviewer understands that the participants were Korean university students. On the other hand, the authors used the English version of the MDMQ. Application of the psychological test in a foreign language might affect the subjects' responses depending on the proficiency of English.\n\nThe reviewer feels that the study will become more significant analyzing the data of the second fMRI experiment after the mental training rather than focusing on the first fMRI experiment. Do we expect the report of the second fMRI experiment come up in the future?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5042", "date": "28 Nov 2019", "name": "Angelo Belardi", "role": "Author Response", "response": "Point by point responses to the comments by Reviewer R. HashimotoReplies are preceded by “*” General comment:This is a well-done study of fMRI-neurofeedback, which is gaining considerable attention as a potential new method for clinical intervention and increasing mental functions. The study is commended for its rigorous sampling and other procedures for the randomized controlled trial. The reviewer is particularly impressed by rigid applications of statistical tests and all the information necessary for assessing the effect of neurofeedback was clearly presented, including effect size and 95%CI. Overall, the study provides significant information for researchers who are interested in the application of fMRI-neurofeedback for various purposes. * We thank the reviewer for the thoughtful and supportive feedback on our publication. Below, we individually address all major and minor points. *************** Major points: The reviewer would like to raise some concerns in the hope of improving the clarity of the study evenmore as follows:Although other parts of the study designs are well written, I feel that the procedures of neurofeedback are less satisfactory. 1: The authors seemed to have identified ROI in the individual brain using a contrast map of incongruent > congruent. How was the statistical threshold determined? How different were identified ROIs between individuals? Probably mean and SD of the cluster sizes should be described. How were ACC and IC combined? * We thank the reviewer for this insightful feedback that we used to add relevant parts to the methods section on the description of the neurofeedback procedures: Once a t-contrast map (i.e., incongruent > congruent) was obtained from the functional localizer run, a default statistical threshold of p < 0.01 was used to select the significantly active voxels from the incongruent compared to the congruent condition and consequently, these voxels entailed the functional region-of-interest (ROI). The intersection of the functional ROI and anatomical ROI was used as the ROI for the rtfMRI-NF runs. Before the rtfMRI-NF run, the default statistical threshold (i.e., p < 0.01) was adjusted to make sure reasonable number of voxels were included in the ROI for the NF runs. The average number of voxels (+/- standard deviation) in the ROIs were 250.7 +/- 83.9 for the experimental group and 289.2 +/- 80.5 for the control group. The number of voxels between groups were not statistically different (t-score = -1.27 from two-sample t-test; uncorrected p = 0.22; 95% confidence interval = [-100.7, 23.8]). Regarding the combination of the pre-determined ROIs, an anatomical ROI to include the ACC and IC was defined from the automated anatomical labeling (AAL) map and Brodmann's area (BA) map atlases available in MRIcron (https://www.nitrc.org/projects/mricron). The intersection of the AAL 29/30 and the BA 48 were defined as the anatomical ROI for the IC and the intersection of the AAL 31/32 and BA 24/32/33 was defined as the anatomical ROI for the ACC. The use of the both the AAL and BA atlas was because the intersected areas from the two atlases has provided a functionally distinct area compared to the area defined from either one of the two atlas [1]. We added this information in the revised version of the manuscript. 2: Subjects were instructed to apply one of the 4 mental strategies that were given to them before the study. What were they actually? The author stated that each subject selected the best one that worked for him/herself (page 5 left column), how was the \"best\" one determined? The reviewer would like to know whether and how the 4 mental strategies are expected to change ACC and IC activation. * We thank the reviewer for this comment, allowing us to clarifying on this relevant aspect of the methods: The four mental strategies are described in detail in an earlier publication (open access) by the authors, from which the written instructions for each strategy are available in detail [2]. We now highlight more clearly where this information on the instructions can be found. The participants were free to choose one of the strategies. There was no instruction on how they had to determine which strategy worked “best” for them. It was a subjective choice based on the experience the subjects made while trying out all four strategies in the scanner, during the “neurofeedback without Stroop” phase, as we assumed that subjective evaluation of the strategies would a) best indicate which strategy indeed worked best for the individual subject and b) lead to the best adherence of the subject using this strategy later on.Based on previous studies on the strategies’ potential in relation to stress reduction, we expected that they could reduce these ROIs activity related to the stress response, since both ROIs had been found to be involved in the stress response elicited by the Stroop task [3, 4]. 3: The reviewer could not locate the description of the length of each fMRI run, including functional localizer, NF only, resting-state, NF+Stroop, and Stroop-only. * As suggested by the reviewer, we added the duration of each phase of the fMRI experiment to the “Methods / Experimental procedure” section in the revised manuscript. 4: In page 5, it says that \"We instructed them to modulate the activity of the ROIs ...\" . Does\"modulate\" include both increase and decrease activation, or only decrease activation? In page 9 (left column), it says \"trying to reduce the activity in these brain regions\". * Indeed, the instruction was to decrease the activity. We adjusted the first cited sentence to specify this. 5: Sham condition. In page 5, it says \"sham feedback for the control condition was the recording of the feedback signal from another participant\". How was \"another participant\" selected? Was it selected from the control group or from experiment group or both? * Sham feedback was always the recording from a participant of the experimental group. Each activity recording used for sham feedback was taken from the preceding participant of the experimental group. 6: Were subjects in the experiment group able to increase scores significantly at the end of the day 1. * If we understand correctly, the reviewer here refers to the modulation of brain activity. These data are not reported in the current publication and will instead be included in another publication reporting the results from the main outcomes of the experiment (fMRI and blood pressure data). 7: Was there any relationship between changes in NF scores and psychological scores betweenindividuals? * See reply to point 6 above; We thank the reviewer for pointing this out and added a respective sentence in the discussion section, highlighting that this would be an interesting additional question to be addressed. 8: Probably related to (5) and (6), were the degrees of score changes controlled between the experiment and control groups? Can the authors exclude the possibility that the psychological scores related to stress responses are associated with the NF scores? * We did not control or adjust for the degree of changes in psychological scores between conditions, but took into account the factor condition, by modeling it in our analyses. With regard to any relations with brain responses, see replies to questions 6 and 7. Minor points: 1: The reviewer understands that the participants were Korean university students. On the otherhand, the authors used the English version of the MDMQ. Application of the psychological test in a foreign language might affect the subjects' responses depending on the proficiency of English. * We agree that this might generally be an issue. To minimize this problem, we ensured that only students with sufficient English language skills were included in the study. We added a respective sentence in the discussion section of the manuscript, pointing out this limitation of the study. 2: The reviewer feels that the study will become more significant analyzing the data of the second fMRI experiment after the mental training rather than focusing on the first fMRI experiment. Do we expect the report of the second fMRI experiment come up in the future? *** We agree with the reviewer that the data from the second part of the fMRI experiments would add valuable information; yet habituation of the stress reactivity, the size of the stress-reactivity is getting smaller over time, makes it increasingly difficult to interpret the signals. To focus on the initial question and to keep the publication readable, we hence chose to focus on the first experiment to see the effects on the first responses to the Stroop task and the mental strategies. Further, given that we didn’t see significant short-term differences between the conditions regarding mood, we do not expect them to develop later, even though we agree that this may theoretically happen and be an interesting topic for further study. We added a respective paragraph in the discussion section. REFERENCES:[1] Lee, J. H., O'Leary, H. M., Park, H., Jolesz, F. A., & Yoo, S. S. (2008). Atlas‐based multichannel monitoring of functional MRI signals in real‐time: Automated approach. Human brain mapping, 29(2), 157-166.[2] Meinlschmidt, G., Lee, J. H., Stalujanis, E., Belardi, A., Oh, M., Jung, E. K., … Tegethoff, M. (2016). Smartphone-based psychotherapeutic micro-interventions to improve mood in a real-world setting. Frontiers in Psychology, 7(JUL). https://doi.org/10.3389/fpsyg.2016.01112[3] Gianaros, P. J., May, J. C., Siegle, G. J., & Jennings, J. R. (2005). Is there a functional neural correlate of individual differences in cardiovascular reactivity? Psychosomatic Medicine, 67(1), 31–39. https://doi.org/10.1097/01.psy.0000151487.05506.dc[4] Gianaros, P. J., Derbyshire, S. W. G., May, J. C., Siegle, G. J., Gamalo, M. a, & Jennings, J. R. (2005). Anterior cingulate activity correlates with blood pressure during stress. Psychophysiology, 42(6), 627–635. https://doi.org/10.1111/j.1469-8986.2005.00366.x" } ] } ]	1	https://f1000research.com/articles/8-1031
https://f1000research.com/articles/8-2023/v1	28 Nov 19	{ "type": "Research Article", "title": "Mitigation of methane gas emission in rice by drip irrigation", "authors": [ "Theivasigamani Parthasarathi", "Koothan Vanitha", "Sendass Mohandass", "Eli Vered", "Koothan Vanitha", "Sendass Mohandass", "Eli Vered" ], "abstract": "Background: Rice farming faces major challenges, including water limitation, drought and climate change in the current scenario of agriculture. Among the innovative water-saving techniques, drip irrigation is a forerunner, with maximized water-saving potential, increased grain yield and methane mitigation. Methods: A field experiment was conducted comprising four different drip irrigation practices: (i) sub-surface drip irrigation (SDI) with 1.0 litre per hour (lph) discharge rate emitters (DRE) (SDI+1.0 lph DRE) (ii) SDI+0.6 lph DRE, (iii) surface drip irrigation (DI) with 1.0 lph discharge rate emitters (DI+1.0 lph DRE), (iv) DI+0.6 lph DRE and were compared with (v) a conventional flood aerobic irrigation (considered conventional). Results: The estimated grain yield of rice was found to be 23.5%, 20.3%, and 15.1% higher under SDI+1.0 lph DRE, SDI+0.6 lph DRE and DI+1.0 lph DRE practices, respectively, than the conventional method. A water saving of 23.3% was also observed for all drip practices compared with conventional practices. Seasonal methane emission flux declined 78.0% in the drip methods over the conventional irrigation: better mitigation than previously reported values (alternate wetting and drying (47.5%) and system of rice intensification (29.0%) practices). Continuous soil aeration and enhanced soil methanotrophs (P<0.05) limit the peak methane emission in rice during the flowering phase in drip irrigation, which is reflected in the methane emission flux values. Consequently, the equivalent CO2 (CO2-eq) emissions and yield-scaled CO2 eq-emission were found to be significantly lower in SDI (43.8% and 49.5%, respectively), and DI (25.1% and 26.7%, respectively) methods as compared with the conventional that ensures better methane mitigation and future climate-smart rice production systems. Conclusions: Drip irrigation could reduce the cumulative methane emission in aerobically grown rice. SDI + 1.0 lph DRE practice can be applied in areas with inadequate water availability and effective in reducing the CO2-eq emission with better yield than conventional.", "keywords": [ "Aerobic rice", "Drip irrigation", "Methane", "CO2 eq-emission", "Water productivity" ], "content": "Introduction\n\nAgriculture was estimated to account for 10–20% of manmade greenhouse gas (GHG) emissions (5.1–6.1 Gt CO2-eq year-1 by 2030) worldwide (Smith et al., 2008; Tubiello et al., 2013). Rice fields are a major source of agricultural methane (CH4) emissions (Malyan et al., 2016), contributing 20–40 Tg CH4 year-1 with a global emission of 52% (Sun et al., 2016). In India, rice cultivation covers about 44 million ha, the largest rice-producing area in Asia. To ensure food security for the exponential population, expanding the cropping area will increase methane emission. Therefore, reducing methane gas emission from the rice eco-system is the foremost preventive measure for a check-in global warming. Altering water level maintenance, an important parameter in measuring methane emission rate, by shifting water level from 15 cm to 10 cm could reduce 26.8% of total methane emissions in the rice field and supports a green eco-system (Sandin, 2005). So, effective water management practices, like midseason drainage, intermittent irrigation, system of rice intensification, alternate wetting and drying, direct dry seeding and aerobic rice cultivation, have the possible potential to mitigate methane emission for irrigated rice cultivation (Bronson et al., 1997; Feng et al., 2013; Trost et al., 2013). The system of rice intensification (SRI) practice reduces total methane emissions by 29.0% (Rajkishore et al., 2013), the value for alternate wetting and drying method was 44.0% (Bouman et al., 2005; Bouman et al., 2007; Oo et al., 2018a; Oo et al., 2018b; Setyanto et al., 2018) and for aerobic rice practice was 51.0% (Joshi et al., 2009; Jain et al., 2014; Keppler et al., 2006; Sharma et al., 2016) compared with flooded rice cultivation. Although above-water management strategies show reduced methane emissions, consumption of more water for initial field setup and surface mode flood irrigation during an entire rice-growing cycle reduces the water productivity (Geethalakshmi et al., 2011) of the rice crop. By 2025, Asia’s 130 million ha of irrigated rice area may experience physical and economic water scarcity (Tuong & Bouman, 2003). In addition, India would need to produce up to 156 million tonnes of rice by 2030 (Dass et al., 2016) to feed its 1,523 million population. So, it is necessary to develop alternative irrigation strategies to mitigate methane emission as well as to improve the rice yield with limited water (Bouman et al., 2005; Reis et al., 2018; Yang & Zhang, 2010).\n\nThe drip irrigation system for rice is a water-saving concept that allows the rice farmers to utilize water effectively through root-zone irrigation (Parthasarathi et al., 2018), which may lead to more rice crop seasons in a year. Also, drip irrigation has scope to mitigate methane emissions in the rice ecosystem. Drip irrigation to rice altered root traits (Parthasarathi et al., 2017), improved the water productivity (He et al., 2013; Parthasarathi et al., 2013; Parthasarathi et al., 2018) and nutrient use efficiency (Rajwade et al., 2018), and reduced pollution of the environment (Adekoya et al., 2014). Drip irrigation kept the field in the condition similar to aerobic/upland throughout the growing season (Adekoya et al., 2014). Rice ecosystems managed by drip irrigation have been scarcely reported with regards to methane emissions and mitigation potential. The effect of drip irrigation on soil environment, growth, yield and water productivity of rice remains unexplained.\n\nWe, therefore, hypothesized that the drip irrigation practice would allow for improved water saving, increased yield and the potential to mitigate methane release in the rice ecosystem. We tested this hypothesis and examined the drip irrigation practices (i.e. sub-surface drip irrigation, surface drip irrigation, 1.0 and 0.6 lph discharge rate emitters) by conducting the field experiment in aerobic rice. We measured the methane emission, soil environment, rice growth, yield, water productivity, and microbial abundance. The results demonstrated that drip irrigation can be adapted to maintain more oxidative aerobic soil condition in rice crop.\n\n\nMethods\n\nThe drip irrigation with methane emission experiment was conducted during summer season 2014 in the wetlands of Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India (located at 110 N latitude, 770 E longitude and at an altitude of 426.8 m above mean sea level). The prevailing agro-ecological conditions during the cultivation period were an average temperature of 34.2/23.3°C (max/min), sunshine hours of 7.3 hrs day-1 and total evaporation of 750.4 mm with total precipitation of 118.6 mm (Figure 1). Soil samples were collected in the field and soil physio-chemical properties were analysed and given in Table 1. Drip experiment was carried out using ADT (R) 45 rice variety (parentage: IR 50/ADT 37) that grows widely in the Cauvery delta zone of Tamil Nadu, India.\n\nThe drip system was installed in the field with the help of Netafim Irrigation, Israel. The drip irrigation was supplied through 40 mm OD PVC pipes by 7.5 HP motors from bore well and pressure maintained in the system was 1.2 kg cm-2. The drip treatments tested were sub-surface drip irrigation (SDI) with 1.0 litre per hour (lph) discharge rate emitters: i) SDI+1.0 lph DRE (SDI with 0.6 lph discharge rate emitters); ii) SDI+0.6 lph DRE (surface drip irrigation (DI) with 1.0 lph discharge rate emitters); iii) DI+1.0 lph DRE (DI with 0.6 lph discharge rate emitters) and iv) DI+0.6 lph DRE.\n\nThese were arranged in a randomized block design with three replications per method. Drip irrigation lateral pipes were laid out at a distance of 0.8 m, emitters placed at 0.3 m distance for DI and SDI. Besides, the laterals placed at a depth of 15 cm below the soil surface for the SDI. Rice plants under drip irrigation system were irrigated at 125% pan evaporation (PE) using the Open Pan Evaporation (PE) values from a USWB Open Pan Evaporimeter. Scheduling of irrigation for the drip methods was conducted by working out effective rainfall using water balance method (Dastane, 1974). A conventional flood aerobic irrigation practice was considered the conventional method; these plots were kept unsaturated and the surface irrigated at 30-mm depth when irrigation water/cumulative pan evaporation (IW/CPE) ratio reached 1.25. A graphical description of irrigation practices, crop evapotranspiration (ET) and soil moisture prevailed during the experiment is given in the Extended data, Supplementary Figures 1 and 2 (Parthasarathi, 2019). Regarding crop management, recommended cultivation practices for aerobic rice were followed (TNAU Crop production guide, 2014). Application of pre-emergence herbicide, pendimethalin 30% EC at 1.25 kg a.i. ha-1 at 3 days after sowing (DAS) and two-time hand weeding at 30 and 45 DAS have controlled the weeds. A recommended fertilizer dose of 150:50:50 kg ha-1 N:P:K was supplied as fertigation in the form of water-soluble fertilizers. Fertigation was applied through a venturi flume at weekly intervals. For the conventional irrigation method, the entire dose of P was applied basally before sowing. In the case of N, the recommended dose was given at basal, tillering, booting and 50% flowering; K was given in two equal splits at basal and panicle initiation stages.\n\nThe sampling of methane gas was performed using the closed chamber technique (Minamikawa et al., 2015). The chamber with rice plants is illustrated in Figure 2. The chamber was placed in between the laterals and 15 cm far from the emitters. Inside the chamber, an electric fan was installed to circulate the air. Samples were collected from 10:00 to 12:00 at 10, 30, 50, 70, 90 and 110 DAS. The gas samples were withdrawn from the top of the chamber using 50-ml gas-tight syringes at 0, 10, 20 and 30 min after putting the chamber in its place. Air inside the chamber was thoroughly mixed by flushing the syringe five times before collection of the gas sample. The sample gases were transferred to 15 ml vacuum glass vials with a rubber stopper and kept cool and dark until analysis.\n\nThe temporal increment of methane concentration inside the chamber was measured in terms of methane flux (Hutchinson & Mosier, 1981). Collected gas samples were analysed using gas chromatography with flame ionization detector (FID). The following formula used to calculate methane gas emitted and concentration of gas denoted as mg m-2 hr-1:\n\nTotal methane emissions (mg m-2 hr-1) = [(Ps × Cs / Pstd) × Vv / Va] × Vhx A × H.\n\nWhere Ps = peak area for sample in gas chromatography; Cs = standard methane gas concentration (mg L-1 ); Vv = vial volume (ml); Vh = headspace volume of the chamber, i.e., [Chamber lengthbreadthheight] (ml); Va = air volume sampled (ml); A = chamber area covered (m2); H : enclosure period (hr); Pstd : standard peak area in gas chromatograph. The equivalent CO2 (CO2-eq) emission for total methane was calculated using the following modified equation (Oo et al., 2018a): CO2-eq = [TCH4 × 34], where CO2-eq is the total amount of equivalent CO2 emission (kg CO2-eq ha-1), TCH4 is the total amount of methane emission (kg ha-1), 34 is the global warming potentials for methane to CO2 over a 100-year time horizon (IPCC, 2013).\n\nThe soil physical measurements were recorded during methane gas collection. Soil redox potential (Eh) and pH were measured by portable Eh meter (EHS-120; Fujiwara Scientific Company Co., Ltd, Tokyo, Japan) with platinum-tipped electrodes and pH meter (Lutron model pH 212; Sunshine Instruments, India), respectively. Dissolved oxygen content (mg L-1) of rice rhizosphere was measured with the analytical apparatus SJG-203A (Shanghai Leici Manufacturers, Shanghai, China). Enumeration of methanotrophic methane-oxidizing bacteria (MOB) was isolated and quantified during flowering phase based on the method of Graham et al. (1992). The soil samples were collected, diluted as suspension and analysed to 10-2 to 10-7 levels by 1-mL suspension in tubes under ambient air condition.\n\nThe plant height (cm) and total dry matter accumulation (g m-2) were measured during methane gas collection. The plant volume and root oxidizing power were taken from tagged plants during the flowering stage of rice. The total plant volume was calculated as the sum of root and shoot volume. Root and shoot volume was recorded by using the water displacement method (Bridgit & Potty, 2002). The values were expressed in cm3 hill-1. The oxidizing power of roots was determined by the method of Ota (1970) and the activity expressed as μg g-1 hr-1. At the end of the experiment, replication wise harvesting was done for each treatment at the net plot (2.4 × 7.0 m). The yield of rice was measured at 14% moisture level and yield expressed in kg ha-1. Harvest index (HI) was calculated by using the formula of Yoshida et al. (1971) and expressed in percentage.\n\nWater use was calculated by the sum of irrigation water applied (mm) and the effective rainfall (mm) during the cropping period (Dastane, 1974). Water productivity was calculated as the grain weight produced per unit of water input (Yang et al., 2005) and expressed as g grains kg-1 of water.\n\nRandomized block design (RBD) analysis was carried out on various parameters (Water productivity, methane flux rate, cumulative methane, CO2-eq emissions, Yield-scaled CO2-eq emission, Grain yield, Straw yield, Harvest index, plant height, Total dry mass accumulation (TDMA), soil pH, soil Eh, Dissolved oxygen, plant volume, Root oxidizing power, Methanotrophs population) as per the procedure suggested by Gomez & Gomez (1984). The coefficient of determination (R2) was made between methanotrophs population and methane flux. Whenever the treatment differences were found significant, critical differences were worked out at 5% probability level. Analysis of variance (ANOVA) was carried out for the recorded mean data using JMP, 2007 (SAS Institute, Cary, NC) software; appropriate standard errors of the means (SEM) and Fisher’s Least Significant Difference at a significance of P ≤ 0.05 was calculated.\n\n\nResults and discussion\n\nThe SDI and DI systems were installed in the field with 1.0 lph and 0.6 lph DRE to the aerobic rice plants. The drip response results on methane gas emission, CO2-eq emission, soil pH, soil redox potential, soil methanotrophs population from the rice ecosystem, plant height, total dry mass, yield, water productivity of rice were compared with the conventional flood aerobic irrigation practice; this section discusses the notable results. Data concerning methane emissions and weather for each group is available as Underlying data (Parthasarathi, 2019).\n\nSeasonal methane flux pattern was observed to be similar in conventional and drip treatments (Figure 3c), and the flux increased gradually at the early rice-growing stage but declined at the end of the growth period (Figure 3c). A recent report by Oo et al. (2018a) on the alternate wetting and drying (AWD) water-saving method in rice emitted methane gas with two peak emissions during the vegetative phase. Contrarily, drip and conventional aerobic irrigation practices had a single emission peak during the flowering phase (70 DAS), particularly at 10 days before and after anthesis. This is because the aerobic condition lacked the dominant pathway of direct methane emission by lesser aerenchyma pore spaces in the root (see Extended data, Supplementary Information 2 (Parthasarathi, 2019)). This is the reason for the reduced methane transport through rice shoots from soil to the atmosphere and was well explained by Bhattacharyya et al. (2019) in rice. Contrary to the sharp decline in methane flux at harvest phase in flooded paddy (Minamikawa et al., 2015), drip and conventional aerobic irrigated rice showed a gradual and slow decline influx after 100 DAS (shaded area in Figure 3). The decrease in methane transport capacity of rice plants under an aerobic environment is the possible explanation for methane reduction after flowering (Figure 3c) and the response was reverse for flooded paddies (Minamikawa et al., 2015). These different responses to irrigation conditions between flooded and aerobic environment explain the significance of the drip system on methane mitigation potential (Table 2).\n\nError bars indicate standard error of the mean (n = 6). Shading indicates the period after post-flowering phase (100 DAS). SDI, sub-surface drip irrigation; DI, surface drip irrigation; DRE, discharge rate emitters; Conventional, conventional flood aerobic irrigation.\n\nNumbers in the table represent means ± standard deviation (n =3). Letters indicate significant differences (P<0.05) among means according to an ANOVA.\n\nSDI, sub-surface drip irrigation; DI, surface drip irrigation; DRE, discharge rate emitters; Conventional, conventional flood aerobic irrigation.\n\nThe SDI at 1.0 and 0.6 lph DRE practices found a significant reduction in methane emission during vegetative to post-flowering phases due to discontinuous soil aeration near the root zone and lack of carbon substrate for methane production (root exudates) during the growing season. Similar reduction reported in the midseason drained paddy field (Jiao et al., 2006) and these were in agreement with Yan et al. (2005) and Sander et al. (2015), who reported an average methane reduction of 40–60% by multiple soil aerations.\n\nAs hypothesized, a significant reduction (P < 0.05) in the rate and cumulative methane flux was observed under drip irrigation methods over the conventional. Lesser methane flux rate (Table 2), cumulative methane (Table 3) emitted in SDI + 1.0 lph DRE (3.37 mg m-2 h-1 and 97.2 kg ha-1, respectively) treatment with greater mean significance over DI + 1.0 lph DRE (4.50 mg m-2 h-1 and 129.6 kg ha-1, respectively) and conventional (6.00 mg m-2 h-1 and 172.9 kg ha-1, respectively). The drip treatments (SDI+1.0 lph DRE, SDI+0.6 lph DRE, DI+1.0 lph DRE, DI+0.6 lph DRE) might have directed to more oxygen penetration to the root zone that inhibits the methanogenic bacteria by methane oxidation. However, the dissolved oxygen content declined significantly in drip practice over the conventional methods; we discuss the reason for the lower values in the up-coming section. Soil methanotrophic bacteria oxidizing methane gas using molecular oxygen in drained paddy soil (Jiao et al., 2006) was the reason for the above reduction in methane flux rate, cumulative methane flux under drip environment. This response was further confirmed an increased in methanotroph population of 39.8% in SDI+1.0 lph DRE, 32.2% in SDI+0.6 lph DRE (44.7 ×105 cells g-1) followed by 24.8% in DI+1.0 lph DRE over the aerobic zones of the rhizosphere (Figure 4). The regression line (Figure 5) represents a negative slope (y=-0.1097x+11.407, R2 = 46.1) or the relationship between methanotrophs population and methane emission that indicates the drip practice reduces the methane by increased methanotrophs. Similarly, drip practices inhabited the aerobic interfaces of the methanogenic environment, that inhibit methanogenesis by killing the methanogenic bacteria (Hanson & Hanson, 1996) and these processes were influenced by the soil physio-chemical properties, and plant growth parameters (Luo et al., 2013).\n\nNumbers in the table represent means ± standard deviation (n =3). Letters indicate significant differences (P<0.05) among means according to an ANOVA.\n\nSDI, sub-surface drip irrigation; DI, surface drip irrigation; DRE, discharge rate emitters; Conventional, conventional flood aerobic irrigation.\n\nError bars indicate standard error of the mean (n = 6). Different letters denote significant differences among means derived using an ANOVA and student test. SDI, sub-surface drip irrigation; DI, surface drip irrigation; DRE, discharge rate emitters; Conventional, conventional flood aerobic irrigation.\n\nThe black line shows a regression line (y=-0.1097x+11.407). The r2 is the coefficient of determination. SDI, sub-surface drip irrigation; DI, surface drip irrigation; DRE, discharge rate emitters; Conventional, conventional flood aerobic irrigation.\n\nThe drip irrigation practices had a considerable change in rice growth in terms of plant height (29.4%) and total dry mass accumulation (TDMA) (26.9%) than conventional systems. These were significantly increased under SDI and DI methods, irrespective of emitter discharge rates, over the conventional method. These results were consistent with the findings of previous studies, which found that the drip irrigation had improved the production potential of rice under non-flooded irrigation (He et al., 2013 and Parthasarathi et al., 2015). The total dry mass of rice did not differ significantly between drip irrigation and conventional irrigation up to 30 DAS, although after anthesis (70 DAS), it significantly improved under SDI + 1.0 lph DRE practice (Figure 3a). This was mainly related to enhanced dry mass accumulation after anthesis, which explained by Parthasarathi et al. (2015) and Parthasarathi et al. (2018), who reported drip irrigation increased the plant height and leaf photosynthetic rates of rice during post-anthesis. TDMA showed a moderate change during the early growth and vegetative phase of rice under SDI and DI methods (Figure 3b), which reasoned for the lesser emission of methane over conventional. These results corroborated with the findings of Arafa et al. (2009) in wheat under SDI system.\n\nHigher dry mass and lower root volume of rice facilitated oxygen transport into the rhizosphere, stimulating methane oxidation (Ma et al., 2010). Similarly, Figure 4 illustrates unaffected root zone volume and increased shoot volume under drip irrigation favours methane oxidation in comparison with the conventional method. This might be due to the favourable balance between root and shoot translocation of assimilates, nutrients and water (Kludze et al., 1993). Higher shoot volume increased the contact surface between roots that enhanced the methane oxidation rates under drip irrigated rice environment and were in agreement with methane oxidation studies in rice by Zhao et al. (2013). SDI + 1.0 lph DRE treatment significantly improved the root oxidase activity (80.7 µg g-1 hr-1) than the conventional (72.7 µg g-1 hr-1) (Figure 4). This indicated an overall higher oxidation status and might have accelerated methane oxidation in the root rhizosphere, which eventually reduced the methane gas emission (Kong et al., 2009).\n\nAmong the irrigation treatments, drip-irrigated rice had higher (P<0.05) soil Eh than conventional. Superior Eh levels reasoned for the lesser emission (P<0.05) of methane under SDI+1.0 lph DRE drip practice (Figure 3e). Present results followed the study of Oo et al. (2018a), who reported water management practice improved the soil redox potential in flooded rice. Dorau & Mansfeldt (2015) and Minamikawa et al. (2015) reported that higher Eh was resulted by the low pH in soil solution and the similar was observed in SDI+1.0 lph DRE followed by SDI+0.6 lph DRE and DI+1.0 lph DRE practices (Figure 3d). This acidic shift might also be due to the supply of water-soluble fertilizers by drip fertigation. This low soil pH could be the reason for the altered balance between CH4 and CO2 under drip irrigation by soil organic matter decomposition (Kongchum, 2005). Thus, low soil pH had a considerable effect, but this was alongside soil oxygen properties, which are likely to be responsible for reduced methane emissions (Kajiura et al., 2018). SDI + 1.0 lph DRE treatment registered lesser (P<0.05) dissolved oxygen (DO) in soil than the conventional during all the phenophases (Figure 3f). This condition led to an increase in the soil oxygen concentration that leads to inhibition of soil organic matter oxidation which directly and indirectly, inhibited the methanogenesis (Kato et al., 2012; Zhou et al., 2014). Soil organic carbon (SOC) of oxygen-enhanced soil was oxidized into carbon dioxide (CO2) instead of methane (CH4), which ultimately reduced the root zone methane formation (Sun et al., 2016). This contrasted with flooded rice (Sharkawi et al., 2009), where increased DO reduces the methane emission but DO in the rhizosphere was controlled by irrigation water quantity.\n\nUnder drip irrigation, the total water saving compared with conventional flood aerobic irrigation was 23.3% (Table 2). The maximum saving of irrigation water with aerobic irrigation was 50% (Peng et al., 2006), with alternate wetting and drying was 47% (Oo et al., 2018a) and with the system of rice intensification was only 27.4% (Suryavanshi et al., 2013), relative to flooded rice. Higher water savings under drip irrigation over the conventional method was due to the supply of irrigation water being based on the evapotranspiration demand for rice. The water productivity of 0.82 g grains kg-1 of water obtained under drip irrigation (SDI + 1.0 lph DRE) due to higher yield potential and substantially higher water productivity than the previous reports (Bouman et al., 2006; Kato et al., 2009).\n\nDrip irrigation improves the grain yield of rice by 9.3-23.6% over the conventional flood aerobic irrigation. Higher grain yield (4489 kg ha-1), straw yield (7297 kg ha-1) and harvest index (38.1%) were obtained under SDI + 1.0 lph DRE practice, and it was significantly lower using the conventional method (3430 kg ha-1, 6562 kg ha-1 and 34.3%, respectively) (Table 3). The yield of rice was significantly superior under SDI followed by DI over the conventional aerobic rice. Present yield response of rice in drip irrigation was due to better water and nutrients discharge (1.0 lph DRE followed by 0.6 lph DRE) to the root zone. These were in line with the recent work on drip-irrigated rice (Parthasarathi et al., 2018) along with plastic mulching (He et al., 2013). Higher harvest index of rice was due to the better balance between the source and sink under drip irrigation. This might be reasoned for the methane reduction (Denier et al., 2002) under drip environment.\n\nThe impact of methane emission calculated by the CO2-eq emission for the 100-year horizon was observed for each method. The SDI and DI treatments reduced the CO2-eq emission by 43.8% and 25.1% over conventional flood aerobic irrigation (Table 3). This reduction found a better result than the recent report (Oo et al., 2018a) on the alternate wetting and drying method (19%-39% better than the conventional flooded method). The decrease in methane emissions by SDI and DI was due to effective depression in CO2-eq methane emission. Yield-scaled CO2-eq emission provides a measure of agronomic efficiency to mitigate climate change and future food production concerns (Grassini & Cassman, 2012). The yield-scaled CO2-eq emissions were higher (594.2 kg CO2-eq t-1) using the conventional method than SDI + 1.0 lph DRE practices (300.1 kg CO2-eq t-1) due to their respective levels of methane emission rate. Therefore, the SDI + 1.0 lph DRE practice is recommended for efficient reduction in CO2-eq methane emission along with increased grain yield and water productivity, rather than the conventional flood aerobic irrigation.\n\n\nConclusion\n\nThe present study demonstrated that drip irrigation practice can mitigate methane emissions and improved the growth and yield of rice when compared to conventional aerobic methods. The drip combinations SDI + 1.0 lph DRE, SDI + 0.6 lph DRE, DI + 1.0 lph DRE and DI + 0.6 lph DRE could reduce the cumulative methane emission in aerobic rice by diminishing the flux rate. So far, it has been impossible to control the soil redox condition of conventional aerobic irrigated rice soil, but the drip irrigation practice (SDI or DI with 1.0 or 0.6 lph DRE) may offer the solution. The better performance of drip irrigation (i.e. SDI+ 1.0 lph DRE) under aerobic conditions was also evident from their higher TDMA, root oxidase activity, soil methanotrophs population along with higher methane mitigation in comparison to the conventional. The SDI + 1.0 lph DRE practice can be applied in areas with inadequate water availability (i.e. where there are water shortages) for flooded rice production and the same practice is effective in reducing the CO2-eq emission and better grain yield than the conventional flood aerobic irrigation. In the case of climate change, drip irrigation systems have promise to ensure food security, while preserving irrigation water and mitigating methane gas emissions in rice. Further studies are required to test the methane-mitigating oxygen-nanobubble water (Minamikawa et al., 2015) under rice crop drip-irrigation, and to evaluate its mitigation potential regarding methane and other greenhouse gases.\n\n\nData availability\n\nOpen Science Framework: Methane Emission. https://doi.org/10.17605/OSF.IO/ZDY6U (Parthasarathi, 2019).\n\nThis project contains the following underlying data:\n\nRaw data methane emission (data concerning methane emission for each experimental group)\n\nRaw data weather (data concerning the weather for each experimental group)\n\nOpen Science Framework: Methane Emission. https://doi.org/10.17605/OSF.IO/ZDY6U (Parthasarathi, 2019)\n\nThis project contains the following extended data:\n\nSupplementary Information (1) (containing Supplementary Figures 1 and 2)\n\nSupplementary Information (2) (containing supplementary Figure 3 and Supplementary Table 1)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nAuthors should like to thank Dr. V.P. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "57226", "date": "03 Jan 2020", "name": "Prashantkumar S. Hanjagi", "expertise": [ "Reviewer Expertise My research program is multidisciplinary in nature", "combining physiology", "molecular biology", "and proteomics. Currently my research focuses on understanding the cellular and molecular mechanisms mediating the response(s) of plants to abiotic stress (Salt", "drought", "heat", "lodging", "etc). I have expertise in automated high-throughput plant phenotyping in getting insights into genomic performance and plant-environment-interactions through non-destructive analysis of phenotypic traits of the major crops like maize", "rice", "wheat", "and pulses. Within my group", "during past years", "we have attempted to characterize the role of candidate genes (NHX1 & AVP1) in improving tolerance to salt stress tolerance in rice." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for asking me to review this article. The authors of the study have generated very good data to communicate the key message on mitigation of methane emission in rice by different irrigation methods. My recommendation would be in and around \"Minor revision\".\n\nMy minor comments to the authors are:\nIn the Introduction text, the previously reported value for reduction in methane emissions due to alternate wetting and drying is 44.0%. But, in abstract it is being mentioned as 47.5%. Right value may be mentioned here.\n\nIn methods section, treatments can be mentioned in a tabular form for better understanding of readers instead of writing in running text.\n\nFor figure 2, Authors could have presented the real set-up of closed diffusion chambers used to collect methane emission in the present study instead of diagrammatic representation.\n\nRewrite this sentence \"This response was further confirmed an increased in methanotroph population of ...\" in results section (page no 7).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "87960", "date": "09 Jul 2021", "name": "Gerard Arbat", "expertise": [ "Reviewer Expertise Irrigation Engineer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper deals with methane emission, water use, and water productivity of drip irrigated rice compared with conventional flooded aerobic irrigation. The study includes surface and subsurface drip irrigation with two different flowrates. The main results of the paper are that drip irrigation reduces methane emissions and increases water productivity. The best option for this irrigation system is subsurface drip irrigation at a flowrate of 1 lph.\nMy recommendation would be a \"Minor revision\". This revision may include the following changes:\nInclude the soil texture (%sand, silt and clay) in table 1, as it can affect the results obtained\n\nDefine 'standard weeks' in Fig. 1.\n\nShow in Fig. 2 the exact location of the chamber, as it is described in Methane sampling and analysis section: “…placed in between the laterals and 15 cm from the emitters.”\n\nIn the section soil characteristics, the measurements are not only related to physical characteristics, please them describe accordingly.\n\nIn the section plant characteristics, define what a hill is.\n\nIn Fig.3, the vertical titles of figs. B and E are in different location than the other figures. Please be consistent.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2023
https://f1000research.com/articles/8-2021/v1	28 Nov 19	{ "type": "Case Report", "title": "Case Report: Successful use of fondaparinux in a case of heparin intolerance during pregnancy", "authors": [ "Mohammed AlSheef", "Noura Shafi", "Bakhitah Aleid", "Abdul Rehman Zia Zaidi", "Ohoud AlArfaj", "Noura Shafi", "Bakhitah Aleid", "Abdul Rehman Zia Zaidi", "Ohoud AlArfaj" ], "abstract": "Heparin is the anticoagulant of choice during pregnancy. However, in cases of intolerance or adverse effects, another anti-coagulant agent should be administered. Here, we describe a case of hypersensitivity skin reaction seen in a 37-year-old pregnant patient at 11 weeks of gestation who used low-molecular-weight heparin (LMWH). Fondaparinux was used as an alternative during her pregnancy with a successful outcome.", "keywords": [ "Fondaparinux", "Heparin Intolerance", "Pregnancy", "LMWH", "Hypersensitivity", "Anticoagulant" ], "content": "Introduction\n\nLow-molecular-weight heparin (LMWH), such as enoxaparin, is the preferred anticoagulant for pregnant women due to its effectiveness, safety, and availability. However, if the pregnant patient experiences an allergy, side effect or intolerance, she should be switched to alternative anticoagulation medication. Unfortunately, in such circumstances, the other anti-coagulation options are limited due to teratogenicity, lack of literature support, or cross-reactivity with LMWH.\n\nFondaparinux, which is a synthetic polysaccharide inhibitor of activated factor X (FXa), has been reported to be a successful alternative anticoagulant in pregnant patients who develop heparin intolerance, such as hypersensitivity skin reaction, which is frequently seen in pregnant patients1. Although it crosses the placental barrier and results in low measurable anti-factor Xa activity in umbilical-cord blood2, it is considered relatively safe since there are no significant reported unfavorable side effects for the mother or child during pregnancy or the postpartum period.\n\n\nCase report\n\nA 37-year-old Saudi female homemaker (G6P4+1) with a history of hypothyroidism on thyroxin and a history of miscarriage and intrauterine fetal death (IUFD) presented to our thrombosis clinic. She explained to us that this is an important pregnancy for her and that she wished to deliver a healthy baby. Her gynecological history comprised of: preeclampsia during her first pregnancy, resulting in premature labor; fetal distress during her second pregnancy; a third pregnancy resulting in premature labor at 30 weeks; a fourth pregnancy resulting in IUFD at 25 weeks; fetal distress in the 31st week of her fifth pregnancy in 2012; and a sixth pregnancy resulting in IUFD in 2015. Therefore, she was referred to our tertiary care hospital and to our clinic to prevent morbidity and mortality in the current pregnancy. She presented to our clinic in 2016, 11 weeks pregnant, and on physical examination, cardiac and respiratory exams were normal. Abdominal examination showed a gravid uterus. Thrombophilia work up including protein C, protein S, antithrombin III, factor V Leiden mutation, prothrombin gene mutation G20210A, and antiphospholipid antibodies were within normal limits (Table 1).\n\nThe patient was started on aspirin 81 mg once daily, and low-molecular-weight-heparin (LMWH) 4000 units via subcu- taneous injection once daily. We explained to the patient the rationale for using heparin was to prevent placental microvascular thrombosis and therefore, prevent placental mediated complications such as preeclampsia and IUFD. This is an internationally recommended evidence-based practice.\n\nFive days later, she developed a severe rash, as depicted in Figures 1A–1D. Hence, LMWH was discontinued, and she was asked to resume taking aspirin 81 mg once daily. She was found to be allergic to LMWH (such as enoxaparin and tinzaparin), resulting in symptoms such as swelling, itching, and erythema, which slowly resolved one week after discontinuation of heparin. One month later, on her scheduled follow-up appointment with us, she no longer had any skin rash or any other hypersensitivity reaction symptoms. We suggested fondaparinux 2.5 mg delivered subcutaneously once daily. We explained to the patient that fondaparinux was an alternative anticoagulant for patients intolerant to heparin, with no reported hypersensitivity reactions or adverse effects on the fetus. She agreed, and hence we discontinued aspirin and started her on fondaparinux. We monitored her health through monthly follow-up clinic visits with regular laboratory tests and ultrasounds in the maternal fetal medicine and thrombosis clinics (Table 1). The ultrasounds had no significant findings, with a single viable fetus and normal growth. She was well with no allergic symptoms or discomfort, and kept taking fondaparinux for five months until her planned induction of labor at 38 weeks gestation with cessation of fondaparinux for 24 hours. She delivered a normal healthy baby and we followed up regularly with the patient for six weeks postpartum (Table 1).\n\nA–D) The composite figure illustrates clinical findings, showing the patient’s rash, when she was administered low-molecular weight heparin.\n\n\nDiscussion\n\nHeparin has always been the anticoagulant of choice to prevent and treat a thrombotic event during pregnancy1. However, its use is limited when adverse reactions such as skin rash (type I hypersensitivity reaction) or heparin-induced thrombocytopenia occurs. Hence, in the setting of heparin intolerance with a high risk of thrombosis, alternative choices for anticoagulation becomes limited.\n\nCurrent evidence shows that fondaparinux is a safe and effective alternative option in the circumstances, such as seen in our case2–4. For instance, a retrospective study comparing the efficacy of fondaparinux to enoxaparin in terms of pregnancy success rate, gestational age, birth weight, and major bleeding complications concluded that both anticoagulants have comparable results5. In addition, a prospective study evaluating the effect of a prophylactic dose of fondaparinux in pregnant women with a history of venous thromboembolism reported 100% uneventful pregnancies without thromboembolic complications6. However, there is limited experience in the use of fondaparinux in pregnancy, but it has been used in patients with heparin intolerance with no reported adverse effects to the fetus or the mother7.\n\nIn concordance with the literature, our patient had an uneventful pregnancy without developing an adverse reaction to fondaparinux. With regards to the long-term safety of fondaparinux, several studies in the literature reported no significant difference in safety profile compared to enoxaparin over a period of therapy ranging between a few weeks to eight months.\n\nOn the other hand, umbilical blood sampling showed a detectable level of anti-factor Xa, indicating the passage of fondaparinux to fetal circulation. However, the accumulative level is measured and found to be subtherapeutic, and no study reported any complications during pregnancy or post-partum8.\n\n\nConclusion\n\nIn conclusion, our case demonstrates that fondaparinux is a safe and effective anticoagulant option in the presence of heparin intolerance.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nMazzolai L, Hohlfeld P, Spertini F, et al.: Fondaparinux is a safe alternative in case of heparin intolerance during pregnancy. Blood. 2006; 108(5): 1569–1570. PubMed Abstract \| Publisher Full Text\n\nCiurzyński M, Jankowski K, Pietrzak B, et al.: Use of fondaparinux in a pregnant woman with pulmonary embolism and heparin-induced thrombocytopenia. Med Sci Monit. 2011; 17(5): CS56–CS59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoyal College of obstetricians and gynaecologists reducing the risk of venous thromboembolism during pregnancy, April 2015. Reference Source\n\nKnol HM, Schultinge L, Erwich JJ, et al.: Fondaparinux as an alternative anticoagulant therapy during pregnancy. J Thromb Haemost. 2010; 8(8): 1876–1879. PubMed Abstract \| Publisher Full Text\n\nWiger EE, Reed JL: A retrospective analysis of fondaparinux versus enoxaparin treatment in women with infertility or pregnancy loss. Am J Reprod Immunol. 2009; 62(4): 253–260. PubMed Abstract \| Publisher Full Text\n\nGerhardt A, Zotz RB, Stockschlaeder M, et al.: Fondaparinux is an effective alternative anticoagulant in pregnant women with high risk of venous thromboembolism and intolerance to low-molecular-weight heparins and heparinoids. Thromb Haemost. 2007; 97(3): 496–497. PubMed Abstract \| Publisher Full Text\n\nMazzolai L, Hohlfeld P, Spertini F, et al.: Fondaparinux is a safe alternative in case of heparin intolerance during pregnancy. Blood. 2006; 108(5): 1569–70. PubMed Abstract \| Publisher Full Text\n\nNagler M, Haslauer M, Wuillemin WA: Fondaparinux - data on efficacy and safety in special situations. Thromb Res. 2012; 129(4): 407–417. PubMed Abstract \| Publisher Full Text" }	[ { "id": "57377", "date": "03 Dec 2019", "name": "Saskia Middeldorp", "expertise": [ "Reviewer Expertise VTE" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report on fondaprinux in a pregnant woman with LMWH allergy does not lead to new insights into the knowledge of heparin allergy or fondaparinux.\nImportant references about the occurrence of type IV (?) allergy to LMWH and how to deal with this, have not been referenced (for instance, Schindewolf, Lancet) It is therefore surprising that the authors immediately chose to use fondaparinux and not other types of LMWH.\nThe authors imply causal inference between use of anticoagulants and the successful pregnancy outcome, which cannot be made on a single case.\nReference to guidelines regarding the use of LMWH to improve outcome in IUFD is missing.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "65571", "date": "30 Jul 2020", "name": "Maha Othman", "expertise": [ "Reviewer Expertise Haemostasis", "coagulopathies in placenta mediated complications", "Women's Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report successful use of fondaparinux in managing heparin intolerance during pregnancy in a 37 y old pregnant lady at her 11 w of gestation.\nHypersensitivity reactions to heparin in pregnancy is not a new issue and is reported in ~ 20% of patients. The successful use of fondaparinux is not new either. So I am not sure if this is still worth reporting. Also, I am not sure I would agree with authors that the experience fondaparinux use in pregnancy is still limited.\nThe most interesting part of this case in my view is to identify the pathology underlying this poor obstetric history (repeatedly complicated pregnancies, labours and multiple IUFDs). The thrombophilia screen is all normal. The authors included APS work up, however these antibodies were only measured once; against the standard diagnostic guidelines. The possibility of APS still exists in this case and requires further investigation.\n\nI encourage the authors to shift focus towards diagnosis of the pathology for this poor obstetric history; perform further investigations and rewrite the case report. This will provide some novelty and help educate the scientific community in similar cases.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-2021
https://f1000research.com/articles/8-665/v1	15 May 19	{ "type": "Opinion Article", "title": "On the explicit use of experimental images in high resolution cryo-EM reﬁnement", "authors": [ "Jacqueline Cherfils", "Jorge Navaza" ], "abstract": "Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the models.", "keywords": [ "cryo-EM", "X-ray crystallography", "computational method", "model refinement", "structural biology" ], "content": "Discussion\n\nSingle-particle cryo-EM has recently joined the circle of techniques that allow macromolecular structure determination at almost atomic resolution. The technique presents a clear advantage over X-ray crystallography in that it allows the structural analysis of particles that are difficult or impossible to grow into crystals, such as very large complexes, membrane proteins or proteins with flexibles portions, and is therefore gaining a prevalent role in the determination of biological macromolecular structures1. As a relatively young method, computational methods to interpret images and derive atomic models continue to be developed and optimized2,3. In that regard, a recent comparison of X-ray and cryo-EM maps calculated at the same resolution, together with the corresponding atomic models, showed that although the appearance of the maps was quite comparable between the two techniques, X-ray crystallography maps were more detailed and the atomic models fitted into them were more accurate4. To make the comparison on a fair basis the X-ray electron densities were calculated with experimental phases (SAD, heavy-atom) and improved by density modification. In this way the maps produced by both techniques were model-free. The accuracy and level of detail of the maps were assessed by fitting the already determined atomic structures into them. These results begged for microscopists to continue to improve the accuracy and performance of methods for map improvement and model refinement, in order to produce atomic models that meet the same quality standards as in X-ray crystallography.\n\nThe cryo-EM and X-ray crystallography experimental techniques are very different but the final stages of the structure determination process are similar. An important difference between crystallography and cryo-EM as techniques to reconstruct scattering densities is that in cryo-EM it is possible, in principle, to obtain a reconstruction starting from the raw experimental data without imposing any model excepting the assumption that the data are projections of similar particles. If we look at the cryo-EM data in reciprocal space the similarities and differences with X-ray data become apparent. The actual experimental data in cryo-EM are two-dimensional images related to the projections of the particle electrostatic potential, along different directions. According to the projection-slice theorem, the Fourier transform of the two-dimensional experimental image corresponds to a central section of the three-dimensional particle Fourier transform multiplied by the contrast transfer function (CTF), a well-defined mathematical function that introduces a modulation in reciprocal space and restricts the data to annular domains delimited by the zero-crossings of the function. So, while in X-ray crystallography the diffraction experiment gives amplitudes of Fourier coefficients for points in a known reciprocal lattice, in cryo-EM the images give, after CTF correction, complex Fourier coefficients in central planes whose relative orientations in reciprocal space are unknown. The accurate determination of the CTF and the assignment of orientations and centers to the images is the task of the reconstruction procedure which, in principle, does not depend on any model. In this regard, the cryo-EM map is somehow equivalent to an experimentally phased X-ray crystallography map in the sense that the required orientations and phases are determined from the sole experimental data. With near atomic resolution data, these maps allow the construction of initial atomic models. In X-ray crystallography, model phases are used to update the map during model refinement, keeping the experimental structure factors—the data—unchanged. Thereby, the crystallographic model is improved as the map becomes clearer in both the particle and solvent regions based on feedback from calculated phases. In cryo-EM, the reconstruction is considered as experimental data and kept unchanged during model building and refinement5. In keeping with elementary ideas of data, it seems natural that the central sections, rather than the reconstructed map or its associated Fourier coefficients, should play the role of data in model refinement.\n\nThe perspective that atomic structures should be refined against raw cryo-EM images was suggested in conclusion of an excellent recent review on cryo-EM refinement6. We propose here directions for the explicit use of experimental images in cryo-EM refinement. We call Gobsk the two-dimensional Fourier coefficients of the kth image and Gcalck the corresponding calculated quantities, given by\n\n\n\nwhere F is the three-dimensional Fourier transform of the model electrostatic potential, Rk the rotation that specifies the section orientation, ok the origin shift that centers the section, χk the section’s associated CTF and q is a two-dimensional reciprocal vector. The mismatch between Gobsk and Gcalck may be used to define the experimental component of a refinement target function. The refinement target function should thus allow to couple the improvement of the model to that of the orientations, centers and CTF. Accordingly, the mismatch between Gobsk and Gcalck could be used to calculate an R-factor for structure assessment. Note that such criteria remain meaningful even in situations where maps are not of high quality, for example when the distribution of central sections is pronouncedly uneven.\n\nBy using experimental images as data, cryo-EM refinement procedures become thus quite similar to those used in X-ray crystallography, in spirit, in that the reconstructed maps are allowed to improve as model building and refinement proceed. A proof-of-principle assessment of the above proposal could be feasible based on available refinement software originally developed by X-ray crystallographers. Such software is mainly implemented in cartesian coordinates, but it can be anticipated that spherical coordinates will be more appropriate not only to handle rotations and interpolation of Fourier coefficients as required by Equation 1, but also to produce more accurate results7,8. Clearly, other not well understood aspects throughout the determination procedures will also need to be improved, such as dealing with errors on the detectors or multiple conformations of molecules6, some of which may benefit from the proposed refinement target to yield statistically more robust structures. Ultimately, this may allow to better exploit the potential of the cryo-EM method and lead to a significant gain of accuracy of the high-resolution refinement protocol.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Grant information\n\nThis work was supported by grants from the Fondation pour la Recherche Médicale (DEQ20150331694) and from the Agence Nationale de la Recherche (ANR-14-CE090028) to J.C.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank our colleagues at the “Conference on methods and applications in the frontier between MX and CryoEM” held in Barcelona in 2017 for discussions, which motivated this communication.\n\n\nReferences\n\nFernandez-Leiro R, Scheres SH: Unravelling biological macromolecules with cryo-electron microscopy. Nature. 2016; 537(7620): 339–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLyumkis D: Challenges and opportunities in cryo-EM single-particle analysis. J Biol Chem. 2019; 294(13): 5181–5197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVinothkumar KR, Henderson R: Single particle electron cryomicroscopy: trends, issues and future perspective. Q Rev Biophys. 2016; 49: e13. PubMed Abstract \| Publisher Full Text\n\nWlodawer A, Li M, Dauter Z: High-Resolution Cryo-EM Maps and Models: A Crystallographer's Perspective. Structure. 2017; 25(10): 1589–1597.e1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfonine PV, Klaholz BP, Moriarty NW, et al.: New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr D Struct Biol. 2018; 74(Pt 9): 814–840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurshudov GN: Refinement of Atomic Structures Against cryo-EM Maps. Methods Enzymol. 2016; 579: 277–305. PubMed Abstract \| Publisher Full Text\n\nLiu H, Cheng L, Zeng S, et al.: Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions. J Struct Biol. 2008; 161(1): 64–73. PubMed Abstract \| Publisher Full Text\n\nEstrozi LF, Navaza J: Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way. J Struct Biol. 2010; 172(3): 253–60. PubMed Abstract \| Publisher Full Text" }	[ { "id": "48578", "date": "17 May 2019", "name": "Ignacio Fita", "expertise": [ "Reviewer Expertise Structural biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work by Cherfils and Navaza proposes to use the central sections from cryo-EM images, rather than the reconstructed maps or its associated Fourier coefficients, as the data for model refinement and validation. To achieve it, the section orientation and the origin shift that centers the section are incorporated in the evaluation of the two-dimensional Fourier coefficients of the model. In this way cryo-EM refinement can become similar to X-ray crystallography refinement. This is a very interesting proposal that could provide (implementation pending) an elegant approach to overcome major limitations in the accuracy and quality of the cryo-EM models obtained with the available methodologies. It might be good if authors could add some considerations about:\nNoise in the individual cryo-EM sections could be high, which would degrade the accuracy of the observed two-dimensional Fourier coefficients. How these (large) errors could affect to the proposed refinement approach? With orientation and translation of individual sections applied to the model, how the “improved” experimental maps can be efficiently computed?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "50124", "date": "25 Jun 2019", "name": "S. Saif Hasan", "expertise": [ "Reviewer Expertise Structural biology", "cryoEM" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article by Cherfils and Navaza provides an important path forward to model refinement in cryoEM, which deals with noisy data. The authors draw on rather rigorous methods of refinement and validation of coordinates in X-ray crystallography to propose the use of central section (or particles) in cryoEM as experimental data rather the reconstructed map.\nI would like to state that Michael Rossmann (Purdue University) would have been an appropriate reviewer for this paper as he was thinking on the same lines for the last few years. Unfortunately, Michael passed away recently. Nevertheless, this is a very interesting idea and provides a direction to incorporate true experimental data rather than depending on reconstructed maps, which could potentially have their own problems.\n\nWhile I support indexing, I have a strong recommendation for the authors: the word \"model\" often does not translate well between crystallography and cryoEM. It could refer to coordinates in crystallography but in cryoEM, it could be the actual map or in later stages, the model of the atomic coordinates. Could the authors please ensure that they clarify this in the paper? On similar lines, \"cryo-EM refinement\" becomes ambiguous because it could refer to the reconstruction process (i.e., refinement of centers, orientation angles, defocus etc) or it could indicate the refinement of atomic coordinates against the map (or images as in this case). It would be extremely helpful to the readers if the authors could ensure that such confusion does not arise.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-665
https://f1000research.com/articles/8-2016/v1	28 Nov 19	{ "type": "Clinical Practice Article", "title": "Treatment of Buerger’s disease (Thromboangiitis obliterans) with autologous adipose tissue-derived mesenchymal stem cell: Report of three cases", "authors": [ "Sun Teak Jeong", "Jeong Chan Ra", "Sun Teak Jeong" ], "abstract": "Buerger’s disease or Thromboangiitis obliterans is an orphan vascular disease that most commonly affects nerves, small or medium-sized vessels in the upper and lower extremities, and is characterized by a non-atherosclerotic, segmental, inflammatory disorder. The etiology and the pathogenesis of the disease have not been fully elucidated. Although various interventions have been adopted recently, there is still no effective treatment for the prevention of the progression of the disease. This report presents three clinical cases that show the efficacies of autologous adipose tissue-derived mesenchymal stem cell (AdMSC) treatment in Buerger’s disease. Three male patients diagnosed with Buerger’s disease were between 46 and 55 years and had a smoking history. AdMSCs (5X106 cells/kg body weight) were injected intramuscularly into at least 38 points of the ischemic legion of the lower limb at one time. The patients were checked for safety and efficacy at one, three, and six months after AdMSC injection. No severe adverse events and no adverse drug events were observed in physical examination, vital signs, and laboratory tests for all three patients. Ulcers in the affected legs of the patients were healed completely after the treatment. Visual Analogue Scale scores and all the criteria (activities, emotional, pain, social, symptoms and total) of the King's College Hospital's Vascular Quality of Life Questionnaire (VascuQOL) of all the patients were improved from baseline to six months follow-up. Digital Infrared Thermal Imaging showed the gradual alleviation of lesions in the leg. Angiogenesis in the affected limbs was identified by CT-Angiography after AdMSC injection. The present cases show the improvement in patients with Buerger’s disease with the observation of angiogenesis after intramuscular injection of autologous AdMSCs. This suggests that autologous AdMSC can be an effective alternative treatment for Buerger’s disease.", "keywords": [ "Buerger’s disease", "autologous adipose tissue-derived mesenchymal stem cell", "intramuscular injection" ], "content": "Introduction\n\nBuerger’s disease or Thromboangiitis obliterans is an orphan vascular disease that most commonly affects small/medium-sized vessels and is characterized by a non-atherosclerotic, occlusive, thrombotic, segmental, inflammatory disorder in the upper and lower extremities1–3. The etiology is unknown, and the pathogenesis of the disease has not been fully elucidated. Typical Buerger’s disease patients are mostly men (<45 years) with a history of smoking, and exhibit various symptoms, such as progressive claudication, ischemic ulcers and rest pain, which are thought be due to peripheral vascular disorders. In severe cases, lesions in the affected limbs are exacerbated and lead to tissue death, and eventually amputation.\n\nCurrently, the cessation of smoking is known to be the most effective treatment, however, the management of smoking is considered difficult and cessation has been known to be insufficient for severe ischemic states in patients with Buerger’s disease2,4–6. There have been various treatments based on the symptoms and pathophysiology of Buerger’s disease, including medications for pain-relief, vascular inflammation, vasodilation or antiplatelet effect, and medical (surgical) intervention, such as sympathectomy, endovascular angioplasty and bypass surgery2. However, these are only palliative for symptoms and there is no treatment or interventions reported to be effective for the prevention of the progression of the disease. Recently, mesenchymal stem cells (MSCs) from various sources emerge as a promising alternative for Buerger’s disease treatment1,7. Angiogenesis is considered as a crucial condition to preserve the affected limbs and prohibit the aggravation of the disease, and previous studies report that MSCs has an angiogenic nature7.\n\nWe previously studied that the safety and the efficacy of adipose-derived mesenchymal stem cells (AdMSC) on the treatment of Buerger’s disease8. Here, we report three patients diagnosed with Buerger’s disease where treatment with AdMSCs brought about angiogenesis in the affected limbs.\n\n\nTreatment with AdMSCs\n\nAdministration of autologous AdMSCs for Buerger’s disease was approved by the Korean Ministry of Food and Drug Safety with Investigational New Drug Application for Emergency Use (Approval Nos. 20170072755, 20170072828, 20170072872). The protocol for administration of AdMSCs was conducted in compliance with the Helsinki declaration and approved by the Institutional Review Board of Bethesda Hospital, Yangsan, Korea (Approval No. 2016-6). Written informed consent to take part in the treatment was acquired from all patients before the initiation of treatment.\n\nThe three cases presented herein were male patients with smoking history (currently non-smoking) and aged between 46 and 55 years. All patients showed the corkscrew appearance or luminal obstruction of the medium-sized or smaller arteries by angiogram, and were diagnosed Buerger’s disease at least six months before the start of AdMSC treatment (Table 1). These patients were suggested for treatment with AdMSCs (between April 2017 and April 2018) as they were classified as Rutherford class III-59 by clinical description, experienced ischemic rest pain and ulcers, and showed the recurrence or no improvement after previous treatments. The baseline characteristics including previous treatment and medications are shown in Table 1.\n\nThe isolation and characterization of the autologous AdMSCs were performed using a previously established culture protocol8 under good manufacturing practice conditions in the Stem Cell Research Institute of R Bio (Seoul, Republic of Korea). Briefly, abdominal subcutaneous adipose tissue was obtained through liposuction three weeks before administration, and digested with collagenase I (Gibco/Life Technologies, Grand Island, NY, USA). After centrifugation, the pellet was resuspended in DMEM (Invitrogen, Carlsbad, CA, USA)-based media containing 0.2 mM ascorbic acid and 10% fetal bovine serum (FBS; JR Scientific, Woodland, CA, USA). The cell fraction was cultured overnight at 37°C/5% CO2, and cell adhesion was checked after 24 h. Cells were maintained for 4 to 5 days until confluent (passage 0). When the cells reached 90% confluency, they were subcultured to expand in keratinocyte SFM-based media (Invitrogen, USA) containing 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng/ml rEGF, and 5% FBS until passage 3. Before transporting the cells for administration, aliquots of the AdMSCs were tested for cell viability, fungal, bacterial, endotoxin, and mycoplasma contamination and immunophenotype for MSCs. Cell viability evaluated by trypan blue exclusion was >91%, and no evidence of bacterial, fungal and mycoplasma contamination was observed. The AdMSCs showed a homogenous population of cells with high positive marker expression levels of CD73 and CD90 at a high level of >92% and >99%, respectively. Negative markers of CD31, CD34, and CD45 were expressed at a very low level of <0.08%.\n\nSince most patients showed allodynia, intramuscular (IM) injections were carried out under spinal anesthesia. According to our previously reported protocols8, AdMSCs were prepared at a concentration of 1X107 cells/0.5 ml saline/syringe before IM injection. Finally adjusted AdMSCs were administered at the dose of 5X106 cells/kg (based on body weight of the patient) were injected into multiple sites (at least 38 points) of the ischemic zone of the lower extremities (the feet of the three patients) at one time. Before the IM injection, the ischemic legions on the affected limbs were identified by Digital Infrared Thermal Imaging (DITI). To assess safety and efficacy, all the patients were followed up at one, three, and six months after the IM injection.\n\nSafety was assessed during follow-up by looking at vital signs, physical examination, laboratory tests, adverse events, and serious adverse events as described in our previous reports8. For the evaluation of the efficacy, the following assessment were performed at every follow-up: Visual Analogue Scale (VAS) for rest pain, designated as 1 (best) to 10 (worst); King’s College Hospital’s Vascular Quality of Life Questionnaire (VascuQoL), consisting of 25 questions grouped into 5 domains (activity, emotional, pain, symptoms, social), for the disease-specific quality of life assessment10,11; assess ulcer size and wound healing; assess the risk of additional amputation; DITI for the identification of ischemic legions before and after the injection of AdMSC. Computed Tomography (CT)-Angiography was carried out for the evaluation of angiogenesis at baseline and the last follow-up.\n\n\nCase 1 (patient 001)\n\nA man, aged 48 years, whose onset of Buerger’s disease symptoms had begun 18 years ago, was admitted to the hospital and identified for treatment with AdMSCs. The patient had a smoking history from age of 24 to 30, and had stopped smoking after the diagnosis of Buerger’s disease. At the time of visiting the hospital for treatment with AdMSCs, the patient had partial amputation in the left 1, 2, 3, 4th toes and total amputation in the right 3rd toe, with no ulcers. The patient already had a history of angioplasty four times and received allogenic stem cell therapy, but the effect of this treatment disappeared two months after the treatment. The patients showed severe pain, allodynia, rest pain, claudication (30 m), and pains in the left thigh, both calves, both feet, and both hands. The patient was on the following medication, which was maintained during treatment with AdMSCs: Aspirin (100mg, qd), clopidogrel (75mg, qd), beraprost (0.06mg, tid), Vytorin (10mg, qd), Mypol (as codeine phosphate 20mg, qd) (summarized in Table 1, with other characteristics). The analgesics were decreased in dose (Mypol 20mg per three days) during the follow-up period as symptoms improved.\n\nNo severe adverse events and no adverse drug events were observed during follow-up after treatment with AdMSCs regarding physical examination (including ulcer size check, capillary refill test), vital signs (temperature, pulse, and blood pressure respiration), and laboratory tests (hematology, biochemistry, urinalysis). Vas and VascuQoL scores showed improvement one month after the treatment (Figure 1A, B). No additional ulcers were observed and no amputations were required. Rest pain and allodynia disappeared, and quality of sleep was improved as night pain disappeared during the follow-up period. Claudication was also improved from 30 m at baseline to 100 m at the final follow-up. DITI images in Figure 2 showed gradual alleviation in the affected lower limbs after three months from the treatment, and also improvement in the non-affected opposite limb six months after AdMSC injection. The persistence of this alleviation effect was identified by DITI images in the additional visiting the hospital one year after AdMSC injection. Angiogenesis in the affected limbs was identified by CT-Angiography after AdMSC injection. The formation of collateral arteries in the legions was newly observed in the non-injected right leg (Figure 3).\n\nChanges in Visual Analogue Scale (A) and the King’s College Hospital’s Vascular Quality of Life Questionnaire (VascuQoL) scores (B–D) of the individual patients from baseline to last follow-up (six months) after the injection of AdMSCs.\n\nDITI images of patient 001 showing the improvement of the left leg at baseline (A), six months (B), one year (C) after injection of AdMSCs.\n\nCT-angiography images of the left leg of patient 001 at baseline (A, C) and at six months (B, D) after injection of AdMSCs showed the increased numbers of collateral arteries (arrows). The arrowheads (B) indicated the newly formed collateral arteries in the right leg that was not injected.\n\n\nCase 2 (patient 002)\n\nA man, aged 55 years, was diagnosed with Buerger’s disease 10 years ago. The patient had 34 years of smoking history from the age of 20 to 54 and stopped smoking one year ago prior to treatment with AdMSCs. Both hands and legs were affected and amputations had been performed partially in both big toes and right 2nd toe. Ulcers were observed in the right big and 2nd toes. The patient had a history of hyperbaric oxygen therapy and no angioplasty. After the partial amputation of affected toes, the following medication was taken by the patient, and was continued during the follow-up period after AdMSC treatment: sarpogrelate (100mg, tid), ginkgo leaf extract (80mg, bid), aceclofenac (100mg, bid), pregabalin (75mg, bid), and ciprofloxacin (250mg, bid). There were pains in both calves, feet, and hands, and stiffness in the feet and lumbodynia after long-distance walking, but no signs in the thigh.\n\nNo severe adverse events and no adverse drug events were observed during follow-up after treatment with AdMSCs regarding physical examination(including ulcer size check, capillary refill test), vital signs (temperature, pulse, and blood pressure respiration), laboratory tests (hematology, biochemistry, urinalysis). Vas and VascuQoL scores showed improvement one month after the treatment (Figure 1A, C). Claudication was improved from 300 m at baseline to 600 m at the final follow-up. The ulcers on the right big and 2nd toes at baseline showed bone exposure and after six months exhibited a complete healed state with no additional ulcers observed and amputations required (Figure 4). This complete healed state was identified in the reinspection one year after AdMSC injection. Rest pain had disappeared, and most symptoms were improved, and all medications were stopped one month after treatment with AdMSCs. DITI images showed alleviation in the affected right limb one month after treatment and also showed improvement in the non-affected opposite limb three months after treatment. The improved state was maintained six months after treatment. Angiogenesis in the right limb was identified by CT-Angiography and the formation of new collateral arteries was observed in the right leg after AdMSC injection (Figure 5).\n\nThe healing process of a toe ulcer in patient 002. The patient’s right second and big toe showed ulcers and exposed bone on the second toe at baseline (A, B). At six months of AdMSC treatment (C, D), the big toe ulcer was completely healed and the second toe is shown just after the crust was removed during healing. The ulcer remained completely healed at one year after AdMSC treatment (E, F).\n\nCT-angiography images of the right leg of patient 002 at baseline (A, C) and at six months (B, D) after the injection of AdMSCs showed increased numbers of collateral arteries (arrows).\n\n\nCase 3 (patient 003)\n\nA man, aged 45 years, whose onset of Buerger’s disease symptoms had begun 19 years ago, was admitted to the hospital. The patient had 25 years of smoking history from the age of 20 to 45 and stopped smoking after angioplasty 8 months ago prior to the treatment with AdMSCs. Both legs were affected and amputations had been carried out on the right 4 and 5th toes. There were no ulcers at the time of treatment with AdMSCs. The patient had bypass graft on the right leg 5 years previous to treatment. The patient reported pain in both calves and right foot, allodynia, rest pain, claudication (50 m.), Raynaud’s symptom in both hands, and slow capillary filling in both fingers and toes. The patient was on the following medication which was maintained during treatment with AdMSCs: Cilostazol (200mgm qd), aspirin (100mg, qd), warfarin (5mg, qd), oxycodone (10mg, tid).\n\nNo severe adverse events and no adverse drug events were observed during follow-up after treatment with AdMSCs regarding physical examination (including ulcer size check, capillary refill test), vital signs (temperature, pulse, and blood pressure respiration), laboratory tests (hematology, biochemistry, urinalysis) during the study. Vas and VascuQoL scores showed improvement one month after treatment (Figure 1A, D). Rest pain, allodynia, and Raynaud’s symptoms disappeared and quality of sleep was improved as night pain disappeared during the follow-up period. Most of the symptoms were improved and the analgesics have decreased the dose (oxycodone 5 mg, tid) during the follow-up period as pains alleviated. Claudication was also improved from 50 m at baseline to 300 m at final follow-up. DITI images showed the gradual alleviation process in the affected lower limb three months after treatment, and also showed improvement in the non-affected opposite limb at the final follow-up (Figure 6). Angiogenesis in the affected left limb was identified by CT-Angiography after AdMSC injection. Newly formed collateral arteries in the injected lesions was observed in the non-injected right leg at the final follow-up (Figure 7).\n\nDITI images of patient 003 showing the improvement of the right leg at baseline (A), three months (B), six months (C) after injection of AdMSCs.\n\nCT-angiography images of the right leg of patient 003 at baseline (A, C) and six months (B, D) after the injection of AdMSCs showing thicker and more abundant arteries (arrows and arrowheads).\n\n\nDiscussion\n\nEtiology, e.g. genetics, and pathophysiology of Buerger’s disease still remain uncertain, with the exception of its high correlation with smoking1. There are no standard diagnostic criteria and no treatment guidelines or protocols for Buerger’s disease2. Treatment and assessment of its efficacy remain debatable for these reasons. However, recent studies and clinical trials have indicated that the restoration of angiogenesis is the key for alleviation of symptoms and the fundamental therapy for Buerger’s disease7.\n\nThe present cases reported the improvement of patients diagnosed with Buerger’s disease after the administration of AdMSCs. Ulcers present in some of the patients on affected limbs were completely healed, a major symptom of Buerger’s disease, and rest pain and claudication were alleviated. In addition, assessment of the patients using VAS scale and VascuQoL indicated treatment satisfaction without any adverse events. VascuQoL is vascular disease-specific, and is a reliable and validated assessment10–11. Along with our previous findings demonstrating the safety and functional improvement in the patients with Buerger’s disease8, the present cases suggest that AdMSCs are involved in modulating inflammation and pain.\n\nAngiogenesis in the ischemic limb in the present cases were identified by non-invasive CT-angiography after the injection of AdMSC, and corresponded with the results of the previous study using the AdMSC provided by our previous established protocols12. Moreover, angiogenesis was also found in the counterpart limb, which had not been injected. These findings demonstrate that focally injected AdMSCs could conduct systemic angiogenetic characteristics in patients with Buerger’s disease. The outcomes of the present clinical cases are considered as a result of the synergy between angiogenetic properties and anti-inflammatory/immunomodulatory action of AdMSC. These various functions of the MSCs are known to be exerted by paracrine actions with the release of extracellular vesicles, exosomes13–14. Previous studies report that AdMSCs secret soluble angiogenetic factors, such as vascular endothelial growth factor (VEGF), fibroblast grow factor-2 (FGF-2), interleukin-6 (IL-6)13–15. AdMSCs have been reported to show various advantages over MSCs from other sources, including a less invasive sampling procedure, higher cell numbers from tissue harvested, higher capacity for proliferation and higher capacity of angiogenesis13,14,16–18. The angiogenetic potential is essential for recovering damaged tissues14. Newly formed blood vessels are extremely helpful for the migration of versatile stem cells to affected lesions and for the transportation of various factors, which is vital for the regeneration of tissues and tissue function13,14.\n\nTaken together, these cases would suggest that AdMSCs have potential advantages for regenerative medicine, and especially AdMSCs may be promising alternatives in orphan disease or emergency cases, such as Buerger’s disease. However, the restricted numbers of patients presented here and the short period of follow-up limit the assessment of the long lasting angiogenetic potential of AdMSCs. Nonetheless, the present clinical cases show improvement and safety of IM injection of AdMSCs in patients with Buerger’s disease, leading to an alleviation of symptoms and observation of angiogenesis in the affected limbs. Further studies are needed for continuous follow-up to optimize the treatment protocol. A precise assessment of the efficacy of AdMSCs in larger clinical trials will also be needed.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of the patients’ clinical details and associated images was obtained from each patient.", "appendix": "References\n\nCacione DG, do Carmo Novaes F, Moreno DH: Stem cell therapy for treatment of thromboangiitis obliterans (Buerger's disease). Cochrane Database Syst Rev. 2018; 10: CD012794. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFazeli B, Dadgar Moghadam M, Niroumand S: How to Treat a Patient with Thromboangiitis Obliterans: A Systematic Review. Ann Vasc Surg. 2018; 49: 219–28. PubMed Abstract \| Publisher Full Text\n\nKlein-Weigel PF, Richter JG: Thromboangiitis obliterans (Buerger's disease). Vasa. 2014; 43(5): 337–46. PubMed Abstract \| Publisher Full Text\n\nModaghegh MS, Hafezi S: Endovascular Treatment of Thromboangiitis Obliterans (Buerger's Disease). Vasc Endovascular Surg. 2018; 52(2): 124–30. PubMed Abstract \| Publisher Full Text\n\nFazeli B, Rezaee SA: A review on thromboangiitis obliterans pathophysiology: thrombosis and angiitis, which is to blame? Vascular. 2011; 19(3): 141–53. PubMed Abstract \| Publisher Full Text\n\nFazeli B, Ravari H: Mechanisms of thrombosis, available treatments and management challenges presented by thromboangiitis obliterans. Curr Med Chem. 2015; 22(16): 1992–2001. PubMed Abstract \| Publisher Full Text\n\nZhao L, Johnson T, Liu D: Therapeutic angiogenesis of adipose-derived stem cells for ischemic diseases. Stem Cell Res Ther. 2017; 8(1): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRa JC, Jeong EC, Kang SK, et al.: A Prospective, Nonrandomized, no Placebo-Controlled, Phase I/II Clinical Trial Assessing the Safety and Efficacy of Intramuscular Injection of Autologous Adipose Tissue-Derived Mesenchymal Stem Cells in Patients With Severe Buerger's Disease. Cell Med. 2016; 9(3): 87–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRutherford RB, Baker JD, Ernst C, et al.: Recommended standards for reports dealing with lower extremity ischemia: revised version. J Vasc Surg. 1997; 26(3): 517–38. PubMed Abstract \| Publisher Full Text\n\nMazari F, Carradice D, Rahman M, et al.: An analysis of relationship between quality of life indices and clinical improvement following intervention in patients with intermittent claudication due to femoropopliteal disease. J Vasc Surg. 2010; 52(1): 77–84. PubMed Abstract \| Publisher Full Text\n\nMorgan M, Crayford T, Murrin B, et al.: Developing the Vascular Quality of Life Questionnaire: a new disease-specific quality of life measure for use in lower limb ischemia. J Vasc Surg. 2001; 33(4): 679–87. PubMed Abstract \| Publisher Full Text\n\nLee H, An S, Lee H, et al.: Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: a pilot study. Circ J. 2012; 76(7): 1750–60. PubMed Abstract \| Publisher Full Text\n\nDufrane D: Impact of Age on Human Adipose Stem Cells for Bone Tissue Engineering. Cell Transplant. 2017; 26(9): 1496–1504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJayaram P, Ikpeama U, Rothenberg J, et al.: Bone Marrow-Derived and Adipose-Derived Mesenchymal Stem Cell Therapy in Primary Knee Osteoarthritis: A Narrative Review. PM R. 2019; 11(2): 177–91. PubMed Abstract \| Publisher Full Text\n\nSalgado A, Reis R, Sousa N, et al.: Adipose tissue derived stem cells secretome: soluble factors and their roles in regenerative medicine. Curr Stem Cell Res Ther. 2010; 5(2): 103–10. PubMed Abstract \| Publisher Full Text\n\nLi C, Wu X, Tong J, et al.: Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy. Stem Cell Res Ther. 2015; 6: 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim Y, Kim H, Cho H, et al.: Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia. Cell Physiol Biochem. 2007; 20(6): 867–76. PubMed Abstract \| Publisher Full Text\n\nVériter S, André W, Aouassar N, et al.: Human Adipose-Derived Mesenchymal Stem Cells in Cell Therapy: Safety and Feasibility in Different \"Hospital Exemption\" Clinical Applications. PLoS One. 2015; 10(10): e0139566. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62806", "date": "15 May 2020", "name": "Pawan K. Gupta", "expertise": [ "Reviewer Expertise Hematology", "Stem cells and Regenerative Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments:\nOnce the MSCs are being isolated from the adipose tissue, the following details can be added: a. The passage number or doubling time of the MSCs. b. Time period of storage of the cells and the stability time of these cryopreserved cells. c. Any in vitro studies potency assay done for this indication for use of Adipose tissue-derived MSCs. d. Was Karyotyping analysis done as it is not mentioned in the text? e. Please mention the values of HLA DR during the release of the cells, if done.\n\nAll the patients enrolled in the study were labeled as Rutherford III - 5. As per the definition Rutherford III - 5 is defined as \"Minor tissue loss—nonhealing ulcer, focal gangrene with diffuse pedal ischemia\". But patient numbers 001 and 003 do not have ulcers at baseline. Please reconsider the grade of these patients.\n\nWas ABPI, ankle pressure or TcPO2 measured in these patients as they are very important indirect findings of increased oxygenation of tissues.\n\nCase number 002, the hands were also involved. Whether any injections of MSCs were given in the upper limbs also.\n\nIn the discussion section more can be discussed on the advantage of CT anigiography on MRA or DSA as CT angiography was done in the three patients.\n\nNo new data is coming out from this small study of three patients as it is now a well-known fact that stem cells improved angiogenesis in these patients.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "5636", "date": "26 Jun 2020", "name": "Jeong Chan Ra", "role": "Author Response", "response": "First of all, I'm very sorry for the late reply and Thank you for your careful reading and kind comments on our manuscripts. 1. Once the MSCs are being isolated from the adipose tissue, the following details can be added:a. The passage number or doubling time of the MSCs.b. Time period of storage of the cells and the stability time of these cryopreserved cells.c. Any in vitro studies potency assay done for this indication for use of Adipose tissue-derived MSCs.d. Was Karyotyping analysis done as it is not mentioned in the text?e. Please mention the values of HLA DR during the release of the cells, if done. Because the present study was carried out for Emergency use, not for the formal clinical trials, it was not needed to include the analysis of karyotyping and HLA DR. So, the karyotyping analysis and HLA-DR analysis was not performed. Also, for emergency use, the fresh AdMSC was injected within hours after testing as written in the text and the passage number was described in the text. The AdMSC in the present study was routinely manufactured by previously established culture protocol under good manufacturing practice conditions(https://pubmed.ncbi.nlm.nih.gov/24449146/ , https://pubmed.ncbi.nlm.nih.gov/28713639/ ) There have been considerable reports on the in vitro angiogenetic potency of adipose tissue-derived mesenchymal stem cells. The angiogenetic property of AdMSC has been well-known and has been suggested as one of the underlying cure mechanisms for various ischemic diseases in many previous studies including the present study, which is basically a clinical case report.The present study was dealt with the emergency use of AdMSC under the approval of related government ministry, and there was no need to carry out additional in vitro potency assay of AdMSC. 2. All the patients enrolled in the study were labeled as Rutherford III - 5. As per the definition Rutherford III - 5 is defined as \"Minor tissue loss—nonhealing ulcer, focal gangrene with diffuse pedal ischemia\". But patient numbers 001 and 003 do not have ulcers at baseline. Please reconsider the grade of these patients. Rutherford classification deals with both chronic and acute limb ischemia. The enrolled patients in the present study were not acute cases but chronic cases with no improvement after previous treatment in other hospitals for over 6 months. According to Rutherford classification, the patients with only pain could be grouped into the grade 0-2 (category 0-4), and the patients starting to show the wounds could be grouped into grade 3(category 5, 6). The patients 001 & 003 had already toe amputation and had shown the continuous aggravating process at the time of the recruitment for the present study. Considering that Buerger’s disease is a progressive and worsening disease rather than healing, it could be assumed that the patients 001 & 003 had the amputations as a result of the progression of low ankle/toe pressure and worsening of toe circulation. Even if the toe amputation was carried out due to necrosis, it could not mean that ABI and AP/TP showed improvement after the treatment. Thus, it is difficult to say that the patients diagnosed with gangrene and ulcer at first diagnosis which requires the amputation had improved in AP/TP and in the Rutherford classification after the amputation. In other words, considering that the amputation is a common solution for non-healing ulcers in the therapeutic strategy in the Buerger’s disease, it could be said that the patients with previous amputations and severe ischemic rest pain at the time of enrollment are relevant to Rutherford III-5 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232437/table/TB00869-2/).As referred to Hardman et al. (Semin Intervent Radiol. 2014; 31(4): 378–388. Overview of Classification Systems in Peripheral Artery Disease), Rutherford did not include the strict temporal division in the definition. So, it can say that there is no clear temporal distinction on the occurrence of the ulcer at the time of diagnosis. 3. Was ABPI, ankle pressure or TcPO2 measured in these patients as they are very important indirect findings of increased oxygenation of tissues. Ankle (ABI) or toe (TBI) brachial indices were not applied as ABI cannot predict walking distance and has difficulty accurately describing the disease state (VASA Zeitschrift fur Gefasskrankheiten. 2002;31(2):107-10. Leder et al. Exercise capacity and Doppler pressure measurements in symptomatic peripheral arterial obstructive disease). Moreover, these were not considered as efficacy measures because of measurement difficulties owing to wounds or the lack of toes in some patients. TcPO2 was not measured because we do not have the device. 4. Case number 002, the hands were also involved. Whether any injections of MSCs were given in the upper limbs also. As an extension of our previous report (Cell Med. 2017;9(3):87-102, Ra et al., Prospective, Nonrandomized, no Placebo-Controlled, Phase I/II Clinical Trial Assessing the Safety and Efficacy of Intramuscular Injection of Autologous Adipose Tissue-Derived Mesenchymal Stem Cells in Patients With Severe Buerger's Disease), the present study was carried out under the approval of Korean Ministry of Food and Drug Safety with Investigational New Drug Application for emergency Use. As required by the KMFDS, the administration of AdMSC injection was carried out once in the affected lower limb, not the upper lime, the same as the previous report. 5. In the discussion section more can be discussed on the advantage of CT angiography on MRA or DSA as CT angiography was done in the three patients. Although angiography is considered to be the most accurate method for precise identification of the vascular condition, this method is invasive, difficult to perform, and cumbersome and was determined to be inappropriate for the comparison before and after stem cell administration. However, as the previous patient status check was performed using angiography, it was possible to identify the existing disease state. Compared to conventional angiography, CT-angiography has a disadvantage with regard to accuracy and locating small vessels but provided good results of 99% sensitivity, 98% specificity, and 98% accuracy in a comparative study in patients with critical limb ischemia (Clinical radiology. 2011;66(10):945-52, Fotiadis et al., 64-section CT angiography in patients with critical limb ischemia and severe claudication: comparison with digital subtractive angiography), and thus was deemed suitable for noninvasive examination in the present study. Nevertheless, as conventional CT angiography constitutes a 3D reconstruction of the cephalic to a caudal axis, it is difficult to compare the axial image in the case of a 90° vertical axis on the vertical axis of the human body, such as the foot, indicating the need to improve the imaging and reconstruction methods 6. No new data is coming out from this small study of three patients as it is now a well-known fact that stem cells improved angiogenesis in these patients. Buerger's disease is a rare disease to have difficulty recruiting enough patients for collecting clinical data which could be analyzed for research. The patients in the present report visited the hospital for the treatment of the aggravated condition and enrolled in the case study. We tried the single focal injection of autologous AdMSC to the patients with almost incurable symptoms and identified the systemic angiogenesis in the improvement status of the patients. The present study is a clinical practice article consisting of 3 clinical case reports according to the submission guideline of F100research, rather than a novel research article for investigation. We hope that our study would be promising opportunities to adapt to AdMSC therapy for the treatment of rare diseases" } ] }, { "id": "78688", "date": "16 Feb 2021", "name": "Kenji Yanishi", "expertise": [ "Reviewer Expertise Therapeutic angiogenesis", "Chronic limb threatening ischemia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for the opportunity of paper review. Cell therapy using autologous adipose tissue-derived mesenchymal stem cells (AdMSCs) is expected as a new therapy for patients with no-option chronic limb-threatening ischemia (CLTI). In addition, it is very interesting that this manuscript shows that cell therapy using AdMSCs tended to be effective. However, I have some concerns below follows to accept these results.\n\nThe number of patients in this study is small. So, it is difficult to judge the efficacy of this cell therapy in patients with CLTI based on these results.\n\nThermography imaging technics may be effective for screening of severe ischemic limb, but this finding is insufficient as a clinical endpoint compared with skin perfusion pressure or transcutaneous oxygen tension.\n\nIt is interesting to use CT-angiography to evaluate the peripheral angiogenesis after this cell therapy. However, the protocol of the CT-angiography is unclear in three cases. Did authors undergo CT-angiography under uniform conditions before and after this cell therapy? In addition, it is unclear where is the CT value cut-off set. Authors should describe the protocol of CT-angiography.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [ { "c_id": "6852", "date": "08 Jul 2021", "name": "Jeong Chan Ra", "role": "Author Response", "response": "First of all, I am very sorry for the too late reply to your kind comments, and Thank you for your careful reading and considerate comments on our manuscripts. I hope our late reply hasn’t caused any inconvenience to you 1. The number of patients in this study is small. So, it is difficult to judge the efficacy of this cell therapy in patients with CLTI based on these results. I would like to mention that this clinical study is not written as explorative research nor designed clinical trials, but a case report reporting the treatment result of 3 emergency cases with the approval of the Korean Ministry of Food and Drug Safety for Emergency Use. The condition for the approval of the KMFDS for Emergency Use is restrictive. In these cases, the number of enrolled patients was small, because they were to be satisfied with several conditions, such as the term after diagnosed with TAO (over 6 months) and showing the recurrence or no improvement after previous treatments. The present study is a case report on the improvement status of the patients with TAO, a rare disease having difficulty recruiting enough patients for collecting clinical data. 2. Thermography imaging technics may be effective for screening of severe ischemic limb, but this finding is insufficient as a clinical endpoint compared with skin perfusion pressure or transcutaneous oxygen tension. Yes, ABI(ankle-brachial index) or TBI(toe brachial index) have been used in many CLTI studies. However, the ABI, or ankle pressure may not predict a walking distance or reflect the status of the disease (PMID: 12099140). In the present study, the patients were not applied ABI or TBI for the difficulty of the adequate comparison; the patients with partial amputation of the big toe or with complete amputation of the 4th and 5th toes could not be considered for measuring TP (toe pressure). The hospital to which the enrolled patients were referred had not equipped TcPO2 (Transcutaneous oximetry) and the measuring TcPO2 was not considered. Of course, there may be lots of difficulties to apply DITI (Digital Infrared Thermal Imaging) as an assessment standard for clinical status. DITI instrument is set up by the relative temperature, not absolute temperature, and, even in the identical individual, the coloring information from DITI usually reflects intrinsic & extrinsic variables, which may influence the blood circulation, such as coffee, nicotine, seasonal effect, use of vasodilator, or patients’ exercise status. However, DITI has the advantage to detect and comparing abnormal thermal distribution by measuring thermal symmetry (PMID: 3418388). DITI easily allows to assessing thermal difference and thermal change by comparing affected and unaffected limbs, or by systemic thermal distribution before and after administration, so allows determining improvement or aggravation of the patients’ status. Thus, though DITI may not be unsuitable for measuring the quantitative comparison of the thermal change, it can be used for evaluating the patients’ status, progressing to whether improvement or worsen by the comparison of the change in the legions before and after administration of AdMSC. 3. It is interesting to use CT-angiography to evaluate peripheral angiogenesis after this cell therapy. However, the protocol of the CT-angiography is unclear in three cases. Did authors undergo CT-angiography under uniform conditions before and after this cell therapy? In addition, it is unclear where is the CT value cut-off set. The authors should describe the protocol of CT-angiography. DSA (digital subtraction angiography) would be an effective tool for comparing or predicting the prognosis of TAO patients. However, DSA is known for an invasive procedure and might burden patients who already suffering from angiopathy. CT-angiography was determined to be adapted for the patients in the present case report for alleviating the burden of the patients and with reference that CT-angiography could show the adjacent results to DSA data (PMID: 21658691). In addition to the comparison of the thermal distribution, the improvement status of the patients after the treatment was checked by the findings of the newly formed collateral arteries. The neogenesis of collateral arteries was macroscopically identified using the anatomical landmarks in the images which were taken in the same condition (the same regions, the same radio-intensity, and non-manipulated contrasts) and the same protocol before and after administration. The present emergency case estimated the efficacy of the administration of AdMSC by the findings of the newly formed collateral arteries after the treatment, not by its quantitative comparisons with the statistical meaning, as in the efficacy test of the clinical trials or research articles, which could be assessed by the Cut-off set or the Hounsfield unit (HU) scale. Again, I would like to comment that the present case study reported the improvement status of 3 TAO cases based on the clinical observations from the point of view of a clinician for emergency patients. Of course, further studies should be needed with an intensive evaluation of the protocol assessment on larger scales." } ] } ]	1	https://f1000research.com/articles/8-2016
https://f1000research.com/articles/8-1728/v1	08 Oct 19	{ "type": "Systematic Review", "title": "Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review", "authors": [ "Goran Medic", "Melodi Kosaner Kließ", "Louis Atallah", "Jochen Weichert", "Saswat Panda", "Maarten Postma", "Amer EL-Kerdi", "Melodi Kosaner Kließ", "Louis Atallah", "Jochen Weichert", "Saswat Panda", "Maarten Postma", "Amer EL-Kerdi" ], "abstract": "Background: Clinical decision support (CDS) systems have emerged as tools providing intelligent decision making to address challenges of critical care. CDS systems can be based on existing guidelines or best practices; and can also utilize machine learning to provide a diagnosis, recommendation, or therapy course. Methods: This research aimed to identify evidence-based study designs and outcome measures to determine the clinical effectiveness of clinical decision support systems in the detection and prediction of hemodynamic instability, respiratory distress, and infection within critical care settings. PubMed, ClinicalTrials.gov and Cochrane Database of Systematic Reviews were systematically searched to identify primary research published in English between 2013 and 2018. Studies conducted in the USA, Canada, UK, Germany and France with more than 10 participants per arm were included. Results: In studies on hemodynamic instability, the prediction and management of septic shock were the most researched topics followed by the early prediction of heart failure. For respiratory distress, the most popular topics were pneumonia detection and prediction followed by pulmonary embolisms. Given the importance of imaging and clinical notes, this area combined Machine Learning with image analysis and natural language processing. In studies on infection, the most researched areas were the detection, prediction, and management of sepsis, surgical site infections, as well as acute kidney injury. Overall, a variety of Machine Learning algorithms were utilized frequently, particularly support vector machines, boosting techniques, random forest classifiers and neural networks. Sensitivity, specificity, and ROC AUC were the most frequently reported performance measures. Conclusion: This review showed an increasing use of Machine Learning for CDS in all three areas. Large datasets are required for training these algorithms; making it imperative to appropriately address, challenges such as class imbalance, correct labelling of data and missing data. Recommendations are formulated for the development and successful adoption of CDS systems.", "keywords": [ "sepsis", "hemodynamic instability", "respiratory distress", "infection", "machine learning", "clinical trials", "critical care." ], "content": "Introduction\n\nCritical care, including intensive and emergency care, is the most expensive and human resource intensive area of in-hospital care. Despite having the most technologically advanced devices, it is the area associated with the highest morbidity and mortality rates1. Decision-making for clinical teams in this area is complex due to variability in procedures and data-overload from the plethora of existing devices. In fact, misdiagnosis in the intensive care unit (ICU) is 50% more common than other areas2, and errors, especially medication errors which account for 78% of serious medication errors3, can have a long lasting effect even after patients are discharged.\n\nComputerized decision support (CDS) systems have emerged as tools providing intelligent decision making based on patient data to address many of the challenges of critical care. CDS systems can be based on existing guidelines or best practices; and can also utilize machine learning as a means of compiling several data inputs to provide a diagnosis, recommendation, or therapy course. CDS systems can improve medication safety by providing recommendations relating to dosing4–6, administration frequencies5, medication discontinuation6 and medication avoidance5. Moreover, these novel systems can improve the quality of prescribing decisions by triggering alerts or warning messages on drug duplication, contraindications, drug interaction errors7, side-effects and inappropriate medication orders5. CDS system notifications can be applied during the prescribing, administering or monitoring stages to detect and prevent medication errors8. These systems can also target patients to facilitate shared decision-making to empower as well as to motivate them9–11. The need for such systems stems from hospitals having to deal with strict guidelines to improve outcomes, document care cycles (raising the need for administrative tasks) and reduce readmissions. This is combined with the need to cope with financial constraints, such as staff shortages and increased pressure to reduce the length of stay12,13.\n\nStrategies for bringing CDS to clinics have been the topic of several workshops, conferences and focus groups14. Factors for success in designing CDS include providing measurable value, producing actionable insights, delivering information to the user at the right time, and demonstrating good usability principles14.\n\nEarly warning systems (EWS) are CDS systems designed for initial assessment and identification of patients at risk of deterioration in in-patient ward areas15–17. These systems have shown that they can enable caregivers and rapid response teams to respond earlier – in time to make a difference18. By alerting clinicians to higher risk patients, treatments can be administered early or harmful medications can be stopped, potentially leading to improved outcomes. Early recognition and timely intervention are also critical steps for the successful management of shock19, cardiorespiratory instability20 and severe sepsis. In sepsis management, adequate timing of administration of antibiotics is directly associated with survival rates21, and incidence, severity and duration of infections.\n\nAccording to the Society of Critical Care Medicine (SCCM)22, the five primary ICU admission diagnoses for adults are respiratory insufficiency/failure with ventilator support, acute myocardial infarction, intracranial hemorrhage or cerebral infarction, percutaneous cardiovascular procedures, and septicemia or severe sepsis without mechanical ventilation. SCCM also highlights other conditions involving high ICU demand such as poisoning and toxic effects of drugs, pulmonary edema and respiratory failure, heart failure and shock, cardiac arrhythmia and renal failure. Given the above, three high-impact areas were selected for the current research where early detection and treatment could impact outcomes for patients in the ICU. The first is that of hemodynamic instability, where early detection could help patients prevent deterioration into shock. The second is that of respiratory distress, affecting many ventilated patients (up to 40% are ventilated according to SCCM)22. The third area selected is that of infection, with a focus on sepsis. Sepsis is the most common cause of death among critically ill patients, with occurrence rates varying from 13.6% to 39.3%23,24. All three areas are major areas of concern with relatively high prevalence in critical care having long term effects on patients.\n\nThe study focuses on both detection, which alerts the clinician to the presence of these specific conditions, as well as prediction of deterioration by alerting the clinician in advance that a patient will deteriorate into one of these disease states. The aims of this study were to perform and report a systematic review of the utilization of CDS systems in the three selected disease areas and summarize the methodological aspects of identified studies.\n\n\nMethods\n\nA systematic literature review was carried out to identify evidence-based study designs, methods and outcome measures that have been used to determine the clinical effectiveness of CDS systems in the detection and prediction of three populations representing the variety and majority of morbid conditions in a critical care setting: Shock (hemodynamic (in-)stability), respiratory distress/failure and infection/sepsis. The search strategy combined ‘intervention terms’ and ‘disease terms’ to identify primary research evaluating the diagnostic performance of CDS systems and other machine learning algorithms in three different populations of any age, sex, and race. Systematic literature reviews were also included for locating further relevant primary research. The search was conducted in MEDLINE (PubMed), ClinicalTrials.gov and Cochrane Database of Systematic Reviews (CDSR); and limited to studies published or registered between January 1, 2013 and November 8, 2018 and reported in English. Publication dates were limited to focus results on the most recent developments in this fast-evolving research domain. The strategy employed in PubMed is provided as Extended data, Table 1–Table 325–27.\n\nStudies conducted in US, Canada, UK, Germany or France with more than 10 subjects per arm were included. These countries were selected because they are known to be active in CDS development. The inclusion and exclusion criteria for selecting abstracts and subsequent full-text publications were based on the population, interventions, comparators, outcomes, and study design (PICOS). These criteria are listed in Table 1.\n\n* Systematic Literature Reviews and (network) meta-analysis are excluded from data extraction since the pooled results cannot be used in our analysis. However, good quality (network) meta-analysis and systematic literature reviews (i.e. Cochrane reviews) will be used for cross-checking of references if the search did not omit any articles.\n\n If studies are conducted in multiple countries and at least 1 of the included countries is included – the study will be included in the selection.\n\n* Mathematical and logistic regression models – can be used to validate and evaluate Interventions of interest (that are listed as included intervention), but the texts discussing these models without any “learning potential” or artificial intelligence potential will be excluded. Therefore, these models can be the foundation of the included listed interventions but will not be included in the Data Extraction Files unless they have also machine learning or artificial intelligence or some other form of “learning potential” on top of the statistical mathematical model. Researchers will pay special attention and caution when screening these abstracts and/or full-text articles.\n\nAUC = Area under the curve; ED = Emergency department; ELISA = Enzyme-linked immunosorbent assay; HR = Hazard ratio; ICU = Intensive care unit; IQR = interquartile range; NPV = Negative predictive value; OR = Odds ratio; PPV = Positive predictive value; RCT = Randomized controlled trial; ROC = Receiver Operating Characteristic; SD = Standard deviation; SE = Standard error; UK = United Kingdom; US = United States.\n\nStudy selection and data extraction was carried out by a single reviewer (MKK or SP). In cases of uncertainty, a second, or even third reviewer, was consulted. Data extraction was performed using a standard data extraction form (DEF). Key data from each additional eligible study were extracted by recording data from original reports into the DEF. The DEF included information on study design, inclusion/exclusion criteria, sample size and characteristics, interventions, outcome measures (measures of predictability like: sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), likelihood ratio, accuracy, odds ratio (OR), hazard ratio (HR), median, receiver operating characteristic (ROC) area under the curve (AUC); and length of hospitalization among others).\n\nStudies identified from the ClinicalTrials.gov registry that did not report results were also included in the extraction to give some indication of the outcomes being collected.\n\nThis research was not aimed at summarizing study results and assessing the relative effectiveness of CDS systems. Therefore, an appraisal of study quality was not deemed necessary.\n\n\nResults\n\nThe search yielded 1588 hits. Screening the titles and abstracts led to 1502 being excluded. The full texts of the remaining 86 titles were obtained and assessed against the PICOS criteria. Studies were excluded due to irrelevant study design (n=22), population (n=1), intervention (n=5), and outcomes (n=38). A total of 20 studies were finally included in this systematic literature review. This included 5 trials identified from ClinicalTrials.gov. The study selection process is depicted in Figure 1.\n\nPop. = Population.\n\nStudy characteristics. Of the 15 published studies, five were conducted by research groups outside the USA28–32. Ten studies were conducted in the US19,33–41, Thirteen studies were retrospective19,28–33,35,37–41 and only two were prospective34,36. Nine studies were single-center28,30,31,33,37–41 and six studies were multi-center19,29,32,34–36. Five studies were time-series28,30–32,40 and nine were case-series19,29,33–35,37–39,41.\n\nAcross all studies, three had sample sizes ≤10029,30,36; three had sample sizes of 101-100028,31,32; four studies had sample sizes of 1001-10,00019,33,34,37,42; and another five studies, four retrospective single-center studies and one multi-center, had sample sizes larger than 10,00035,38–41. The three largest studies included patients admitted to various wards of a specified hospital. The majority of the studies did not restrict their sample to a specific in-patient hospital setting. Five studies reported on patients in the ICU19,28,32,40,41 and one study reported on patients admitted to the surgical ward33.\n\nThe characteristics of the published studies are summarized in Table 2.\n\nUSA: United States of America. UK: United Kingdom. NR: Not reported. ICU: Intensive care unit. EHR: Electronic health records.\n\nCDS systems. Machine learning algorithms were developed to detect or predict septic shock28,33,35,40,41, various heart arrhythmias29,30,34, heart failure37–39, hemodynamic instability and hypovolemia19,36, myocardial infarction31, as well as hypotension32.\n\nAll studies, except one, trained a single algorithm. Ebrahimzadeh et al. 201830 trained and compared support vector machine (SVM), instance-based and neural network models to predict paroxysmal atrial fibrillation. SVMs were the most frequently used algorithms, followed by least absolute shrinkage and selection operator (LASSO) regularization. In one study, the SVM was trained using sequential minimal optimization37. An overview of the investigated machine learning algorithms is presented in Table 3.\n\nCHMM: clustered hidden Markov model. LR: Logistic regression. SVM: Support vector machine. kNN: k nearest neighbor. RF: Random forest. Conv.: Convolutional.\n\nOutcome measures. Three of the 15 papers measured a single outcome of model performance. In two studies the preferred measure was accuracy28,34; whereas in another study this was the ROC AUC. This study was large and based their algorithm on EHRs33. Across all studies, accuracy was reported in about half of the instances and the ROC AUC was one of the most frequently reported outcomes.\n\nSensitivity and specificity were reported together in 10 studies. Blecker et al. 201638 reported sensitivity together with PPV. Sensitivity and specificity were not measured in the study by Sideris et al. 201637, instead model accuracy and the ROC AUC were preferred. This study was concerned with developing an alternative `comorbidity` framework based on disease and symptom diagnostic codes to cluster individuals at low to high risk of developing chronic heart failure.\n\nPPVs were reported in six studies and accompanied with negative predictive values in two studies. These studies developed and validated machine-learning algorithms for the early detection of less investigated health conditions, these being hemodynamic instability in children19 and acute decompensated heart failure39. The highest number of outcome measures, including likelihood ratios, was observed in Calvert et al. 201640 who investigated an under-represented population of patients with Alcohol Use Disorder.\n\nThe outcomes measured are summarized in Table 4.\n\nNPV: Negative predictive value. PPV: Positive predictive value. LR: Likelihood ratio. OR: Odds ratio. RR: Risk ratio. ROC AUC: Receiver operating characteristic area under the curve.\n\nOngoing studies. Five studies are currently ongoing, one in Germany43 and the others in the USA44–47. Two studies are prospective case series44,47, two studies are prospective cohort studies43,45 and one is a RCT46. Two of the studies are concerned with developing prediction models, and the others are concerned with implementing machine learning algorithms into clinical practice as early warning systems.\n\nThe details of these trials are summarized in Table 5.\n\nUSA: United States of America. NR: Not reported. ED: Emergency department. ICU: Intensive care unit. GI: Gastroenterology.\n\nThe search yielded 1279 hits. Screening the titles and abstracts lead to 1142 being excluded. The full texts of the remaining 137 titles were obtained and assessed against the PICOS criteria. Studies were excluded due to irrelevant study design (n=42), population (n=6); intervention (n=18) and outcomes (n=47), and conference proceeding from before 2017 (n=2). A total of 22 studies were finally included in this systematic literature review. None of the trials retrieved from ClinicalTrials.gov were included. The study selection process is depicted in Figure 2.\n\nPop. = Population.\n\nStudy characteristics. Of the included studies, 17 were conducted in the US33,48–63. Five studies were conducted outside the US; two in Canada64,65 by the same research group, two in France66,67 and one in the UK68. In total, 17 studies were retrospective33,48–50,52–55,58–66 and five were prospective51,56,57,67,68. Of these studies, 12 were single-center33,48,49,51,52,54,55,58,59,64–66 and 10 studies were multi-center50,53,56,57,60–63,67,68. Five studies were time-series48,52,55,56,64, 14 studies were case-series33,49,51,53,54,57–62,65,66,68, one was case-control50 and one was case/time series study63.\n\nThe smallest sample of 100 patients came from two single-center retrospective studies48,66. Ten studies had sample sizes of 101–100033,49–53,57,63,67,68; seven studies had sample sizes of 1001–10,00054,55,59,60,62,64,65; and three had sample sizes larger than 10,00056,58,61. The largest study included more than 50,000 patients admitted to the ED of two centers over a 3-year period61. Several published studies did not report their in-patient setting. When reported, some evaluated data from different wards56,59,64,65,68, and some included patients admitted only to the ED53,54,61,63, the ICU48,60,67 and the surgical ward33,51,55.\n\nThe characteristics of all published studies are given in Table 6.\n\nNA: Not applicable. NR: Not reported. USA: United States of America. COPD: Chronic obstructive pulmonary disease. ECLIPSE: Evaluations of COPD Longitudinally to Identify Predictive Surrogate Endpoints. UK: United Kingdom. CABG: Coronary artery bypass grafting. PCI: Percutaneous coronary intervention. ICD: Implantable cardioverter defibrillator. ICU: Intensive care unit. ED: Emergency department.\n\nCDS systems. About half of the studies developed machine-learning algorithms, whereas the other half focused on natural language processing (NLP) algorithms. One study differed from the rest by developing a computer-aided detection (CAD) system to measure the axial diameter of the right and left pulmonary ventricles, aiding in the diagnosis of pulmonary embolisms49. Many learning algorithms were concerned with detecting pulmonary embolisms and deep vein thrombosis53,54,58,59,64–67 as well as pneumonia33,48,57,60–63. Three studies developed machine-learning algorithms to detect COPD50,56,69. One study developed a machine learning algorithm to detect acute respiratory distress syndrome52; while other studies developed machine learning algorithms to detect respiratory distress or failure following a pressure support ventilation trial67, cardiovascular surgery55 and pediatric tonsillectomy51.\n\nThe classifiers used in the NLP-based studies were various. However, some commonalities emerged between the studies developing machine-learning algorithms. Multiple studies applied SVM, logistic regression, random forests, K- nearest neighbor (kNN), gradient boosting and neural network models. Various classifiers were explored in 5 studies. An overview of the developed learning algorithms is provided in Table 7.\n\n*A computer aided detection system was developed for measuring the right ventricular/left ventricular axial diameter ratio and detecting pulmonary embolism.\n\nOne study, Reamoroon et al. 201852, used a novel sampling technique to accommodate for inter-dependency in longitudinal data. Model accuracy and ROC AUC with this method was <5% better than random sampling and 4–11% better than no sampling.\n\nOutcome measures. The majority of the studies reported multiple outcome measures of model performance. The most frequently reported outcome measure was sensitivity, followed by specificity and ROC AUC. Likelihood ratios, on the other hand, were only reported in one study: Silva et al. 201767 reported eight outcome measures of their novel machine learning model to predict post extubation distress. The outcomes measured across all studies are summarized in Table 8.\n\nNLP: Natural language processing. ML: Machine learning. CAD: Computer aided detection. NPV: Negative predictive value. PPV: Positive predictive value. LR: Likelihood ratio. OR: Odds ratio. RR: Risk ratio. ROC AUC: Receiver operating characteristic area under the curve.\n\nMany of the studies that developed NLP-based algorithms reported negative and positive predictive values, as well as sensitivity and specificity. In contrast, the ROC AUC was the most frequently reported outcome measure of machine learning algorithm performance. It was also the single preferred outcome in three studies33,50,55. About half of the studies additionally reported sensitivity, specificity, and accuracy. One study reported specificity with sensitivity set at 90% and 95% to ensure that few disease positive cases were missed52. The single study that developed a CAD system measured the ROC AUC and model accuracy49.\n\nThe search yielded 2659 hits. Screening the titles and abstracts lead to 2562 being excluded. The full texts of the remaining 97 titles were obtained and assessed against the PICOS criteria. Studies were excluded due to irrelevant study design (n=41), population (n=4); intervention (n=6) and outcomes (n=14). A total of 31 studies were finally included in this systematic literature review. Four of these were ongoing trials. The study selection process is depicted in Figure 3.\n\nPop. = Population.\n\nStudy characteristics. Of the included studies, 24 were conducted in the US. Three studies were conducted outside the US; one in France; one in the Netherlands and one in the UK. In total, 21 studies were retrospective33,35,70–88 and six were prospective89–94. There were 21 single-center studies33,70–75,77–83,86–88,90–92,94 and six multi-center studies35,76,84,85,89,93. Seven studies were time series71,78,82,84–86,92, 18 studies were case series33,35,70,72–76,80,81,83,87–91,93,94, one was a case-control77 and one was a matched-controlled study79.\n\nThe smallest studies included patients with leukemia89 and combat casualty patients90. Four studies had a sample size below 100070,72,73,79, three had a sample size between 1001–10,00033,71,87 and 12 had a sample size larger than 10,00035,74,77–78,80–82,84–87,88. Eight studies had samples even larger than 50,00035,74,77,78,82,84,85,88. Large samples were achieved by less restrictive inclusion criteria where all patients admitted to specific ward(s) or hospital(s) over a given time were defined.\n\nMajority of the published studies evaluated data from different wards; several studies included patients admitted only to the ICU70,72,81,84–86,93 and surgical ward73,76,78,87,91,92, less often the General ward33 and Emergency Department74. Of these, 23 studies included data collected at their own hospital; and four utilized previously collated databases76,81,84,86.\n\nThe characteristics of all published studies are given in Table 9.\n\nNA: Not applicable. NR: Not reported. USA: United States of America. UK: United Kingdom. ICU: Intensive care unit. ED: Emergency department.\n\nCDS systems. The machine learning algorithms evaluated in the studies were developed to predict a range of diseases. These included sepsis33,35,72,78,81,85,93,94, acute kidney injury70,78–80,82,84,91, surgical site infections33,73,76,87,92, central line-associated bloodstream infections77,86, Clostridium difficile83,88, pulmonary aspergillosis89, bacteremia90, fibrosis71, urine tract infection33,74 and infections in general75.\n\nAlmost half of the studies compared different machine learning algorithms, while the others focused only on Bayesian algorithms73,92, decision tree algorithms84, ensemble algorithms35,71,82,83,90,93, regression algorithms33,78,85, regularization algorithms81,88 and rule learning70. The most frequently applied model was random forest (15 studies) followed by logistic regression (10 studies), support vector machines (5 studies), naïve Bayes (5 studies) and gradient tree boosting (5 studies).\n\nOne study compared three different sampling methods for handling class imbalance; under-sampling the majority class (RANDu), over-sampling the minority class (RANDo) and synthetic minority over-sampling (SMOTE). This was a very large study including more than 500,000 patients to predict the onset of infections75. The authors found that SMOTE outperformed the other techniques and improved model sensitivity. Two other very large studies used the RANDu method80 and mini-batch stochastic gradient descent with backpropagation85. No other studies were concerned with imbalance in disease positive and negative classification.\n\nMachine learning models were developed and validated in 26 studies and subsequently tested in an independent dataset in four studies35,72,75,77.\n\nThe machine learning algorithms used are illustrated in Table 10.\n\nAKI: Acute kidney injury. SSI: Surgical site infection. UTI: Urinary tract infections. CLABSI: Central line-associated bloodstream infections. NB: Naive Bayes. AODE: Averaged one dependence estimators. CART: Classification and regression tree. RF: Random forest. MARS: Multivariate Adaptive Regression Splines GPS: Generalized path seeker algorithm. LR: Logistic regression. SVM: Support vector machine. GLM: Generalized linear model. PH: Proportional hazards.\n\nOutcome measures. The most frequently reported outcome measure was the ROC AUC. Three studies did not report this measure: Ahmed et al. 201570 developed an algorithm based on decision rules; Legrand et al. 201391 was primarily interested in identifying risk factors of AKI after cardiac surgery; and Scicluna et al. 201793 was primarily concerned with identifying genetic biomarkers of sepsis.\n\nSensitivity and specificity were reported together in 14 studies35,70–72,74,75,78,81–84,87,90,92. When specificity was not reported, sensitivity was reported together with PPV; and when sensitivity was not reported, this was due to sensitivity being set at a fixed value to report other diagnostic performance measures. In relation to the prior observation, more studies reported PPV than NPV. Four studies reporting likelihood ratios reported both negative and positive likelihood ratios70,74,81,84.\n\nAn overview of measured outcomes is illustrated in Table 11.\n\nNPV: Negative predictive value. PPV: Positive predictive value. LR: Likelihood ratio. OR: Odds ratio, RR: Risks ratio. ROC AUC: Receiver operator curve area under the curve.\n\nOngoing studies. Four trials are currently ongoing, one in Germany and the others in the USA, all concerned with the prediction of sepsis. Three of them are prospective studies and one is retrospective. The retrospective study aims to develop a prediction algorithm based on claims data, EHRs, risk factors and survey data of an estimated 50,000 adult patients admitted to the ED. The German study NCT0366145095 is a single-arm trial evaluating the utility of a CDS system to identify SIRS or sepsis from EHRs in a pediatric ICU population. Another single-arm trial NCT0365562647 is concerned with implementing a sepsis prediction algorithm in clinical practice as an early warning system. NCT0364494046 is comparing two versions of InSight introduced into clinical practice as an early warning system.\n\n\nDiscussion and conclusions\n\nThis systematic literature review shows that over the last 2 decades, there has been an increased interest in CDS as means of supporting clinicians in acute care. CDS has been investigated for several applications ranging from the detection of health conditions60,61, to the prediction of deterioration or adverse events40,55,76,81,83,84. Applications also include therapy guidance, as well as updating clinicians on new or changed recommendations96. CDS can also provide guidance by predicting clinical trajectories for different patient profiles over time97.\n\nFrom rule-based algorithms and simple regression models, CDS has evolved to encompass a multitude of techniques in Machine-Learning98. These techniques can be dependent on the problem selected and the data types used. Across the three disease areas investigated, the frequent use of random forest classifiers (28.1%), support vector machines (21.9%), boosting techniques (20.3%), LASSO regression (18.8%) and unspecified logistic regression models (10.9%) were observed. The use of more complex modeling such as maximum entropy, Hidden Markov Models (for temporal data analysis) as well as Convolutional Neural Networks has also emerged over the last few years. In the respiratory distress area, the use of NLP models is more common as radiology reports and clinical notes are the main source of input. Different image analysis techniques have been developed to aid in the prediction and diagnosis of respiratory events from radiology images.\n\nTypical measures of NLP model performance include sensitivity, specificity and predictive values. In measuring ML algorithm performance, sensitivity, specificity and ROC AUC are more common. A wide range of outcome measure were reported in research on less-investigated health conditions40,67; and also when uncommon, more complex algorithms were compared to basic algorithms74,78,81,84. This is not surprising given the novelty of these applications.\n\nMany of the ML algorithms and all of the NLP models covered in this work were based on medical data collected in certain clinical sites rather than publicly available data. Datasets from national audits, completed studies or other online sources can additionally play a role, particularly in model validation and testing. This could aid in the adoption and wider use of CDS systems. In this SLR, publicly available datasets were mainly utilized for developing prediction models of heart arrhythmias29–31, hypotension32, septic shock28,33,40,41, COPD50, pneumonia33 and a range of infections33,76,78,81,84,86. In only three cases were they used for testing model performance in sepsis and septic shock prediction; this included the Insight algorithm35,85,93.\n\nMost of the studies identified in this SLR were retrospective and originated in the USA where electronic health records (EHR) are commonly used. This makes it easier to access and compile large amounts of patient-level information. Many of the studies on shock and infection/sepsis based their models on data extracted from EHRs and utilized large sample sizes. The diversity in the identified CDS systems makes it challenging to draw conclusions on methodology. The lack of comparisons between different classifiers within studies, especially for the indication of shock, adds to this challenge. To assess the effectiveness of ML algorithms, future research should evaluate multiple algorithms on standard well-labeled datasets.\n\nClass imbalance can be an important issue when training classifiers on datasets for the conditions highlighted in this work. Unequal distributions can arise naturally between disease negative and positive classes when forming validation sets, particularly when disease prevalence is low75. We refer the reader to several machine learning reviews that have addressed this issue99–101. Another important issue in forming disease positive classes relates to the analysis of repeated-measures within subjects, for example, when clinical records are available for each hospitalization day. Several studies have approached this by selecting the first record indicating positive for a health condition. Few researchers have utilized all records and corrected for within-subject variation. An example is the selection of cases depending on observed correlation decay52.\n\nIn all three areas investigated, the number of retrospective studies exceeded by far the number of prospective studies conducted in a clinical setting. This highlights the challenges in substantiating clinical performance while bringing new clinical decision tools to routine in-hospital patientcare. Examples of algorithms that can be integrated in clinical practice include InSight45,46 and Sepsis Watch47 which are intended for predicting sepsis and septic shock.\n\nThe current systematic literature review did not search multiple bibliographic databases or clinical trial registers; and focused on diagnostic performance rather than other outcomes. In fact, during study screening, trials that evaluated the impact of early warning systems on measures of clinical workflow, rate of re-admissions and/or mortality were discarded as they are somehow out of the focus of this work. This implies that there may be more CDS systems used in practice for the three populations investigated within this research, where the outcomes measured are different. Limiting the search to publications in English and to studies conducted in particular countries may have further limited the studies selected. Nevertheless, studies identified within each population represented a diverse range of models applied in different hospital settings trained to predict a range of health conditions. The most widely researched conditions were sepsis and septic shock, venous thromboembolisms, acute kidney injury and surgical site infections.\n\nSpecific challenges were identified in collecting sufficient data for training CDS systems on hemodynamic instability. Patients who are, for example, at risk of hemorrhage due to a traumatic injury need to be carefully monitored; and the speed by which they reach a critical state may influence data and study management. It may also be difficult to find healthy volunteers who are willing to undergo procedures like lower body negative pressure which can be unpleasant36. Identification of cases in need of hemodynamic interventions can lend towards larger sample size19. Other conditions that need further attention are clostridium difficile and CLABSI. Prediction models were driven by almost perfect specificity and very low (<10%) sensitivity77,83,86,88. Considering that these studies used a wide range of features from the EHRs and a large number of patients, except LaBarbera, Nikiforov83, there is a need to better understand the risk factors to improve sensitivity.\n\nBased on the literature reviewed in this work, as well as several recent surveys and workshops, we would recommend the following points to be addressed when bringing a new CDS tool to critical care14,102–104:\n\nIntegrating CDS in clinical workflows without adding unnecessary extra work to busy clinical teams. The CDS101 toolbox by HIMMS highlights the “CDS five rights”, which are certainly applicable to critical care105: Providing the right information in the right intervention format, to the right person at the right point in their workflow, and through the right channel.\n\nDeveloping tools and concrete proof-points able to assess CDS efficacy in the clinic. This also highlights the importance of providing continuous feedback to clinicians.\n\nThe importance of easy to use user interfaces and focusing on human-computer interaction during deployment.\n\nEfficient training that is available when needed.\n\nBeing aware of alert or alarm fatigue and not overloading clinicians with alerts due to CDS. The intensive care unit is already plagued with alarms, and if anything, CDS should help in reducing alarms by bundling alerts according to underlying conditions.\n\nDisplaying the rationale for decisions as well as the underlying data to clinical users would lead to improved adoption.\n\nUnderstanding ethical challenges for CDS, as well as a careful risk assessment in every site before deployment106.\n\nBeing able to repeat/standardize implementation across organizations – most prospective studies reviewed in this work covered single centers. Only a few were multi-center studies.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required\n\nFigshare: Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review. Extended data - Table 1-Search strategy for shock (hemodynamic (in-stability) in MEDLINE.docx. https://doi.org/10.6084/m9.figshare.9892109.v125.\n\nFigshare: Working title: Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review. Extended data - Table 2-Search strategy for respiratory distress or respiratory failure in MEDLINE.docx. https://doi.org/10.6084/m9.figshare.9892112.v126.\n\nFigshare: Working title: Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review. Extended data - Table 3-Search strategy for infection or sepsis in MEDLINE.docx. https://doi.org/10.6084/m9.figshare.9892115.v127.\n\nFigshare: PRISMA checklist for ‘Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review’. https://doi.org/10.6084/m9.figshare.9894107.v1107.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank Mark Connolly from Global Market Access Solutions Sàrl for his contribution during the whole project.\n\n\nReferences\n\nMolina JA, Seow E, Heng BH, et al.: Outcomes of direct and indirect medical intensive care unit admissions from the emergency department of an acute care hospital: a retrospective cohort study. BMJ Open. 2014; 4(11): e005553. 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Crit Care. 2013; 17(5): R220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanger PC, van Ramshorst GH, Mercan E, et al.: A Prognostic Model of Surgical Site Infection Using Daily Clinical Wound Assessment. J Am Coll Surg. 2016; 223(2): 259–270.e2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScicluna BP, van Vught LA, Zwinderman AH, et al.: Classification of patients with sepsis according to blood genomic endotype: a prospective cohort study. Lancet Respir Med. 2017; 5(10): 816–826. PubMed Abstract \| Publisher Full Text\n\nTaneja I, Reddy B, Damhorst G, et al.: Combining Biomarkers with EMR Data to Identify Patients in Different Phases of Sepsis. Sci Rep. 2017; 7(1): 10800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCT03661450: Evaluation of the Accuracy of a Clinical Decision-Support System (CDSS) to Support Detection of SIRS and Sepsis in Paediatric Intensive Care Patients Compared to Medical Specialists. Reference Source\n\nVan de Velde S, Kortteisto T, Spitaels D, et al.: Development of a Tailored Intervention With Computerized Clinical Decision Support to Improve Quality of Care for Patients With Knee Osteoarthritis: Multi-Method Study. JMIR Res Protoc. 2018; 7(6): e154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinaire J, Azé J, Bringay S, et al.: Patient healthcare trajectory. An essential monitoring tool: a systematic review. Health Inf Sci Syst. 2017; 5(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddleton B, Sittig DF, Wright A: Clinical Decision Support: a 25 Year Retrospective and a 25 Year Vision. Yearb Med Inform. 2016; (Suppl 1): S103–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLongadge R, Dongre S: Class Imbalance Problem in Data Mining Review. arXiv e-prints. 2013. Reference Source\n\nBuda M, Maki A, Mazurowski MA: A systematic study of the class imbalance problem in convolutional neural networks. Neural Netw. 2018; 106: 249–259. PubMed Abstract \| Publisher Full Text\n\nNanni L, Fantozzi C, Lazzarini N: Coupling different methods for overcoming the class imbalance problem. Neurocomputing. 2015; 158: 48–61. Publisher Full Text\n\nKindle RD, Badawi O, Celi LA, et al.: Intensive Care Unit Telemedicine in the Era of Big Data, Artificial Intelligence, and Computer Clinical Decision Support Systems. Crit Care Clin. 2019; 35(3): 483–495. PubMed Abstract \| Publisher Full Text\n\nRumsfeld JS, Joynt KE, Maddox TM: Big data analytics to improve cardiovascular care: promise and challenges. Nat Rev Cardiol. 2016; 13(6): 350–9. PubMed Abstract \| Publisher Full Text\n\n(NQF), NQF: Driving Quality and Performance Measurement—A Foundation for Clinical Decision Support: A Consensus Report. 2010; NQF: Washington, DC. Reference Source\n\nHIMMS: Clinical Decision Support 101. 2019; [cited 2019]. Reference Source\n\nChar DS, Shah NH, Magnus D: Implementing Machine Learning in Health Care - Addressing Ethical Challenges. N Engl J Med. 2018; 378(11): 981–983. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedic G, Kließ MK, Atallah L, et al.: Evidence-based Clinical Decision Support Systems for the prediction and detection of three disease states in critical care: A systematic literature review. PRISMA Checklist. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9894107.v1" }	[ { "id": "56199", "date": "06 Nov 2019", "name": "Stavros Nikolakopoulos", "expertise": [ "Reviewer Expertise Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report on a systematic review in order to assess the state-of-the -art in the field of Clinical Decision Support (CDS) systems in the last 5 years (2013-2018). They review and report on study designs, outcomes and methods employed in CDS in the scientific literature as well as in study databases (like Clinicaltrials.gov).\nThe paper is clearly written and organized. The methodology for the systematic review is solid and comprehensive. The topic is also very relevant and timely. I do have some concerns which are mentioned below:\nThe authors could potentially include in the study (as described by the inclusion criteria), conference abstracts that were published only as abstracts in 2017 or 2018, even without subsequent publication. I assume they do that in order to somehow keep up with later developments even if they are not published elsewhere, given the very fast pace of the research area. However, they exclude protocols of studies that were published in the same (or more extended) time frame, which seems slightly inconsistent. Some discussion concerning this choice would be enlightening.\n\nThere seems to be some confusion with terminology, with unknown consequences on the review's results. The authors seem to separate \"machine learning\" methods, from \"statistical\" methods ( Table 1: \"Multivariable hierarchal logistic regression models*** (models which are based only on statistics - but there is no machine learning)\", as an exclusion criterion ). This is clearly not the suitable platform to resolve this issue, but, the distinction between machine learning and statistics is not at all that clear. Specifically, under the term \"supervised learning\", any regression method (statistics) could be classified. So, logistic regression IS a machine learning method. So is LASSO and several other methods reported. Again, this is not the appropriate place for going into further details, but there is certainly some confusion, especially when in the results Logistic regression keeps appearing as a preferred method.\n\nAgain concerning terminology, the term \"accuracy\" appears often in the results section. Sometimes it is reported as a different outcome than i.e. ROC AUC, sensitivity and specificity. All the latter methods are quantifying \"accuracy\" in some way and some clarification is needed.\n\nMinor comments:\nTable 1: Treatment/Intervention, a parenthesis is missing.\n\nTables 7 & 10: Maybe reverse the orientation of the column titles, it is impossible to read on a screen.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "56200", "date": "18 Nov 2019", "name": "Milena Kovacevic", "expertise": [ "Reviewer Expertise Pharmacokinetics and Clinical Pharmacy", "Patient outcomes." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review summarizes the utilization of clinical decision support (CDS) systems in three selected states in critical care – shock/hemodynamic (in-)stability; respiratory distress/failure; and infection/sepsis. The background of the study has a strong rationale.\nThe study comprised the results from primary sources, describing models/algorithms used to detect and alert clinicians to the presence of these conditions, as well as models/algorithms developed to predict deterioration in an individual patient state, leading to these selected conditions.\nThe systematic review was performed and the findings are presented in line with the PRISMA guidelines. Variables for which data were sought were clearly stated (PICOS) in Table 1.\n\nSpecific comments:\nWhat I found especially beneficial for the readers and future research in this area, is Table 2 with the presented collected data used for training algorithms.\n\nIt would be beneficial to provide additional information whether an internal or external validation was performed - within Table 4 (measured outcomes in studies on shock), Table 8 (measured outcomes in studies on respiratory distress/failure) and Table 11 (measured outcomes in studies on infection/sepsis).\n\nWhat was the rationale for including the studies predicting acute kidney injury within the Infection/sepsis results section? If it is about the decline in glomerular filtration rate due to hypotension seen in sepsis, it might have been presented within the Shock section.\n\nTable 7: include the abbreviations for ARDS (Acute respiratory distress syndrome), ARDE (Acute respiratory disease events) and DVT (deep vein thrombosis) below the Table.\n\nTable 9: include the abbreviation for AKI (Acute kidney injury) below the Table.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1728
https://f1000research.com/articles/8-2003/v1	26 Nov 19	{ "type": "Method Article", "title": "Adapting the 3D-printed Openflexure microscope enables computational super-resolution imaging", "authors": [ "Stephen D. Grant", "Gemma S. Cairns", "Jordan Wistuba", "Brian R. Patton", "Stephen D. Grant", "Gemma S. Cairns", "Jordan Wistuba" ], "abstract": "We report on a 3D printed microscope, based on a design by the Openflexure project, that uses low cost components to perform fluorescence imaging. The system is sufficiently sensitive and mechanically stable to allow the use of the Super Resolution Radial Fluctuations algorithm to obtain images with resolution better than the diffraction limit. Due to the low-cost components, the entire system can be built for approximately $1200.", "keywords": [ "Light Microscopy", "Super-resolution", "Open Science", "3D Printing" ], "content": "Introduction\n\nThe Openflexure microscope is an open-access, 3D printable microscope which has three dimensional movement of the specimen stage provided by a flexible plastic mechanism1. It uses easily obtainable components and the fine adjustment and stability of the 3D printed microscope allows high quality optical images to be collected. Standard versions of the microscope use medium to low-resolution lenses, enabling observation of cellular features of sub-micron size.\n\nSuper-resolution radial fluctuations (SRRF) allows super resolution information to be extracted from a series of fluorescence images taken with a high numerical-aperture (NA) lens (typically NA>0.8). SRRF is a post-processing method that generates a radiality map for each frame and then looks for temporal correlations across frames to create a final image of greater resolution than the original images2. The final resolution achievable by SRRF depends on multiple factors, including the strength of the fluctuations used to generate the correlations, the labelling density and the photo-stability of the fluorophores; however, in the best-case scenario it shows resolution approaching that of localisation based super-resolution methods while utilising a wider range of hardware and fluorophores. Low illumination intensities also avoid photo-bleaching samples3.\n\nCombining SRRF with an adapted version of the Openflexure 3D printable microscope allowed for high quality super resolution imaging on low cost hardware. SRRF analysis was carried out using ImageJ4,5 plugins meaning all aspects of this work (SRRF algorithms, Microscope schematics, etc.) are freely available and open-access.\n\n\nHardware design\n\nWe give links to the designs for all key components at the end of this paper (See underlying data6 and hardware design). Measurements were performed using an adapted version of the open source 3D printable microscope (v5.17.2-LS75-M) designed by the Openflexure project. The upper image in Figure 1 shows the microscope as constructed and mounted on a 30×30 cm aluminium breadboard for stability and portability. The colour scheme is arbitrary and does not contain any information about which parts are custom. The custom parts for this project consist of a mirror and camera holder beneath the microscope along with taller legs to allow space for the new optical path shown in the middle of Figure 1. The mirror and camera holder are combined into a single piece to control the distance from the objective to the camera and make the optical alignment more robust. This new piece is shown in the lower image of Figure 1. We also designed a cover for the camera unit to block external light and thereby improve signal to noise. The microscope was printed using polylactic acid material from Ultimaker (Red PLA - Ultimaker part no. 1618 and Green PLA - Ultimaker part no. 1608) on an Ultimaker S5 printer. As our modifications are based on a standard version of the Openflexure stage, stepper motors can be attached to the microscope if required. In this instance all stage movement and focus adjustments were made by hand with the attached adjustment gears. A compact laser module from Thorlabs (CPS532a: 4.5 mW @ 532nm) was used as the light source. This was focused using a Thorlabs plano-convex lens (LA1461-A: f= 250 mm), which gives a minimum spot size of 19 μm at the focus. We purposely defocussed this lens to give a wider illumination spot. The excitation wavelength was chosen to allow us to image the fluorescent Nitrogen-Vacancy (NV) defect centre in samples containing nanodiamond.\n\nTop - image of microscope as constructed. It is mounted on an aluminium breadboard for portability and stability. The ruler is 15cm and shown for scale. The colours of individual parts are arbitrary. Middle - beam path showing key design parameters. Illumination is from a laser mounted above the sample and the camera is mounted at the primary imaging plane of the microscope objective with an emission filter directly attached. The turning mirror allows a more compact design. Bottom: integrated mirror and camera mount to allow imaging with high sensitivity camera. We used the slots in the base of this part to pass mounting screws to the breadboard for additional stability.\n\nA Nikon oil immersion objective (NA=1.25, 100×, Edmund Optics part number #59-938) was used to image the sample directly - this objective is not an infinity corrected lens, and so the camera is placed at the primary image plane. The objective was mounted beneath the microscope stage. Focus adjustments are performed by moving the objective vertically using the adjustment gears as is standard with the Openflexure system.\n\nA Thorlabs silver mirror (PF10-03-P01) was placed beneath the objective in our custom holder and directs the beam onto the camera. The microscope uses a UI-3060CP-M-GL camera from IDS connected to a laptop computer to capture images. The IDS camera sensor (Sony IMX174LLJ-C) has dimensions of 11.345 mm × 7.126 mm with pixel size 5.86 × 5.86 μm. As printed, our mirror and camera mount places the camera sensor 170mm from the mounting flange of the lens. This is not in accordance with the DIN standard of 150mm. However, for the samples used we found that it did not create significant imaging errors but does require calibration of the image field of view at the sensor. By imaging a chrome grid sample with 10 μm grid spacing, we were able to determine that we have a field of view of approximately 95.8×60.2 μm within the sample. This corresponds to a 49.5nm pixel size at the sample; we are sampling above the Nyquist criterion for our optical system. With the known physical pixel dimensions for the camera, we therefore have a magnification of 118x rather than the 100x from the lens specifications.\n\nTo reject excitation laser light and allow only the emission from the NV centres to be detected, we used a 650nm longpass filter (Thorlabs FELH0650) mounted directly to the camera’s c-mount hardware.\n\n\nSample preparation\n\nWe used two types of sample for this paper and their preparation is as follows:\n\nWe used samples previously described in 7 and the following preparation protocol is from this reference. The nanodiamonds (ND) used in these experiments were produced by Adamas (Catalogue numbers ND-NV-40nm-Bio-2mg and ND-NV-100nm-COOH-2ml). We prepared slides for imaging from a mix of two monodisperse suspensions (both 0.1%w/v) of 40nm and 100nm diameter ND. The 40nm ND each contain approximately 10 NV while there are closer to 400 NV per 100nm ND as per the manufacturers calibration information. To prepare a suspension suitable for deposition on a coverslip we first sonicated each source of NDs at room temperature for approximately 30 minutes (we have not found this step to be time critical), using a Grant Ultrasonic Bath XUBA3, to break up larger aggregates before adding 10 μl of each ND suspension to 100 μl of distilled water. The resulting suspension was then deposited onto a #1.5 microscope glass cover slip and allowed to dry to ensure some ND adhered to the coverslip before being mounted on to a microscope slide using a small amount of distilled water as a mountant medium and finally sealing the sample with nail polish.\n\nWhole blood samples were obtained, after written informed consent, from healthy donors on the Centre for Inflammation Research Blood Resource (approved by AMREC, reference number 15-HV-013). Peripheral blood mononuclear cells (PBMCs) were first isolated from these samples before being plated onto #1.5 square glass coverslips (22×22mm) in 6 well plates at a concentration of 9 × 106 cells/coverslip. The PBMCs were then cultured in media (RPMI (Sigma Aldrich catalogue R8758) containing 10% low lipopolysaccharide (LPS) fetal calf serum (FBS Good Forte, Pan Biotech, P40-47500) in an incubator at 37°C with 5% carbon dioxide (CO2). They were cultured for 14 days with media changed twice per week to allow for the differentiation of PBMCs into monocyte-derived macrophages (MDMs).\n\n90nm nanodiamonds (Sigma-Aldrich 798150) were sonicated for 1 hour to break up aggregates and then added to media at a dilution of 1:100. After 14 days of cell culture, MDMs were incubated with 1 ml of the nanodiamond/media solution at 37°C with 5% CO2 for 2 days. After 2 days, media was removed from the MDMs before washing three times with pre-warmed Hank’s balanced salt solution (with calcium and magnesium) to remove any remaining media. MDMs were then fixed using 2% paraformaldehyde (PFA) for 20 minutes at room temperature. Finally, the MDMs were washed three times with phosphate buffered saline (PBS) and once with deionised water (dH2O) before mounting the coverslips onto microscopy slides with ProLong Diamond Antifade Mountant(Thermofisher catalog number P36970).\n\n\nData acquisition and analysis\n\nData was collected using the supplied IDS software (IDS Software Suite 4.92) with gamma, auto exposure, and auto gain switched \"off\". Exposure time and frame rate were adjusted to produce an image with signal in the middle of the 8-bit greyscale range. Stacks of 200 images were captured as 200 frame videos. These stacks were then processed using nanoJ-Core V2.1 RC18 to determine the drift correction and nanoJ-SRRF v1.14Stable13 to preform super resolution radial fluctuations analysis. For the analysis of all the data presented here we used the following processing parameters within the Fiji plugins.\n\nDrift Correction: Averaged drift over 5 frames and corrected position to first frame\n\nSRRF: Ring radius 0.5, Radiality Magnification 6, Axes in Ring 7. Other parameters were default for plugin.\n\nImages are presented using the Cube Helix colormap9 and were generated for publication using OriginLab Origin.\n\nAll raw (unprocessed) image data is available along with the microscope design files (Underlying data6).\n\n\nResults\n\nFigure 2 shows the results of imaging ND directly mounted on a coverslip. In the standard fluorescence image, Figure 2a), we see good contrast, particularly after correcting for sample drift and summing the frames. A significant increase in resolution is visible after processing the stack using the SRRF algorithm, Figure 2b). To better demonstrate the effect of the image processing, we have expanded the region in the red dashed box and present it in Figure 2c) and d). We can now clearly see individual NDs, particularly at the edges of the region highlighted. In the central region, it is likely that some of the structure shown is due to single ND; however, we cannot currently unambiguously rule out the presence of artefacts from the image processing algorithm.\n\na) Drift corrected and summed stack of 200 individual frames. b) Superresolution radial fluctuations (SRRF)-processed image of same stack of images. c),d) zoom on equivalent region marked by red dashed box in a),b) showing increased resolution enabled by SRRF.\n\nIn order to better understand the resolution enhancement, we processed the images using the Fourier Ring Correlation technique (FRC)10. To estimate resolution, FRC processes pairs of images that differ only in noise: at low spatial frequencies which are dominated by the (identical) structure of the sample there is a high degree, FRC ≈ 1, of correlation, which decreases with increasing spatial frequency until reaching a point at which the images are dominated entirely by uncorrelated noise. The spatial frequency at which this occurs is considered to be equivalent to the effective resolution of the image. There are two important caveats here:\n\nThe image may possess no significant features at the resolution of the system generating the image. In this case, the FRC will return an apparent resolution worse than the actual system resolution.\n\nThe FRC does not actually measure resolution directly, but maps spatial frequency correlations to resolution.\n\nTo consider why the second point is of relevance for our paper here, consider performing the FRC of an image, consisting entirely of random pixel values (pixel noise), with itself. In this case, the FRC would correctly return a perfect correlation at all spatial scales, and any implications on the resolution of the system would be meaningless. This incorrect resolution estimation can also arise as the signal to noise ratio (SNR) can itself influence the apparent resolution returned by the FRC algorithm. In most fluorescent microscopies, this is not a significant issue, as the fluorophores degrade and so put a limit on the maximum SNR obtainable from the sample. However, the fact that the ND emission does not degrade means that we can achieve arbitrarily high SNR by either increasing the exposure time of our sensor, or summing more sub-frames. When calculating the FRC for Figure 2a) we ran into this problem with the SNR; if you calculate the FRC by performing the correlation on the summed, drift-corrected odd versus even frames the FRC implies super-resolution imaging in wide field mode! By reducing the number of sub-frames used for the FRC, we obtained a resolution of 238 nm, which is in line with the expected diffraction limited resolution for a wide-field microscope using a 1.25NA objective lens, albeit implying a higher resolution than the 280nm expected for the ND emission peak at 700 nm. An alternative approach to estimating the resolution is to measure the full width half maximum (FWHM) of individual emitters within the image. Due to the background, it is difficult to unambiguously determine the resolution in this sample. However, measuring the resolution in this manner consistently returned values for the FWHM in the range of 300–400 nm, i.e. close to diffraction limited performance.\n\nThe FRC of the SRRF processed images generated correlation curves that followed the expected behaviour for reliable resolution estimation. In this case, we recover an FRC resolution of 115 nm. Again, this is slightly higher than the resolution returned by FWHM measurements, which tended to the range 120–140 nm. Nevertheless, it supports our assertion that the modifications to the microscope allow computational super-resolution imaging using the SRRF technique.\n\nImaging bright, stable fluorophores that are confined to the surface of a cover slip can be considered to be the optimal sample for the characterisation of a microscope system and so somewhat unrealistic. We therefore also used ND in MDM as described above, and the resulting widefield and SRRF images are shown in Figure 3 a)-d). Again we see that the microscope performs well, generating high quality fluorescence images that process well with the SRRF algorithm. The slightly lower SNR in these images returned more reliable FRC estimates, and we see FRC resolution of 322nm for the widefield images and 114nm for the SRRF images.\n\nThe nanodiamonds were suspended in the growth medium before uptake by the macrophages. a),b) large field of view encompassing multiple macrophages in both wide-field fluorescence and super-resolution radial fluctuations (SRRF) imaging modes. c),d) detail view of single macrophage in both widefield and SRRF modes.\n\n\nConclusions\n\nSRRF imaging is possible using low cost, open-access components and software on a 3D printed microscope, achieving resolutions of 115 nm, compared to the unprocessed (standard) resolution of 322 nm.\n\nWhen compared to traditional optical systems capable of super resolution, SRRF presents a much simpler method while the instrumentation shown here allows for its implementation at much lower costs.\n\n\nData availability\n\nA zip file containing the 3D printable .STL files and the raw (unprocessed) data used in the images in this paper is available from the University of Strathclyde institutional repository\n\nUniversity of Strathclyde Knowledgebase: Data for “Adapting the 3D-printed Openflexure microscope enables computational super-resolution imaging” https://doi.org/10.15129/df032aa8-2b85-4ec8-adf8-ad435806b81b6\n\nThis project contains the following underlying data:\n\nFolder: Figure-Layout (Contains files used for Figure 1)\n\n– BetterMicroscopeImage.JPG (A JPEG photograph of the microscope\n\n– Cam_MirrorSupportWithLegs_V3.1.png The rendering of the custom mirror and camera unit in PNG format)\n\n– Figure-LayoutV2.pdf (The file used to generate Figure 1, in PDF format)\n\n– Figure-LayoutV2.svg (The file used to generate Figure 1, in SVG format)\n\nFolder: Figure NV (Contains files used for Figure 2)\n\n– FigureNDonCoverslip4.pdf (The file used to generate Figure 2 in PDF format)\n\n– FigureNDonCoverslip4.png (The file used to generate Figure 2 in PNG format)\n\n– NV200Frames4.avi (The movie (in AVI format) containing the raw data for Figure 2)\n\nFolder: Figure-Macrophages (Contains files used for Figure 3)\n\n– Figure-Macrophages.png (The file used to generate Figure 3, in PNG format)\n\n– MacrophageFrames3.avi (The movie (in AVI format) containing the raw data for Figure 3)\n\nFolder: Microscope STL Files (This folder contains multiple .stl files that can be directly printed on a 3D printer that can print files in this widely used file format. All parts should be printed and assembled using the instructions on the https://openflexure.org/ website, along with the additional details given in this paper)\n\nAll figures and raw data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe .stl files for the 3D printer, including the new components described in this paper, are available under the terms of the CERN Open Hardware licence (CERN OHL).\n\n\nHardware design\n\nThe microscope was constructed using an adapted version of the open source 3D printable microscope (v5.17.2-LS75-M) designed by the Openflexure project, https://openflexure.org/, with modifications as described above.", "appendix": "References\n\nSharkey JP, Foo DC, Kabla A, et al.: A one-piece 3D printed flexure translation stage for open-source microscopy. Rev Sci Instrum. 2016; 87(2): 025104. PubMed Abstract \| Publisher Full Text\n\nGustafsson N, Culley S, Ashdown G, et al.: Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations. Nat Commun. 2016; 7(1): 12471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCulley S, Tosheva KL, Matos Pereira P, et al.: SRRF: Universal live-cell super-resolution microscopy. Int J Biochem Cell Biol. 2018; 101: 74–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRueden CT, Schindelin J, Hiner MC, et al.: ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017; 18(1): 529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatton B: Data for: \"Adapting the 3D-printed Openflexure microscope enables computational super-resolution imaging\". 2019. http://www.doi.org/10.15129/df032aa8-2b85-4ec8-adf8-ad435806b81b\n\nJohnstone GE, Cairns GS, Patton BR: Nanodiamonds enable adaptive-optics enhanced, super-resolution, two-photon excitation microscopy. R Soc Open Sci. 2019; 6(7): 190589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaine RF, Tosheva KL, Gustafsson N, et al.: NanoJ: A high-performance open-source super-resolution microscopy toolbox. J Phys D Appl Phys. 2019; 52(16): 163001. Publisher Full Text\n\nGreen DA: A colour scheme for the display of astronomical intensity images. Bull Astr Soc India. 2011; 39: 289–295. Reference Source\n\nNieuwenhuizen RP, Lidke KA, Bates M, et al.: Measuring image resolution in optical nanoscopy. Nat Methods. 2013; 10(6): 557–562. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "58499", "date": "14 Jan 2020", "name": "Richard Bowman", "expertise": [ "Reviewer Expertise My expertise is in optical instrumentation", "particularly microscope design and optomechanics. I am not an expert in superresolution", "as stated in my report", "but am comfortable assessing the instrumentation aspects of the work." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes an open, low-cost, super-resolution imaging system, using nanodiamond probes and the SRRF algorithm to localise individual emitters. It uses the OpenFlexure Microscope as the critical optomechanical mount, responsible for holding the microscope objective and the sample - consequently, most of the mounting and micropositioning hardware in the system can be 3D printed from open, documented designs; the major expenses are the camera, microscope objective, and laser - totalling around $1200.\nI have led the OpenFlexure Microscope project for some years now, and am very excited to see it used for super-resolution imaging. It is particularly pleasing that this imaging is also enabled by open-source software in the form of ImageJ, nanoJ-Core, and nanoJ-SRRF. The authors have done a great job of producing a clear, concise manuscript that describes the system and their characterisation data, that proves the principle of accessible, DIY super-resolution microscopy. I am very happy to endorse the manuscript as scientifically sound.\nI should be clear about what I don't know; I can't claim to be an expert on super-resolution techniques, and so I'm not able to state authoritatively whether I would trust the FRC figures. The authors are commendably clear about the fact that FRC can be confused by excessively high signal-to-noise ratios. While they do state the FRC figures, they also provide the more direct FWHM measure of the point spread function. FWHM gives only slightly worse resolution figures than FRC; the system is still clearly beating the diffraction limit by some margin.\nOverall, I am delighted to see this manuscript, which really advances what is possible using an inexpensive, home-built microscope. I very much hope that others are able to build on this development, and use some of the techniques shown to make novel microscopy instruments accessible, as well as reproducible.\nI have detailed below a few minor improvements to the manuscript that might help someone reproducing the system. I don't believe any of these affect scientific soundness, but they would nonetheless be helpful.\nFirst paragraph of \"hardware design\": \"insamples\" is missing a space in the PDF version. There's also a \"fromthe\" later on, though both are OK in the web view.\nFigure 1: it would be nice to add the laser diode and emission filter to the middle panel. I understand the laser enters the sample from the top, and it is stated in the text that the emission filter is mounted to the camera's C-mount hardware - but it would be clearer if they appeared in the diagram. Also, I don't think it's stated anywhere how the laser diode is mounted - is that the half-inch post I see in the background of the photo? While it's hardly a scientifically critical detail, it's helpful for anyone reproducing the system.\nSample Preparation, final paragraph: were the 90nm nanodiamonds functionalised in any way to label specific features of the MDMs? I don't think that's specified if so. If not, do you know where they tend to localise within the cell? It doesn't affect the imaging performance either way, but I'm curious.\nData acquisition and analysis: it would be nice to know the total elapsed time of the 200-frame video, and ideally the exposure time if that's available. A comment on how far the stage drifted laterally over that time might also be helpful (particularly because there will, presumably, be axial drift that is not corrected for). Do the authors believe drift was the limiting factor in the resolution that was achieved?\nReference [1] is the most up-to-date paper on the OpenFlexure Microscope currently published, and the authors are absolutely correct in referencing openflexure.org as the most up to date information on the project. However, we have described the microscope recently in a pre-print and I have taken the liberty of including the DOI below1. This version of the microscope is much closer to the one used in the present article than the earlier version described by Sharkey et al.2.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "63807", "date": "26 May 2020", "name": "Romain F Laine", "expertise": [ "Reviewer Expertise My expertise is in the development of Super-resolution microscopy techniques", "including hardware and software-based solutions. This includes the SRRF super-resolution technique and I currently work in the lab (Henriques lab) which pioneered the method." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGrant et al. provides a clever open-source microscopy platform capable of SRRF super-resolution microscopy. Open-source and cheap microscopy developments are currently a hot topic in the field and a number of contributions are available from various labs across the world. My expertise is in the development of Super-resolution microscopy techniques, including hardware and software-based solutions. This includes the SRRF super-resolution technique and I currently work in the lab (Henriques lab) which pioneered the method. Typical open-source microscopes designed for high resolution can suffer from a number of drawbacks:\nMechanical stability can be poor and compromise the image acquisition by adding motion blur and sample drift\n\nUse of typical high NA microscope objectives can be expensive and require alignment of full optical train (objective, tube lens, camera)\n\nSample movement and focus control can be either bulky and/or expensive\n\nThe implementation of Grant et al. nicely combines a number of open-source components (3D printed elements, simple opto-mechanics and cheap CMOS camera) that makes it uniquely simple and powerful and provide interesting trade-offs for these common drawbacks:\nUse of the cheap and flexible OpenFlexure system for sample movement control and objective focus control\n\nSimple, single 3D-printed block assembly for the detection path of the fluorescence microscope\n\nUse of finite conjugate objective, doing away with tube lens altogether and simplifying significantly the assembly\nThe super-resolution capability is enabled by SRRF, an approach based on picking up on radial symmetry of single emitter fluorescence pattern and correlated temporal fluctuations of these emitters. I do not know about the photophysics of NV and their blinking properties but a preprint from Narayanasamy et al.1 shows their compatibility with super-resolution microscopy. Furthermore, the resolution improvement quoted and the images shown here are convincing.\n\nUsing SRRF as a super-resolution microscopy technique is a great way to address the aspect of drift since SRRF requires only relatively short acquisition times compared to other super-resolution microscopy techniques such as SMLM and also allows the correction of sample drift at post-acquisition level. Although SRRF does not provide as high a resolution as single-molecule localization microscopy (SMLM), the short acquisition times are more compatible with the plastic stage of OpenFlexure, which may introduce more sample drift than typical metal body assemblies.\nI agree however with Reviewer 1 that some measurements of stability and amplitude of drift on the order of a typical acquisition would be helpful. This could be easily done on single emitters such as commonly used TetraSpeck beads or the single emitter NV that the authors describe in the paper.\n\nThe use of a finite objective (which is surprisingly cheap: £203 as of May 2020) with a high NA and a decent plan field of view for the price provides a great compromise between price and performance. This work constitutes the first use of finite conjugate objective to super-resolution microscopy to my knowledge.\nI am especially impressed by the quote of a FWHM of 300-400 nm on single emitters, especially since the objective is not quite working at the nominal conjugate. Is there any reason why the system was not designed to be at the nominal conjugate distance? It would be interesting to get some insight into some of the design choices made here for whoever would like to replicate the system (which I may very well do myself at some point in the future).\n\nThe discussion about FRC is absolutely valid and is a nice addition to the overall idea of the paper. A few additional comments however:\nIt is true (as reviewer #1 highlighted) that the illumination system is described only superficially, yet it is unavoidable to build an efficient illumination system for a good quality fluorescence microscopy. Also, the downside of using an illumination configuration coming from the top of the objective and therefore contributing to a larger amount of background light onto the detector should be discussed/mentioned\n\nSince one of the novelty is the use of finite conjugate objective, it would be interesting to characterise it a little further in terms of PSF size and shape (as preliminary done), flatness of the field, sensitivity (I suspect that transmission is poorer than high end objectives) and multi-colour capability (although the latter might be out of scope)\n\nIs the rationale for developing the new method (or application) clearly explained? Yes, it is a nice set of trade-offs introduced here, truly reducing the cost of high resolution microscope to the bare minimum, taking advantage of 3D printing especially, which I have not seen in other open-source microscopy implementations out there.\nIs the description of the method technically sound? Yes, the authors present a couple of experiments showing proof-of-concept and some performance.\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes, generally. STL files and other technical details are all there. Maybe a bit more info about how the illumination was set up would be nice for completeness.\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes.\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes, apart from the couple of suggestions of characterisation of mechanical stability and microscope objective performance which would really nail down a full characterisation of the system presented here.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2003
https://f1000research.com/articles/8-1353/v1	05 Aug 19	{ "type": "Research Article", "title": "On the evaluation of research software: the CDUR procedure", "authors": [ "Teresa Gomez-Diaz", "Tomas Recio" ], "abstract": "Background: Evaluation of the quality of research software is a challenging and relevant issue, still not sufficiently addressed by the scientific community.\n\nMethods: Our contribution begins by defining, precisely but widely enough, the notions of research software and of its authors followed by a study of the evaluation issues, as the basis for the proposition of a sound assessment protocol: the CDUR procedure.\n\nResults: CDUR comprises four steps introduced as follows: Citation, to deal with correct RS identification, Dissemination, to measure good dissemination practices, Use, devoted to the evaluation of usability aspects, and Research, to assess the impact of the scientific work.\n\nConclusions: Some conclusions and recommendations are finally included. The evaluation of research is the keystone to boost the evolution of the Open Science policies and practices. It is as well our belief that research software evaluation is a fundamental step to induce better research software practices and, thus, a step towards more efficient science.", "keywords": [ "Scientific Software", "Research Software", "Research Software Citation", "Research Software Evaluation", "Open Science", "Research evaluation" ], "content": "1 Introduction\n\nScientific software is a key component in today’s science and engineering development 1. As described in detail in the Research software definition section a particular, yet fundamental, subset of scientific software is the research software (RS)1 that is developed and used by researchers in the process of doing scientific research in public institutions or publicly funded projects2.\n\nComputer software was included among the positive spillovers of knowledge production in 2 (p.508):\n\nWhat is alluded to here is that there may be important positive spillovers across projects in the form of ‘learning effects’. [...] which often remain in the region of tacit knowledge [...] including the development of generic computer software for performing data processing, storage, retrieval and network transmission.\n\nHenceforth, its importance to the scientific enterprise is generally accepted 3:\n\nModern science depends on software. Software analyzes data, simulates the physical world, and visualizes the results; just about every step of scientific work is affected by software.\n\nSimilarly, most of the cited references in this work highlight the central role of software development in science nowadays. As this central role is increasingly assumed, it is also noticeable the emergence of some serious drawbacks. For example, finding scientific software can be a quite hard enterprise 4,5; and difficulties can also arise when dealing with software citations 6,7. Moreover, in 3 we can find:\n\nSoftware is increasingly important to the scientific enterprise, and science-funding agencies are increasingly funding software work. Accordingly, many different participants need insight into how to understand the relationship between software, its development, its use, and its scientific impact.\n\nResearch quality evaluation is an intrinsically embedded component of research itself, with deep impact ranging from the enhancement of academic careers to boosting new knowledge production 8. Accordingly, in the Key evaluation issues section we discuss and develop the intricate notion of evaluation in the context of research software, considering both the perspective of the evaluators and of the evaluated researchers. In particular, we clarify in the CDUR proposal section whether we are evaluating research, software, or research software as a scientific output. Likewise, in the same section we settle the basis to decide, within a research software evaluation scheme, when and how we are evaluating some software or its associated research.\n\nOur goal is then to set up a basis for a simplified and flexible protocol concerning research software evaluation: what we have named as the CDUR procedure. It includes four stages, labelled as Citation, Dissemination, Use and Research, that are thoroughly developed in the CDUR proposal section. The procedure is meant to help all the key actors involved in the evaluation process, and it applies to any scientific area, as the considered research software aspects are quite transversal. CDUR will provide insight in the relationship between software, its development, its use, and its scientific impact, as we will show in this work.\n\nWe are aware that there are plenty of references in specialized journals regarding software quality, free/open source or educational software assessment methodologies (e.g. the related Wikipedia pages or some of the Software Sustainability Institute (SSI) documents3). The testing, validation and verification of such types of software are well established concepts that proceed in a direction that we are not going to pursue in this work. There are also different publications concerning research evaluation (e.g. 8 for a recent study with more than 50 references on the subject) and a very complete review on the literature for scientific software testing can be found in 1.\n\nMoreover, in the context of Open Science career assessment, the European Commission report 9 considers as an evaluation criteria the use and the development of free/open source software, although without proposing any concrete method for achieving such criterium; and similar considerations can be found in the NASA report concerning open source software policies and career credit 10 (p.74). Besides, the importance of the evaluation step in the context of future scholarly communication and Open Science is stressed in 11: The conclusion is actually simple: the evaluation of research is the keystone.\n\nNevertheless, we have not been able to find publications addressing, in particular, evaluation of software developed for scientific research, (not scientific software in general), or concerning the evaluation of research software as a whole process (and not just testing). Thus, in our opinion, there is a clear need to approach the issue we are dealing with in this paper, concerning a more precisely determined object (research software) although in a wider evaluation context (as a global process).\n\nOur contributions are distributed along this article as follows: next, the Research software section is devoted to discuss the different aspects related to the concept of research software and its associated issues concerning the notions of authorship, publication and citation. For the sake of completeness, a panoramic report of the international scientific community that has grown around research software has been included in the section entitled A snapshot on the international RS landscape. Then, the section Key evaluation issues develops a similar analysis of the key facts concerning research software scientific evaluation, where we study the evaluation methods and the key evaluation actors, as well as the concepts of success and value of research software. Finally, the CDUR proposal section is devoted to the presentation of the proposed CDUR protocol. To facilitate the reader to reach a global perspective of this proposal, this section begins with a summary description of CDUR’s four components, and then describes and studies in detail the main points of each of these components, discussing the pros and cons. The article ends with some conclusions and recommendations for the consideration of the scientific community.\n\n\n2 Research software: definition, publication and citation\n\nIn this section we examine a first block of the essential components that are involved in the scientific software evaluation, such as its definition, or more precisely, the definition of research software, and what does it mean to be an author or a contributor to this software. In our work here we would like to highlight the widespread importance of RS production as a research output, and the relevance of the publication of software papers – as publication is an essential part of the evaluation protocol. We study the relationship as well between references and citations. Note that to cite software in science is different to citing scientific (or more precisely, research) software, which is the issue here.\n\nUnder these premises, the following section entitled Key evaluation issues will be devoted to discuss some main points concerning the evaluation of RS in its own (methods, key actors, paradigms).\n\nGenerally speaking (e.g. 1) authors consider scientific software as the one widely used in science and engineering fields. More precisely, in 12,13 we can find the following definition:\n\nScientific software is defined by three characteristics: (1) it is developed to answer a scientific question; (2) it relies on the close involvement of an expert in its scientific domain; and (3) it provides data to be examined by the person who will answer that question ...\n\nOr, as concisely described by 14, scientific software is software developed by scientists for scientists. Note that 12 excludes the following software types from the scientific software definition:\n\n... control software whose main functioning involves the interaction with other software and hardware; user interface software that may provide the input for and report of scientific calculations; and any generalized tool that scientists may use in support of developing and executing their software, but does not of itself answer a scientific question.\n\nbut, on the contrary, all these types could fit in the larger definition given in 1. On the other hand, the more precise term of “research software” (RS) is also employed in the literature, a definition can be found in 15:\n\nResearch software (as opposed to simply software) is software that is developed within academia and used for the purposes of research: to generate, process and analyse results. This includes a broad range of software, from highly developed packages with significant user bases to short (tens of lines of code) programs written by researchers for their own use.\n\nand the NASA report 10 (p.26) mentions\n\nResearch software – that is, the software that researchers develop to aid their science...\n\nThe concept of RS is equally studied in 16 in the context of a (French) research laboratory’s production:\n\nLogiciel d’un laboratoire : tout programme ou fragment de programme utile pour faire avancer la recherche et qui a été produit avec la participation d’un ou plusieurs membres du laboratoire.\n\n[Software of a laboratory: every program or part of a program useful to make research advance and that has been produced with the participation of one or several members of a laboratory.]\n\nThese RS definitions include some software productions that would be excluded according to the framework described in 12, such as, for example, software tools that scientists may use in support of their own developments and that could be, as well, object of research in other scientific lab’s.\n\nA complete study of a lab’s RS production is achieved in 16 through the comparison of software and publications, considering the legal aspects and the governance main issues in both cases. This comparison: software/publications, reconciles the different views. For instance, among scientific publications we can find preprints, articles published in conference proceedings or journals, book chapters and books. Similar diversity appears in the large spectrum that begins with research software specifically done by researchers as part of their research tasks, and that includes as well the ample concept of scientific software, widely used in science and engineering, or the notion of academic software, developed to fit education or management needs or that is developed in public institutions or in publicly funded projects.\n\nFinally, let us mention that, as in the case of publications, the research software production of a laboratory is decided and proposed by the lab’s members, and it is approved by the leading institutions during the usual laboratory evaluation and funding procedures.\n\nWe remark that these definitions do not take into account the status of the software: project, prototype, finished, publicly available; nor its quality, scope, size, or existing documentation; it can be used by a team, just for the purpose of achieving a publication or it can be soundly installed in several labs, where it is used regularly. Moreover, we think that these considerations equally apply to software developed in any scientific area. Figure 1 shows some of the interrelations between the different concepts involved in this article.\n\nAlthough we think that software development skills have improved in the last decades in the scientific community, and more and more research software developments arise from well-organized teams with well-established testing, management, documentation and dissemination procedures, the paradigmatic model that we have here in mind is one that we feel it is still largely present 12,13,16,17. Software is developed by small (perhaps international) teams, or individually, usually with few development skills, where the main goal is the research activity itself. That is, software that mainly aims to give correct scientific results of whatever kind 14,18 and not necessarily a sound software “product”, well documented, tested and reusable. Note that If the software gives the wrong answer, all other qualities become irrelevant 12.\n\nAs we can see in detail in the section Publication of Research software, there are several possibilities to publish software papers. Software publications, similarly to research data papers, are becoming progressively installed in some scientific areas. However, in our opinion, there is still not a real RS publication procedure of comparable status as the one achieved for research articles, that is, well established and widely adopted by the scientific community. Thus, we cannot rely on the concept of a research software paper to fix some features towards a precise RS definition.\n\nIncidentally, note that research software can contain knowledge that is not otherwise published or just difficult to extract 19 (even from the associated publications analyzed in the section about Publication of research software) as for example: negative results, or modifications for algorithms, either to make their implementation faster and sounder or to correct problems that are arising during the coding process and that can generate new scientific understanding, etc. Code and theory can evolve side by side 20.\n\nBearing all these facts into account, we conclude that the definition of research software we will deal with in this paper must lie in the context of a research work, ongoing or already done, and, thus, in relation with a research result that is published or in construction.\n\nTherefore, we will consider here that a research software (RS) is a well identified set of code that has been written by a (again, well identified) research team. It is software that has been built and used to produce a result published or disseminated in some article or scientific contribution. Each research software englobes a set (of files) that contains the source code and the compiled code. It can also include other elements as the documentation, specifications, use cases, a test suite, examples of input data and corresponding output data, and even preparatory material. Note the role of the preparatory material to set initial dates to the software itself (see for example the Dates paragraph in 16).\n\nThe concept of author is an essential part of the definition of RS: developed by scientists for scientists 14 or, simply, written by members of a research lab 16.\n\nThis is a point where legal issues are important, as the concept of author from the legal point of view might differ from the author concept in the usual scientific practice. The author of a painting is the one who holds the paintbrush, and the author of a novel is the one who writes it, which also applies to code writing. However, it is very usual that the teams involved in RS development include researchers that do not write much code, but the software will simply not exist without their scientific contribution. On the other hand, should we consider that the person who corrects a few code lines is an author? What happens if the code of a contributor has been later on fully rewritten or translated from, say, Fortran to C? In 16 we can find a complete study of the concept of RS authoring in the scientific context, as well as a study of the involved legal aspects (under the French authorship law). Note that authorship roles can also be discriminated in scientific publications, see for example 21.\n\nAs mentioned above, the role of contributors to a RS can be manifold, for example, as responsible or head of the RS developer team, as main developer or as minor contributor, as writer of an old part of the code or as the researcher supplying scientific expertise, etc. In larger teams there can be specialized roles for management, documentation writing or for testing issues. In order to simplify the analysis of the evaluation aspects implied by this concept, we have selected three main roles: (i) responsible or leader for the RS, (ii) main contributor or (iii) minor contributor. We should bear in mind that it may happen that RS leaders or scientific experts do not participate in writing the code, or just participate with minor contributions to the code while having other important roles in design, management, etc. They may also participate in the code writing as the main contributors. A very detailed view of several RS contribution roles can be found in 22, see also 23.\n\nIn conclusion, in this paper we will consider the concept of RS author as describing someone who fulfills any of the three selected roles presented above. When the contribution is about code writing, some percentage of code participation can be estimated. It can be a decision of the RS team to associate a contribution percentage to the RS responsible as well, in the case of no code writing.\n\nIn this section we would like to reflect the human component, in the current landscape, that is most directly concerned with research software, even if it is not always specifically related with the evaluation issues that are the object of this work. Please recall that this presentation only attempts to provide a partial view of a much larger panorama.\n\nAs the RS activities evolve in the scientific community, there is a growing organization among the RS developers and more and more initiatives of different nature build up the current RS community landscape. We can find national initiatives or institutes, national or international networks and workshops. These initiatives deal with RS as a scientific output, usually without focusing in specific scientific topics (that are out of the scope of this study). In what follows, we will mention a few examples from North America and Europe to give a glimpse of a very rapidly evolving panorama.\n\nThe first one we will like to bring here is the Research Software Engineers (RSE) initiative4, an international association that has been launched by the UK. The last International RSE leaders meeting has taken place in January 20185, gathering members from Africa, America (North and South), Europe and Australia.\n\nAgain, in the UK, the Software Sustainability Institute (SSI)6 has been launched by the EPSRC7 in 20108 to help researchers build better software 24. Let us recall that the SSI is at the origin of the UK-RSE community (see 25) and has launched many other initiatives like the Journal of Open Science Research (JORS) publication9, or the Fellowship Programme10. It also organizes many workshops regularly.\n\nA similar structure to SSI is currently under construction in the USA, the US Research Software Sustainability Institute (URSSI)11 that aims to develop a pathway to research software sustainability.\n\nFrom a different perspective, the Workshop on Sustainable Software for Science: Practice and Experiences (WSSSPE)12 is an international community-driven organization that promotes sustainable research software by addressing challenges related to its full lifecycle, through shared learning and community action. It has organized workshops in USA and Europe since 2013.\n\nThe Software Citation Group has published the Software citation principles in 2016 7. The group is now closed and has evolved to the Software Citation Implementation Working Group13. They are specialized communities, focusing on the particular issue of software citation.\n\nIn France, the PLUME project (2006–2013)14 was launched by the CNRS15 to promote economical, useful and maintained software for the higher education and the research community, and to promote the community’s own developments 26. PLUME had a platform to publish software descriptions and reference cards (fiches in French) and organized many workshops and training activities around research software, see for example ENVOL 200816 or the Journées PLUME 22-23/09/09 : Pourquoi et comment diffuser un développement logiciel de laboratoire ou d’université en libre ?17. PLUME has also published several studies regarding RS issues that can be found under the title Patrimoine logiciel d’un laboratoire18.\n\nIn France there is also the developers network named Réseau des acteurs du DÉVeloppement LOGiciel au sein de l’Enseignement Supérieur et de la Recherche (DevLOG)19 that was launched in 2011 to gather the actors of the software development in the academic community. It has regularly organised the JDEV conference since 201120.\n\nCanarie21 is Canada’s National Research and Education Network. It is a funding agency with a Research Software Program22 that has organized the Canadian Research Software Conference in 201823.\n\nIn Netherlands, the NL-RSE (Netherlands Research Software Engineer community)24 was formed in April 2017, an initiative launched by the Netherlands eScience Center25 and ePLAN26 and had its first meeting in 2017. In Germany, the deRSE27 organized its first Conference for Research Software Engineers in 201928.\n\nBesides these initiatives, we can mention many different conferences or workshops around research or academic software, such as the Engineering Academic Software (Dagstuhl Perspectives Workshop 16252)29 (June 2016) 27 or the DANS30/SSI-Sustainable Software Sustainability Workshop31 (March 2017) with a follow up in 201932.\n\nWe consider that these references provide a relevant, albeit partial, snapshot of a situation evolving towards an increasingly internationalized structuration33.\n\nIn this section we will present a partial panorama of the RS publication world. Research software papers, like research data papers, are publications directly related with the description and functioning of the RS, published in a scientific area’s specific journal or in generic journals.\n\nIn order to give a glimpse of the current panorama for these publications, we can begin with the list that N. Chue Hong keeps in the SSI platform34. Among the general scope journals mentioned in N. Chue Hong’s list, Wiley’s Journal of Software: Practice and Experience35 has published articles about software in science since 197936 and seems to be one of the oldest journals for this subject. On the other hand, the Research Ideas and Outcomes (RIO) Journal37 has published, at the time of writing this paper, three software descriptions and one software management plan, and seems still too novel concerning this kind of publication. The recent Software Impacts38 journal foresee its first volume in August 2019.\n\nIn a similar mood regarding RIO’s RS descriptions, we can mention the software publications by The Journal of Open Source Software (JOSS)39 28, a “developer friendly” journal launched in 2016 that has review policies taking into account good dissemination practices (see for example 29,30). Reviewers are expected to install the software they are reviewing and to verify the core functionality of the software40.\n\nAnother journal considering RS submissions is the Elsevier SoftwareX Journal41, launched in 2015, where the peer review procedure requests the referees, among others tasks, to build, deploy, install and run the software following the provided documentation42. Similarly, JORS43 has published software metapapers since 2013 and also has a precise software peer review procedure44.\n\nOn the other hand, the French PLUME project (2006–2013) 26,31 published RS description cards45 containing the software metadata, a short description, and links to related publications (see the Treecloud46 example in Figure 2).\n\nThe objective of these publications was to promote and increase the visibility of RS, in order to favor scientific collaboration47 :\n\nCes fiches sont destinées à valoriser les productions logicielles de la communauté ESR et à les faire connaître auprès d’un public plus large dans un but d’augmenter les collaborations scien tifiques.\n\n[These description cards are intended to promote the software production of the research community and to make it known to a larger public in order to increase scientific collaboration.]\n\nThe description or reference cards48 (named fiches in French) are classified by subject (maths, network, biology...) and by keyword tagging. For example, a set of institutional keywords was used to identify software produced with the joint participation of members of a laboratory or a research establishment. Both approaches (subject classification, keywords) facilitate software searching, as it is very difficult in general to find a specific RS if one does not know its name or its developer’s team.\n\nNote that the difficulties of finding software of interest for scientists and engineers have been thoroughly studied in 5, yielding relevant inconveniences such as, for example, the risk of work duplication, of reduced scientific reproducibility and of poor return of funding agencies’ investment. Of course, the most serious drawback is the real reduction of the RS’s potential scientific impact.\n\nWe can emphasize that, although PLUME did not have a software peer review procedure, in the case that the software was known to be used regularly in at least three laboratories or institutions, the RS software got the status of validated by the academic community in the sense of PLUME and had a more detailed publication note (fiche) written and reviewed by identified users49. See, for example, the three RS publications associated to the Unitex software, developed at the Gaspard-Monge Computer science laboratory (LIGM)50: the Unitex English reference card51 is a translation of the French card52 and both indicate that there is a more detailed description card as a validated software53 (see Figure 3).\n\nAlthough it does not fall in the category of general journals, we would also like to mention the Image Processing On Line Journal (IPOL)54 (see 32), an Open Science and Reproducible Research journal. IPOL publishes image processing and image analysis articles, and the source code associated to the described algorithms. Both the submitted article and the corresponding software are reviewed together and published simultaneously in the IPOL platform. The software can be tested to verify the correctness of the published results or to generate new knowledge with other data.\n\nFinally, as a general reflection regarding peer review procedures specifically suited for research software articles, we would like to highlight the “Peer Community in”55, a non-profit scientific organization that aims to create specific communities of researchers reviewing and recommending, for free, unpublished preprints in a scientific area. This initiative seems to us an interesting experience that could be well adapted to develop RS recommending and review.\n\nComparative software reviews are other forms of reviewing that could be useful to improve RS assessment inside scientific communities or for specific targeted topics, see 33 for counsel in writing such reviews.\n\nBefore the emergence of a specific approach to software papers publication, as described in the previous section about Publication of Research software, scientists used to present research software through the usual scientific article media. Many of these traditional articles did (and still do) present scientific software to some extent, from a light mention of the involved software within a more or less detailed description of the core scientific work, to a very thorough explanation of the RS, including its name, version and retrieval indications. Thus, quite a few of these papers would have been considered today as RS papers, as it might have happened that the corresponding reviewers have had the opportunity to carefully check the involved RS or, at least, they have had some first-hand knowledge about it (as users from the corresponding research community, for example). That is, the reviewers may actually have behaved, albeit informally, as true RS reviewers, meeting current requirements of journals like JOSS or JORS (see previous section).\n\nWhat is, yet, less usual is to set a reference to one of these papers as the reference for the RS being described in the article. And, even when a reference is treated in this way, it is still rare that it assigns, to the corresponding RS, the status of a research output on its own, since the usual situation is to understand that the most valuable contribution is the article itself. That is, the researcher, and its evaluators, still consider that the value remains in having another article added to the curriculum vitæ publication list, mostly forgetting the relevance of adding a RS to the list of the research production outcomes, or to take it into account in the evaluation process. This is a serious drawback, both towards standardizing RS citation procedures as well as for its full recognition as a research output.\n\nNowadays, as the community value perception switches towards including RS, research data and other outputs as fully recognized elements of research production, the issue of how to properly cite these works sharply raises. Many scientific groups currently deal with this question, mainly for research data. For the case of research software we would like to mention, for example, the Software Citation Group 7, the Software Citation Implementation Working Group56 and the Dagstuhl Perspectives Workshop 16252: Engineering Academic Software 27.\n\nIn general, we consider that the first step required to facilitate RS citation is to have associated, from the origin, each RS with a clear reference. As it is explained in 34:\n\n... la différence entre référence et citation : l’acte de référence relève d’un auteur donné alors que la citation est une nouvelle propriété, éventuellement calculable, du texte source. Selon P. Wouters (1999), ce renversement a radicalement modifié les pratiques de référencement et littéralement créé une nouvelle “culture de la citation”.\n\n[… the difference between reference and citation: the act of reference is the responsibility of a given author while the citation is a new property, possibly calculable, of the source text. According to P. Wouters (1999), this reversal has radically altered the practice of referral and has literally created a new \"culture of citation\".]\n\nThat is, to establish a precise reference is the authors’ task, so that the interested public can use this reference to cite the corresponding work. As stated in the above quotation, reference usage is radically evolving and is creating a new citation culture.\n\nIn this paper we will consider a RS reference or citation form as (see 35, section 6.1):\n\n– the reference to a research software paper or other kind of scientific publication that includes, and relies on, a software peer review procedure, or\n\n– the reference to a standard research article that includes a description of the RS and the implemented algorithms, explaining motivations, goals and results. . ., or\n\n– a typical label, associated to the RS itself, and that identifies it as a research output, specifying its title, authors, version, date, and the place the software can be recovered from. In this respect it can be relevant to make the code citable57 via repositories such as Zenodo 36.\n\nNote that a RS can have simultaneously more than one of these types of reference forms, and, in fact, all three types can coexist for the same RS. Moreover, concerning the second reference type, we remark that there can be several classic articles associated to a single RS, and that could be used as its reference. Thus, in order to facilitate RS citation by others, it is advisable to choose the simplest reference form, whenever this is feasible. This can be easy for a small RS that has been produced and used for a unique purpose and described in just one research article and that will not evolve any further. But RS can be a living object with many evolutions, versions and different references associated to it, making it harder to decide on a single citation form.\n\nA further issue concerning RS citation arises from the observation that, in the case of research papers, the classic publication model attaches only one version and only one reference to each paper. Thus, the paper and its reference act also as a timestamp for a published scientific result. This model is still largely set in place, but it is evolving and now journals like F1000 Research58 manage quite a few versions of the same article as part of its open peer review practice 37. Furthermore, in 38, the sixth key principle claims that the idea of the journal article as a monolithic object that will stand for all time unless formally retracted has gone. Indeed, unlike articles, software development has never been associated to a monolithic object, and there exists RS with a long life, involving several international collaborations, with a large number of versions and of related publications that could act as a reference. Moreover, some RS users would prefer to cite a specific version of the RS, as the one that has been included in other RS, or that has been used to obtain the published result.\n\nThe use of persistent identifiers59 such as DOIs facilitate RS access and it is advisable to include these identifiers in the citation formula.\n\nA more complex way for RS identification than a citation form is the use of metadatasets. The Software Citation Implementation Working Group is working over several possibilities for software metadatasets60. The PRESOFT (Preservation for REsearch SOFTware) metadataset proposed for RS in 35 is built over the skeleton of the RS reference cards that where published between 2008 and 2013 by the PLUME project 31. This metadataset benefits from the PLUME experience, which validates the proposed model, and sets a reasonable level of standards to manage RS identification.\n\nFurther discussions on software reference and citation issues can be found in 3,4,6,23,31,39–41 and a thorough digression on software metadata appears in 42 section 6. We can also mention that 43 addresses a related topic, namely, the proposition of a usage format for CITATION files.\n\nThese works, among many others, as well as the contributions of different software and data discussion groups, and the previous reflections in this section show the complexity of the concept of RS citation and its actual evolution towards a more well-established future model(s).\n\n\n3 Key evaluation issues\n\nThis section outlines the second block of issues that appear in our conception of the CDUR procedure for RS evaluation, as are the possible evaluation methods, the main evaluation actors and the study of the concept of a successful RS and its comparative value regarding other kind of research contributions.\n\nIf we follow the academic life of a standard researcher, we can appreciate that research evaluation starts with the doctoral thesis, followed by the recruitment period and continues, along the years, with the career evolution. Subject to this research evaluation are, in particular, articles and other publications with peer review procedures, participation at congress and workshops requiring invitations or with refereed submission procedures, applications at different competitive calls for project funding, proposal and/or involvement in different research projects (perhaps with the collaboration of other colleagues, institutions or technological enterprises...) as well as the establishment and consolidation of a network of students and of reputed colleagues, usually in an international context and within a scientific area.\n\nCollaborations tend to be more and more interdisciplinary, which raises difficulties in the evaluation process, as evaluators can be experts in one area but have little knowledge in the other involved areas. As remarked in 44, there is also a lack of widely agreed quality principles in interdisciplinary research, and reviewers might lack cross-disciplinary experience and, maybe, do not have shared understanding of quality. Interdisciplinary aspects can appear in RS evaluation, as software conceived and developed in a specific research area can be easily used and adopted (including with new developments) in many other areas. This aspect is part of the usual research evaluation considerations.\n\nGenerally speaking, we can identify two main methods to conduct research evaluation, both of which usually take into account only the “paper” production (articles, books, presentations, project proposals, etc.): a qualitative approach (sit and read to evaluate, following a subjective criterion, the quality of the presented documents) and a quantitative estimation, by using established external metrics such as the citation index, impact factor, number of publications and other indices, and then adopting some models of evaluation 8. Nevertheless it is widely known that indicators and bibliometrics should be used with careful attention, as remarked in 11,34,45–48.\n\nMoreover, the community’s “social knowledge” can also occur and influence evaluation practice, as stated by 49 (p.8) regarding the review of mathematical statements in research papers: the methods of checking the proof are social rather than formal. That is, evaluation of the quality of a work can rely, although maybe not intentionally, upon a community perceived knowledge.\n\nThe extent of these social practices is very difficult to assess but could be neutralized by increasing openness and transparency in the evaluation policies and by exercising special caution during the evaluation committee selection, as recommended by 11 (p.9):\n\nUniversities and research institutions should:\n\n4. Strive for a balanced and diverse representation (including, but not limited to, gender, geography and career stage) when hiring, seeking collaborations, when organizing conferences, when convening committees, and when assigning editors and peer-reviewers, and building communities such as learned societies.\n\nResearch funders and policy-makers should:\n\n2. When evaluating researchers, ensure that a wide range of contributions (scholarly publications, but also data, software, materials, etc.) and activities (mentoring, teaching, reviewing, etc.) are considered, and that processes and criteria of evaluation are both appropriate to the funder’s research programme, and transparent.\n\nAs we have seen in the previous section, there are, all along a research career, different key actors performing the evaluation tasks at different stages: for example, the research community as a whole, through its experts, regarding peer review procedures for publications and journal editorial activities; the academic community of colleagues from universities, laboratories or research centers, involved in evaluation processes for recruitment and career progress; the committees nominated by funders of scientific activities at local, national and international level; and, finally, the policy makers at any level, that set the policies that will be applied by the selection, recruitment or the funding committees.\n\nOn the other hand, community evaluation appears while setting new collaborations or in the gathering of a team looking for some project funding. Besides, informal evaluation also happens each time the reader of an article or a RS user weighs if further attention to the research object is worth it: is this paper or software interesting for my work/research?\n\nA researcher aiming to achieve success during any evaluation of whatever kind needs, first to submit good research outputs (articles, software…) and, then, to make public this work adequately in order to facilitate the evaluator’s task. But it is not the same to face a journal peer review procedure, a grant selection for project funding, or to be involved in a recruitment process, etc. Similarly, it is not the same to be subject to an evaluation by qualitative or quantitative methods. As a consequence, consciously or not, author’s adaptations occur when facing evaluation procedures and requirements 46.\n\nOn the side of policy makers, it is necessary to foresee and to adapt to science evolutions, and these challenges also ask for new evaluation policies and criteria. We would like to mention here three examples of the preparation of such policies. The first one corresponds to the Expert group of the European Commission that has produced the report 11. This Expert group has been set up to support the policy development of the European Commission on Open Science, in order to assess the current situation with regard to scholarly communication and publishing mechanisms, as well as to propose the adoption of some new, general, principles for the future. The second example corresponds to the Committee on Best Practices for a Future Open Code Policy for NASA Space Science that has produced the report 10. This Committee was charged to investigate and recommend best practices for the NASA, as it is under study whether to establish an open code and open model policy, complementary to its current open data policy. Both of these two reports do provide recommendations regarding evaluation, RS and Open Science (as we include open code in the general framework of Open Science policies). The third example that we would like to mention is slightly different, as it corresponds to the Symposium organised by the French Académie des sciences (April 2nd 2019) for Foresighting Open Science61. Among others, the goal of the symposium was to look into the issues that the current Open Science acceleration raises, such as science and researchers’ evaluation. The Académie acts as an expert and advisory body for the French public authorities. These examples show us how the policy makers set expert committees or organize events to study a particular subject and to seek counsel for the new policies to be defined.\n\nAnother important role of the policy makers is to set the evaluation committees, as there is a fundamental distinction between who establishes the norms, policies or habits in the evaluation procedures, and the evaluator or the evaluation’s committee, who has to apply them. Yet, it can also happen that the roles of the policy maker and the evaluator are concentrated in the same person or persons. Policy makers set not only the evaluation methods to be applied, but also the characteristics and criteria of the jury’s selection and whether the committees are totally independent and have the final decision or are just an advisory board, etc.\n\nIn particular, issues such as gender, age, race, geography, etc. biases can be better dealt with through committees with a balanced representation of diversity 11 (p.9):\n\nResearch funders and policy-makers should:\n\n4. Consider how funding policies affect diversity and inclusivity of research on a global scale. In particular, funders should work to ensure that review boards, committees, panels, etc., are diverse - in terms of gender, geography, and career stage.\n\nFurther considerations on the evaluation role of universities, scientific establishments, funders and policy makers are addressed in the next section dedicated to the CDUR protocol.\n\nFinally, let us to point out that when evaluating a publication or when performing peer review of an article, the evaluator is expected to have the necessary knowledge to recommend if the document should be published. At the end of the review process, the evaluator is expected to have a fair amount of knowledge about the reviewed work. Similarly, in a recruitment procedure, the evaluator is expected to have the necessary knowledge in order to decide the best candidate for the position. But these arguments are not obvious concerning RS evaluation, as we will detail in the CDUR proposal section.\n\nObviously, a good scientific software is one that has been written by a scientific team to produce good, sound scientific results. This is quite a circular definition, and other, more precise, criteria should be taken into account. For instance we could consider as a positive feature the RS availability, and the fact that it is adequately documented, licensed, version controlled, and tested 15,50. Other qualifying principles that are currently under discussion are the Software Seal of Approval 51 or those involved in the notion of FAIR (Findable, Accessible, Interoperable and Re-usable) software, as FAIR is already a very popular concept among the research data communities.\n\nWhat is less obvious is to determine how these criteria will be concretely used in a specific evaluation context. In fact, setting the list and the weight of the different criteria to determine what should be understood as a good RS depends on three different aspects:\n\n– the evaluation context (peer review, funding, career...),\n\n– the evaluation committee,\n\n– the policy makers.\n\nTo continue the study of the criteria that could be considered to declare a RS as successful, we can recall that the scientific community has clearly established the difference between an article and a preprint through the peer review process that is part of the publication step and that is missing in the preprint case. As a result, an article has a quality label that does not exist in the preprint case.\n\nNow, in the same way as preprints are made publicly available through their deposit in ArXiv62 or in many other popular platforms, software can be also made publicly available in platforms like Zenodo63, GitHub or many others. Although there may be some control of what is deposited in these platforms, there is not (as far as we know) a real scientific control of the deposits, or something that approaches peer review procedures.\n\nAgain, a distinction similar to the one existing between a preprint and a published article can be claimed for software: there is a clear difference between RS publicly available through well known platforms (or personal web pages) and RS that has been the object of a publication of the kind detailed in the section of Publication of Research software. As we have seen there, RS reviewers are generally expected to have both sound scientific knowledge and enough software knowledge to be able to build, deploy, install and run the software following the provided documentation. This is, then, a dual context where the evaluation of purely software aspects has to go in parallel or, perhaps, get mixed with the evaluation of scientific aspects.\n\nThis level of evaluation of software aspects can be adequate for a RS paper, but it could be less adapted to recruitment or a career evaluation process, where evaluators have got to achieve a global vision of a curriculum vitæ.\n\nAnother relevant remark is related to the assessment of those RS products which are already well known and popular within a scientific community. Here, it could be more adequate to assess the RS quality or its impact in some indirect way, by assessing the quality of the related publications, or by the number of users and collaborators that have been attracted, by the number of funded projects, etc. (16, p.134). Yet, this quality test is to be carefully considered, as a RS considered as successful from the software point of view is not necessarily good from the scientific perspective, and vice versa (16, p.134).\n\nIn 52, a RS is considered as successful when it is delivered with a base code that produces consistent, reproducible results, when it is usable and useful, when it can be easily maintained and updated, and has a reasonable shelf life. The French Inria institute uses a set of criteria for software “self-assessment” in career evaluations in order to determine software quality 23,53. On the other hand, as seen in section about Publication of Research software, PLUME handles the concept of “validated” software, based in the verification of its regular use in at least three different laboratories (avoiding in this case the need for a careful analysis of the code). Going beyond PLUME’s concept of validated software, a very successful RS could be simply defined as one that is widely adopted and used by a scientific community.\n\nWhether we are evaluating a RS or a contribution to a RS, the RS needs to be well identified with a version number and a date, that is, with a reference of the kind proposed in the section about Referencing and citation of Research software. The role of the different agents contributing to the RS should also be clearly presented, as seen in the section about RS authors.\n\nFinally, concerning the role of policy makers and of evaluation committees in setting and applying the different criteria used in RS evaluation, we would like to emphasize that they should clearly state the precise balance between the scientific and the purely technical aspects that has been considered in the evaluation process, as this is a key consideration to understand the concept of successful RS that is behind the evaluation outcome.\n\nFurther considerations on this manifold notion are the object of the CDUR procedure proposed in the next section.\n\nOnce we have analyzed the different criteria to determine when a given RS could be considered as good or successful, an important issue that remains to be settled is how this success should be reflected in the more general value scale of a research evaluation process. To our knowledge, the question of the comparative weight between a relevant RS and a good research article arises repeatedly: should the evaluators assign to the RS the same value as to the article?\n\nIn this context, it seems relevant to analyze a similar situation that happens in the well-established publication evaluation scheme64 when considering different publication outputs: preprint, conference proceedings, journal published article, book chapter, book... It seems to us that, in this case, the value scale for the different products is widely accepted in whatever scientific community, although idiosyncratic variations can apply (for example, to manage a blog can be taken into consideration in Humanities and Social Sciences evaluations, but not in Mathematics). For instance, journal published articles are usually considered better if there is a rigorous peer review procedure behind the acceptance procedure, but, again, the specific scientific ecosystem determines the criteria to declare which journals are better than others (leaving aside, voluntarily, the arguable journal impact factor consideration).\n\nIn a similar way, we could tentatively try to set a RS value scale, backed by the standard uses of a specific community. Say, proposing a scale that would start with a RS with only one associated article, followed by a RS with several associated articles by the same team that has produced the RS, and then a RS that is used by other teams to produce new published results, a RS that has passed a strict software peer review (as in the section about Publication of Research Software), and finally, up to a RS being the object of international collaborations during months or years... We think that a way to solve this problem of comparing the value of publications and RS is to give each of them the value that they have achieved in the corresponding, specific, scale. Likewise, for other research outputs (data, prototypes...) that are not publications or RS, they need to have their own scale of value too. The policy makers should build the corresponding scales and explain how they will be applied, mainly in comparison to the publication scale, while respecting the traditions and functioning of the involved community.\n\n\n4 The CDUR proposal\n\nIn this section we detail our proposal for the evaluation of research software (RS). We have labelled our proposal with the acronym CDUR, that stands for Citation, Dissemination, Use and Research. In what follows we will introduce each of these items from multiple perspectives, referring to the policy makers, to the evaluators and to the evaluated researchers, that is, to the key evaluation actors, as seen in section about the Key evaluation actors. Moreover we have chosen to present our proposal, first, in a summarized way, followed, then, by an extended description that develops in detail a multiplicity of choices for a very flexible application of the CDUR protocol.\n\nThe CDUR protocol contains four steps to be carried out in the evaluation of a RS. These steps are to be applied in the following chronological order: Citation, Dissemination, Use and Research. For example, we consider that, to facilitate dissemination, a RS should be, first, a well identified object; and in order to be correctly cited, the RS reference should be clearly indicated, as argued in the section about Referencing and citation. Let us introduce a resumed version of these four steps.\n\n(C) Citation. This step measures to what extent the evaluated RS is well identified as a research output, as a research object in its own. It is also the step where RS authors are correctly identified as well. We have seen in the RS publication section three different ways to establish a RS reference, in order to facilitate its citation. Moreover, a more evolved RS identification level could be provided in the form of a metadataset. Reference and metadata include, among other information, the list of the RS authors and their affiliations, as seen in the section about RS authors.\n\n(D) Dissemination. This step measures the quality of the dissemination plan for the RS, involving actions such as (see 29,30):\n\n– Choosing a license, with the agreement of all the rights’ holders and authors. Consider, preferably, using free/open source software licenses.\n\n– Choosing a web site, forge, or deposit to distribute the product; stating clearly licensing and conditions of use, copy, modification, and/or redistribution.\n\n– Creating and indicating a contact address.\n\nThis is the step related to legal issues dealing with the authors and rightsholders (as established in the Citation step) deciding and installing the license(s) for the RS dissemination 16,29,54,55. This is also the step concerning Open Science, as the RS license expresses its sharing conditions; and the step where policy makers should establish the Open Science policies that will be applied in the evaluation process.\n\nFinally, let us recall that the inclusion of the list of related publications, data sets and other related works in the dissemination procedure helps to prepare the reproducible science issues that are to be taken into account in the Use step.\n\n(U) Use. This step is devoted to the evaluation of the technical software aspects. In particular, this step measures the quality of the RS usage, considering that a performing RS is one that is both correct and usable by the target scientific community.\n\nThe RS usability does not only refer to the quality of the scientific output but also can deal with other matters, such as the provided documentation, tutorials and examples (including both inputs and outputs), an easy and intuitive manipulation, testing and version management, etc. 50.\n\nThis is the reproducible science step, where the published results obtained with the RS should be replicated and reproduced 32,56–58.\n\n(R) Research. This step measures the impact of the scientific research that has required in an essential way the RS under consideration.\n\nThe evaluation of this item should follow whatever standards for scientific research quality in the concerned community (e.g. 8,46,59,60).\n\nThis is the step where the RS related publications (as described in the RS definition section) come into play, and where the evaluation should consider the difficulty of the addressed scientific problems, the quality of the obtained results, the efficiency of the proposed algorithms, etc. The RS impact can also be assessed through the research impact of the related publications, and through its inclusion (or use) as software component in other RS.\n\nFinally, the CDUR procedure is meant to assist the evaluated researchers, the evaluator committees and the evaluation policy makers. It can be easily adapted to many different RS evaluation situations. It applies equally to any scientific area, as we concentrate our evaluation protocol in the general RS aspects, concentrating in the Research step those aspects specifically related to some particular areas.\n\nAs we have seen in the previous summary, the CDUR’s RS evaluation procedure considers a RS as a scientific output, measures the way it is identified and disseminated, and then takes into account the specific software and research aspects. We should remark that the realization of some Software Management Plan (SMP), such as those proposed in 35,61, can help the development team to reflect on and to prepare the different evaluation points. These plans can be made public and released jointly with the RS. In this way, SMPs can also help evaluators to achieve a better RS assessment. Note that the provision of such SMPs can be part of the evaluation process policies, as it is already the case of Data Management Plans when applying for EC funded projects 62.\n\nRegarding precedents to the CDUR protocol we can mention the Inria software description form 63, that was proposed in 2007 for RS assessment, where we can find points in common with the CDUR protocol. Both, the Inria form and CDUR have common ground with the software peer review methods mentioned in the RS publication section. In fact, to fill forms like the one proposed at Inria can help in the preparation of the CDUR evaluation. And, what is more relevant, as it happens with the SMPs, such Inria forms might help yielding and guiding some key reflection issues over the evaluated RS that could be, otherwise, somehow forgotten by the RS development teams. The generalized use of these or other similar forms should be decided by the RS evaluation committees or by the policy makers to set some standards in the evaluation procedure.\n\nIn what follows we will analyze and develop in detail the different points of the CDUR protocol.\n\n(C) Citation. In order to facilitate the RS citation, authors must set a reference. As we have seen in the Referencing and citation section, there are at least three possibilities for RS referencing and, moreover, they can cohabit. In order to facilitate the citation by others, one reference form should be put forward.\n\nThe reference should indicate, among others, the RS authors and their affiliations. In the case of a large list of contributors, it should give the list of the main contributors and refer to other documents (web page, RS documentation...) for the complete list of contributors. This basic step could be completed with the inclusion of DOIs or other persistent identifiers in the reference form.\n\nA more complex way for RS identification is the use of metadata sets. Note that the existing RS metadata sets can be adapted or completed in order to fit many different evaluation situations.\n\nWe consider that it is the RS authors’ role to set the best way to cite or identify their RS. On the other hand, and following the software citation principles in 7 (see also 27), authors should cite correctly other related works, with thorough attention to cite other research software components that could have been included in the RS or on which the RS depends upon.\n\nCitation and metadata are the tools to measure how easy is the access to RS. It is the role of the policy makers to set the required citation or metadata level that is best adapted to the evaluation context at stake, either by fixing a reference format or with adapted metadata sets.\n\nFinally, let us recall that evaluators should verify that the RS under evaluation complies with the citation or metadata characteristics required in the evaluation, and they should also check the correctness of the citations, in the given RS, to other external RS works.\n\n(D) Dissemination. RS dissemination should take into account that it can target different levels and types of public: from a very restricted set of persons, such as the closest collaborators of the RS team or the evaluation committee itself, up to the most general collection of addressees, through the dissemination of the RS via a web page, a software forge or deposit, either oriented to a scientific area or to a very general public. Moreover, in the case of a restricted dissemination of a RS having a widely available reference (because of the existence of related publications, for example), it is advisable to include a RS mail contact address to facilitate potential scientific collaborations.\n\nIn any dissemination procedure (as the ones proposed in 29,30) the RS sharing conditions should be clearly stated, as the running, loading, reproducing, translating or arranging of a computer program can only be done upon the corresponding (written) authorization, as stated by the law65. Thus, RS dissemination documents should include a license66 (or a written agreement, in the case of a restricted public dissemination) establishing the sharing conditions and describing the legal framework where the RS can be used, compiled, reproduced, copied etc. 16,29,54,55. Only the right holders can decide and set the RS license, hence the importance of including the list of authors and its affiliations, as described in the Citation step. Note that the license information could be included in the citation formula, and it is usually included in the RS metadata (using for example standards like SPDX (Software Package Data Exchange)67.\n\nThe license will determine if the software is free68 and open source69, and whether, as a research output, its dissemination fits the Budapest Open Access Initiative guidelines70. In this direction, the report 9 considers as a positive criteria in the evaluation of research careers, the regular use and development of free/open source software, fully acknowledging Open Science practices.\n\nAs part of the RS dissemination procedure, it should be taken into account the establishment of a list of related publications, data sets and other related works, that could be disseminated together with the RS, or that could be deposited elsewhere. In the latter case, let us remark that the links among all these objects will facilitate research reproducibility 32,56–58.\n\nStrong dissemination methods can include the deposit of the RS in places like the Agence pour la protection des programmes71 and the realization of Software Management Plans 35,61.\n\nFinally, let us mention that it is the task of the policy makers to set the Open Science policies that should be applied in the evaluation procedure, as well as to establish the required dissemination level and related characteristics that are best adapted to the evaluation context at stake, as mentioned in the above considerations. Furthermore, evaluators should verify and check that the presented RS complies with the established requirements regarding free/open access and Open Science issues, as well as with the dissemination practices set by the policy makers.\n\n(U) Use. This is the step devoted to the more technical software evaluation issues. The goal of this Use step is not to propose some software performance evaluation estimations, as for RS the most important characteristic is its scientific correctness, but different levels of software quality can be taken into account here.\n\nEvaluators considering a particular RS can have several approaches in mind. For instance, to check published results obtained through the use of the RS, in order to be able to find bugs or defects in the program that could affect the published results; to explore the scientific issues behind the RS computations (for example, to get better understanding of the implemented algorithms and the corresponding theoretical framework); to compare with other RS products; to take into account reproducible research issues 32,56–58 to measure the potential of the RS for the production of new scientific results...\n\nIn all these cases it is evident that the Use evaluation first goal should be to assess how much the RS team has facilitated its use, in concordance to the level of requirements set up by the policy makers. This basic step involves (i) checking how easy/difficult is to retrieve and install the RS, (ii) verifying if the RS has the necessary documentation and instructions to install, run and test the software, (iii) mentioning the requirements of the RS to some computing environments and to components that should be pre-installed, and (iv) providing the necessary examples and links to articles and other data, in order to help users to launch the first computations and to be able to verify and reproduce the already published results. In this regard, RS development teams preparing for a tough software examination procedure can find help in bibliographic references like 12,13,17,50,52,64–70 and the citations therein.\n\nEvaluators should launch the RS, run and verify some examples and compare the output with the expected results. However, they must also have a look to the code. In our vision, running some examples do not dispense the evaluators from looking the RS source code. This calls for evaluation committees provided with some reasonable software skills. This Use evaluation basic step could be facilitated if the RS is installed in platforms like IPOL 32, where the software can be launched and the source code is also available for its study in the same platform.\n\nBut the notion of usability is also related to the concept of scientific validation (16, p.133) and also to software verification and validation, including the verification of the code correctness, clarity and simplicity 71. RS verification and validation and RS code correctness can be assessed, for example, by using software inspection techniques see 72 and also 73, with a comprehensive survey of “software inspection” (sometimes referred to as software review or software peer review) literature during 1980–2008. Correctness is the highest priority for scientific software, as scientists do science rather than software 18,71.\n\nThus, before applying this Use step, it is required to establish the level of exigence for the RS testing, that can go from launching a few examples, to verifying if the RS provides a test suite and has installed testing procedures 1, up to inspecting carefully each line of the code.\n\nAs mentioned above, other points to be evaluated concern the level of documentation of the RS, the management of versions, the portability, bug tracking, user interfaces, governance, user support... 50. In this Use step, software assessment procedures can go quite way up, including, for instance, the verification of the application of ISO/IEC standards72. See also 51 for a discussion on FAIR software and the Software Seal of Approval.\n\nFinally, another issue that could be taken into account here concerns the relevance of the involved RS for technology transfer and industrial applications.\n\nLast, let us recall that policy makers should set the definition of good/validated/successful RS that should be applied in the corresponding evaluation context, and they should as well indicate the expected level of reasonable/good/best software development practices. Besides, it is recommended that RS evaluators consider setting an evaluation matrix taking into account the different software aspects to be evaluated and the rate scale to be applied in each case.\n\n(R) Research. This is the last step, the one where the standard research evaluation issues are to be taken into account 8,44,59,60 the place where the RS scientific value is to be assessed. Hence, it is also the point where the scientific software ecosystem requires full consideration 3 and where the related RS publication(s) that appear in the RS definition given in the definition dedicated section come into play, as their number, quality and impact reflects the quality and impact of the RS.\n\nAs this is the step for the evaluation of the research carried out with the RS and that is published in the related articles, the evaluation of both, software and articles, can be confounded in this global vision. Nevertheless, it is the intention of our proposition to put the RS at the center of the research evaluation that is under consideration, and policy makers and evaluators should decide the right balance between associated articles and software evaluation.\n\nMoreover, this step includes the evaluation of the difficulty of the addressed scientific problems, the quality of the obtained results and theories, the efficiency of the proposed (and coded) algorithms, the participation in funded projects, the measure of the potential of the RS for the production of new scientific results, etc.\n\nWe consider that the basic level of this Research step should rely on the number and quality of the related publications, and on the number of their citations. In fact, the dissemination levels of such documents provide indications about up to what point the RS is being widely adopted by the scientific community, yielding, therefore, an estimation of the impact of the whole research work (articles and software).\n\nActually, citation of software alone is still not a fairly well adopted behavior by the scientific community, so we must rely on the citations of the publications directly related to the RS. As a rough approximation to quality, we can measure publications’ impact through the number of citations. Indeed, as concluded in 74, citation numbers approximate with good accuracy the perceived impact of scientific publications. On the other hand, as we can see in 75, results in both studies [...] indicate that papers with code available online are more highly cited than those without. Thus, impact of some RS and impact of its related articles are, again, confounded.\n\nIn this Research step we can also take into account another evaluation item, namely to consider the estimated number of RS users. A widely used software product can also attract new funding and new collaborations 16 (point about quality and evaluation):\n\nLa qualité d’un logiciel peut se mesurer par celle des articles associés au logiciel, mais aussi par le nombre d’utilisateurs qu’il est capable d’attirer, de collaborations et de contrats qu’il est capable de générer.\n\n[The quality of a software can be measured by the quality of its associated articles, but also by the number of users that it is able to attract, and the collaborations and contracts that it is able to generate.]\n\nNote that the citation number of a publication provides somehow an estimation of the number of its users, that is, the readers of the publication. Likewise, the citations of the RS related publications can provide another (rough) measure of the number of RS users. Moreover, a RS can also be included as a component in other RS, but this kind of impact is difficult to assess as, again, we cannot rely on pure RS citation issues yet.\n\nThus, the basic level of this Research step should rely on the number and quality of the related publications, the estimation of the number of their citations and the estimation of the RS citations (as a research output or as an included component) whenever possible. Higher levels of research quality evaluation should assess up to which point the RS is widely adopted by the scientific community and the impact of the whole research work (articles and software).\n\nSimilarly, to the previous Use step, the policy makers should set the definition of good/validated/successful RS that would be applied in the corresponding evaluation context as well as to indicate the expected level of reasonable/good/best research practices. Besides, it is recommended that RS evaluators set an evaluation matrix taking into account the different research aspects to be evaluated and the rate scale to be applied.\n\nAs we have already seen in the extended presentation of the CDUR procedure, each step in the protocol proposes to consider different elements of achievement. Each of these elements can reach different levels and the corresponding scale is to be set up by the policy makers considering a particular evaluation event. Thus, our protocol can be easily adapted to different circumstances: career evolution, recruitment, funding, RS peer review or other procedures to be applied by universities and other research institutions, research funders, or scientific journals, and it can also be adapted to different situations arising in different scientific areas. As mentioned before in the detailed explanation, each CDUR step is associated to some RS important issues:\n\n(C) Citation. This step considers the citation issues that require setting a reference, and the identification of RS authors. The legal issues that appear in here correspond to the intellectual property associated to the authors and their affiliations.\n\n(D) Dissemination. If the RS is disseminated, it should be under a correct free/open source software license, and following best dissemination practices. This is the step where Open Science issues are most relevant.\n\n(U) Use. This step is devoted to software use and correctness, and it is also the step associated to reproducibility issues. It can be enhanced with best software practices.\n\n(R) Research. This is the step associated to research quality and its impact.\n\nIn CDUR, each of these issues has been put in a particular place in the whole protocol, that is to be applied as a set of chronologically ordered steps.\n\nLet us remark here that the CDUR protocol is clear in discriminating the diverse roles of the evaluators, the policy makers and the evaluated researchers, and identifies the level of policies and the different actions to be put into place by the different key evaluation actors.\n\nIn CDUR, policy makers should determine the relevance and the balance between the pure technical software aspects and the research aspects. RS development can involve high levels of both, technical software skills and research expertise, and in our view, it is important to recognise when the evaluation process is dealing with software skills or with research. For example, it is not the same to detect poor software practices in the recruitment of a mathematics researcher than to detect that the produced RS presents a severe lack of correctness. Besides, if good citation practices are missing, this matter could be easily improved, mostly if candidates know in advance which will be the good practices that are to be taken into account.\n\nSo, in CDUR, not only can each step be applied with flexibility, but also the whole protocol can be adapted to different situations by the evaluation committees, following the policy makers’ requirements. The only drawbacks that we have found are the necessary transparency in the establishment of the applied protocol as well as the necessity of balanced committees with both research and software skills. On the other hand, to raise these drawbacks and to understand when they are relevant in the evaluation protocol also helps to tackle them correctly, which becomes another of the CDUR’s benefits.\n\nFinally, to complete the list of the protocol advantages, let us consider CDUR in the context of the world brain vision of 11 (chapter 2). This chapter lists ten principles that can guide the future of scholarly communication. But we note that, when comparing article and RS dissemination, a fundamental difference arises. Up to now, RS dissemination is predominantly in the hands of the RS producers, a rather different key actor than those ruling journals in scholarly publishing. This is one of the reasons why sound evaluation procedures are capital for the progress of RS issues in the scientific ecosystem. Consequently, it is our belief that the adoption of evaluation protocols like CDUR will contribute to support the above mentioned principles, such as:\n\n– maximize RS accessibility and usability,\n\n– support and expand range of contributions with equity, diversity and inclusivity criteria,\n\n– support community building, and\n\n– promote high-quality research with heightened integrity;\n\nwhich will have repercussions in the whole scholarly communication system.\n\n\n5 Conclusion\n\nIn this paper we have analysed the concepts of research software (RS), its authors, and the issues related to RS publications, referencing and citing. Then we have studied the evaluation issues such as the existing methods and its key actors. Regarding more specifically RS evaluation, we have detailed the ideas around the concept of successful software and its value scale in a scientific community. These preliminary steps open the path to the proposition of the CDUR protocol for RS evaluation, that comprises four steps dealing with Citation, Dissemination, Use and Research, and that are to be applied in this chronological order. This protocol and its advantages have been thoroughly investigated, including the different actions and decisions of the key actors (policy makers, evaluators, evaluated researchers) and the wide flexibility for its application in several contexts.\n\nResearch Software production is already part of the daily activities of many researchers, but it is still not sufficiently recognized in the research evaluation procedures that are being currently applied in the scientific world, as far as we know. On the other hand, the difficulties of scientists and engineers in finding software of their interest have been studied in 4,5 and involve a collection of serious drawbacks affecting RS development such as, for example, work duplication, reduced scientific reproducibility and poor return of funding agencies’ investment. Indeed, RS limited visibility means that incentives to produce high-quality, widely shared, and codeveloped software may be lacking.\n\nThus, we consider that it is in the interest of the research communities and institutions to adopt clear and transparent procedures for the evaluation of research software. Procedures like the proposed CDUR protocol facilitate RS evaluation and will, as a consequence, improve RS sharing and dissemination, RS citation practices and, thus, RS impact assessment. This is an important step for the recognition of RS production and, therefore, to help scientists towards better, more efficient research.\n\nClearly, a policy is only as good as its enforcement 4 (p.15). Procedures such as CDUR are exigent regarding transparent decisions. When seeking quality results, it is generally advisable to avoid having social factors to take a relevant role in the evaluation process. Indeed, social evaluation methods could be as good as any other, if they would drive a similar levels of quality, as with the qualitative or quantitative methods. But the social methods should be applied quite consciously rather than unconsciously, and the moment when these practices come into play should be clearly detected and highlighted. The main drawback is that, in this case, it is usually difficult to refer to transparent policies and decisions. In other words, when transparency is at stake, social influence in evaluation procedures should be neutralized, as seen in the evaluation section.\n\nMany of the RS points discussed here have common issues with research data evaluation. For example, as we remark in 29, research data and RS could be disseminated following the same procedure. Therefore, it is easy to conceive a similar CDUR evaluation protocol for research data, suitably modified to take into account some of its specific features, mainly by adapting the Use step to data use. The other steps, Citation, Dissemination, and Research can have a pretty similar presentation by changing RS for research data, but taking into account the fundamental differences that appear between software and data legal issues.\n\nAs proclaimed in 11, the evaluation of research is the keystone for the evolution of the Open Science policies and practices. It is our belief that research evaluation is also the keystone for the evolution of research software practices and for the full consideration of its role towards a more efficient science.\n\nAs a final conclusion, we hope that the adoption of protocols such as CDUR will motivate and consolidate evaluation policies and practices. Yet, as warned in 10 (p.2), Enacting any new policy that requires a shift in culture also will require community support for successful and efficient implementation. Evolutions will come, some are already there. Therefore, the future roles of the RS key evaluation actors are likely to evolve and change current practices, which carries with it challenges and opportunities. The RS roles could evolve as part of the whole scholarly communication or on their own, probably both at the same time. There will be movement backwards and forwards, but, in our view (and in agreement with the EC Expert Group report 11) the success of the foreseen evolutions will be associated to initiatives that put researchers’ aims at the center of the various interests. Success will come if the different actors participating to build the future will work closely with the researchers, to create and to provide procedures and services that are valued and trusted by them.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nPublication of this article is supported by the Gaspard-Monge computer science laboratory (LIGM) at the University of Paris-Est Marne la Vallée.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis work is partially in debt to the research software producers of the Gaspard-Monge computer science laboratory (LIGM) at the University of Paris-Est Marne-la-Vallée, where LIGM RS production has been studied by the first author since 200673.\n\n\nFootnotes\n\n1In what follows we will use often the acronym RS to refer to research software in order to facilitate the reading of this article.\n\n2Our work can be also extended to deal with software driven by technological development – the development (D) in research and development (R&D), as observed in 8 (p.595) – or with scientific software being developed in private institutions or commercial enterprises, albeit requiring some specific adjustments, as, for example, the adaptation of the Research step.\n\n3https://software.ac.uk/\n\n4https://rse.ac.uk/about/\n\n5https://rse.ac.uk/rse-international-leaders-meeting/\n\n6https://software.ac.uk/\n\n7The Engineering and Physical Sciences Research Council, https://epsrc.ukri.org/\n\n8https://software.ac.uk/news/2010-06-01-future-software\n\n9https://openresearchsoftware.metajnl.com/\n\n10https://software.ac.uk/programmes-and-events/fellowship-programme\n\n11http://urssi.us/\n\n12http://wssspe.researchcomputing.org.uk/\n\n13https://www.force11.org/group/software-citation-implementation-working-group\n\n14PLUME stands for Promouvoir les Logiciels Utiles, Maîtrisés et Economiques dans la communauté de l'Enseignement Supérieur et de la Recherche. Although the project and the platform are frozen, all the produced information is online, see https://projet-plume.org/ and https://projet-plume.org/en.\n\n15Centre National de la Recherche Scientifique, http://www.cnrs.fr/\n\n16ENVOL stands for dEveloppemeNt et la ValOrisation des Logiciels en environement de recherche [software development and valorization in research environment] and was a full training week organized by the PLUME team and funded by the CNRS, see https://projet-plume.org/ENVOL_2008\n\n17https://projet-plume.org/ressource/journees-plume-diffuser-en-libre\n\n18http://www.projet-plume.org/patrimoine-logiciel-laboratoire\n\n19http://devlog.cnrs.fr/\n\n20JDEV stands for Journées nationales du DEVeloppement logiciel and is a 4 day national workshop that gathers developers in the research environment. See for example JDEV 2017 at http://devlog.cnrs.fr/jdev2017\n\n21https://www.canarie.ca/\n\n22https://www.canarie.ca/software/\n\n23https://www.canarie.ca/software/canadian- research- software- conference/canadian- research- software- conference- 2018/program/\n\n24http://nl-rse.org/pages/community.html\n\n25http://www.esciencecenter.nl/\n\n26https://escience-platform.nl/\n\n27https://www.de-rse.org/en/\n\n28https://www.de-rse.org/en/conf2019/index.html\n\n29https://www.dagstuhl.de/de/programm/kalender/semhp/?semnr=16252\n\n30DANS is the the Netherlands institute for permanent access to digital research resources, see https://dans.knaw.nl/en\n\n31https://dans.knaw.nl/nl/actueel/software-sustainability-workshop-7-9-march\n\n32https://software.ac.uk/wosss19/agenda\n\n33https://researchsoftware.org/\n\n34https://www.software.ac.uk/resources/guides/which-journals-should-i-publish-my-software\n\n35https://onlinelibrary.wiley.com/journal/1097024x\n\n36Unfortunately, we have no free access to this journal, which hinders our goal of presenting a short comparison with more recent approaches to RS publishing.\n\n37https://riojournal.com/\n\n38https://www.journals.elsevier.com/software-impacts/\n\n39http://joss.theoj.org/\n\n40https://joss.readthedocs.io/en/latest/review_criteria.html\n\n41https://www.journals.elsevier.com/softwarex/\n\n42https://www.elsevier.com/__data/assets/pdf_file/0010/97066/ReviewerForm.pdf\n\n43https://openresearchsoftware.metajnl.com/\n\n44https://openresearchsoftware.metajnl.com/about/editorialpolicies/\n\n45http://www.projet-plume.org/en\n\n46https://projet-plume.org/en/relier/treecloud\n\n47https://www.projet-plume.org/types-de-fiches#dev_ens_sup_recherche\n\n48Similar descriptions can be also found at the Netherlands eScience Center, see https://research-software.nl/\n\n49Validated software descriptions are only available in the French side of the platform and were done for well-known software used within the academic community. Part of these validated software were developed by researchers, see https://www.projet-plume.org/fiches_logiciels_dev_internes for the list of RS validated software descriptions published in French.\n\n50http://ligm.u-pem.fr/\n\n51https://projet-plume.org/en/relier/unitex\n\n52https://projet-plume.org/relier/unitex\n\n53https://projet-plume.org/fiche/unitex\n\n54http://www.ipol.im/\n\n55https://peercommunityin.org/\n\n56https://www.force11.org/group/software-citation-implementation-working-group\n\n57https://integrity.mit.edu/handbook/citing-your-sources/avoiding-plagiarism-cite-your-source\n\n58https://f1000research.com\n\n59http://en.wikipedia.org/wiki/Persistent_identifier\n\n60https://www.force11.org/group/7784/google-forum\n\n61Agenda and videos are available at https://www.academie-sciences.fr/en/Colloquia-Conferences-and-Debates/foresighting-open-science.html\n\n62http://www.arxiv.org\n\n63https://zenodo.org/\n\n64For a recent analysis of different behaviors regarding publication practices and Open Science issues, you can see the (French) presentation of Jean-Pierre Bourguignon, President of the European Research Council, the 2nd April 2019, at the Académie de Sciences in Paris, https://public.weconext.eu/academie-sciences/2019-04-02/video_id_010/index.html\n\n65See for example the Directive 2009/24/EC of the European Parliament and of the Council from 23 April 2009 on the legal protection of computer programs, https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A32009L0024\n\n66Please note that no license means All rights reserved.\n\n67https://spdx.org/\n\n68Free software is defined by the Free Software Foundation (FSF) in https://www.gnu.org/philosophy/free-sw.en.html\n\n69Open source software is defined by the Open Source Initiative (OSI) in https://opensource.org/docs/osd.\n\n70https://www.budapestopenaccessinitiative.org\n\n71https://www.app.asso.fr/en\n\n72See for example ISO/IEC 9126 Software engineering - Product quality at https://en.wikipedia.org/wiki/ISO/IEC_9126.\n\n73http://igm.univ-mlv.fr/~teresa/2013octPostersAERES/PatrimoineLogicielLIGM/LogicielsLIGMPlume2013_EN.pdf\n\n\nReferences\n\nKanewala U, Bieman JM: Testing Scientific Software: A Systematic Literature Review. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelly D, Sanders R: The challenge of testing scientific software. Proceedings 3rd Conference for the Association for Software Testing (CAST), Toronto, 2008; 30–36. Reference Source\n\nKelly D, Shepard T: Eight maxims for software inspectors. Softw Test Verif Rel. 2004; 14(4): 243–256. Reference Source\n\nKollanus S, Koskinen J: Survey of Software Inspection Research. The Open Software Engineering Journal. 2009; 3: 15–34. Reference Source\n\nRadicchi F, Weissman A, Bollen J: Quantifying perceived impact of scientific publications. J Informetr. 2017; 11(3): 704–712. Publisher Full Text\n\nVandewalle P: Code sharing is associated with research impact in image processing. Computing in Science and Engineering. 2012; 14(4): 42–47. Publisher Full Text" }	[ { "id": "53711", "date": "13 Sep 2019", "name": "Francisco Queiroz", "expertise": [ "Reviewer Expertise Digital and interactive design", "UX", "UI", "Scientific Software usability." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is a clear, well-written article outlining a novel procedure that could impact, in positive ways, policies and practices regarding scientific software development and impact. However, I believe there are some aspects of the article that could be either improved or clarified.\nThe study seems to be mostly based on secondary research – more specifically, arguments and ideas are developed based on findings from literature. Moreover, it can be assumed, the study is informed by the proximity of the authors to the academic/professional environment it investigates. However, there is no clear indication on how the material that serves as basis for the analysis was selected (indexed databases? specialized resources such as publications and conferences?), in which case it would be interesting to include a justification on why those sources are relevant.\n\nAdditional material that could, potentially, be referenced are Chue Hong`s position paper on the need for a framework for comparing software1; and also ESRC's plans of developing a software accreditation framework2.\n\nIn section 2, the authors write: \"Note that to cite software in science is different to citing scientific (or more precisely, research) software, which is the issue here\". Perhaps the difference could be clarified?\n\nIn 2.1, the authors write: \"in 12,13 we can find the following definition\". Given that the definition`s authorship is originally from 12, it would be probably more appropriate to write something along the lines of \"a definition elaborated by 12 was summarized by 13 as\"\n\nThere are several passages in both French and English. Wouldn't it be enough to keep the English versions (along with an indication that they were translated from French by the authors?)\n\nThe authors write: \"Finally, let us mention that, as in the case of publications, the research software production of a laboratory is decided and proposed by the lab’s members, and it is approved by the leading institutions during the usual laboratory evaluation and funding procedures\". Is that true for every case of research software production and are there any references supporting that?\n\nFigure 1, illustrating concepts appearing in the study, could be improved, possibly by matching label colors to the colors of the rectangles for easier identification.\n\nIs not clear why there are two screenshots of Plume (one in French and one in English). One would probably be enough?\n\nThe authors write: \"(...) we remark that there can be several classic articles associated to a single RS, and that could be used as its reference\". Could references to some of those classic articles be provided?\n\nThe study ends without (i) considerations on future research, (ii) clear indications on initiatives/pilots for the procedure, (iii) a call to action for the community to test it. Overall, it's not clear what the next steps are.\n\nRegarding the applicability of the procedure, the article would benefit from including a table or diagram (probably in Section 4) explaining the procedure, preferably featuring a clear, step-by-step description of the process. That should clarify how CDUR could be adopted, increasing the potential for its adoption and use.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "52525", "date": "23 Sep 2019", "name": "Jean-Pierre Merlet", "expertise": [ "Reviewer Expertise computer science", "robotics", "mathematics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper gives a good definition of what is a research software (RS) and I agree with the authors that an extensive version of what is a RS. I have however various remarks:\nA career of a researcher has extensively changed in the recent years: more chairs available for a relatively short period of time (typically 5 years) are given so that in this limited amount of time a relatively young researcher cannot afford to stand by the dissemination and maintenance work required by the evaluation scheme provided by the authors\n\nThe authors still favor a specific type of RS, with availability, documented, licensed and with versioning. This is dangerous as many RS do not enter in this category: software that is produced to check a specific scientific result, software specifically designed to manage a particular system etc.. These software are not intended to be disseminated either as they are used only for checking and benchmarking or because they are very specific (for example a real-time software where the purpose is to save as much as possible computation time so that it cannot be general by essence as any general procedure will require tests that will penalize the performances) and therefore cannot be reproducible. As developing software is time intensive having too strict rule for the evaluation of software (such as documentation and availability) may discourage researchers producing them although they provide some good idea of the efficiency of the underlying theoretical work.\n\nCitations of publications as a measure of impact is already questionable and I will say this is even worse for RS. It is quite current in the computer science community to say that the only good software is the one produced by the authors so that citations is limited and usually not very positive.\n\nI fully agree that software development may be a major component in the daily activity of a researcher and therefore must be taken into account in our evaluation. However the specificities of the RS and of the domain must also be taken into account in this evaluation. The CDUR procedure proposed by the authors is very fine but can be applied only on a limited number of RS types and his application will be disastrous for other types. For example it is easy to find RS for which the CDU part is not relevant while the R part is the only one of importance: for example a RS that performs the control of a specific robotic system cannot be reproducible (unless the reviewer has at hand an exactly similar robot), its use is limited to a specific prototype (and the RS has been designed for it) and has to bey tailored to manage another system so it cannot be disseminated. Hence the only evaluation criteria is the R part which is illustrated by the performance of the whole system, being given the performance of the hardware.\n\nI recognize that the authors are right in term of objectives: our current approach of RS development may lead to a waste of time with researchers programming again and again the same algorithms instead on focusing on their specific objectives. Some kind of mutualization will be beneficial provided that he RS is sufficiently disseminated and open to the community. But a researcher has to find the right balance between his/her research activity and the time devoted to maintenance, documentation and dissemination, a balance that greatly depends upon the domain. Furthermore good software practices for development is also dependent of the domains.\nIn summary although the authors have been very careful in their definition and evaluation rules and the absolute necessity of flexibility in the evaluation rules, they ended up with an evaluation procedure that may lead researcher to avoid going into the business of RS development. But this is nice paper that is worth being indexed for opening a discussion on RS evaluation according to the specific domain and context in which it has been developed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1353
https://f1000research.com/articles/8-1993/v1	26 Nov 19	{ "type": "Brief Report", "title": "Identification of Mycoplasma genitalium from clinical swabs by direct PCR", "authors": [ "Robinson M. Irekwa", "Perpetual Ndung'u", "Peter Kipkemboi", "Tonny Teya", "Anne Wanjiru Mwangi", "Matthew Mutinda", "Caroline Njoroge", "Joanne Yego", "Irumva Vanessa", "Samson Muuo Nzou", "Perpetual Ndung'u", "Peter Kipkemboi", "Tonny Teya", "Anne Wanjiru Mwangi", "Matthew Mutinda", "Caroline Njoroge", "Joanne Yego", "Irumva Vanessa", "Samson Muuo Nzou" ], "abstract": "Mycoplasma genitalium is one of the smallest self-replicating organisms. It is an obligate parasite found in the human genital tract. In men, the bacteria cause both acute and chronic non-gonococcal urethritis (NGU). In women, it has been associated with pelvic inflammatory disease and cervicitis among other related infections. Treatment of M. genitalium related infections has been effective using antibiotics such as the macrolides (e.g. azithromycin) and fluoroquinolones. However, there have been recorded cases of resistance to these antibiotics in various parts of the world as a result of a mutation in the 23SrRNA gene, although the antibiotic resistance has not been well established. The aim of this study was to detect M. genitalium in 352 swab samples collected from a clinic for sex workers in Nairobi, Kenya. DNA was extracted from the swabs and stored as a crude extract at -31°C. The swab lysates were subjected to direct polymerase chain reaction using primers that specifically target the 16S rRNA gene for M. genitalium. A total of 29 samples tested positive for M. genitalium. The data results showed a M. genitalium prevalence of 8.24% among sex workers in Nairobi, Kenya.", "keywords": [ "Direct PCR", "Mycoplasma genitalium" ], "content": "Introduction\n\nMycoplasma genitalium is an emerging sexually transmitted disease that was first identified and isolated in 19801 from men with non-gonococcal urethritis (NGU). Its epidemiology in connection to other STI syndromes been established since nucleic acid amplification assay development in the early 1990s2. The bacteria have been detected in substantial amounts from men with urethritis and women with cervicitis3. M. genitalium prevalence in the general population has been studied and found to be ranging between 1–3%4.\n\nM. genitalium is found in roughly 15% of men with NGU and in 22% of men with non-chlamydial NGU. However, the associated infections do not have unique clinical symptoms, making it difficult to use clinical signs as a mode of identification5. Cervicitis has been described as the female version of male urethritis. M. genitalium is found in 10% of women with cervicitis6. Chlamydial coinfections in women with cervicitis are also common in some settings7. M. genitalium is a very fastidious bacterium and culturing of the bacterium is exhaustive and time consuming.\n\nThe introduction of polymerase chain reaction (PCR) assays has provided the necessary data for its clinical prevalence8. Many assays have been developed for the detection of M. genitalium in human specimens8–17. Most of these assays are mainly based on the PCR detection technique. Use of these PCR tests have shown that the disease spectrum is similar to those caused by Chlamydia trachomatis and Neisseria gonorrhoeae in both males and females13. However, these assays differ in their target DNA sequences, specimen preparation and amplicon detection methods. Many of these detection methods target the 16S rRNA and the MgPa protein genes. Conventional and, more recently, real-time PCR assays have been applied. Most of the detection studies have been conducted in the U.S.A, Europe and Australia, with various strains being discovered. In line with the detection of the bacterial species and its related infections in Africa, more studies need to be conducted on possible strains and their epidemiology. Whether the bacteria have links with other sexually transmitted infections can also be investigated. In this study, it is shown that direct PCR can be applied to the detection of M. genitalium from crude DNA extracts. M. genitalium prevalence and characteristics among female sex workers have been studied in Kenya and Uganda18–21. Its prevalence has also been studied among males who underwent circumcision in order to prevent HIV acquisition in Kisumu, Kenya22. However, most of these studies have focused on conventional, real-time PCR or transcription-mediated amplification assays for the detection of M. genitalium. This study reports the use of direct PCR for M. genitalium detection from crude DNA extracts using specific primers that target the 16S rRNA gene.\n\n\nMethods\n\nThis study was approved by the Jomo Kenyatta University of Agriculture and Technology Institutional Ethics Review Committee (JKUAT-IERC): reference number JKU/2/4/896B. The swab samples were collected with written informed consent for the performance of further analysis.\n\nThe samples used in this study were collected as part of the sex workers outreach program (SWOP) central business district clinic in Nairobi, Kenya. As part of this program, patients who showed STI symptoms and consented to the study were sampled by taking vaginal swabs. The specimens were then put into sterile containers and transported to the Pan Africa Hub Laboratory (NUITM-KEMRI) within an hour and stored at -80°C. Anonymized samples were retrieved for use in this study.\n\nThe 352 swab lysates were prepared using the MightyPrep reagent for DNA (TAKARA BIO INC, Kusatsu, Shiga Prefecture, Japan; Cat No: 9182) using the manufacturer’s protocol with a slight modification. Swabs were cut and put into 1.5ml Eppendorf tubes. A total of 200uL of the MightyPrep reagent was added to the tubes and later centrifuged at 15krpm for one minute. The tubes were then transferred to a heated block at 95°C with shaking at 800rpm for 15 minutes. Later, the tubes were cooled down by lowering the heat block temperature to 25°C, followed by hard vortexing of each tube for one minute and, finally, centrifugation at 15krpm for two minutes before storage at -31°C.\n\nThe master mix was prepared using the manufacturer’s protocol with slight modifications (Hotstar Taq® Master Mix Kit 2.5 Units, Qiagen; Cat No: 203443). 100μM primer concentration was achieved by adding 303μl and 353μl of Tris EDTA (Nippon Gene Company Ltd, Japan, Cat No: 314-90021; 10mM Tris-HCl [pH 8.0], 1mM EDTA [pH 8.0]) to the forward and reverse primers (Table 1; Sigma-Aldrich, Darmstadt, Germany), respectively. The primers (Table 1) targeted the 16S rRNA gene23 giving 433bp amplicon size fragments. Master mix components were: RNase- free water (1x = 7.84μl); primer mix (1x = 0.08μl, 0.2μM of each primer); and HotStar Taq® master mix (2x), comprised of 2.5 units HotStarTaq DNA polymerase (1x = 10μl),1x PCR buffer (1x, contains 1.5mM MgCl2) and 200μM of each dNTP.\n\nAfter vortexing the master mix for five seconds, 18μl was aliquoted to each of the labeled 96 PCR tubes. 2μl of the swab lysates was added to each tube to make a final reaction volume of 20μl and the tubes were finger tapped for five seconds to mix the contents. A positive control (M. genitalium positive sample) and negative control (PCR water) were used. The PCR tubes were placed in the SimpliAmp™ Thermal Cycler (Applied Biosystems) and run under the following reaction conditions.\n\nAn initial antibody inactivation step was carried at 95°C for 15 minutes, followed by 35 cycles of: denaturation at 94°C for 60 seconds, annealing at 67°C for 60 seconds and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes, followed by the final hold at 4°C for ∞.\n\nThe PCR products were subjected to gel electrophoresis using 2.5% agarose gel SeaKem® GTG® agarose (Lonza, Rockland, ME, USA; Cat No: 50074) at 100V for 45 minutes. 6x loading dye (Nippon Gene; Cat No: 314-90261) was diluted with sample to make 1x and loaded onto the gel. The 100bp GelPilot® Ladder marker (Qiagen; Cat No: 239035) was used. The gels were stained with 2x GelRed™ Nucleic Acid Gel Stain (Biotium; Cat No: 41003) for one hour on a shaker. The image was viewed using the UltraSlim UV Transilluminator (Maestrogen).\n\nThe PCR products were subjected to another PCR. Master mix components were as described above. 18μl was aliquoted into the PCR tubes. 1μl of the sample products was added to the tubes to make a 19 μl final volume. The PCR tubes were placed in the SimpliAmp™ Thermal Cycler (Applied Biosystems) and run under the following reaction conditions.\n\nAn initial antibody inactivation step was carried out at 95°C for 15 minutes, followed by 30 cycles of: denaturation at 94°C for 60 seconds, annealing at 69°C for 60 seconds and extension at 72°C for 60 seconds. A final extension step was carried out at 72°C for 10 minutes, followed by the final hold at 4°C for ∞.\n\nThe products were run on a 2.0% agarose gel at 100V for 40 minutes. A 3000bp ladder (Solis BioDyne, Tartu, Estonia) was used. The gels were stained using 2x GelRed for one hour and viewed under an UltraSlim UV Transilluminator.\n\n\nResults\n\nA total of 352 lysates were analyzed in this study. The results show evidence for the presence of M. genitalium from swabs taken from the female sex workers who were sampled. 352 lysates were prepared using the MightyPrep reagent.\n\nM. genitalium was detected among the 352 swab lysates. Examples of M. genitalium detection are shown in Figure 3 to Figure 4. Figure 1 shows clear bands at positions 1, 2, 9, 10 and 17 on a 26-well agarose gel. The same kind of bands can be seen in Figure 2 at positions 9, 10, 11 and 18. The positive and negative controls are at positions 24 and 25, respectively, on each gel. A 600bp ladder was used at positions 1 and 26 to track the M. genitalium amplicon sizes of interest.\n\nUltraviolet camera gel image showing 22 samples run on a 26-well gel. The ladder is at positions 1 and 26 (Gel Pilot®). Clear Mycoplasma genitalium positive bands can be seen at positions 1, 2, 9, 10 and 17 (Sexually transmitted infection lysates S099, S100, S108, S109 and S116, respectively). The positive control (PC) and negative control (NC) are at positions 24 and 25, respectively.\n\nUV camera gel image showing the 22 samples run on a 26-well gel. The first and last wells represent the ladder (GelPilot®). Clear Mycoplasma genitalium positive bands can be seen at positions 9, 10, 11 and 18 (lysates S137, S138, S139 and S146, respectively). The positive control (PC) is shown at position 24 and the negative control (NC) at position 25.\n\nThe selected PCR products were amplified: the above image shows the first nine PCR products run on a gel after the amplification was conducted. The ladder (Solis BioDyne) is at the first and last lanes. The positive control (PC) is at lane 11, while the negative control (NC) is at lane 12.\n\nThis gel shows PCR products of the samples that were selected for amplification. The first and last lane contain the ladder marker (Solis BioDyne), the PCR products run from lanes 2 to 10 and the positive control (PC) is at position 11, while the negative control (NC) is at position 12.\n\nThe PCR products were subjected to another amplification reaction. After the reaction, the products were run on a 13-well agarose gel. A 3000bp ladder was loaded on positions 1 and 13, with the positive and negative controls at positions 11 and 12, respectively, as can be seen in Figure 3 and Figure 4. Out of the 352 swab lysates used, 29 tested positive for M. genitalium.\n\nM. genitalium prevalence among the cohort of female sex workers was found to be at 8.24% (29/352), showing one out of eight patients had M. genitalium related infections.\n\n\nDiscussion\n\nRecently, DNA amplification protocols using PCR have been employed in the detection of M. genitalium. To investigate the presence of M. genitalium from the clinical swab samples collected from a clinic for sex workers in Nairobi, Kenya, a direct PCR technique was used for the detection of M. genitalium. This technique involves the use of target-specific primers to select the DNA of interest from a crude extract. The study detected M. genitalium using primers that bind to the 16SrRNA gene from the crude DNA extract. Jensen and his colleagues23 developed a wide range of primers that target the 16S rRNA gene, producing different amplicon sizes. A novel PCR was used24 to detect M. genitalium using oligonucleotide primers that corresponded to sequences along its 16S rRNA gene.\n\nThe study was able to detect 29 M. genitalium positive samples out of the 352 lysates. However, the challenge experienced with this method was non-specific amplification, realized from the multiple fragments produced. A possible solution to this is in the use of more precise target-specific primers to prevent the amplification of genes with closely related sequences. Application of this method can be a remedy to the constant loss of DNA due to long extraction processes, at the same time maintaining its quality for further downstream analysis.\n\nM. genitalium prevalence was shown to be at 8.24%. This shows that one out of every eight patients sampled was positive for M. genitalium related infections. Balkus and colleagues in 2018 were able to detect M. genitalium from 25 out of 221 (11.3%) women from Kenya and the US25. Prevalence rates of 12.9%18 and 16%19 have also been reported among sex workers in Nairobi, Kenya. The prevalence obtained in this study therefore does not show any significant drop in M. genitalium infections. Despite better and improved access to healthcare, M. genitalium infections seem to continue to be a burden. Possible reasons might be due to having multiple sex partners26 or antibiotic resistance to drugs of choice such as macrolides and fluoroquinolones27,28\n\nOverall, the prevalence results suggest that more measures need to be taken to control M. genitalium infections. Awareness campaigns need to be carried out to sensitize people on preventive measures rather than taking potential risks that may lead to exposure to the infection. Studies need to be done to investigate M. genitalium drug resistance. This will be helpful in informing policy and practice. As a result, screening can be done in patients to check for resistance before prescribing medication.\n\n\nData availability\n\nFigshare: Detection of Mycoplasma genitalium using Direct PCR. https://doi.org/10.6084/m9.figshare.10282691.v129.\n\nThis project contains the following underlying data:\n\n- Direct PCR 1.docx – Direct PCR 4.docx (lists of the samples tested for Mycoplasma genitalium in four sets of 88 samples)\n\n- Exp 1 Gel 1.JPG - Exp 4 Gel 4.JPG (gel electrophoresis of PCR products; 100bp GelPilot® Ladder marker [Qiagen] at positions 1 and 26, samples from positions 2 to 23, positive control at position 24 and negative control at position 25)\n\n- Amplification Gel 1.JPG - Amplification Gel 3.JPG (gel electrophoresis of the amplified products; 100bp ladder [Solis BioDyne] at positions 1 and 13, samples from positions 2 to 10, positive control at position 11 and negative control at position 12)\n\n- Amplification Gel 4.JPG (gel electrophoresis of the amplified products; 100bp ladder [Solis BioDyne] at positions 1 and 13, samples from positions 2 to 7, positions 8, 11 and 12 contain no samples [blanks], positive control at position 9 and negative control at position 10).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank the Nagasaki University Institute of Tropical Medicine in collaboration with the Kenya Medical Research Institute (NUITM-KEMRI), The Pan Africa University Institute of Science Technology and Innovation hosted at the Jomo Kenyatta University of Agriculture and Technology, Kenya (PAUSTI-JKUAT) for the research conceptualization and support.\n\n\nReferences\n\nHuengsberg M: Sexually Transmitted Diseases. 3rd ed. Ed King K Holmes, P Frederick Sparling, Per-Anders Mardh, Stanley M Lemon, Walter E Stamm, Peter Piot, Judith N Wasserheit. $170.50. New York: McGraw Hill, 1999. Sex Transm Infect. 2000; 76(6): 498. Publisher Full Text\n\nTully JG, Cole RM, Taylor-Robinson D, et al.: A Newly Discovered Mycoplasma In The Human Urogenital Tract. Lancet. 1981; 1(8233): 1288–91. PubMed Abstract \| Publisher Full Text\n\nTaylor-Robinson D, Jensen JS: Mycoplasma genitalium: from Chrysalis to Multicolored Butterfly. Clin Microbiol Rev. 2011; 24(3): 498–514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersen B, Sokolowski I, Ostergaard L, et al.: Mycoplasma genitalium: prevalence and behavioural risk factors in the general population. Sex Transm Infect. 2007; 83(3): 237–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWetmore CM, Manhart LE, Lowens MS, et al.: Ureaplasma urealyticum Is Associated With Nongonococcal Urethritis Among Men With Fewer Lifetime Sexual Partners: A Case-Control Study. J Infect Dis. 2011; 204(8): 1274–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunham RC, Paavonen J, Stevens CE, et al.: Mucopurulent cervicitis--the ignored counterpart in women of urethritis in men. N Engl J Med. 1984; 311(1): 1–6. PubMed Abstract \| Publisher Full Text\n\nGaydos C, Maldeis NE, Hardick A, et al.: Mycoplasma genitalium as a Contributor to the Multiple Etiologies of Cervicitis in Women Attending Sexually Transmitted Disease Clinics. Sex Transm Dis. 2009; 36(10): 598–606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalmer HM, Gilroy CB, Furr PM, et al.: Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium. FEMS Microbiol Lett. 1991; 61(2–3): 199–203. PubMed Abstract \| Publisher Full Text\n\nWroblewski JK, Manhart LE, Dickey KA, et al.: Comparison of transcription-mediated amplification and PCR assay results for various genital specimen types for detection of Mycoplasma genitalium. J Clin Microbiol. 2006; 44(9): 3306–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBjörnelius E, Lidbrink P, Jensen JS: Mycoplasma genitalium in non-gonococcal urethritis--a study in Swedish male STD patients. Int J STD AIDS. 2000; 11(5): 292–6. PubMed Abstract \| Publisher Full Text\n\nDeguchi T, Gilroy CB, Taylor-Robinson D: Comparison of two PCR-based assays for detecting Mycoplasma genitalium in clinical specimens. Eur J Clin Microbiol Infect Dis. 1995; 14(7): 629–31. PubMed Abstract \| Publisher Full Text\n\nDutro SM, Hebb JK, Garin CA, et al.: Development and performance of a microwell-plate-based polymerase chain reaction assay for Mycoplasma genitalium. Sex Transm Dis. 2003; 30(10): 756–63. PubMed Abstract \| Publisher Full Text\n\nJensen JS: Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseases. J Eur Acad Dermatol Venereol. 2004; 18(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nJensen JS, Uldum SA, Søndergård-Andersen J, et al.: Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples. J Clin Microbiol. 1991; 29(1): 46–50. PubMed Abstract \| Free Full Text\n\nJurstrand M, Jensen JS, Fredlund H, et al.: Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay. J Med Microbiol. 2005; 54(Pt 1): 23–9. PubMed Abstract \| Publisher Full Text\n\nYoshida T, Deguchi T, Ito M, et al.: Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR. J Clin Microbiol. 2002; 40(4): 1451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshida T, Maeda S, Deguchi T, et al.: Rapid Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum Organisms in Genitourinary Samples by PCR-Microtiter Plate Hybridization Assay. J Clin Microbiol. 2003; 41(5): 1850–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomih-Alakija A, Ting J, Mugo N, et al.: Clinical characteristics associated with Mycoplasma genitalium among female sex workers in Nairobi, Kenya. J Clin Microbiol. 2014; 52(10): 3660–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen CR, Nosek M, Meier A, et al.: Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya. Sex Transm Dis. 2007; 34(5): 274–9. PubMed Abstract\n\nVandepitte J, Muller E, Bukenya J, et al.: Prevalence and correlates of Mycoplasma genitalium infection among female sex workers in Kampala, Uganda. J Infect Dis. 2011; 205(2): 289–96. PubMed Abstract \| Publisher Full Text\n\nVandepitte J, Weiss HA, Kyakuwa N, et al.: Natural history of Mycoplasma genitalium Infection in a Cohort of Female Sex Workers in Kampala, Uganda. Sex Transm Dis. 2013; 40(5): 422–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehta SD, Gaydos C, Maclean I, et al.: The effect of medical male circumcision on urogenital mycoplasma genitalium among men in kisumu, kenya. Sex Trans Dis. 2012; 39(4): 276–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen JS, Borre MB, Dohn B: Detection of Mycoplasma genitalium by PCR Amplification of the 16S rRNA Gene. J Clin Microbiol. 2003; 41(1): 261–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEastick K, Leeming JP, Caul EO, et al.: A novel polymerase chain reaction assay to detect Mycoplasma genitalium. Mol Pathol. 2003; 56(1): 25–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalkus JE, Manhart LE, Jensen JS, et al.: Mycoplasma genitalium Infection in Kenyan and US Women. Sex Transm Dis. 2018; 45(8): 514–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPepin J, Labbé AC, Khonde N, et al.: Mycoplasma genitalium: an organism commonly associated with cervicitis among West African sex workers. Sex Transm Infect. 2005; 81(1): 67–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson T, Coughlan E, Werno A: Mycoplasma genitalium Macrolide and Fluoroquinolone Resistance Detection and Clinical Implications in a Selected Cohort in New Zealand. J Clin Microbiol. 2017; 55(11): 3242–3248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTagg K, Jeoffreys N, Couldwell DL, et al.: Fluoroquinolone and macrolide resistance-associated mutations in Mycoplasma genitalium. J Clin Microbiol. 2013; 51(7): 2245–2249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIrekwa R, Nzou SM: Detection of Mycoplasma genitalium using Direct PCR. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10282691.v1" }	[ { "id": "59417", "date": "25 Feb 2020", "name": "Hans Fredlund", "expertise": [ "Reviewer Expertise Infectious diseases and microbiology", "epidemiology especially in the area of Neisseria sp and STI." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA commecial PCR method to detect M.genitalium was used in this study and a prevalence of 8% was found in female sex workers in Nairobi, Kenya. The manuscript is well written. It is of interest to perform such studies in many countries and cities globally to follow and understand the epidemiology of this STI. Of that reason the study can be accepted for indexing as it is. What is not studied is the antibiotic sensitivity of M.genitalium. In coming studies this has to be performed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7301", "date": "13 Oct 2021", "name": "Robinson Irekwa", "role": "Author Response", "response": "Thank you Dr. Huns Fredlund." } ] }, { "id": "66861", "date": "14 Jul 2020", "name": "Mihaela Laura Vică", "expertise": [ "Reviewer Expertise molecular biology", "microbiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a laboratory method used for detection of Mycoplasma genitalium in swab samples collected from sex workers. The authors provide descriptions of the method used and the results obtained. I consider that, in order to be indexed, the paper needs some revisions and explanations.\nAbstract The paragraph on treatment and antibiotic resistance is not necessary because they are not the subject of this article.\nIntroduction The phrase \"Most of the detection studies have been conducted in the U.S.A, Europe and Australia, with various strains being discovered\" requires citations from literature to exemplify what is stated. The phrase “In this study, it is shown that direct PCR can be applied to the detection of M. genitalium from crude DNA extracts.” should not be placed in this section. It must be moved to the Discussions section.\nMethods In Sample preparation and Direct PCR sections, the authors declare that they used the manufacturer’s protocol “with slight modifications”. What are these changes and for what purpose were they made? A positive control (M. genitalium positive sample) was used. Where does it come from? Why two PCR amplifications were made? The authors should explain why they have amplified PCR products.\nResults I think two Figures are enough: a representative gel for direct PCR and a representative gel for the amplification of the PCR products. There are mistakes in the legends of the Figures regarding the positive positions: e.g. in Figure 1 “The ladder is at positions 1 and 26” and “Clear Mycoplasma genitalium positive bands can be seen at positions 1, 2, 9, 10 and 17”. How do you explain that in Figure 4 the band corresponding to sample S163 is at a different level than the others?\nDiscussion There is too little discussion about the technique used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7300", "date": "13 Oct 2021", "name": "Robinson Irekwa", "role": "Author Response", "response": "Thank you for the comments given, will work on the sections. Truly appreciate" } ] } ]	1	https://f1000research.com/articles/8-1993
https://f1000research.com/articles/8-971/v1	26 Jun 19	{ "type": "Research Article", "title": "Preprints and Scholarly Communication: Adoption, Practices, Drivers and Barriers", "authors": [ "Andrea Chiarelli", "Rob Johnson", "Stephen Pinfield", "Emma Richens", "Rob Johnson", "Stephen Pinfield", "Emma Richens" ], "abstract": "Background: Since 2013, there has been a dramatic increase in the number of preprint servers available online. To date, little is known about the position of researchers, funders, research performing organisations and other stakeholders with respect to this fast-paced landscape. In this article, we explore the benefits and challenges of preprint posting, along with issues such as infrastructure and financial sustainability. We also discuss the definition of a ‘preprint’ in different communities, and the impact this has on further uptake. Methods: This study is based on 38 detailed semi-structured interviews of key stakeholders based on a purposive heterogeneous sampling approach. Interviews were undertaken between October 2018 and January 2019. These were recorded, transcribed and subjected to thematic analysis to identify trends. Interview questions were designed based on Innovation Diffusion Theory, which is also used to interpret the results of this study. Results: Our study is the first using empirical data to understand the new wave of preprint servers and found that early and fast dissemination is the most appealing feature of the practice. The main concerns are related to the lack of quality assurance and the ‘Ingelfinger rule’. We identified trust as an essential enabler of preprint posting and stress the enabling role of Twitter in showcasing preprints and enabling comments on these. Conclusions: The preprints landscape is evolving fast and disciplinary communities are at different stages in the innovation diffusion process. The landscape is characterised by significant experimentation, which leads to the conclusion that a one-size-fits-all approach to preprints is not feasible. Cooperation and active engagement between the stakeholders involved will play an important role in the future. In our paper, we share questions for the further development of the preprints landscape, with the most important being whether preprint posting will develop as a publisher- or researcher-centric practice.", "keywords": [ "preprints", "scholarly communication", "peer-review", "innovation diffusion theory" ], "content": "Introduction\n\nThe period since 2013 has seen a marked rise in the number of preprint servers set up for different communities in order to facilitate the rapid dissemination of pre-refereed research outputs. Tennant et al. (2018) list 18 servers launched between 2013 and 2018, variously set up by disciplinary communities, countries, research funders and publishers. One of the first in this new wave was the discipline-based server, bioRxiv – set up by the Cold Spring Harbor Laboratory in 2013 to cover the life sciences – which has been a focus of discussion and debate (Abdill & Blekhman, 2019; Luther, 2017; Vale, 2015). However, there are a considerable number of other disciplinary servers, including several set up by the Center for Open Science, such as SocArXiv, engrXiv and PsyArXiv (all of which were launched in 2016), as well as platforms such as ESSOAr, set up by the American Geophysical Union in 2018. At the same time, national servers have been launched, including ChinaXiv (for China), IndiaRxiv (for India) and INA-Rxiv (Indonesia) (Mallapaty, 2019). Funders of research have also set up platforms that allow the sharing of articles before peer-review, including, in 2016, Wellcome Open Research, for Wellcome-funded researchers. Finally, a number of journal publishers have added the dissemination of preprints to their workflows. The open access (OA) publishers PeerJ and MDPI both have their own preprints services; and PLOS now deposits submissions to its journal PLOS ONE in bioRxiv on behalf of authors. The F1000Research publishing platform has promoted a novel publication model involving preprints, in which immediate release of author submissions as preprints is followed by open peer review, with revised versions of a paper (alongside author responses to reviewer comments) published in the journal as they are made.\n\nOf course, preprint servers as a venue of scholarly communication are not new. arXiv, the preprint server for physics, mathematics, computer science and related subjects, and often regarded as an exemplar preprint server, was set up as early as 1991 (Larivière et al., 2014) – it is considered by some as the origin of the open access movement (Gajdacs, 2013). RePEc, housing ‘working papers’ in economics, was launched in 1997. There have also been unsuccessful attempts in the past to develop preprints services, including for biology, in the late 1990s (Ginsparg, 2016), and chemistry, in the early 2000s (Warr, 2003).\n\nThe move to set up servers since 2013 signals a new level of interest in preprints and a number of recent studies (e.g. Abdill & Blekhman, 2019; Balaji & Dhanamjaya, 2019; Luther, 2017; Tennant et al., 2018) have provided useful overviews of what Tennant et al. (2018, p. 5) call the “explosion of preprint platforms and services”. Significantly, this new interest has often come from disciplinary communities not previously associated with adoption of preprints. Biomedical disciplines served by bioRxiv, for example, have traditionally been associated with ‘Gold’ open access (publication in journals) rather than ‘Green’ OA (deposit of copies of papers in archives or repositories), and have typically not favoured dissemination of papers in pre-refereed form (Martín-Martín et al., 2018; Wang et al., 2018).\n\nThe launch of these new preprint servers has led to discussion and debate, and some have suggested that preprints may become a disruptive force in scholarly communication (Luther, 2017; Velterop, 2018). Green (2019) has argued for a digital transformation of publishing into a two-step process: articles would first be posted as preprints, and then invited to formal peer review only if they receive sufficient attention. He argues that this would not only represent a cost‐effective model for OA and drive out predatory journals, but could also resolve the so-called ‘serials crisis’, under which growth in research budgets (which produce articles) consistently outstrips that of library budgets (which are used to purchase articles).\n\nThe case has been made for preprints in a number of disciplines, including biology (Desjardins-Proulx et al., 2013; Vale, 2015), medicine (Lauer et al., 2015) and chemistry (Carà et al., 2017) . Some funders have signalled support for preprints being used in grant applications, including National Institutes of Health (NIH) and Zuckerberg Foundation in the USA, and the Wellcome Trust in the UK. However, sceptics have questioned the value of preprints and even suggested they may be dangerous – circulating versions of articles before they have been quality controlled by peer review may lead to unnecessary risk, particularly in disciplines like medicine (Sheldon, 2018).\n\nThis paper aims to explore the current and potential future role of preprints as a vehicle for scholarly communication by investigating current practices, drivers and barriers to their use. The overall objective of the study was to explore the place of preprints in the research lifecycle from the points of view of key actors, including:\n\nresearch funders;\n\nresearch performing organisations;\n\npreprint servers and service providers; and\n\nresearchers (engaged and unengaged).\n\nThe topics in focus included usage of preprints, perceived benefits and challenges, policy positions, values and strategies. The research took the form of a set of 38 detailed interviews with representatives from these groups.\n\nThe study was funded by, and co-produced with, Knowledge Exchange (a group of national organisations from six European countries supporting research infrastructure), as part of their work on open-access policy and service development. It was, therefore, important that the research should not merely have a descriptive purpose but also a prescriptive one, involving setting out possible directions for future action. The study is the first using empirical data focusing on the new wave of preprint servers set up since 2013, as such it aims to make a significant contribution to knowledge in this dynamic area.\n\nApart from recent discussion on the growth of preprint services (Abdill & Blekhman, 2019; Balaji & Dhanamjaya, 2019; Tennant et al., 2018), consideration of preprints in the formal academic literature, as well as in the scientific press and other online venues (such as blogs and other social media commentary), has tended to concentrate on four main issues: firstly, defining preprints; secondly, their benefits and dis-benefits; thirdly, disciplinary differences; and fourthly, policy developments. We discuss these in turn in what follows. There are, however, still a relatively small number of peer-reviewed studies focusing on preprints – much of the literature is still to be found in editorials and opinion pieces rather than data-driven research.\n\nDefining preprints. Different definitions of preprints in the academic literature typically relate to a number of key components: (1) genre, (2) timing, (3) versioning, (4) accessibility, (5) responsibility and (6) value (see Table 1).\n\nWith regard to (1) genre, Berg et al. (2016, p. 899) state, “a preprint is a complete scientific manuscript”, and Bourne et al. (2017) observe, “typically, a preprint is a research article, editorial, review, etc.”. Whilst the latter widen the scope also to include, “a commentary, a report of negative results, a large data set and its description, and more” (p. 1), most of the discourse on preprints tends to assume they are conventional research papers and therefore follow the academic conventions of that ‘genre’.\n\nWith regard to (2) timing, the key point made by most commentators is that a preprint is made available before formal publication, which Carà et al. (2017) describe as “prepublication”.\n\nFor (3) versioning, the relationship of a preprint to peer review is central. Desjardins-Proulx et al.’s (2013, p. 1) observation is typical in stating that preprints are made available, “before, or in parallel to, submitting them to journals for traditional peer review”. Suber (2012, p. 102) points out that this is not to “bypass peer review”, but that it applies to “works destined for peer review but not yet peer reviewed”. However, Bourne et al. (2017) controversially extend the definition to include “a paper that has been peer reviewed and…was rejected, but the authors are willing to make the content public”.\n\nAccessibility (4) is crucial, with a preprint normally defined as being (or assumed to be) openly available: it “can be viewed without charge on the Web” (Berg et al., 2016, p. 899). Therefore, the venue for distribution of preprints is often assumed to be a freely-accessible server of some kind, a point highlighted by Berg et al. (2016, p. 899), who include in their definition that a preprint “is uploaded by the authors to a public server”.\n\nThe above phrase, “by the authors” here is important and relates to (5) responsibility. Responsibility for distribution of preprints is traditionally assumed to be that of the author, a component of the definition that is often implicit in the verbs used to describe dissemination of preprints, such as, “sharing”, “posting” and “self-archiving”.\n\nThe final component of (6) value is summarised by Bourne et al. (2017): “a preprint is a research output that has not completed a typical publication pipeline but is of value to the community and deserving of being easily discovered and accessed”. To say that an output is “deserving” of dissemination is, of course, a value judgement and difficult to demonstrate for each deposit as it is made, but it is one that is implicit in much of the discourse on preprints.\n\nWith ambiguities associated with each of these six definitional components, Neylon et al. (2017) are right that “no universal definition of preprints exists”. The ‘pre’ of ‘preprint’ has sometimes been defined in relation to formal publication, with a preprint characterised as “prepublication”, leading to the controversial question of whether a preprint can itself be considered a ‘publication’ in its own right (Larivière et al., 2014). More commonly, however, the ‘pre’ of ‘preprint’ specifically refers to peer-review and is contrasted with ‘postprint’, a version produced after peer review. The conflation of peer review and publication in some discussions is a reflection of their close association in scholarly communication. It is interesting that the use of the terminology of ‘postprint’ in contradistinction to ‘preprint’, and with both termed generically as ‘eprints’, has declined in recent years. However, Tennant et al. (2018) have proposed its revival for reasons of clarity. Of course, the ‘print’ part of ‘preprint’ is largely anachronistic, but like terms such as ‘paper’ and ‘manuscript’, has continued to be used even in a digital environment.\n\nBenefits and dis-benefits of preprints. Of the advantages of preprints discussed in the literature, perhaps the most prominent are the early and rapid dissemination of research results (Khera, 2018). Using preprints has the potential to “accelerate” science, something that is particularly useful, for example, in combatting outbreaks of diseases (Johansson et al., 2018). The formal scientific publication process is often seen as frustratingly slow, particularly in a context where final versions of articles may be little different from preprints (Klein et al., 2016). Preprints allow authors to assert priority early – a preprint is date-stamped in a way widely recognised by many communities (Ginsparg, 2016; Mallapaty, 2019; Tennant et al., 2019). Preventing researchers being ‘scooped’ is a major priority in many fast-moving disciplines, but applies, at least to some extent, in all areas of academic research, where novelty is prized. Early dissemination can be useful to some particular members of the scholarly community, with early career researchers (ECRs) commonly being identified as particular beneficiaries, as preprints can allow them to rapidly achieve “visibility” (Desjardins-Proulx et al., 2013; Sarabipour et al., 2018; Tennant et al., 2019).\n\nAs well as being a fixed point in the scholarly discourse (date-stamped, etc.), another benefit of preprints emphasised in the literature is in the fact they are still subject to change. Authors can benefit from what Pinfield (2004) has called “informal peer review” of versions of their papers. Ginsparg calls this “crowdsourced peer review”, in contrast to “journal-mediated peer review”, and states, “authors benefit greatly from feedback from interested readers, contributing to improved versions of articles, which are then uploaded” (Ginsparg, 2016, p. 5). There are, however, few empirical studies of such feedback and its value. There also appears to be little acknowledgement that the claim stands in tension to the one cited earlier that preprints often differ little from final published versions.\n\nOther key advantages of preprints include wider and fairer distribution of research results, both within and beyond the academic community, something fundamental to many arguments for openness in general (Ginsparg, 2016; Sarabipour et al., 2018). Access to preprints for machine-based crawling in order to facilitate text mining is also seen as an advantage by some commentators (Chodacki et al., 2017). Preprints, partly as a result of wider dissemination, can also increase numbers of citations of papers (Davis & Fromerth, 2007) and create opportunities for collaborations (Kleinert et al., 2018). Finally, preprint servers can sometimes usefully also house research outputs that might otherwise be ‘homeless’, including items that do not end up being published in peer-reviewed journals (Bourne et al., 2017).\n\nPerhaps the most prominent criticism of preprints relates to this last issue: the lack of quality assurance through peer review (Sheldon, 2018). As well as a general concern about lowering quality standards, lack of quality control has been seen as potentially dangerous as “reports that have not undergone formal peer review [organised by a journal] could be misleading” (Lauer et al., 2015). Furthermore, uncertified science might be reported prematurely in the media and might even give rise to ‘fake news’ (Sheldon, 2018). Some insist that, at the very least, the opportunity to disseminate knowledge rapidly without peer review may encourage academics to produce low-quality outputs on fashionable topics (Teixeira da Silva, 2017).\n\nDespite claims of the value of informal peer review enabled by preprints, some have pointed to the limited use of commenting and feedback features on preprint servers (Sarabipour et al., 2018). Others have gone further and questioned the value of self-appointed reviewers, as opposed to those selected by journal editors (see the issue of “self-policing” highlighted by Harnad, 1998). Preprint posting, however, is unlikely in any case to substitute for the valuable role played by selective journals in filtering content.\n\nA number of authors report the concern that dissemination of a preprint may be considered ‘prior publication’, thereby jeopardising acceptance of the paper by a journal – the so-called ‘Ingelfinger rule’ (Nallamothu & Hill, 2017). Whilst this convention has come under criticism and been withdrawn by some publishers, it still exists for some journals, e.g. in medicine and chemistry (Lauer et al., 2015; Teixeira da Silva & Dobránszki, 2019).\n\nDisciplinary differences. Disciplinary differences have been a prominent feature of the literature on preprints. Neylon et al. (2017) in their seminal work conceptualising preprints usefully distinguish between the “state” and “standing” of preprints. “State” relates to, “external, objectively determinable, characteristics” of preprints; “standing” refers to the “position, status, or reputation” of preprints. Neylon et al. (2017) discuss in detail how preprints of similar states can have very different standings in different disciplinary communities, using the example of the contrast between physics and life sciences. For example, it is not universally agreed when an output should be citable (in the literature, funding proposals or promotion cases) or when it can be used to establish a claim of precedence.\n\nIn disciplines where a preprint is not considered appropriate to establish precedence, it has also been suggested that making a preprint available may actually encourage research to be scooped by rival researchers who publish in a recognised journal before the preprint authors (the ‘flip side’ of the priority claim argument above) (Kaiser, 2017).\n\nIt is commonly observed that physics has a well-established preprint tradition unlike many other Science, Technology and Medicine (STM) disciplines. Lauer et al. (2015, p. 2448) note, “biology…has trailed behind, whereas clinical research remains well behind.” Carà et al. (2017, p. 7924) characterise chemistry as being “late in embracing preprints”. Such language (“behind”, “late”) seems to represent an assumption that all disciplines will eventually come to use preprints, but different disciplines are now simply at different points in the adoption process. Such a view has been disputed, however, with some arguing disciplinary differences in communication practices are likely to exist in the long term and therefore that preprints will not be adopted universally across disciplines (Kling & McKim, 2000).\n\nPolicy developments. Of course, disciplinary practices do not operate in a vacuum. They are influenced, amongst other things, by the policy environment in which researchers work. The ‘policy stack’ consists of several interlocking layers: publisher policies, funder policies and institutional policies. The first of these is critical, with the Ingelfinger rule and deposit embargoes at its core. To this position of publishers rejecting preprints may now be added the contrasting recent development of some publishers embracing preprints, even setting up their own preprints services (Callaway, 2013). This development is not completely unprecedented, however, since it does build to some extent on well-established processes in areas like high-energy physics of integrating preprints into the journal submission process (as an example, some physics journals allow submission by simply pointing to an arXiv preprint).\n\nPerhaps the most noticeable shift recently in terms of policy is that of funder policies. Some funders have now explicitly signalled support for use of preprints, including allowing citation of preprints in funding bids, and support their inclusion in cases for academic advancement (Berg et al., 2016; Bourne et al., 2017). Very few funders, however, currently mandate use of preprints, although this has been proposed by preprints advocates (Sever et al., 2019). Institutional policy in this area shows some limited movement, with examples of institutions rethinking (usually rather cautiously) their approaches to criteria for career advancement in relation to the shifting scholarly communication environment, with some explicitly allowing submission of preprints (ASAPbio, n.d.). It appears that, at present, many organisations still rely on metrics such as the journal impact factor when it comes to review, promotion and tenure of their staff (McKiernan et al., 2019).\n\nSome have argued that initiatives such as Declaration on Research Assessment (DORA), with its emphasis on the quality of the output rather than venue of publication, promote use of preprints (Polka, 2018). Another interesting area of institutional policy is the positioning of the institutional repository (IR) in relation to preprints. IR policies and practices differ in this area, with many to date having focused on versions of outputs following peer review, although this is not a universal position (Baughman et al., 2018).\n\n\nMethods\n\nAnalysis framework. With preprints and preprint servers still innovative developments for most disciplines, using a qualitative research approach is an appropriate way to gain an in-depth understanding of multiple perspectives, including motivations, challenges and opportunities, and taking account of disagreements and uncertainties. We therefore chose to carry out detailed interviews of key actors in this space who could explain their perceptions, attitudes and practices in relation to preprints. Bearing in mind that perspectives of stakeholders from disciplines where preprints are established are well represented in the literature (e.g. physics, computer science, and economics), we focused our research on disciplines where preprint services are relatively new but growing rapidly in order to gauge the way in which preprint servers are now making an impact. These were biology, chemistry and psychology, corresponding to preprint servers, bioRxiv, ChemRxiv and PsyArXiv.\n\nAs a way of framing our research design, we used innovation diffusion theory (IDT), a well-established theoretical framework for explaining the way innovations are adopted in different contexts (Rogers, 2003). IDT has been tested and deployed widely and proved to be a robust explanatory model in a wide range of contexts, including OA (Hampson, 2014; Jones et al., 2006; Pinfield & Middleton, 2016; Xia, 2012). It is designed to describe “the process by which an innovation is communicated through certain channels over time among members of a social system” (Rogers, 2003) (original emphasis). An innovation is defined as “an idea, practice, or object that is perceived as new by an individual or other unit of adoption” (Rogers, 2003). Preprints are both cultural innovations, as they aim to change practices in scholarly communication, and technological innovations, in terms of changes to infrastructures and processes. IDT offers ways in which these aspects of preprints as innovation can be understood, particularly in relation to two key issues: the “innovation decision process”, and the “rate of adoption”.\n\nThe innovation adoption decision process is seen as going through a number of consecutive steps:\n\n1. Knowledge, when the decision-making unit is exposed to the innovation’s existence and gains an understanding of how it functions;\n\n2. Persuasion, when the decision-making unit forms a favourable or unfavourable attitude towards the innovation;\n\n3. Decision, when the decision-making unit engages in activities that lead to a choice to adopt or reject the innovation;\n\n4. Implementation, when the decision-making unit puts an innovation into use; and\n\n5. Confirmation, when the decision-making unit seeks reinforcement for an innovation-decision already made but may reverse the decision if exposed to conflicting messages about it. (Rogers, 2003)\n\nA particularly important concept to understand the success of innovations is their rate of adoption (see Table 2). This was used as the initial basis of the design of the interview questions. From the key factors associated with the rate of adoption, we selected a range of features that appear appropriate for the scope of the present investigation and that were suitable to discuss via interviews for the different stakeholder groups:\n\nPerceived attributes, are what stakeholders feel are the benefits arising from an innovation, in this case the introduction of preprints. Perceived attributes can be split into relative advantage, compatibility, complexity, trialability and observability.\n\nNature of the social system, including “norms”, which are the established behaviour patterns for the members of a social system. They define a range of accepted behaviours and serve as a guide or standard for the behaviour of members of a social system. Norms tell individuals what behaviour they are expected to adopt and are affected by the introduction of an innovation. These relate to the level of interconnectedness or cohesiveness of the community.\n\nChange agents’ promotion efforts, which are the efforts made by individuals with influence in the system to promote the adoption of an innovation deemed desirable by a change agency (e.g. funders and institutions, service providers, publishers). Change agents often use opinion leaders in a social system as their lieutenants in diffusion activities.\n\nType of innovation decision, which describes how the uptake of preprints is affected when individuals or communities support them, or authorities mandate their posting.\n\nThe topic of communication channels (additionally part of IDT theory on the rate of adoption) also arose organically from the discussions with our interviewees.\n\nInterview sampling and approach. Interview questions were developed based on the factors outlined in Table 2 (see Extended data (Chiarelli et al., 2019a) for more information). From an initial long list of possible questions, areas for investigation were prioritised based on the different stakeholder groups involved, and our review of the literature. We also incorporated questions associated to current policy-related issues, agreed in consultation with the Knowledge Exchange steering group, taking into account the innovation adoption process. This ensured that the approach taken was both theoretically robust and sufficiently grounded in practice to be useful in generating actionable insights.\n\nThe study adopted a heterogeneous purposive sampling approach, aiming to include a wide range of perspectives from actors in the area. Participants comprised senior representatives from research funders, research-conducting organisations (universities and research institutes), preprint services, other related service providers (such as infrastructure providers), as well as researchers, both researchers demonstrably engaged with preprints (they had themselves posted a preprint) and non-engaged (there was no evidence of them having posted a preprint). Participants were based in eight countries: Denmark, Finland, France, Germany, Netherlands, Switzerland, UK and USA, all apart from the USA and Switzerland being KE member countries. Participants were identified from the literature and from their associations with relevant services or organisations. Snowball sampling was also used as the research progressed and appeals for participation on social media were also shared (particularly to identify non-engaged researchers). Participants gave their informed consent and agreed to be named as participants in any reporting on the understanding that particular views or quotations reported would not be linked to them or their organisation and that the full text of transcripts would be kept confidential. The research approach adopted by the project was given ethical approval by the University of Sheffield. A full list of participants is available in Chiarelli et al. (2019b).\n\nWe undertook 38 semi-structured interviews, with participants distributed across the targeted stakeholder groups as illustrated in Table 3. Interviews, which took place between October 2018 and January 2019, and ranged from 32–75 minutes in length, were conducted via GoToMeeting, recorded and fully transcribed using the intelligent verbatim method (including minor edits e.g. removing ‘fillers’ etc). Two interviews took the form of an email Q&A because of restrictions around the participants’ availability. The transcripts were then subjected to thematic analysis (Braun & Clarke, 2006) which took place in several stages. Initially, the research team independently read a sample of transcripts from different stakeholder interviews, including some in common, and then discussed key topics arising from the transcripts. This formed the basis of the initial coding approach then undertaken by E.R. This was reviewed as analysis proceeded, with coding being checked and validated by A.C. and S.P. as it progressed and amended as necessary in light of their comments. The codes were then grouped into themes, which form the basis of the findings reported below.\n\nParticipants are listed by Chiarelli et al. (2019b).\n\nLike many kinds of qualitative research, this study was designed to be exploratory, in this case to map out key aspects of the policy environment. Its tentative conclusions will need testing. Interviewees selected for this study were not necessarily representative of the overall community and outlying results may be over-represented. Wider consultation, using other methods, will be needed in future. Furthermore, some stakeholder groups, such as publishers (who only have very limited representation in this study), and other groups (such as non-academic users of the research literature) could usefully be included in future studies.\n\n\nResults\n\nThe analysis of the data identified nine major themes arising from the interviews, which can be grouped into four thematic areas (Table 4). These themes are used as the framework for presenting results and explored in more detail in what follows.\n\nDefinitions of preprints. In view of the ambiguities and disagreements in the literature around definitions of ‘preprints’, one key aim of the interviews was to ask our participants about their understanding of the term. Whilst all of our participants agreed with the broad definition that a preprint is a research output made available in a form before it has been peer-reviewed and published, there was considerable variation in the specifics of what that means. Some participants were themselves aware that the term was being used in different ways by different people and there were discrepancies (and in some cases confusion) about its precise meaning. One expressed dislike for the term, saying that it “presupposes…that you are in a print era”. However, other alternatives used by participants in this space, such as “manuscript” and “paper”, as has been observed, are equally anachronistic. There was evidence of participants struggling to find a clear language for the innovation being discussed.\n\nMany saw a preprint as being in a form that was ready to be submitted to a journal, “at the point of submission” (Research performing organisation), as one participant put it. Referring to their own experience, one participant stated:\n\n“the preprint…was only submitted or uploaded at a stage where it was essentially submission-ready for a journal.” (Engaged researcher)\n\nOther participants saw preprints as earlier versions of outputs made available in order to receive comments (e.g. working papers in economics). One participant acknowledged that a preprint was commonly thought of as the submitted version of an article but also discussed possible earlier versions being shared:\n\n“…in terms of quality, almost like [the] thing that will appear in the journal later on but if you consider a preprint like a working paper, you…definitely can see it as a step earlier in the whole research process in which there is still the possibility to enrich and improve the later formal publication on the same time.” (Research funder)\n\nAnother participant referred to the benefit of this: “it gets feedback from the community” before “official peer review” (Unengaged researcher). A key point made by one participant, but implicit in the comments of many, was that that a preprint is a version of an output that has not been peer reviewed but that the author is “committing to get it peer reviewed” (Other service provider).\n\n“…the term itself includes the idea that you’re building it just towards something. That it’s only the preprint and then something will come later after from it.” (Research funder)\n\nAnother author said of their own approach to posting preprints: “I always had the intent to publish this in a…proper journal subsequently” (Engaged researcher). A preprint is part of a “continuum” of different research stages, one participant argued, and authors should deposit all versions of their papers (and data) in a repository as part of contributing to that continuum. However, one participant did question this emphasis on a work flow with the preprint being provisional, since it appeared to devalue preprints:\n\n“…preprint means there’s something you know, there’s…a paradise afterwards, there’s a better life, and that it’s not a publication of its own.” (Other service provider)\n\nEither as a provisional version of a forthcoming publication or as a “publication” in its own right, a preprint was seen as part of a recognisable genre of scientific output, and which was planned to be part of the formal published literature, but was made available earlier than formal publication.\n\n“they’re not that radical, the concept is radical, but when you look at them, they look like articles.” (Other service provider)\n\nSeveral participants included post-refereed versions of articles within the definition (although some acknowledged these were not “pure” preprints), and others recognised that in reality many authors deposited post-refereed versions on preprint servers, usually to enhance their accessibility. A small number of interviewees acknowledged that some papers posted as preprints might not end up being formally published, although this might raise questions about the value of the output:\n\n“If there’s nothing to follow the preprint then I would start to wonder what did happen. Why was the work dropped and left on this preprint level?” (Research funder)\n\nSome questioned whether a paper which did not end up being published could legitimately be considered a preprint, with one interviewee asserting, “it’s not a preprint; it’s just a manuscript” (Other service provider). That a preprint is basically a research paper was the assumption of the majority of participants, but one questioned this:\n\n“You know, it’s basically anything, you know between a tweet…or a poster presentation and an actual paper…” (Engaged researcher)\n\nMany acknowledged there was uncertainty about preprints and some researchers in particular were themselves cautious in committing to a definition. In some cases, this related to disciplinary differences – something acknowledged by several interviewees. One interviewee discussed definitional differences between his own discipline, chemistry, and that of physics, mainly in terms of community acceptance. Another participant from the humanities stated:\n\n“I don’t think there’s any one perspective that scholars in the humanities have on preprints and I think that there’s some confusion about the terminology.” (Other service provider)\n\nDisciplines, cultures and practices. Disciplinary differences were evident not only in perceptions of what preprints are but also in terms of their acceptance. This was important throughout the interviews. There were firstly differing levels of awareness and, following this, adoption. Some interviewees recognized physics, mathematics and computer science to have well-established preprints practices with very high levels of awareness and adoption, but in other disciplines awareness was often still low:\n\n“I think in chemistry it’s small but growing and biology is being helped a lot by bioRxiv …there’s certainly some areas where there is still a kind of much less awareness. I would say really outside the math and physics [communities] the awareness is much lower of what preprints are about.” (Preprint server provider)\n\nOne service provider summarized confusions (about definitions and processes) even applying to people trying to submit their work as preprints:\n\n“So there’s definitely a growing awareness but it’s still a minority. And we still find that there are some who are confused by the process and when they submit a preprint they don’t really understand. Despite everything we try and make them aware of, they don’t really understand the process.” (Preprint server provider)\n\nFurthermore, where there was evidence that awareness was rising, this did not necessarily result in uptake. Some unengaged researchers interviewed were aware what preprints were but had not been motivated to use them to date. Some researchers clearly saw practical barriers:\n\n“I am not entirely sure of the process, that’s the reason why I haven’t done it. I’m sure I could work it out but I’m not entirely sure which preprint server I would use, whether one would be better for my type of work than another…” (Unengaged researcher)\n\nHowever, there were signs this was beginning to change. One chemist described the situation in their discipline as moving from a position where there had previously been no use of preprints to where use was beginning to happen:\n\n“Almost all that is changing now and also from the chemistry part, which might be the related field, there was as far as I know ChemRxiv, which is like the main repository for chemistry data. It has been going on for one or two years maybe.” (Unengaged researcher)\n\nOne participant described the process by which awareness of this new development diffuses through the community via informal channels and how they had become more personally aware of it during the course of doing a PhD:\n\n“I didn’t get any information on anything like, you know, I found out about it myself, you know, something as simple as that. Most of it’s just through word of mouth, like as you go through a PhD, as you’re talking to people, a lot of meetings, as you hear these terms come up more and more… It’s just accidental.” (Unengaged researcher)\n\nOne participant representing a preprint server saw growing awareness and willingness to experiment in different subject communities:\n\n“And the momentum behind [name of the preprint server] – and I would not call it a success yet, I would call it momentum – but I think that momentum has given encouragement to other groups of scholars to investigate the possibility of developing a preprint platform for their own discipline. Whether that’s in earth sciences or anthropology or psychology, sociology and so on.” (Preprint server provider)\n\nThis was confirmed by another service provider, who stated that before 2016, preprints “were not used much” in their discipline and many researchers “were unaware of what a preprint was”. But this was changing: “the popularity of preprints within the field…is rising” and this was partly attributable to the preprint server the provider represented which had been a “driving force” for change (Preprint server provider).\n\nThe subject specificity of servers was seen as a natural way for preprints services to develop as it was seen as in line with the way researchers worked. However, even within disciplines, some referred to what they saw as significant differences between sub-disciplines, with some engaging in preprints and others not. Participants were agreed, however, that in disciplines where preprints were not established, although there might be some willingness to experiment, there was still a great deal of resistance:\n\n“I think lots of my colleagues use them just as much as I do but then I think there are some colleagues that would never post to a preprint.” (Engaged researcher)\n\nFinally, we note that adjacency between disciplines appears to be playing an important role. For example, disciplines close to those which posted preprints (e.g. those close to some areas of computer science or physics, which use arXiv consistently) may be more favourably disposed to the practice compared to those from other disciplines.\n\nPreprints’ position in the landscape. Perceptions of the role of preprints in the scholarly communication landscape were partly derived from understandings of and sympathy for open science and open access developments. It was clear that those who were supporters of wider open science developments generally supported increased use of preprints.\n\nWe note that peer-reviewed journals are still seen as essential. As such, preprints were for most interviewees “part of a new ecosystem” (Research performing organisation), but not a radical departure from or replacement for selective journals, although their potential to prompt more fundamental change was highlighted by some (see below). The level of integration of preprints in processes associated with submission to a journal was generally seen as low, but there was some awareness of provisions, for example, where a preprint could be transferred to a journal’s submission system. For most disciplines, these were not seen as fundamentally important, however, in determining usage or take up decisions.\n\nOne researcher referred to ongoing “scepticism” in “many fields” (Engaged researcher). One of the key reasons for this was that the ‘standing’ of preprints in different fields was seen to be very different. This was reflected, for example, in different perceptions of the value of citing preprints. In some cases, researchers believed that citing preprints was not acceptable in either papers or grant proposals.\n\nBenefits. Participants highlighted a number of (potential) benefits and challenges of preprints. For the most part, these correspond to those identified in the literature, but it is useful to see how these are articulated and prioritized by our participants. Table 5 summarises the main benefits of preprints as highlighted in the interviews, comparing them with the literature. We have attempted to rank these by the prevalence of certain points across the entire dataset of interviews and have also classified them as to whether they create benefits at an individual or systemic level, that is whether they benefit individual researchers who practice them or the scholarly communication system as a whole.\n\nMentions across the entire dataset: ✔* =over 20 mentions; ✔ =between 10 and 20 mentions; ✔* =fewer than 10 mentions.\n\nOf the benefits highlighted by participants, early and rapid dissemination of research was the most frequent. As one engaged researcher succinctly put it:\n\n“The primary purpose of preprints is to communicate scientific knowledge as early as possible to as wide an audience as possible.” (Engaged researcher)\n\nPreprints were described by one researcher as enabling “science in real time” (Engaged researcher), particularly with reference to the lengthy period of peer review and publication that often applied to article publishing. One participant representing a preprint server described the peer review process as, “often long and tortuous” (Preprint server provider), and a university representative described it as a kind of “limbo” (Research performing organisation) in which the research was not being read or used.\n\nAchieving rapid and wide dissemination could in turn “accelerate” the pace of research itself (Other service provider). This might mean being able to “see a result that somebody else did and I can start working on it” (Research funder), even though this type of behaviour was described as appropriate only in very fast-moving sub-disciplines. Research could also make progress thanks to the reduction of “redundant work”, which makes it more likely for researchers to identify “the next big research question” (Research performing organisation) more quickly.\n\nThe benefits discussed were mostly seen in terms of communicating with a particular disciplinary community, but some interviewees also mentioned reaching a wider audience, including policymakers or clinicians, in a timely way – research reflecting the latest thinking in an area has more “news value” (Research performing organisation). One engaged researcher also emphasized the benefit of making the latest research available in the case of an outbreak of disease.\n\nParticipants were conscious of the importance of dialogue and interchange as part of the research process and so valued the potential for preprints to create opportunity for feedback. Some participants conceived of this from the point of the researcher receiving comments on their paper in order to make corrections – a kind of “debugging” (Engaged researcher) – or other improvements:\n\n“…feedback from others to help you with your thinking and to improve your ideas.” (Research performing organisation)\n\nIt was commented that some preprint servers facilitate this in various ways, by for example allowing authors to solicit feedback or providing commenting or annotation features. Many respondents saw this in wider systemic terms, rather than just personal: enabling community engagement and discussion – what one funder called “community-oriented discourse on research results” (Research funder). The language of ‘community’ was particularly strong amongst many participants here. Some provided stories of their experience of this, with one researcher commenting on a particular paper that,\n\n“the feedback from the community which we received through the preprint was at least as constructive and helpful as the official reviews from the journal.” (Engaged researcher)\n\nMany of the participants agreed that preprints servers could be a useful outlet for otherwise ‘homeless’ research outputs, even though this goes counter to the emphasis of many of preprints as early versions of outputs later formally published elsewhere. The most common sorts of outputs mentioned were null results and replication studies, which would not satisfy journal requirements of novelty or significance. However, participants also mentioned older or under-developed papers that had not been formally published but could easily be deposited on a preprint server and would be of value.\n\nSuch an approach could be particularly useful for early career researchers, who benefit in general from posting preprints in order achieve greater “visibility” relatively quickly. This could be particularly useful in the case of funding proposals or job applications in order to demonstrate productivity, although several participants were quick to emphasise that formal publications would be preferable. For all researchers, in fact, preprints were seen as possible evidence of productivity but no real substitute for formal publications.\n\nThe benefit of preventing scooping was also prominent – preprints were a way to establish priority:\n\n“If you’re an author the benefit of having a preprint in the public domain is it identifies the ideas as being yours, so it prevents you from being scooped because the ideas are down in time stamp against your name.” (Research performing organisation)\n\nHowever, a small number of participants observed that this might cut both ways, since in disciplines where preprints did not have the standing of a citable resource, making research available in this way might cause the researcher to be scooped: “if you put something online and it’s not yet published [I worry] that it would be stolen by competitors” (Unengaged researcher). The strength of the language (“stolen by competitors”) is telling of the intensely competitive culture in which many researchers work and is in marked contrast to the language of community noted above. However, other researchers were sceptical about whether this was likely to happen.\n\nThis was partly because a preprint is available so widely. Breadth of availability was certainly a benefit seen by many participants, primarily in reaching as many members of their own research community as possible. However, many participants also mentioned access by a wider readership, including the general public. Either way, the benefit of increased usage of papers was seen as particularly important. However, once again, the benefits were qualified, with greater value being placed on formal publications or accepted author manuscripts:\n\n“I think preprints are a very good kind of second option if you can’t access the published version and you can’t find the author’s accepted manuscript.” (Research performing organisation)\n\nA number of participants were confident making work available as a preprint would increase citations, with the paper being citable in preprint form if available on a preprint server. This applied particularly in fast-moving areas. Getting work out into the community at an early stage also, it was suggested, increased the opportunity to form new collaborations. This is in many respects the more optimistic side of the idea of competitors stealing ideas – the possibility that researchers may be encouraged to develop a collaboration as a result of seeing the developing work of peers.\n\nChallenges. Participants also recognised problems with preprints (summarised and compared with the literature in Table 6, with an indication of prevalence). It is worth noting, however, that participants often had a nuanced view of the challenges, commonly stating potential disadvantages but then themselves qualifying their criticisms or citing possible solutions.\n\nMentions across the entire dataset: ✔* =over 20 mentions; ✔ =between 10 and 20 mentions; ✔* =fewer than 10 mentions.\n\nOf the problems, the lack of quality of assurance was most widely discussed by participants, with a set of related issues clustering around this theme. In some cases, there was a concern preprints could simply mean lower quality:\n\n“…so my worry would be with rapid publication by preprint that there would be an increase in the amount of poor quality science that’s available…” (Unengaged researcher)\n\nHowever, more commonly, there was the view that a lack of quality assurance meant greater uncertainty and that readers had a greater responsibility to approach preprints critically. Words such as “caution” and “sceptical” were often used. The filtering role of selective journals was valued by many participants. Peer review, for all of its faults (and participants were not slow to point out its possible failings), was still highly valued:\n\n“It’s really important that good reviewers have looked at the article and at the results section.” (Research funder)\n\nPeer review was thought of not just as a safety net but as a process which often improved the quality of a paper. Most of the supporters of preprints, did not therefore see them as an alternative to or replacement for peer-reviewed papers but as a complement to them. Whether or not they valued informal comments, participants commonly pointed out that the use of feedback functionality of preprint servers was still limited. One preprint service provider observed that only 10% of preprints received comments.\n\nParticular concerns around quality related to the media or members of the public latching onto unreliable findings, and on the harm a preprint containing errors could create in sensitive areas, especially associated with medicine or perhaps law. Several participants observed that people from non-academic contexts may not know the difference between a preprint and peer-reviewed article, and could therefore be more easily misled. Science journalists might potentially play a negative role. One participant said it was common for journalists to report findings from a peer-reviewed paper but “misinterpreting the results…and spreading the news without actually…understanding” the research (Engaged researcher). The risks associated with this were perceived to increase with preprints. Other participants acknowledged the responsibility of journalists, but were more optimistic, citing evidence of journalists using preprint servers responsibly and adding appropriate qualifiers to reports on preprints.\n\nSeveral participants commented researchers themselves need to be aware of these problems and be cautious about how they published on controversial issues. Peer review did not mean that research papers were immune from such problems in any case.\n\nThere was a view expressed that basic screening provided by preprint servers, which was seen as very important by many participants, could address some of these concerns, but was still limited. There was consciousness that such basic checks themselves differed across different preprint servers and also that they would need strengthening in the case of servers dealing with medical outputs. One service provider working in this area suggested that preprint servers in the medical field might have to consider, among other things,\n\n“conflict of interest, financial disclosures, assurance that the work reported has been cleared by appropriate ethical review boards and assurance that data underpinning the article is available in an appropriate repository.” (Preprint server provider)\n\nThis is a very interesting development which would involve preprint servers undertaking additional quality checks than just the current basic screening, potentially blurring the boundaries between them and peer-reviewed journals.\n\nParticipants were conscious of preprints creating what might be called a trust barrier. Whilst some of the determinants of trust for peer-reviewed papers might transfer to preprints (such as the overall shape of the paper, its authors, etc), some of the key determinants, the brand of the journal and its associated peer review processes, were missing. Participants were clear that preprints should be clearly marked as “not peer reviewed” and therefore treated with caution and handled responsibly. However, interviews also indicated possible contributors to trust that might apply to preprints, including: the preprint being widely discussed on social media, receiving comments online (e.g. on the preprint server), being cited, reported on by a magazine or newspaper, recommended by a colleague, and housed in a recognised preprint server.\n\nWhilst there was enthusiasm amongst many participants for “community” based review and commentary on papers facilitated by preprint servers, there was some scepticism about the value of reviewers who “self-select themselves” (Unengaged researcher). This could either be because an author might invite comments from people who are expected to be positive about their work, or people commenting may not understand the area or use commenting to pursue personal agendas. The view was also expressed by some participants that the practice of commenting on preprints could cause difficulty with the formal peer review process, since people who had made public comments may be barred from undertaking blind peer review because of a conflict of interest.\n\nThere was concern also about information overload expressed by some participants, some of whom believed that preprints may inflate the number of papers being made available. However, other participants expressed scepticism of this, emphasising that the same number of papers were simply being made available earlier. There was also an acknowledgement that the filtering role played by selective journals was being removed from the process: some participants suggested that this could be at least partially solved with technology-based solutions, improving discoverability and filtering of content:\n\n“I think there is a lot of information out there but I think there’s also the potential to find technical solutions that will avoid the information overload.” (Preprint server provider)\n\nA number of participants pointed out that the solutions were better enabled by open, interoperable content, avoiding what one researcher called, “technical and legal restrictions put on by the publishers” (Engaged researcher).\n\nOf these restrictions, the Ingelfinger rule was still the most common mentioned (although not by that name) by participants. However, there was considerable uncertainty and confusion amongst some participants (particularly researchers) about what authors could or could not do with regard to depositing preprints, and where they might find reliable information on what was permissible. Several researchers voiced such doubts:\n\n“I think there’s always the concern that people are worried that if I put something up there then it restricts where they can submit their paper.” (Engaged researcher)\n\n“I have a duty to make sure that the work is peer reviewed in the best journals that we can get it into and if I rule certain journals out because I’ve submitted it as a preprint then I’ve kind of done a disservice to myself and those who I work with.” (Unengaged researcher)\n\nSuch “fears” themselves acted as a considerable barrier to uptake. Interestingly, one engaged and one unengaged researcher did show an awareness of SHERPA RoMEO (a database of copyright and OA self-archiving policies of academic journals) as a source of information, but this was not common.\n\nApart from permissions barriers, there were also fears that reputational damage could arise from premature release of preprints. The consequence of this was that sharing a preprint would be delayed until the authors were confident in it to avoid the possibility of any reputational damage. One researcher commented:\n\n“I don’t think people in my field would just post off stuff that’s…terrible…because you’re still being judged on what’s going up there.” (Engaged researcher)\n\nIn addition, where work was produced by a team of co-authors, gaining agreement from the team was itself an important quality barrier to overcome before disseminating the research in preprint form.\n\nWe note that some of the challenges arising from the posting of preprints were only perceived to be substantial in cases where researchers or re-users behave unprofessionally or unethically; however, this does not just apply to preprints, but also to other areas of scholarship and publishing. A key feature of many of the interviews was that participants were rarely able to cite empirical evidence of either benefits or challenges of preprints. At best, personal experience or anecdotes about the experience of colleagues was being cited. As one engaged researcher said:\n\n“I don’t have a lot of examples here, but certainly, you know, I hear anecdotes.” (Engaged researcher)\n\nAnother said, “I have heard stories on Twitter” (Engaged researcher). It was apparent from many of the interviews that there is a need for further empirical work in this area in order to provide an evidence base for practice.\n\nInfrastructure. The view was commonly expressed by participants who commented on infrastructure that many technologies were already available to support use of preprints. Those mentioned by participants include repository and publishing solutions (open source, such as OSF Preprints, Eprints or DSpace and proprietary technology, such as Figshare), but also the broader scholarly communication infrastructure (e.g. indexing via Crossref). However, several infrastructural issues were emphasised as being important by participants, including technology considerations (such as interoperability, search and discovery), as well as process considerations (including licensing, versioning management, and digital preservation). Functionality and usability considerations for individual services were also mentioned, such as search and annotation of preprints.\n\nInteroperability was regarded as a priority by many participants, with standards often being seen as key. Use of digital object identifiers (DOIs) for preprints (which could then be linked to later versions of the paper) was seen as a particular priority, but other standards such as ORCID, and service providers, such as Crossref, were also mentioned as being important.\n\nUse of standards was seen as an important enabler of effective search and discovery of preprints. Discovery was seen as being achievable largely through network-level discovery services, such as Google Scholar, but interestingly, even greater emphasis was placed on social media, particularly Twitter. It was common for researchers to report finding out about preprints of interest from Twitter, either by following particular individuals or signing up to Twitter feeds set up and managed by preprint servers (including automated Twitter posting when preprints are released). Twitter was reported to be the main way several participants found out about preprints in their field. Several researchers also mentioned gaining feedback on their own preprints as a result of posting links to them on Twitter, with comments sometimes being posted on Twitter itself or other social media rather than the preprint server. The importance of Twitter was also emphasised by service providers, with one representative of a preprint server stating:\n\n“I would say that the momentum behind [name of the preprint server] owes a great deal to Twitter, and to Facebook, a bit less so. But nevertheless the effect is there. These are means of amplifying work once it has been posted.” (Preprint server provider)\n\nThis is a significant finding. The fact that part of the infrastructure upon which preprint services are currently reliant includes generic discovery services, such as Google Scholar, and social media, particularly Twitter, needs to be taken into account in considering future developments of preprint services. Usage (or at least availability) of Twitter was assumed by participants to be widespread – a reasonable assumption in most Western countries, but not in others, particularly China.\n\nOpen licensing was also seen as an important infrastructural issue, which enabled interoperability and discovery. Preprint servers typically offer depositors Creative Commons (CC) licence options and there was some discussion in our interviews of the best one among these. Some authors were clearly confused about the options available to them when depositing their work. Some were aware of various requirements (including from funders) but were also wary of the possible consequences of signing particular licenses on a preprint when it comes to formal publication at a later stage.\n\nManagement of versioning was mentioned by a number of researchers although experience of this was mixed, with many authors not taking advantage of the facilities offered by preprint servers in terms of tracking versions. Preprint service providers were divided about whether these services allowed for withdrawal of items, but this was regarded as important by a number of participants in the case of misleading results or disputes between co-authors, for example.\n\nDigital preservation, the final infrastructural issue discussed by participants at any length, was regarded as important in principle but often de-prioritised in practice because of its costly nature.\n\nPolicy. Participants identified key policy issues at all levels of the policy stack: publisher, funder and institutional. A cross-cutting issue applying to all policy levels discussed by many participants was, however, the value of preprints as reflected in and recognised by policy. Preprints were recognised to be valuable in providing early open access to research but their value in the scholarly record was commonly qualified by participants. Put simply, they were not considered as valuable as the author accepted manuscript (AAM) or the version of record (VoR), a view reinforced in the minds of many by the fact that many funder and government OA mandates did not have any requirements relating to preprints, but focused rather on AAMs and VoRs. This affected the extent to which different actors regarded preprints as a policy or operational priority. The concept of “standing” (Neylon et al., 2017) is relevant here, with, as an example, standing relating to whether preprints are seen as an appropriate object for evaluation in exercises such as the UK’s Research Excellence Framework, about which there remains ambiguity. One representative of a university stated:\n\n“they’re not acceptable for REF, so they’re not even part of the equation. So it’s the author’s accepted manuscript is the currency we deal with.” (Research performing organisation)\n\nThe value of preprints was seen rather in the extent to which they are a component of a more open research system. Whilst they were seen by some as a useful counterbalance to expensive OA publishing funded by APCs (article processing charges), mostly they were seen as important in terms of their contribution to the overall open science agenda:\n\n“…in terms of preprints, I’m more interested in a different problem, which is the problem of the opening up the whole research endeavour throughout the whole research process through the collection of data to the analysis of data to the curation of data through the writing research protocols. And then analysing the…the results and writing the software and all that stuff. Each of those things themselves should be considered a research output. The preprint is towards the end of that.” (Research performing organisation)\n\nThis was important at all levels of the policy stack. Institutional-level policy was seen by many participants in this light. Preprints might be encouraged in a general way by institutional policy, but deposit of preprints would not normally be mandated. In fact, some participants reported that institutional policy was silent on the matter, and in practice preprints were not accepted as deposits to the IR.\n\nFunders, similarly, often encouraged use of preprints and allowed them to be cited in grant applications, but they were not included as an acceptable form of an output which should be made available OA as part of a funder OA mandate. One preprint provider suggested funders might in future mandate use of preprints but pointed to only one small US funder who was currently doing so. There were other possible funders policies that could encourage change:\n\n“The other thing that funders really could do is to make very public the fact that they will allow a grantee to cite preprints in her progress reports and then her grant renewal application.” (Preprint server provider)\n\nPublishers’ policies were criticized by many participants as preventing authors from depositing preprints either before submission (the Ingelfinger rule) or after acceptance (contractual exclusions or embargoes). The environment was often seen as a confusing one for authors, something which was itself discouraging.\n\nFinancial sustainability and business models. Four models for delivering preprint services emerged from the data, each of which has different implications for sustainability and funding, an issue which was emphasised as being concern by many participants:\n\n1. Standalone preprint servers using in-house technologies e.g. bioRxiv, arXiv;\n\n2. Standalone preprint servers using third-party technologies e.g. ChemRxiv using Figshare infrastructure;\n\n3. Publisher-supported preprints e.g. PeerJ, F1000; and\n\n4. Publisher posting preprints to a preprint server e.g. PLOS partnership with bioRxiv.\n\nModels 1 and 2 involve an ‘author-driven’ mode of posting preprints; Models 3 and 4 are a new ‘publisher driven’ mode.\n\nModel 1 is the ‘classic’ model of preprints, apparently assumed by most participants in their interviews. Model 2 is a version of 1 in which some infrastructure is outsourced to a third party, something which might help to enable sustainability. Models 1 and 2 also include publisher-operated services such as Preprints.org and SSRN, which are owned by MDPI and Elsevier, respectively. These are not classified as publisher-supported (Models 3 and 4) but as standalone servers, because they still follow a paradigm of individual authors being responsible for posting their own preprints.\n\nIn this respect, Models 3 and 4 are different, since they involve the publisher rather than then author driving the preprint submission process, either through the publisher itself providing a preprints service or by depositing on the author’s behalf. Models 3 and 4 move away from the traditional ‘author-driven’ preprints practices – where the author voluntarily deposits a paper as part of their own workflow, separately from submission to a journal – to a ‘publisher-driven’ preprints model, where the publisher to whom a manuscript has been submitted makes it available as a preprint. This is a fundamental shift which has major implications for the way preprints are conceived and the way preprints services are configured.\n\nFinancial sustainability was a concern for many of the participants with one describing the financial sustainability of a number of preprint services as “fragile” (Research performing organisation), often being based on short-term project funding. One preprint service provider described the work they did in partnership with the Center for Open Science as based largely on the goodwill of “volunteers”. One participant commented that independent preprint servers have no business model associated with them other than grant funding. Although this was sometimes seen as a mark of immaturity, with experimentation in funding and sustainability models ongoing, some participants did point to problems in the funding of arXiv, a well-established preprint server, which had been through several periods of uncertainty with regard to its funding during its history. These kinds of concerns, raised by many of the participants, relate to models 1 and 2 particularly.\n\nDespite this, most participants seemed to favour a not-for-profit approach to preprint servers, some for reasons of sustainability, others emphasising the need for independence. Developments associated with models 3 and 4, and even 2, were, therefore, viewed with suspicion or even hostility by some participants. A number expressed concerns about the consolidation of services associated with academic workflows into the hands of a few commercial companies, including Elsevier ownership of SSRN, and Digital Science’s ownership of Figshare. This was seen as potentially jeopardising the independence of the relevant preprint services, even if they were currently operating as standalone. The language used by some participants to describe this development was in some cases strong, with one describing the sale of SSRN to Elsevier as “a huge betrayal of trust” (Engaged researcher) and another observing that this and similar developments have led to “power concentration” (Research performing organisation) in a small number of commercial providers. On the other hand, one representative from the preprint provider observed, “I don’t think that your average researcher thinks about that” (Preprint server provider); and it was indeed evident that many researchers amongst our participants did not show an awareness of such issues.\n\nThere was considerable uncertainty amongst participants about the future role of preprint servers. When questioned whether preprint servers could form a significant part of a system of scholarly communication which would be an alternative to or replace peer-reviewed journals, most were sceptical. There was, however, some discussion of the potential value of ‘overlay journals’ – where virtual journals are created from content held in preprint servers, having been peer-reviewed and selected after their circulation. Some suggested that automated filtering rather than human-based peer review might have a role to play in creating overlay services. There was some awareness of experimental work in the overlay area, but few were able to identify working examples.\n\nIt was clear from comments of participants that if preprints were to play a more significant role in scholarly communication, major improvements to the preprints infrastructure would be needed. This would include incorporation of preprints into scholarly and publisher workflows, provision for production of preprints in standards-based formats (e.g. XML) and greater consideration of preservation services. All of this would require major investment. However, even in a system where preprints did not replace existing channels of communication, such as journals, many of these developments were still considered necessary in order to make the preprint infrastructure more robust.\n\nWhilst uptake of preprints was seen by many to be increasing, the role of policy in this area was uncertain. It was seen as particularly problematical for use of preprints to be mandated, as opposed to encouraged, by funders. There was a clear perception that preprints should be adopted by researchers who see the benefits themselves, rather than in response to a mandate:\n\n“There needs to be an intrinsic interest of the research community to communicate via preprints. I don’t think preprint posting can be enforced top-down or from anyone other than the research community and specifically the disciplinary communities themselves.” (Research funder)\n\nIt is noteworthy that this view was expressed by a policy maker.\n\n\nDiscussion and conclusions\n\nThe findings of this research clearly relate to the innovation adoption decision process identified in IDT, beginning with developing knowledge of innovation to confirmation of its adoption (Table 7).\n\nIt was evident that our participants were at different stages in the adoption process and this is a reflection of their peers’ and subject communities’ practices. Knowledge of preprints is rising but many are still not beyond the persuasion stage. Community norms remain crucial and have not shifted in many cases, therefore constraining individuals’ decisions. There was, nevertheless, some willingness to experiment, particularly amongst general OA supporters. There was some awareness of potential benefits becoming evident in practice but still at low levels. The rate of adoption is influenced in our data by a number of factors highlighted in IDT (Table 8).\n\nIt is worth mentioning that, in some cases, it is not clear whether researchers do not post preprints because there is no discipline-appropriate server for them, or there is no server because researchers in the field do not (want to) post them. The data shows that some researchers are happy to be early adopters often because of sympathy with general open science and open access goals, as well as being motivated by the potential benefits they see in their own use of preprints. Others remain sceptical, fearing that journals may reject their submissions, which is still the case in some areas (Mallapaty, 2019), and questioning the value of circulation of pre-peer-reviewed outputs. The current environment for many disciplinary communities is therefore characterised currently by some experimentation, but also by uncertainty and fragmentation.\n\nAnother key issue is trust. Trust is an essential feature of scholarly communication and was a recurring theme in the data, along with responsible posting and use of preprints. Nicholas et al. (2014) have shown that “researchers play down difficulties of establishing trustworthiness, not because there are none, but because they have well‐developed methods of establishing trust.” However, preprints cut across those methods and create new ambiguities and uncertainties. Many of our participants were conscious that new norms were needed (COPE Council, 2018) in this space and it was often the apparent absence of these that limited enthusiasm for preprints.\n\nThe preprints landscape is rapidly changing, and disciplinary communities are at different stages in the innovation diffusion process. The current high level of experimentation means that a one-size-fits-all approach to preprints is neither feasible or appropriate at present (if it ever will be). Something that will clearly play a role in the success (or lack thereof) of preprints is cooperation between the range of stakeholders involved: though some of the issues we highlighted might appear independent of one another, we note that the majority of these affect multiple stakeholders at once.\n\nOur findings have given rise to a number of key questions that we believe need to be addressed so that preprints can be supported sustainably in the future. Active engagement with these questions should lead to improved clarity and provide solid foundations for policy development and implementation. The questions and their relationships to the different actors involved are illustrated in Figure 1. These include some key questions that need to be addressed by particular stakeholder groups, such as funders or preprint server providers. We have also added publishers as a separate group to this figure, taking account of issues that have arisen from the groups considered in this current study. However, there are also a large number of questions that need to be addressed through dialogue between different stakeholder groups. These are illustrated in the figure. One of the key challenges is to find channels for such dialogue to take place in order to develop solutions which are widely accepted.\n\nThis is in many respects an agenda for further research, discussion and policy design. We expect, following a rapid rise in preprints since about 2013, that many of these questions will come to be seen as increasingly important over the next five years. The urgency with which they are addressed, and the ways in which they are answered by the different stakeholders, will shape the role that preprints play in scholarly communications in the future.\n\n\nData availability\n\nThe authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. Data underlying this study cannot be made publicly available in order to safeguard participant anonymity and that of their organisations. Ethical approval for the project was granted on the basis that only extracts of interviews would be shared (with appropriate anonymisation) as part of publications and other research outputs. In order to share data with other researchers, the participants must be contacted and consent to this data being released. In order to request data release, other researchers should contact the corresponding author or Chair of the University of Sheffield Information School Research Ethics Committee (ischool_ethics@sheffield.ac.uk).\n\nZenodo: Mapping of interview questions to areas of Innovation Diffusion Theory, https://doi.org/10.5281/zenodo.3240426 (Chiarelli et al., 2019a).\n\nExtended data.csv contains our interview questions, split by stakeholder group and mapped to innovation diffusion theory.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis study has been funded by Knowledge Exchange (KE), a group of national organisations from six European countries supporting research infrastructure and services to enable the use of digital technologies to improve higher education and research: CSC in Finland, CNRS in France, DEFF in Denmark, DFG in Germany, Jisc in the UK and SURF in the Netherlands.\n\n\nAcknowledgements\n\nThis article draws on the findings of an initial phase of work on the preprints landscape (Chiarelli et al., 2019b) supported by the KE Task and Finish Group on preprints.\n\n\nReferences\n\nAbdill RJ, Blekhman R: Tracking the popularity and outcomes of all bioRxiv preprints. eLife. 2019; 8: pii: e45133. 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Publisher Full Text" }	[ { "id": "50461", "date": "23 Jul 2019", "name": "Sarvenaz Sarabipour", "expertise": [ "Reviewer Expertise 1) Sarvenaz Sarabipour", "PhD: Systems Biology", "Signal Transduction", "Computational Modeling 2) Humberto Debat", "PhD: Virology", "Viromics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nIn this manuscript Chiarelli et al. assess preprints from the point of view of a diverse group of stakeholders. The authors perform an analysis based on 38 interviews with representatives of research funders, research performing organizations, preprint servers and service providers; and researchers (engaged and unengaged). Additional discussion is provided on the benefits and challenges of preprint posting, along with issues such as infrastructure and financial sustainability and the definition of a ‘preprint’ in different communities, and the impact this has on further uptake. This study provides a thorough investigation on an emerging and relevant topic, giving a platform to the opinions of under-represented stakeholders on the discussion of preprints: a key driver of the transformation of the scholarly publishing landscape.\nComments:\nWe would like to ask the authors to address the following:\nThe introduction may benefit from additional literature to balance the many sections. For instance:\n1. On Page 3: “However, skeptics have questioned the value of preprints and even suggested they may be dangerous – circulating versions of articles before they have been quality controlled by peer review may lead to unnecessary risk, particularly in disciplines like medicine (Sheldon, 2018).” The only negative note on preprints by Sheldon 2018 was well rebutted by at least 5 publications:\nMaintaining confidence in the reporting of scientific outputs - Sarabipour et al. (2018)1. Preprints are good for science and good for the public - Sarabipour (2018)2. Preprints help journalism, not hinder it - Tennant el al. (2018)3. Together scientists and journalists can spot poor preprints - Fraser & Polka (2018)4. Preprints: recall Nature’s nasty past - Cobb (2019)5.\nArticles 1-4 above address the relationship between scientific reporting and journalism. Further to the authors remark on “unnecessary risk particularly in disciplines like medicine” - preprint servers carry a highly visible note on preprinted manuscript stating that these research products are not peer-reviewed.\nRegarding biomedical and medical preprints and risk to public: This is not a compelling argument since care is delivered to patients by physicians. MedRxiv was recently established and is accepting manuscripts to accelerate medical research. The server front page notes that: \"Caution: Preprints are preliminary reports of work that have not been peer-reviewed. They should not be relied on to guide clinical practice or health-related behavior and should not be reported in news media as established information.”\n2. On Page 3: “the current and potential future role of preprints as a vehicle for scholarly communication” - there are and will be many role(s) for preprints in the current and future of the scholarly endeavors and they are summarized here:\nOn the value of preprints: An early career researcher perspective - Sarabipour et al.(2019)6 https://asapbio.org/reading In praise of preprints - Fry et al. (2019)7.\n3. On Page 4: “much of the literature is still to be found in editorials and opinion pieces rather than data-driven research” - the fact is that the number of data driven literature on preprints is growing rapidly and are noteworthy. Examples are:\nTracking the popularity and outcomes of all bioRxiv preprints - Abdill & Blekhman (2019)8. Releasing a preprint is associated with more attention and citations - Fu & Hughey (2019)9. The effect of bioRxiv preprints on citations and altmetrics - Fraser et al. (2019)10. Comparing quality of reporting between preprints and peer-reviewed articles in the biomedical literature - Carneiro et al. (2019)11.\n4. On Page 4: “Accessibility (4) is crucial, with a preprint normally defined as being (or assumed to be) openly available: it “can be viewed without charge on the Web” and “Therefore, the venue for distribution of preprints is often assumed to be a freely-accessible server of some kind”. It is a fact that all preprint servers are freely accessible to any human being that has access to internet around the world: free of charge to submit by authors, free of charge to read by all anywhere on the planet.\n5. On Page 5: “To say that an output is “deserving” of dissemination is, of course, a value judgement and difficult to demonstrate for each deposit as it is made, but it is one that is implicit in much of the discourse on preprints.” - this is not entirely true. The value of preprints are well known/articulated/discussed at this point as preprints accelerate research and altmetric attention to scientific work: Please see references 8-11 above. Preprints have been deposited on arXiv for nearly three decades now and are invaluable to the physical sciences community. Other forms of pre-peer review material such as computer code and protocols have been disseminated open source and their value is well noted in various fields.\n\n6. On Page 5: “dis-benefits of preprints” - unless the authors may provide any evidence of “dis-benefits of preprints” besides one note (Sheldon 2018), it is best to avoid using this word “dis-benefits” and consider rephrasing to “perceived dis-benefits”. Please see reference 1-5 above.\n7. On Page 5: “Early dissemination can be useful to some particular members of the scholarly community, with early career researchers (ECRs)” - preprints and all other forms of scholarly pre-publication material are beneficial to ALL researchers not jut ECRs. Timely release of scientific results in the form of preprints and other forms such as code and data bases have already accelerated research by encouraging sharing of ideas, resources and as discussed in previously suggested references above.\n8. On Page 5: “Perhaps the most prominent criticism of preprints relates to this last issue: the lack of quality assurance through peer review (Sheldon, 2018). As well as a general concern about lowering quality standards, lack of quality control has been seen as potentially dangerous as “reports that have not undergone formal peer review [organised by a journal] could be misleading” (Lauer et al., 2015). Furthermore, uncertified science might be reported prematurely in the media and might even give rise to ‘fake news’ (Sheldon, 2018). Some insist that, at the very least, the opportunity to disseminate knowledge rapidly without peer review may encourage academics to produce low-quality outputs on fashionable topics (Teixeira da Silva, 2017).” The authors should declare that there are no evidences supporting all these bold statements. The Sheldon (2018) news article in fact explicitly states its speculative nature in the title which reads “preprints could promote confusion and distortion”. Please see references 1-5 above.\n9. On Page 5: “Others have gone further and questioned the value of self-appointed reviewers, as opposed to those selected by journal editors (see the issue of “self-policing” highlighted by Harnad, 1998).” This is a misleading claim. Voluntary reviews of preprints are done by researchers in that field or community and are not self-appointed by the authors. The open access nature of preprints allows the potential assessment of manuscripts by more than 2-3 (more than journal reviewers) normally and there is no evidence on these reviews having different quality than journal peer-reviews.\n10. On Page 5: “Preprint posting, however, is unlikely in any case to substitute for the valuable role played by selective journals in filtering content”. This is an unfounded claim as a system of preprint servers and overlay journals may very well replace the outrageously expensive and time-consuming system of for profit journal publishing in under a decade. Please see reference 12.\nA proposal for the future of scientific publishing in the life sciences - Stern & O’Shea (2019)12.\n11. On Page 5: “Whilst this convention has come under criticism and been withdrawn by some publishers, it still exists for some journals, e.g. in medicine and chemistry (Lauer et al., 2015; Teixeira da Silva & Dobránszki, 2019)” - this sentence is misleading. In the current scenario, authors should consider changing “some” to “most” publishers.\n12. On Page 6: “For example, it is not universally agreed when an output should be citable (in the literature, funding proposals or promotion cases)”. It is now becoming more and more agreed upon that preprints should be cited in articles and research grant proposals. Major funding bodies such as NIH, CZI and Wellcome Trust allow and encourage citation of preprints. Many journals allow citation of preprints and other research outputs (eLife and PLOS family of journals are examples).\n\n13. On Page 6 and other pages: “Scooping”, and “a claim of precedence”: Scooping is a perceived concern of researchers and is unfounded. Many researchers present unpublished research at conference. Perceived preprint concerns and claim of precedence have been discussed in reference 6 above.\n14. On Page 6: “or when it can be used to establish a claim of precedence” and “In disciplines where a preprint is not considered appropriate to establish precedence, it has also been suggested that making a preprint available may actually encourage research to be scooped by rival researchers who publish in a recognized journal before the preprint authors (the ‘flip side’ of the priority claim argument above) (Kaiser, 2017).”Regarding these repeated sections on scooping and priority claims:\n2-3 papers published via preprints or journals in close temporal proximity of each other have all been projects 2-4 years in making so it is very difficult to assess who did it first.\n\nWho gives researchers priority? A preprint that is posted online has a date and that gives precedence to the work. In reality, as researchers we all know that priority is given to researcher by their field and their peers. Scientific research is often presented at conferences well in advance of preprints or journal publication. As we know what matters in research is scientific reputation and that is given to researchers by their peers and field during a career in scientific research.\n\nMultiple papers published via preprints or journals in close proximity of each other only show the reproducibility of one another are need to be celebrated not feared so repeated arguments on scooping and priority claims do not move a positive conversation on research forward specially for early career researchers and the future generations of researchers.\n15. On Page 6: “Some have argued that initiatives such as Declaration on Research Assessment (DORA), with its emphasis on the quality of the output rather than venue of publication, promote use of preprints (Polka, 2018)”. It is noteworthy that DORA has now become a major international initiative and is signed by over 1,400 organizations including major universities and other research institutions and 14,000 researchers.\nOther comments to the MS text:\nA) The Title: Authors could consider an alternative title “Preprints and Scholarly Communication: a preliminary/exploratory study on Adoption, Practices, Drivers and Barriers”\nB) The Abstract:\n“Our study is the first using empirical data to understand” - please change to: Our study uses empirical data… (See comments below highlighting other empirical studies on the topic).\n\n“The main concerns are related to the lack of quality assurance and the ‘Ingelfinger rule’.” Please change to: main perceived concerns.\nC) The Introduction:\nOn Page 3: “key actors”. It is puzzling how the authors determined which the key stakeholders are in the preprints ecosystem. Authors should discuss why they did not incorporate “scientific journal publishers” as a relevant actor for this work. It is interesting that they incorporated “publishers” as node in Figure 1 as a stakeholder group but failed to incorporate “representatives” during their interviews.\n\nOn Page 3: “(Carà et al., 2017) .” Please delete the space.\n\nOn Page 3: “The study is the first using empirical data”. Please see our comment on the abstract.\n\nOn Page 3: “Zuckerberg Foundation“ - the correct name is the Chan-Zuckerberg Foundation (CZI).\n\nOn Page 3: “The study is the first using empirical data”. Please see the comments on the abstract above.\n\nOn Page 4: “…relatively small number of peer-reviewed studies focusing on preprints – much of the literature is still to be found in editorials and opinion pieces”. The authors surely know that in general opinion pieces published in scientific journals are also peer-reviewed.\n\n“Defining preprints. Key components. Table 1”. A relevant aspect of preprints that is not contemplated here is that preprints are published open access free of charge. Perhaps that important feature could be incorporated in component 4. Maybe this feature, highly relevant for unprivileged research communities, was neglected given the sampling strategy employed (see comment below).\n\nOn Page 8: “The study adopted a heterogeneous purposive sampling approach, aiming to include a wide range of perspectives from actors in the area. Participants comprised senior representatives…”. If the authors selected senior representatives of each stakeholder categories, one could question how heterogeneous the sampling approach was. Moreover, authors mention the use of snowball sampling, which is linked to homogeneous sampling. In addition, participants correspond to only eight OECD High-income economies according to World Bank (half of them only from UK and Germany), which again appear to be more reflective of a homogenous sampling approach. Authors should also highlight the intrinsic limitations of quality coding in terms of reproducibility.\n\nOn Page 8: “We undertook 38 semi-structured interviews”. Authors did not describe how sample size was determined. Authors should discuss how this small sample size might redound in uncertainty issues. The complete absence of statistical analyses of the data is striking. This affects the “empirical power” of the work, and one could question whether the described “trends” indicated in the manuscript are purely anecdotal, derived from sampling error and/or speculative.\n\n“Like many kinds…” paragraph. This important paragraph highlighting the limitations of the study should be moved to the discussion/conclusion section.\nD) The Results section in general:\nThe reviewers wonder if the results section as a whole, which is relatively long in comparison to the rest of the paper, should be limited to Table 5 and its description and avoid the unending narrative of anecdotal specific responses. In case the authors feel the need to present all these quoted responses, perhaps they could be moved to the supplementary section.\n“Some participants…” “Many saw a preprint as…” “Other participants saw preprints..” “there were signs this…” and elsewhere. This vague and unclear wording could be avoided by the use of absolute numbers or percentages of total responses.\n\nTable 5: Please consider complementing the legend to provide definitions to “systemic” and “individual”.\n\nTable 6: Following Table 5 title, it would be more reasonable to entitle Table 6 as “potential challenges of preprints”, or “possible” as depicted in the same table in Chiarelli et al. 2019.\n\nOn Page 13: “One preprint service provider observed that only 10% of preprints received comments”. While this % is depicted as low in the text by the use of “only”, authors should contextualize this type of assertion. Peer reviewed articles published in journals are far less commented than preprints as reported elsewhere.\n\nOn Page 14: “author might invite comments from people who are expected to be positive about their work”. Again, this and other speculative statements should be leveled when they are employed to contrast with traditional peer-reviewed journal articles. It is a common practice in an important number of journals to ask authors to provide contact information of potential reviewers, which could eventually lead to the same issues expressed in the preceding statement.\n\nOn Page 14: “We note…”. This relevant paragraph, highlighting the limitations and anecdotal nature of the study, should be moved to the discussion/conclusions.\nE) The Discussion/Conclusion: As expressed above, some parts of the results section could be moved here as they reflect more speculative comments based on the interview responses.\nF) The References:\nThis citation “Sarabipour et al.: On the value of preprints: an early career researcher perspective. PeerJ Preprints (2018)” Is now peer-reviewed and hosted as “On the value of preprints: An early career researcher perspective. Sarabipour et al. PLoS biology (2019)[.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5026", "date": "25 Nov 2019", "name": "Andrea Chiarelli", "role": "Author Response", "response": "Summary: In this manuscript Chiarelli et al. assess preprints from the point of view of a diverse group of stakeholders. The authors perform an analysis based on 38 interviews with representatives of research funders, research performing organizations, preprint servers and service providers; and researchers (engaged and unengaged). Additional discussion is provided on the benefits and challenges of preprint posting, along with issues such as infrastructure and financial sustainability and the definition of a ‘preprint’ in different communities, and the impact this has on further uptake. This study provides a thorough investigation on an emerging and relevant topic, giving a platform to the opinions of under-represented stakeholders on the discussion of preprints: a key driver of the transformation of the scholarly publishing landscape. Authors’ response: We are grateful for the comments made by the reviewers. They have been extremely useful for us in reviewing our approach. We have made changes in response to the suggestions of the reviewers wherever possible and have explained our responses in detail below. Reviewers’ comments: We would like to ask the authors to address the following: The introduction may benefit from additional literature to balance the many sections. For instance: 1. On Page 3: “However, skeptics have questioned the value of preprints and even suggested they may be dangerous – circulating versions of articles before they have been quality controlled by peer review may lead to unnecessary risk, particularly in disciplines like medicine (Sheldon, 2018).” The only negative note on preprints by Sheldon 2018 was well rebutted by at least 5 publications: Maintaining confidence in the reporting of scientific outputs - Sarabipour et al. (2018)1. Preprints are good for science and good for the public - Sarabipour (2018)2. Preprints help journalism, not hinder it - Tennant el al. (2018)3. Together scientists and journalists can spot poor preprints - Fraser & Polka (2018)4. Preprints: recall Nature’s nasty past - Cobb (2019)5. Authors’ response: We are happy to comment that this point has been rebutted. Prompted by this comment, we have added a paragraph in the literature review on the fact there has been vigorous debate on this and a number of other aspects of preprints. We believe it is worthwhile explicitly acknowledging this, as follows: “It is noticeable that the literature on the pros and cons of preprints has, like many aspects of open science, given rise to robust discussion and debate. The paper by Krumholz, Ross, & Otto (2018) cited above is itself structured as a debate, with the first two authors making the case for preprints and the third expressing concerns. Sheldon’s (2018) opinion piece in Nature arguing that preprints could have a negative impact beyond the scientific community, was met with vociferous rebuttals in the letters pages of the journal the following month (Fraser & Polka, 2018; Sarabipour, 2018; Tennant, Gatto, & Logan, 2018). On social media, such as Twitter, there have also been vigorous exchanges (e.g. Twitter, 2019).” For completeness, we have also added additional references to sources raising concerns about the adoption of preprints within medicine (Krumholz, Ross, & Otto, 2018 and Leopold et al, 2019). The article by Leopold et al is particularly significant in that it is not just a contribution to the debate but is the (re-)statement of an editorial policy for several medical journals who follow the Ingelfinger rule, clearly stating arguments against preprints. We believe this remains a live debate, and have sought to reflect this in the revised article. Apart from this particular point, a large number of comments made by the reviewers which we respond to in detail below relate to how we present the literature and the wider debate in our introduction and literature review. It is, therefore, worthwhile clarifying our approach in general terms. In our literature review, we report various views on preprints – their features, their perceived advantages and disadvantages etc. We do so in an evaluative way but aim to represent different perspectives. We agree with the reviewers, balance is important. We, therefore, spend as much time discussing perceived benefits as portrayed in the literature as we do challenges – and our account is constructed in a way specifically to create balance. Our introduction and literature review sets the scene for our research – the concern about quality and potential misuse of results features in the literature as well as in our interview data, for example, and so discussing the first of these helps to contextualise the second. Our review of the literature is not an opinion or advocacy piece (although much of the literature we cite fit these categories). Our literature review is not designed to set out a particular position (for or against) in relation to preprints, nor would that be appropriate for a study of this kind. Rather, it aims to report the literature in a balanced way. Krumholz, H. M., Ross, J. S., & Otto, C. M. (2018). Will research preprints improve healthcare for patients? BMJ, 362, k3628. https://doi.org/10.1136/BMJ.K3628 Leopold, S. S., Haddad, F. S., Sandell, L. J., & Swiontkowski, M. (2019). Editorial: Clinical Orthopaedics and Related Research, The Bone & Joint Journal, The Journal of Orthopaedic Research, and The Journal of Bone and Joint Surgery will not accept clinical research manuscripts previously posted to preprint servers. Clinical Orthopaedics and Related Research, 477(1), 1–4. https://doi.org/10.1097/CORR.0000000000000565 Reviewers’ comments: Articles 1-4 above address the relationship between scientific reporting and journalism. Further to the authors remark on “unnecessary risk particularly in disciplines like medicine” - preprint servers carry a highly visible note on preprinted manuscript stating that these research products are not peer-reviewed. Authors’ response: The point we are making here is that criticism of preprints has occurred in the literature based on the argument that they are potentially dangerous. Once again, we are reporting the debate in the literature, we are not writing an advocacy/opinion piece ourselves. We are not saying we agree or disagree with the arguments, rather we are reporting them to be part of the current debate. As noted above, we have added two further citations to our paper to further illustrate the point that this concern has been raised in the literature (Krumholz, Ross, & Otto, 2018 and Leopold et al, 2019). In the article by Krumholz et al (another opinion piece, this time in the form of a debate between supporters and sceptics of preprints), Otto argues, “Medical research preprints are unnecessary for scientific progress and dangerous for patient care”. Interestingly, the guidance on preprints produced by COPE (https://publicationethics.org/files/u7140/COPE_Preprints_Mar18.pdf) acknowledges that such criticisms have been made of preprints (even if it is not an argument that it supports): “Some critics have raised concerns that preprints may have a negative impact on the credibility and public perception towards research, since the information has not been scrutinised and validated via peer review. For example, what are the risks if a preprint with a potential impact on public health is interpreted by some as established evidence?” The fact that preprints are labelled as such, as the reviewers point out, helps to mitigate the possible misuse of preprints, but does not eliminate that danger. In an open environment, there are possibilities that medical research could be accessed by patients, campaigners and others directly. There are well known examples of this happening and of the impact of misunderstandings being magnified by social media. Even if many of the concerns are hypothetical (as our research suggests), they cannot be simply dismissed, and need to be reported in any overview of the literature. Reviewers’ comments: Regarding biomedical and medical preprints and risk to public: This is not a compelling argument since care is delivered to patients by physicians. MedRxiv was recently established and is accepting manuscripts to accelerate medical research. The server front page notes that: “Caution: Preprints are preliminary reports of work that have not been peer-reviewed. They should not be relied on to guide clinical practice or health-related behavior and should not be reported in news media as established information.” Authors’ response: Our point, again, is that the debate in the literature raises this concern. And it needs to be acknowledged, we believe, in any fair reporting of the current debate. We refer to our response above. We would add that open-access medical content can more easily be accessed directly by non-clinicians without expert mediation. In fact, the whole literature on the ‘expert patient’ builds on this as a positive. There is however the risk of misunderstanding. That is one point made by Otto (see above) and also by Leopold et al. Note MedRxiv had not been released when we carried out our data gathering, although one participant anticipated the kind of screening and flagging of preprints mentioned in the reviewers’ comments above. Reviewers’ comments: 2. On Page 3: “the current and potential future role of preprints as a vehicle for scholarly communication” - there are and will be many role(s) for preprints in the current and future of the scholarly endeavors and they are summarized here: On the value of preprints: An early career researcher perspective - Sarabipour et al.(2019)6 https://asapbio.org/reading In praise of preprints - Fry et al. (2019)7. Authors’ response: Agreed. These papers make the case for preprints in the current scholarly communication system. We have cited Sabipour et al (2019). We will happily add reference to the more recent explanation of the advantages of preprints by Fry et al (2019). These papers (for the most part, at least) confine themselves to discussing the role of preprints in the current scholarly communication environment rather than discussing how they might contribute to more radical change in future. We have however added to our Discussion a paragraph with some more information in this area. This discusses the overlay idea amongst others: “The future of preprints servers and their links with the overall scholarly communication process and infrastructure remain unclear. It is possible that the recent rise in preprint services might be reversed and that preprints go through a period of retrenchment, returning to serve the core areas traditionally associated with preprint use, such as high-energy physics. A possible alternative to such a ‘retrenchment scenario’ is what might be called the ‘patchiness scenario’, where different levels of adoption exist across different fields. Patchiness may be an ongoing situation, or may be a transition stage towards a possible ‘ubiquity scenario’. For preprints to become ubiquitous, of course, requires significant cultural and infrastructural change, some of which is indicated by the data presented here. This may partly depend on the integration between preprint services and other parts of the scholarly communication infrastructure and on related cultural norms. Closer integration may give rise the possibility of more radical change in scholarly communication, creating opportunity for developments, such as overlay journals. Overlay journals have been discussed as thought experiments since at least the turn of the 21st Century (A. Smith, 2000; J. W. T. Smith, 1999) and there have been notable experiments in this area, and some ongoing services in specialised areas do exist, such as Discrete Analysis (Ball, 2015). However, we are yet to see their widespread adoption, even though the potential remains.” Reviewers’ comments: 3. On Page 4: “much of the literature is still to be found in editorials and opinion pieces rather than data-driven research” - the fact is that the number of data driven literature on preprints is growing rapidly and are noteworthy. Examples are: Tracking the popularity and outcomes of all bioRxiv preprints - Abdill & Blekhman (2019)8. Releasing a preprint is associated with more attention and citations - Fu & Hughey (2019)9. The effect of bioRxiv preprints on citations and altmetrics - Fraser et al. (2019)10. Comparing quality of reporting between preprints and peer-reviewed articles in the biomedical literature - Carneiro et al. (2019)11. Authors’ response: Agreed. All of these papers have been made available in 2019 in various forms (all of them since we gathered our data, two of them since we submitted our paper to F1000). They comprise a new empirical base for preprints studies. We have adjusted our literature review to take these recent additions to the literature into account, adding a new section on ‘The use and impact of preprints’: “The use and impact of preprints. A noticeable recent development in the literature has been publication of a number of empirical studies on the use and impact of preprints. These include Carneiro et al.’s (2019) study which compared the quality of reporting of findings in preprints from PubMed and bioRxiv against formally-published journal articles based on a number of criteria tested through a questionnaire...” etc. Reviewers’ comments: 4. On Page 4: “Accessibility (4) is crucial, with a preprint normally defined as being (or assumed to be) openly available: it “can be viewed without charge on the Web” and “Therefore, the venue for distribution of preprints is often assumed to be a freely-accessible server of some kind”. It is a fact that all preprint servers are freely accessible to any human being that has access to internet around the world: free of charge to submit by authors, free of charge to read by all anywhere on the planet. Authors’ response: Agreed. And that is why we have included open accessibility in our definition of what constitutes a preprint. When discussing preprints that is the way many authors of the literature define them, or at least that is a characteristic they assume. Sometimes this is taken for granted amongst commentators but is always at least implicit. We have made changes to our discussion of definitions to make this clearer. In Table 1, we have changed the description of this component to, “Preprints are openly available online”. In the accompanying textual discussion, it now reads as: “Accessibility (4) is crucial in definitions. A preprint is normally defined as being (or assumed to be) openly available online: it “can be viewed without charge on the Web” (Berg et al., 2016, p. 899). The idea of openness is fundamental to discussions on preprints. The venue for distribution of preprints is often assumed to be a freely-accessible server of some kind, a point highlighted by Berg et al. (2016, p. 899), who include in their definition that a preprint “is uploaded by the authors to a public server”.” Reviewers’ comments: 5. On Page 5: “To say that an output is “deserving” of dissemination is, of course, a value judgement and difficult to demonstrate for each deposit as it is made, but it is one that is implicit in much of the discourse on preprints.” - this is not entirely true. The value of preprints are well known/articulated/discussed at this point as preprints accelerate research and altmetric attention to scientific work: Please see references 8-11 above. Preprints have been deposited on arXiv for nearly three decades now and are invaluable to the physical sciences community. Other forms of pre-peer review material such as computer code and protocols have been disseminated open source and their value is well noted in various fields. Authors’ response: What we are discussing here is how preprints are defined in the literature. Bourne et al. (2017) extend the definition of a preprint beyond that of a version of a peer-reviewed paper before it is peer-reviewed and formally published to include “a research output that has not completed a typical publication pipeline but is of value to the community and deserving of being easily discovered and accessed”. What we are pointing out is that it is interesting that this definition defines a preprint as something that is “deserving” of dissemination. To state that something is “deserving” of dissemination is a value judgement. To incorporate a value judgement in a definition is unusual. Here we are not discussing whether preprints are valuable or not, rather we are discussing whether value should be part of the definition of what constitutes a preprint. Of course, particular preprints may or may not be valuable. Preprints as a whole may or may not be valuable. But to define preprints as what is valuable is somewhat curious. If an output has “not completed a typical publication pipeline” but is not deemed to be valuable, does that mean it is not a preprint? And if so, who decides what is a preprint or not, and on what basis? By extending the definition in this way Bourne et al have at least created an ambiguity. Reviewers’ comments: 6. On Page 5: “dis-benefits of preprints” - unless the authors may provide any evidence of “dis-benefits of preprints” besides one note (Sheldon 2018), it is best to avoid using this word “dis-benefits” and consider rephrasing to “perceived dis-benefits”. Please see reference 1-5 above. Authors’ response: We have changes “dis-benefits” to “challenges”. We now refer to “Perceived benefits and challenges” of preprints. This is a fairer way to refer to the current debate, especially as most of the outputs we refer to discussing benefits and challenges are opinion pieces which present arguments – some more cogent than others – but only limited empirical evidence. Reviewers’ comments: 7. On Page 5: “Early dissemination can be useful to some particular members of the scholarly community, with early career researchers (ECRs)” - preprints and all other forms of scholarly pre-publication material are beneficial to ALL researchers not jut ECRs. Timely release of scientific results in the form of preprints and other forms such as code and data bases have already accelerated research by encouraging sharing of ideas, resources and as discussed in previously suggested references above. Authors’ response: Agreed. We have rephrased this to make (we hope) the point we are making clearer i.e. some of the literature emphasises the particular benefits to ECRs. This is not to say preprints are not useful for other groups, it is just that the benefits for this particular group are emphasised in some of the literature. The rewording is: “Early dissemination is seen by some as especially useful to a number of members of the scholarly community in particular, with early career researchers (ECRs) commonly identified as specific potential beneficiaries, as preprints can allow them to rapidly achieve “visibility” and demonstrate productivity in job and grant applications (Desjardins-Proulx et al., 2013; Sarabipour et al., 2018; Tennant et al., 2019).” Reviewers’ comments: 8. On Page 5: “Perhaps the most prominent criticism of preprints relates to this last issue: the lack of quality assurance through peer review (Sheldon, 2018). As well as a general concern about lowering quality standards, lack of quality control has been seen as potentially dangerous as “reports that have not undergone formal peer review [organised by a journal] could be misleading” (Lauer et al., 2015). Furthermore, uncertified science might be reported prematurely in the media and might even give rise to ‘fake news’ (Sheldon, 2018). Some insist that, at the very least, the opportunity to disseminate knowledge rapidly without peer review may encourage academics to produce low-quality outputs on fashionable topics (Teixeira da Silva, 2017).” The authors should declare that there are no evidences supporting all these bold statements. The Sheldon (2018) news article in fact explicitly states its speculative nature in the title which reads “preprints could promote confusion and distortion”. Please see references 1-5 above. Authors’ response: As stated above, we have added two other references to articles critiquing preprints to this paragraph – one of these (Otto) uses the term “dangerous” in relation to preprints. The other (Leopold et al) states directly, “We believe that the benefits proposed by advocates of medical preprint servers can be better achieved in other ways, and that medical preprint servers pose serious health and safety dangers to the patients for whom are supposed to be caring”. This is a view evident in the literature (as well as widely held amongst researchers and practitioners). We, therefore, need to report it. We refer to our comments above, that we are not writing an opinion piece here – rebutting arguments point by point we may disagree with; rather, we are reporting the contours of the debate in the literature. It is appropriate, we believe, to report this as an important argument which occurs in the literature. Reviewers’ comments: 9. On Page 5: “Others have gone further and questioned the value of self-appointed reviewers, as opposed to those selected by journal editors (see the issue of “self-policing” highlighted by Harnad, 1998).” This is a misleading claim. Voluntary reviews of preprints are done by researchers in that field or community and are not self-appointed by the authors. The open access nature of preprints allows the potential assessment of manuscripts by more than 2-3 (more than journal reviewers) normally and there is no evidence on these reviews having different quality than journal peer-reviews. Authors’ responses: The phrase “self-appointed reviewers” means reviewers who are self-appointed i.e. they appoint themselves, not that the authors appoint them. We hope this clarifies our point. Reviewers’ comments: 10. On Page 5: “Preprint posting, however, is unlikely in any case to substitute for the valuable role played by selective journals in filtering content”. This is an unfounded claim as a system of preprint servers and overlay journals may very well replace the outrageously expensive and time-consuming system of for profit journal publishing in under a decade. Please see reference 12. A proposal for the future of scientific publishing in the life sciences - Stern & O’Shea (2019)12. Authors’ response: Proposals of overlay journals have been around for at least 20 years (A. Smith, 2000; J. W. T. Smith, 1999). One of us wrote about such possibilities in 2004 (Pinfield, 2004); ideas further developed a decade ago (Pinfield, 2009). We agree, that the overlay model has real potential but are yet to see significant adoption of it (although interesting experiments and even some specialised services do exist). However, even in an overlay model filtering for quality is important and involves peer review. In the short/medium-term this function is normally seen as coming from journals since other peer-review providers are not commonly available. Note again, however, here we are still providing commentary on literature and are not advocating any particular approach ourselves. We have, however, changed the text of this sentence to make that clearer: “Preprint posting, however, is not normally seen as a substitute for peer review, currently managed by journals, in filtering content (Suber, 2012), a process that is commonly valued, even if recognised to be imperfect (Lee, et al., 2013).” Also, we have added a paragraph (as previously mentioned) in the Discussion on possible futures of preprints servers and their connections to the wider scholarly communication infrastructure, including overlay journals, since we agree, there is considerable potential in the overlay model. “The future of preprints servers and their links with the overall scholarly communication process and infrastructure remain unclear… Closer integration may give rise the possibility of more radical change in scholarly communication, creating opportunity for developments, such as overlay journals. Overlay journals have been discussed as thought experiments since at least the turn of the 21st Century (A. Smith, 2000; J. W. T. Smith, 1999) and there have been notable experiments in this area, and some ongoing services in specialised areas do exist, such as Discrete Analysis (Ball, 2015). However, we are yet to see their widespread adoption, even though the potential remains.” Pinfield, S. (2004). Self-archiving publications. In G. Gorman & F. Rowland (Eds.), Scholarly Publishing in and Electronic Era: International Yearbook of Library and Information Management 2004-2005 (pp. 118–145). Retrieved from http://eprints.nottingham.ac.uk/142 Pinfield, S. (2009). Journals and repositories: An evolving relationship? Learned Publishing, 22(3), 165–175. https://doi.org/10.1087/2009302 Smith, A. (2000). The journal as an overlay on preprint databases. Learned Publishing, 13(1), 43–48. https://doi.org/10.1087/09531510050145542 Smith, J. W. T. (1999). The deconstructed journal – a new model for academic publishing. Learned Publishing, 12(2), 79–91. Reviewers’ comments: 11. On Page 5: “Whilst this convention has come under criticism and been withdrawn by some publishers, it still exists for some journals, e.g. in medicine and chemistry (Lauer et al., 2015; Teixeira da Silva & Dobránszki, 2019)” - this sentence is misleading. In the current scenario, authors should consider changing “some” to “most” publishers. Authors’ response: Amended. Reviewers’ comments: 12. On Page 6: “For example, it is not universally agreed when an output should be citable (in the literature, funding proposals or promotion cases)”. It is now becoming more and more agreed upon that preprints should be cited in articles and research grant proposals. Major funding bodies such as NIH, CZI and Wellcome Trust allow and encourage citation of preprints. Many journals allow citation of preprints and other research outputs (eLife and PLOS family of journals are examples). Authors’ response: We agree that it is becoming more common to accept citation of preprints in articles and grant proposals, and we have cited instances of this in our paper: “Perhaps the most noticeable shift recently in terms of policy is that of funder policies. Some funders have now explicitly signalled support for use of preprints, including allowing citation of preprints in funding bids, and support their inclusion in cases for academic advancement (Berg et al., 2016; Bourne et al., 2017).” However, our statement that this is not “universally” the case is still correct and is a measured way of making the point. Reviewers’ comments: 13. On Page 6 and other pages: “Scooping”, and “a claim of precedence”: Scooping is a perceived concern of researchers and is unfounded. Many researchers present unpublished research at conference. Perceived preprint concerns and claim of precedence have been discussed in reference 6 above. Authors’ response: Yes, we agree that many of the concerns expressed in the area of scooping are unfounded. However, the point is that concerns are expressed in this area, and that is what we are reporting. Like many of the other areas we report from the literature, this area is also raised in our dataset. This issue is addressed in the ASAPbio site, which also discusses the EMBO “scoop protection” initiative: “If an author submits a manuscript within 4 months of posting a preprint, EMBO will consider the work novel even if a competitor publishes similar work during that time.” The fact that such protections are put in place in this way demonstrates that the issue is still a concern for many. As an advocate of preprints, Berlin (2018) still states, “While some journals already guarantee a “scooping protection”, this is not common practice and most authors are still at risk of being “scooped””. What we are reporting here is the concern. Berlin, S. (2018). If the papers don’t come to the journal…. EMBO Reports, 19(4). https://doi.org/10.15252/embr.201845911 Reviewers’ comments: 14. On Page 6: “or when it can be used to establish a claim of precedence” and “In disciplines where a preprint is not considered appropriate to establish precedence, it has also been suggested that making a preprint available may actually encourage research to be scooped by rival researchers who publish in a recognized journal before the preprint authors (the ‘flip side’ of the priority claim argument above) (Kaiser, 2017).” Regarding these repeated sections on scooping and priority claims: 2-3 papers published via preprints or journals in close temporal proximity of each other have all been projects 2-4 years in making so it is very difficult to assess who did it first. Who gives researchers priority? A preprint that is posted online has a date and that gives precedence to the work. In reality, as researchers we all know that priority is given to researcher by their field and their peers. Scientific research is often presented at conferences well in advance of preprints or journal publication. As we know what matters in research is scientific reputation and that is given to researchers by their peers and field during a career in scientific research. Multiple papers published via preprints or journals in close proximity of each other only show the reproducibility of one another are need to be celebrated not feared so repeated arguments on scooping and priority claims do not move a positive conversation on research forward specially for early career researchers and the future generations of researchers. Authors’ response: Yes, these are useful and interesting points. It is not clear from them, however, how we might change our paper in response. The key point we are making in our literature review is that scooping is identified as a concern in the literature. It is also expressed as a concern within our interviews, demonstrating it is a live issue in this space. The fact that preprints are often argued to be a way of asserting priority is itself evidence that researchers fear being scooped – in that case using that fear as an argument to deposit preprints. Either way, the key point we are making in our literature review is that scooping is raised as an issue, and that is what we are reporting. Reviewers’ comments: 15. On Page 6: “Some have argued that initiatives such as Declaration on Research Assessment (DORA), with its emphasis on the quality of the output rather than venue of publication, promote use of preprints (Polka, 2018)”. It is noteworthy that DORA has now become a major international initiative and is signed by over 1,400 organizations including major universities and other research institutions and 14,000 researchers. Authors’ response: Yes, once again we agree. We expect that most readers of this paper will be familiar with DORA and as it is not the focus of our study, we do not discuss it in detail. If readers wish to know more, we have now provided a reference in our paper to online information about the initiative. Other comments to the MS text: Reviewers’ comments: A) The Title: Authors could consider an alternative title “Preprints and Scholarly Communication: a preliminary/exploratory study on Adoption, Practices, Drivers and Barriers” Authors’ response: We have changed the title to, “Preprints and Scholarly Communication: An Exploratory Qualitative Study of Adoption, Practices, Drivers and Barriers”. Reviewers’ comments: B) The Abstract: “Our study is the first using empirical data to understand” - please change to: Our study uses empirical data… (See comments below highlighting other empirical studies on the topic). Authors’ response: Noted and changes made as outlined above and below. Reviewers’ comments: “The main concerns are related to the lack of quality assurance and the ‘Ingelfinger rule’.” Please change to: main perceived concerns. Authors’ response: We have added “perceived” to our discussion of benefits and challenges of preprints (as mentioned above). Here we refer to “concerns”. Concerns, by definition, relate to people's perceptions of the challenges. “Concerns” is not being used here as a synonym of “disadvantages” or “challenges” but rather to represent perceived disadvantages. “Perceived concerns” is tautological, and so we have limited our use of “perceived” in relation to benefits and challenges. Reviewers’ comments: C) The Introduction: On Page 3: “key actors”. It is puzzling how the authors determined which the key stakeholders are in the preprints ecosystem. Authors should discuss why they did not incorporate “scientific journal publishers” as a relevant actor for this work. It is interesting that they incorporated “publishers” as node in Figure 1 as a stakeholder group but failed to incorporate “representatives” during their interviews. Authors’ response: In our study, we did acknowledge the important role of academic publishers but did not engage them directly. Our reason for this was that the publishing community is already discussing preprints in a structured way, e.g. via the work of COPE. Furthermore, we engaged directly a representative of MDPI (to discuss preprints.org) and representatives of F1000Research (to discuss their approach to pre-publication sharing), who are running preprint services. We have added an explanation of this in our Methodology and there is a note in the new limitations statement on this as well. Reviewers’ comments: On Page 3: “(Carà et al., 2017) .” Please delete the space. Authors’ response: Corrected. Reviewers’ comments: On Page 3: “The study is the first using empirical data”. Please see our comment on the abstract. Authors’ response: We have removed this sentence. We believe that our study is the first qualitative study on the new wave of preprints, nevertheless. As acknowledged above several important quantitative studies have appeared (most still in preprint form) in 2019. Reviewers’ comments: On Page 3: “Zuckerberg Foundation” - the correct name is the Chan-Zuckerberg Foundation (CZI). Authors’ response: Corrected. Reviewers’ comments: On Page 3: “The study is the first using empirical data”. Please see the comments on the abstract above. Authors’ response: Corrected. Reviewers’ comments: On Page 4: “…relatively small number of peer-reviewed studies focusing on preprints – much of the literature is still to be found in editorials and opinion pieces”. The authors surely know that in general opinion pieces published in scientific journals are also peer-reviewed. Authors’ response: We have changed this sentence to avoid ambiguity: “Nevertheless, much of the literature is still to be found in editorials and opinion pieces rather than data-driven research.” Reviewers’ comments: “Defining preprints. Key components. Table 1”. A relevant aspect of preprints that is not contemplated here is that preprints are published open access free of charge. Perhaps that important feature could be incorporated in component 4. Maybe this feature, highly relevant for unprivileged research communities, was neglected given the sampling strategy employed (see comment below). Authors’ response: This the core of key component 4. We have rephrased the description in Table 1 to make this clearer: “Preprints are openly available online” (as also described above). Reviewers’ comments: On Page 8: “The study adopted a heterogeneous purposive sampling approach, aiming to include a wide range of perspectives from actors in the area. Participants comprised senior representatives…”. If the authors selected senior representatives of each stakeholder categories, one could question how heterogeneous the sampling approach was. Moreover, authors mention the use of snowball sampling, which is linked to homogeneous sampling. In addition, participants correspond to only eight OECD High-income economies according to World Bank (half of them only from UK and Germany), which again appear to be more reflective of a homogenous sampling approach. Authors should also highlight the intrinsic limitations of quality coding in terms of reproducibility. Authors’ response: We respond to the specific point raised by the reviewers below, but before doing so we thought it would be useful to precede our responses with an explanation of our approach to qualitative research in general. We repeat this explanation from our response to the other set of peer review comments received and although lengthy it, we hope, helps to explain our approach. This is the case since some of the comments made by the reviewers in their report are as much about qualitative research in general as they are about our particular study, and so we would like to set out our approach in the round, as context to our detailed comments further below. The open peer review process may be helpful here, as the comments we make are openly available and help to explain our approach, even though not all of the detail can be incorporated in our final paper. However, we thought it would be useful to reproduce it here as it helps to contextualise our specific responses to issues raised by reviewers below. Our general approach in responding to the comments of the reviewers on qualitative methods is to try to achieve a balance between, on the one hand, providing clarity and further detail where necessary, and, on the other hand, avoiding over-extending an already long paper. So we have made some changes to the text of the paper with the aim of achieving greater clarity but provide more detailed explanations here in in this report in order to provide fuller explanations that might be too detailed for the paper itself. Our study uses well-established qualitative research methods. Bazeley (2013) states: \"researchers engaging in a qualitative study focus on observing, describing, interpreting, and analysing the way that people experience, act on, or think about themselves and the world around them”. Qualitative research aims to collect and analyse “rich, deep data” (Bryman, 2015) which enable understandings of issues like motivations, beliefs, and values of participants in the contexts in which they act. It focuses on understanding the perspectives of participants, including where disagreement and conflict may exist. Qualitative research typically attempts not just to identify behaviours but also meanings associated with behaviours. It is often useful for encountering unexpected issues and influences. Qualitative methods are commonly deployed therefore in studying phenomena and contexts which are emerging, where issues need to be mapped out, and theory developed (which can then in turn potentially be tested in various ways, including quantitatively). We chose to explore the preprints space using these methods in order to gain a rich and deep understanding of the issues involved, specifically including varying motivations, differing behaviours, and conflicting perspectives. Qualitative data can take different forms and be gathered in different ways, but often take the form of text, e.g. transcripts of interviews, as in our study. In this case the transcripts are the data being analysed, and there are well-established methods for carrying out such analysis. Analysis is typically conducted inductively, identifying patterns which emerge from the data. This is normally done through a process like thematic analysis (used in this study) involving a number of steps, as explained by Braun & Clarke (2006), who are cited in our study. There are a large number of other texts that describe this process, albeit with variations e.g. Bazeley (2013), Bryman (2015), Maxwell (2013) etc. In the first stage of the process, the interviews are fully transcribed and then read and reread thoroughly by the researchers. Secondly, the entire dataset is coded – key features of the data being labelled at a detailed level. “Codes…serve as shorthand devices to label, separate, compile and organize data” (Charmaz, 1983). This is an iterative process involving constant comparisons across the dataset. It can be carried out in various ways, but in our case, we used the NVivo software which facilitates coding of large amounts of text. Coding is a kind of fracturing process, breaking down the data into small fragments, and so the third stage of thematic analysis involves assembling codes into groups or themes. Themes are identified iteratively through constant comparisons, often involved in mapping out themes in relation to one another in order to define and refine them. These were the processes we followed in this study. In reporting qualitative research, the themes are normally used as a framework, rather than the questions that were initially asked of participants. This is an important aspect of the reporting of an inductive study in order to represent the themes that emerged from the data rather than the initial framework of the questions asked. From analysis of these themes, theoretical insights should emerge. These could take the form of systematic explanations of a context; models of relationships, issues, challenges etc; sets of questions or hypotheses that need to be further investigated; etc. We have tried to provide some of these, albeit tentatively, in our study. Sampling in qualitative research is normally carried out in a purposive (or purposeful) way. This is described by Bryman (2015): “Purposive sampling is a non-probability form sampling. The researcher does not seek to sample research participants on a random basis. The goal of purposive sampling is to sample cases/participants in a strategic way, so that the sample are relevant to the research questions that are being posed. Very often, the researcher will want to sample in order to ensure that there is a good deal of variety in the resulting sample, so that sample members differ from each other in terms key characteristics relevant to the research question.” We describe our sampling approach as a “heterogeneous purposive sampling approach”, which we go on to define as, “aiming to include a wide range of perspectives from actors in the area”. The sample was heterogeneous in a number of respects: it contained representatives of different roles in the scholarly communication system, from different countries (and therefore policy environments), and with different views and levels of experience of using preprints. Participants were selected by us based on an analysis of the field, consultation with our funders and others, and then approached directly and invited to participate. We also advertised on email discussion lists and social media inviting participants, but only selected people to be interviewed who met our sampling criteria. One challenge was in finding researchers willing to be interviewed who were not engaged with preprints, and so in order to do this, we not only used the selection method of approaching people directly but also used “snowball sampling” – asking for recommendations of other people who we might approach from participants. As Bryman (2015) comments: “Purposive sampling often involves more than one of the approaches outlined… For example, it is quite common for snowball sampling to be preceded by another form of purposive sampling. In effect, the process entails sampling initial participants without using the snowball approach and then using these initial contacts to broaden out to a snowballing method.” This is what we did, resulting in a final sample of 38. Sample size is a controversial issue. There is no obvious right answer to the question of, “how many interviews are enough?” The essays addressing that question by well-known qualitative researchers edited by Baker & Edwards (2012) include a wide range of different views, depending on a number of factors. Bryman quotes Mason (2010) as having examined UK and Irish PhD theses using qualitative interviews, which identifies them as using a mean sample size of 31 and a median of 28. Bryman, in his chapter on reporting research, looks in detail at a sample journal article reporting semi-structured interviews which has a sample size of 20 (Jones, Leontowitsch, & Higgs, 2010). Our sample size of 38 is relatively large. One key point to emphasise, however, is that sample size in qualitative research is normally not determined in advance. Rather, the sampling approach will be decided and participants recruited within the sampling frame. The researchers will then make a number of judgements during the data gathering process about when sufficient data has been collected. Chief amongst these is the idea of “theoretical saturation”, where new data “no longer suggests new insights” (Bryman, 2015). This involves a number of judgements, involving a close engagement with the data in relation to the research objectives. Because qualitative research usually involves identifying areas of motivations, perspectives etc, there is commonly a need to allow participant anonymity. Often interviewees are less frank when they are ‘on the record’. Less frank interviewees tends to lead to a rather bland dataset which makes it difficult to understand the real contours of a landscape. In our study, we gained ethical approval to name participants and their organisations (with their permission) but not to associate any particular quotations with individuals. We have adopted a conventional way of dealing with this by identifying various groups amongst our sample which we do relate with each quotation (“Unengaged researcher”, “Preprint service provider” etc). Naming the group from which the interviewee being quoted came helps to contextualise each quotation, but does not compromise promised anonymity. Providing extensive extracts of participant quotations is a critical part of presenting findings from qualitative research. Since these quotations are illustrative of the themes identified, and are examples of the data collected, showing them in detail is an important way of demonstrating the validity of the inferences being drawn from the research. Baker, S. E., & Edwards, R. (2012). How many qualitative interviews is enough? Retrieved from http://eprints.ncrm.ac.uk/2273/4/how_many_interviews.pdf Bazeley, P. (2013). Qualitative data analysis: Practical strategies. London: Sage. Bryman, A. (2015). Social research methods (5th ed.). Oxford: Oxford University Press. Jones, I. R., Leontowitsch, M., & Higgs, P. (2010). The experience of retirement in second modernity. Sociology, 44(1), 103–120. https://doi.org/10.1177/0038038509351610 Maxwell, J. (2013). Designing a qualitative study. In L. Bickman & D. J. Rog (Eds.), The SAGE handbook of applied social research methods (pp. 214–253). https://doi.org/10.4135/9781483348858.n7 To reply to the specific issue on sampling and its heterogeneity raised by the reviewers on heterogeneity and snowball sampling. Our sample was heterogeneous in various ways and we have explained this in text added to our methodology, as follows: “The study adopted a heterogeneous purposive sampling approach, aiming to include a wide range of perspectives from actors in the area, selected in a “strategic way” in order to address the objectives of the study (Bryman, 2015). The sample was heterogeneous in a number of respects: firstly, it contained representatives of different roles in the scholarly communication system; secondly, it included participants from different countries (and therefore policy environments); thirdly, it comprised interviewees and with different views and levels of experience of using preprints.” On snowball sampling; snowball sampling is not linked to either homogenous or heterogeneous sampling in itself. It depends how it is used. However, as we state above, it was used in conjunction with another sampling method, as a way of achieving heterogeneity in the sample, particularly to recruit researchers (and particularly unengaged researchers). Participants from eight different countries is a wide sampling frame for a study of this type. This study does not make any claims to cover issues to do with the Global South but does encompass a wide range of different policy environments present in the different countries covered. We have added more explanation of the limitations of the study in a new section in the methodology, explaining limitations, as previously mentioned. Reviewers’ comments: On Page 8: “We undertook 38 semi-structured interviews”. Authors did not describe how sample size was determined. Authors should discuss how this small sample size might redound in uncertainty issues. The complete absence of statistical analyses of the data is striking. This affects the “empirical power” of the work, and one could question whether the described “trends” indicated in the manuscript are purely anecdotal, derived from sampling error and/or speculative. Authors’ response: We have described issues of sample size in our general explanation above. The comments of the authors about the absence of statistical analysis from our reporting of findings needs comment. Ours is a qualitative study, using qualitative methods and qualitative analysis. It would be inappropriate to apply most quantitative approaches to our data. The “power” of qualitative findings of this sort (to use the term used by the reviewers) is in the richness of the data associated with issues of motivation, attitudes etc, not in any quantitative inferences drawn. We hope our detailed explanation of our approach to using qualitative methods is helpful in contributing to our response on this point. It includes the approach we took to inductively analysing themes in our data. Reviewers’ comments: ”Like many kinds…” paragraph. This important paragraph highlighting the limitations of the study should be moved to the discussion/conclusion section. Authors’ response: Conventions on this differ across journals, and to some extent across disciplines. We have noticed statements of this sort in the method sections of other articles in F1000, and so would prefer to keep this section (in its newly amended form) in the Methods section, since it relates directly to the methods just described. Reviewers’ comments: D) The Results section in general: The reviewers wonder if the results section as a whole, which is relatively long in comparison to the rest of the paper, should be limited to Table 5 and its description and avoid the unending narrative of anecdotal specific responses. In case the authors feel the need to present all these quoted responses, perhaps they could be moved to the supplementary section. Authors’ response: The specific responses of our participants are the data for this study. Presenting them in this way is conventional for this sort of research and doing so in detail demonstrates the validity of the inferences we are drawing. The specificity of the quotations is in the nature of qualitative research – that is where the richness of the data comes from. It is noticeable that in his book on social sciences research, Bryman presents a sample article in order to illustrate presentation of qualitative research (Jones, et al., 2010)). This sample article follows a similar pattern of presentation of thematic findings with commentary by the authors and quotations from participant interviews. Bryman comments, “it is striking that, in presenting their findings, Jones et al. (2010) use verbatim quotations to reinforce the point they are making… They do so by including the quotations as they go along to reinforce or illustrate points they are making about the themes they extracted from that data. This is quite a common approach to the use of verbatim interview quotations.” Jones, I. R., Leontowitsch, M., & Higgs, P. 2010. The experience of retirement in second modernity. Sociology, 44(1), 103–120. https://doi.org/10.1177/0038038509351610 Reviewers’ comments: “Some participants…” “Many saw a preprint as…” “Other participants saw preprints..” “there were signs this…” and elsewhere. This vague and unclear wording could be avoided by the use of absolute numbers or percentages of total responses. Authors’ response: We have addressed this comment by amending wording to be as precise as possible within the constraints of a qualitative study. Once again, there is a balance to struck here. On the one hand, as a general principle, it is essential to recognise that qualitative research should not be analysed quantitatively. Inferences cannot be drawn from the data in any statistically valid way from quantitative analysis of a dataset like this, and it is important not to give the impression that they can. Certainly, including percentages would not be appropriate. In any case, the data is not sufficiently structured to allow for reliable identification of a type of response which could be reported quantitatively. Every datum in a qualitative dataset is actually unique and although it is possible to identify commonalties, it is not normally possible to say that a certain proportion of participants said one thing or another. This likely to lead inappropriate homogenisation of views. Furthermore, it is important not to confuse prevalence with significance. In a qualitative dataset, some ideas may be mentioned by a large number of participants, but this does not necessarily mean they are significant. Significance may not, of course, be easy to judge (and it is a matter of judgement based on careful analysis) but it is not a simple issue of numbers of times an issue is mentioned in the dataset. We have however made some attempts to identify prevalence of issues raised in discussing pros and cons of preprints, something which is possible (simply identifying topics mentioned), with the relevant caveats. On the other hand, it is perfectly reasonable to mention some quantitative terms, “the majority”, or “a minority” or if an issue was only mentioned by one or was mentioned by all participants. That is what we have tried to do within the bounds of what is defensible. Reviewers’ comments: Table 5: Please consider complementing the legend to provide definitions to “systemic” and “individual”. Authors’ response: We have added definitions as follows: ““Systemic” significance relates to those factors with system-wide impact e.g. the broad scholarly communication system or disciplinary community; “individual” relates to those factors primarily affecting individuals or small groups.” Reviewers’ comments: Table 6: Following Table 5 title, it would be more reasonable to entitle Table 6 as “potential challenges of preprints”, or “possible” as depicted in the same table in Chiarelli et al. 2019. Authors’ response: Agreed and done. Reviewers’ comments: On Page 13: “One preprint service provider observed that only 10% of preprints received comments”. While this % is depicted as low in the text by the use of “only”, authors should contextualize this type of assertion. Peer reviewed articles published in journals are far less commented than preprints as reported elsewhere. Authors’ response: The preprint service provider making the statement was making the point that it was relatively low. Interestingly, it is, however, comparable to levels identified in a recent study of PLOS journals, co-authored by one of us: Wakeling, S., Willett, P., Creaser, C., Fry, J., Pinfield, S., Spezi, V., … Medina Perea, I. (2019). ‘No comment’? A study of commenting on PLOS articles. Journal of Information Science, (Online first). https://doi.org/10.1177/0165551518819965 Reviewers’ comments: On Page 14: “author might invite comments from people who are expected to be positive about their work”. Again, this and other speculative statements should be leveled when they are employed to contrast with traditional peer-reviewed journal articles. It is a common practice in an important number of journals to ask authors to provide contact information of potential reviewers, which could eventually lead to the same issues expressed in the preceding statement. Authors’ response: Here we are reporting the views of participants. We have added a comment in our discussion about there being evidence of misunderstandings amongst some researchers. However, stepping in to engage with participants during the reporting of findings in this one particular instance would not be consistent with our approach or appropriate for this kind of research. Reviewers’ comments: On Page 14: “We note…”. This relevant paragraph, highlighting the limitations and anecdotal nature of the study, should be moved to the discussion/conclusions. Authors’ response: We have introduced a new section spelling out the limitations of our research. We again note that this is a qualitative piece of research, based on interviews, with text as the data being analysed. Reviewers’ comments: E) The Discussion/Conclusion: As expressed above, some parts of the results section could be moved here as they reflect more speculative comments based on the interview responses. Authors’ response: We have moved a whole subsection of the results into the Discussion section. This is the section which models different preprint services based on ownership and contribution. “At a systemic level, the issue of sustainability emerged as critical from our work. Four models for delivering preprint services emerged from the data… This is a fundamental shift which has major implications for the way the role of preprints is understood and the way preprints services are configured.” This discussion is derived from our data, but does not directly report data, but rather systematises what is expressed by our participants. It is therefore appropriately located in the discussion. Reviewers’ comments: F) The References: This citation ”Sarabipour et al.: On the value of preprints: an early career researcher perspective. PeerJ Preprints (2018)” Is now peer-reviewed and hosted as “On the value of preprints: An early career researcher perspective. Sarabipour et al. PLoS biology (2019)[. Authors’ response: Updated." } ] }, { "id": "50950", "date": "05 Aug 2019", "name": "Jessica K Polka", "expertise": [ "Reviewer Expertise We have backgrounds in cell biology and neuroscience", "and are currently working to promote the productive use of preprints." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn “Preprints and Scholarly Communication: Adoption, Practices, Drivers and Barriers,” Chiarelli and colleagues present a literature review, commentary, and a summary of attitudes and perceptions towards preprints found by interviewing 38 stakeholders. The interviews convey many commonly-held perceptions about the benefits and disadvantages of preprints, and the authors suggest questions for further exploration and testing. With revisions (clarifying the origin of claims, contextualizing the responses from study subjects, and correcting factual information in the text) this paper will become a very useful addition to the literature.\n\nMajor comments\nDesign and Subjects:\n\nWe encourage authors to rely on a more robust review of published literature (including the grey literature) to complement their interviews, as the published literature contains useful context for comments which are obfuscated by the process of anonymous reporting (For example, the sentence, “One preprint service provider observed that only 10% of preprints received comments.” would be more useful to readers if accompanied by a reference with attribution: https://asapbio.org/biorxiv)\n\nSince this work may be of interest to a wide range of readers, we recommend including more detail regarding the chosen experimental design.\n\nPlease provide justification for the experimental design, for example, why were interviews used instead of a survey? What are the methodological considerations that would be helpful for readers outside of qualitative research to understand: limitations, constraints, advantages over more quantitative methods? Please define semi-structured interviews — how were researchers encouraged to expand on thoughts? Please define “purposive heterogeneous sampling approach.” Please clarify why the sample size is 38.\n\nPlease provide more information about the subjects to help readers assess relevance:\nWhat is their geographical context? How knowledgeable were subjects, and do they bring bias for or against open science into their statements? How did interviewees arrive at their opinions? How much close experience do they have with preprints? Were participants asked to answer only within their own expertise? There are quoted statements that reflect inaccurate understandings and are not always qualified by the author: for example, “relating to whether preprints are seen as an appropriate object for evaluation in exercises such as the UK’s Research Excellence Framework, about which there remains ambiguity. One representative of a university stated: “they’re not acceptable for REF, so they’re not even part of the equation. So it’s the author’s accepted manuscript is the currency we deal with.” (Research performing organisation)” This is inaccurate and misleading (see https://www.ref.ac.uk/publications/guidance-on-submissions-201901/) but there is no further commentary from the authors. Please add clarification that the interviewee responses are not necessarily factual. (It would be interesting to compare interviewee opinions with known facts to understand which topics are well-understood.)\n\nClarity of presentation:\n\nPlease provide more quantitative context to the opinions expressed. For example, for phrases such as “Some researchers” it would be useful to be more specific. Please also be more quantitative in assessment of the themes — it is useful to know if a sentiment was expressed by a majority or minority, as shown clearly in tables of benefits and challenges. Please provide aggregate data of thematic analyses, not just quotes, and use specific numbers and proportionality (Two of Five researchers mentioned X as a benefit).\n\nThe paper combines elements of a literature review, white paper/policy piece, and a qualitative study. It would be helpful to clarify the origin of ideas by separating the work clearly into Results and Discussion sections. (As one example, under the heading “Financial sustainability and business models,” it is unclear whether the proposed taxonomy was expressed by interviewees or generated by the authors. Similarly, it is unclear whether use of the term “seminal” comes from interviewees or authors.)\n\nReadability:\nPlease use active voice and concise language to improve readability. Bring interview questions and list of participants into this report — it is hard to interpret findings while having to continuously refer back to other sources, e.g. the 2019b interview structure paper.\n\nClaims:\n\nThe abstract sets the reader up to expect a review of preprint servers that have emerged since 2013. However, the methods section discloses that coverage of the study is limited to biology, chemistry, and psychology. It would be helpful to readers to state this up front. It is also unclear how this scope was applied in the selection of study subjects. It is difficult to determine if the scope influenced the selection of participants as listed in https://zenodo.org/record/2654832#.XTeUB5NKjs0 – the questions listed at https://zenodo.org/record/3240426 do not seem specific to these three disciplines and the quoted answers in this report suggest interviewees’ responses were not limited to these three disciplines. Please clarify how the scope of the interviews was constrained to the stated subjects, or if not, please revise the stated scope to more accurately reflect the scope employed.\n\nThe abstract does not reflect the content. The interview responses are summarised in general terms, not related to defined scope or the newer servers. A substantial proportion of this article is review material: either in the explicit literature review section, or as author-contributed content interspersed with summaries of interviewee responses. For example, conclusions listed in the abstract appear to be drawn from the literature review.\n\n“Our study is the first using empirical data to understand the new wave of preprint servers” — this is not supported by the study content: no results are specifically related to the newer servers identified in the introduction; quoted excerpts indicate that interviewees responded in general about all servers and disciplines. Furthermore, prior surveys and bibliometric analyses have looked at a subset of these servers, either individually or in groups (eg https://osf.io/5fk6c/).1\n\nMinor comments\nWe recommend making the following amendments to improve the clarity and accuracy of the report.\nIn the Introduction and literature review:\n\nThe summary of the longer history of preprint efforts across disciplines, would benefit from incorporating and citing ‘The Brief Prehistory of Preprints” (Cobb, 2017)2\n\nZuckerberg Foundation should be corrected to Chan Zuckerberg Initiative.\n\nDiscussion of new servers would be improved with a note as to their current size, and with awareness that not all new ‘preprint’ servers are exclusively for preprint content (many COS servers include postprints that are not possible to easily distinguish from preprint versions).\n\n“This paper aims to explore…by investigating current practices, drivers and barriers to [preprint] use.” The interviews indicate perceptions and attitudes but do not reveal actual behaviours or practices. Please clarify how this study examines current practices.\n\nFigures and tables:\nFig 1: To increase accessibility, please provide as raw text with headings or tags to indicate which stakeholder group you believe is most relevant\n\nLiterature review:\nThe authors focus on four main issues — how were these arrived at? Was the literature review systematic in any way?\n\nGiven the emergent phase of preprint infrastructure in biology, chemistry and psychology, and as the authors note that empirical data is lacking, it may be equally valuable to include more grey literature (blogposts, webpages) in this review, given that several of the cited articles are opinion pieces, albeit as editorials or peer-reviewed review articles. Of note, we find personal blogs are a useful source of stories that detail benefits and drawbacks of preprints from personal experiences.\n\n\"Dis-benefits:\" is not a common term, perhaps use “drawbacks,” “concerns,”, “disadvantages” or “potential risks”.\n\nWhen discussing the lack of quality assurance, the authors seem to make an implicit assumption here that journal-led peer review assures quality. It would be fair to contextualise this section with some literature on peer review to understand variety of peer review outcomes on quality of paper and validity of findings.\n\nResults:\nIn the discussion of preprints in the medical field, please note that the concerns raised are being addressed by medRxiv. https://www.medrxiv.org/\n\nIn the table of business models:\n\nNote that bioRxiv uses 3rd party technology, Highwire.\n\n“Publisher driven” models are not new; PeerJ is older than bioRxiv, Nature had Nature Preceedings in the early 2000s, and bioRxiv has had J2B preprints since its inception.\n\nHow does the outsourcing of infrastructure to a third party enable sustainability? Arguably, depending on the third party, this leaves the service vulnerable to changes in service entirely outside its control.\n\nIndividual authors can still submit to PeerJ.\n\nMost publisher-mediated preprint submissions are voluntary for authors (a notable exception being F1000).\n\nRegarding the statement of subject preferences: we have conducted a survey on these preferences that may provide additional context: https://asapbio.org/asapbio-funders-workshop\n\nThe following suggestions may also help improve the paper but are not essential.\nIn the Introduction and literature review:\nOpen Research platforms could be discussed in context of F1000Research, since this is the model they are derived from (in collaboration with F1000).\n\nPlease clarify the age of PeerJ and Preprints.org in comparison with bioRxiv.\n\nPLOS is not the only journal that deposits preprints to biorxiv using the J2B mechanism — several do, please refer to the list of journals offering direct transfers to bioRxiv (https://www.biorxiv.org/about-biorxiv).\n\nRegarding trust in preprints, it would be relevant to mention or cite the current effort by COS to better understand indicators of trust in preprints: https://cos.io/about/news/center-open-science-receives-grant-alfred-p-sloan-foundation-study-trust-and-credibility-preprints/.\n\nFigures and tables:\nFig 1 introduces some interesting questions, however many of these are related to topics that are brought up for the first time in this figure.\n\nTable 6: The concern of reputational damage is indeed in the literature; please see point 5 here: https://smallpondscience.com/2017/07/24/whats-up-with-preprints/\n\nLiterature:\n“However, Bourne et al. (2017) controversially extend the definition to include “a paper that has been peer reviewed and…was rejected, but the authors are willing to make the content public”.” — Please provide reasoning or citation as to why this is controversial.\n\nAccessibility — note that preprints are not necessarily open access, where they are not provided with permissive licensing, and this is certainly the case for bioRxiv.3\n\nOne potential benefit to ECRs that is not mentioned here is being able to demonstrate productivity to funders and hirers. This would be useful to append to the current paragraph, given it is raised in the interview questions.\n\n“There also appears to be little acknowledgement that the claim stands in tension to the one cited earlier that preprints often differ little from final published versions.” — There is only tension if both claims concern all preprints. It is possible for there to be preprints that improve through a lot of feedback as well as preprints that undergo no/very little change.\n\nPlease use unshared or unpublished instead of “homeless”.\n\nWe recommend avoiding the term “policy stack,” as there is no dependent relationship among these different groups/stakeholders.\n\nMethods:\nThis section ignores the efforts of researchers as change agents.\n\nResults:\nPlease providence evidence for this statement: “we note that adjacency between disciplines appears to be playing an important role.”\n\n“Many of the participants agreed that preprints servers could be a useful outlet for otherwise ‘homeless’ research outputs, even though this goes counter to the emphasis of many of preprints as early versions of outputs later formally published elsewhere.” — this attitude may reflect a lack of publication venues for such content, not a lack of desire to publish these other outputs in a peer-reviewed destination.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5027", "date": "25 Nov 2019", "name": "Andrea Chiarelli", "role": "Author Response", "response": "In “Preprints and Scholarly Communication: Adoption, Practices, Drivers and Barriers,” Chiarelli and colleagues present a literature review, commentary, and a summary of attitudes and perceptions towards preprints found by interviewing 38 stakeholders. The interviews convey many commonly-held perceptions about the benefits and disadvantages of preprints, and the authors suggest questions for further exploration and testing. With revisions (clarifying the origin of claims, contextualizing the responses from study subjects, and correcting factual information in the text) this paper will become a very useful addition to the literature. Authors’ response:We are grateful for the reviewers’ summary of and comments on our article, and are pleased that they consider that “with revisions…this paper will become a very useful addition to the literature.” The above summary makes a number of general remarks about revisions that should be made which we address below, responding to the detailed comments of the reviewers.We believe that it may be helpful before addressing the specific comments below to precede our responses with an explanation of our approach to qualitative research in general. Many of the comments made by the reviewers in their report are as much about qualitative research in general as they are about our particular study, and so we would like to set out our approach in the round, as context to our detailed comments further below. The open peer review process may be helpful here, as the comments we make are openly available and help to explain our approach, even though not all of the detail can be incorporated in our final paper. This general explanation is reproduced in both our responses to reviewers’ comments, as similar issues arise in both.Our general approach in responding to the comments of the reviewers on qualitative methods is to try to achieve a balance between, on the one hand, providing clarity and further detail where necessary, and, on the other hand, avoiding over-extending an already long paper. So we have made some changes to the text of the paper with the aim of achieving greater clarity but provide more detailed explanations here in in this report in order to provide fuller explanations that might be too detailed for the paper itself.Our study uses well-established qualitative research methods. Bazeley (2013) states: \"researchers engaging in a qualitative study focus on observing, describing, interpreting, and analysing the way that people experience, act on, or think about themselves and the world around them”. Qualitative research aims to collect and analyse “rich, deep data” (Bryman, 2015) which enable understandings of issues like motivations, beliefs, and values of participants in the contexts in which they act. It focuses on understanding the perspectives of participants, including where disagreement and conflict may exist. Qualitative research typically attempts not just to identify behaviours but also meanings associated with behaviours. It is often useful for encountering unexpected issues and influences. Qualitative methods are commonly deployed therefore in studying phenomena and contexts which are emerging, where issues need to be mapped out, and theory developed (which can then in turn potentially be tested in various ways, including quantitatively). We chose to explore the preprints space using these methods in order to gain a rich and deep understanding of the issues involved, specifically including varying motivations, differing behaviours, and conflicting perspectives.Qualitative data can take different forms and be gathered in different ways, but often take the form of text, e.g. transcripts of interviews, as in our study. In this case the transcripts are the data being analysed, and there are well-established methods for carrying out such analysis. Analysis is typically conducted inductively, identifying patterns which emerge from the data. This is normally done through a process like thematic analysis (used in this study) involving a number of steps, as explained by Braun & Clarke (2006), who are cited in our study. There are a large number of other texts that describe this process, albeit with variations e.g. Bazeley (2013), Bryman (2015), Maxwell (2013) etc. In the first stage of the process, the interviews are fully transcribed and then read and reread thoroughly by the researchers. Secondly, the entire dataset is coded – key features of the data being labelled at a detailed level. “Codes…serve as shorthand devices to label, separate, compile and organize data” (Charmaz, 1983). This is an iterative process involving constant comparisons across the dataset. It can be carried out in various ways, but in our case, we used the NVivo software which facilitates coding of large amounts of text. Coding is a kind of fracturing process, breaking down the data into small fragments, and so the third stage of thematic analysis involves assembling codes into groups or themes. Themes are identified iteratively through constant comparisons, often involved in mapping out themes in relation to one another in order to define and refine them. These were the processes we followed in this study.In reporting qualitative research, the themes are normally used as a framework, rather than the questions that were initially asked of participants. This is an important aspect of the reporting of an inductive study in order to represent the themes that emerged from the data rather than the initial framework of the questions asked. From analysis of these themes, theoretical insights should emerge. These could take the form of systematic explanations of a context; models of relationships, issues, challenges etc; sets of questions or hypotheses that need to be further investigated; etc. We have tried to provide some of these, albeit tentatively, in our study.Sampling in qualitative research is normally carried out in a purposive (or purposeful) way. This is described by Bryman (2015):“Purposive sampling is a non-probability form sampling. The researcher does not seek to sample research participants on a random basis. The goal of purposive sampling is to sample cases/participants in a strategic way, so that the sample are relevant to the research questions that are being posed. Very often, the researcher will want to sample in order to ensure that there is a good deal of variety in the resulting sample, so that sample members differ from each other in terms key characteristics relevant to the research question.”We describe our sampling approach as a “heterogeneous purposive sampling approach”, which we go on to define as, “aiming to include a wide range of perspectives from actors in the area”. The sample was heterogeneous in a number of respects: it contained representatives of different roles in the scholarly communication system, from different countries (and therefore policy environments), and with different views and levels of experience of using preprints. Participants were selected by us based on an analysis of the field, consultation with our funders and others, and then approached directly and invited to participate. We also advertised on email discussion lists and social media inviting participants, but only selected people to be interviewed who met our sampling criteria. One challenge was in finding researchers willing to be interviewed who were not engaged with preprints, and so in order to do this, we not only used the selection method of approaching people directly but also used “snowball sampling” – asking for recommendations of other people who we might approach from participants. As Bryman (2015) comments:“Purposive sampling often involves more than one of the approaches outlined… For example, it is quite common for snowball sampling to be preceded by another form of purposive sampling. In effect, the process entails sampling initial participants without using the snowball approach and then using these initial contacts to broaden out to a snowballing method.”This is what we did, resulting in a final sample of 38.Sample size is a controversial issue. There is no obvious right answer to the question of, “how many interviews are enough?” The essays addressing that question by well-known qualitative researchers edited by Baker & Edwards (2012) include a wide range of different views, depending on a number of factors. Bryman quotes Mason (2010) as having examined UK and Irish PhD theses using qualitative interviews, which identifies them as using a mean sample size of 31 and a median of 28. Bryman, in his chapter on reporting research, looks in detail at a sample journal article reporting semi-structured interviews which has a sample size of 20 (Jones, Leontowitsch, & Higgs, 2010). Our sample size of 38 is relatively large. One key point to emphasise, however, is that sample size in qualitative research is normally not determined in advance. Rather, the sampling approach will be decided and participants recruited within the sampling frame. The researchers will then make a number of judgements during the data gathering process about when sufficient data has been collected. Chief amongst these is the idea of “theoretical saturation”, where new data “no longer suggests new insights” (Bryman, 2015). This involves a number of judgements, involving a close engagement with the data in relation to the research objectives.Because qualitative research usually involves identifying areas of motivations, perspectives etc, there is commonly a need to allow participant anonymity. Often interviewees are less frank when they are ‘on the record’. Less frank interviewees tends to lead to a rather bland dataset which makes it difficult to understand the real contours of a landscape. In our study, we gained ethical approval to name participants and their organisations (with their permission) but not to associate any particular quotations with individuals. We have adopted a conventional way of dealing with this by identifying various groups amongst our sample which we do relate with each quotation (“Unengaged researcher”, “Preprint service provider” etc). Naming the group from which the interviewee being quoted came helps to contextualise each quotation, but does not compromise promised anonymity.Providing extensive extracts of participant quotations is a critical part of presenting findings from qualitative research. Since these quotations are illustrative of the themes identified, and are examples of the data collected, showing them in detail is an important way of demonstrating the validity of the inferences being drawn from the research. Baker, S. E., & Edwards, R. (2012). How many qualitative interviews is enough? Retrieved from http://eprints.ncrm.ac.uk/2273/4/how_many_interviews.pdfBazeley, P. (2013). Qualitative data analysis: Practical strategies. London: Sage.Bryman, A. (2015). Social research methods (5th ed.). Oxford: Oxford University Press.Jones, I. R., Leontowitsch, M., & Higgs, P. (2010). The experience of retirement in second modernity. Sociology, 44(1), 103–120. https://doi.org/10.1177/0038038509351610Maxwell, J. (2013). Designing a qualitative study. In L. Bickman & D. J. Rog (Eds.), The SAGE handbook of applied social research methods (pp. 214–253). https://doi.org/10.4135/9781483348858.n7 Major commentsReviewers’ comments: Design and Subjects: We encourage authors to rely on a more robust review of published literature (including the grey literature) to complement their interviews, as the published literature contains useful context for comments which are obfuscated by the process of anonymous reporting (For example, the sentence, “One preprint service provider observed that only 10% of preprints received comments.” would be more useful to readers if accompanied by a reference with attribution: https://asapbio.org/biorxiv) Authors’ response:We have included a literature review in our study and are happy to update it with other relevant literature (as mentioned below and in response to comments of the other reviewers).We sketch out the literature review and engage with literature again in our discussion, but limit our reporting of our data to doing exactly that – reporting our data. We report therefore that, “One preprint service provider observed that only 10% of preprints received comments” but we have to be careful not to appear to attribute this quotation in a way that makes the interviewee identifiable (see above comments on anonymity). As this quotation is from a “preprints service provider” it is reasonable to assume it is reliable. Reviewers’ comments: Since this work may be of interest to a wide range of readers, we recommend including more detail regarding the chosen experimental design. Please provide justification for the experimental design, for example, why were interviews used instead of a survey? What are the methodological considerations that would be helpful for readers outside of qualitative research to understand: limitations, constraints, advantages over more quantitative methods? Please define semi-structured interviews — how were researchers encouraged to expand on thoughts? Please define “purposive heterogeneous sampling approach.” Please clarify why the sample size is 38. Authors’ response:To respond to the above bullet points in turn:Justification for the approach. As the above general explanation outlines, we chose qualitative methods in order to explore in-depth the “varying motivations, differing behaviours and conflicting perspectives” of key actors in the preprints space – something qualitative methods are often designed to do. We have added some more detail to the method to clarify this, notably in the first paragraph of the Methods section:“As preprints and preprint servers are still innovative developments for most disciplines, it is important to gain an in-depth understanding of the perspectives of different stakeholders. In order to explore issues, such as varying motivations, differing behaviours, and conflicting perspectives, particularly in emerging areas, qualitative research methods are often deployed, since they are well suited to such investigations. We chose to carry out detailed interviews of key actors in this space who could explain in depth their perceptions, attitudes and practices in relation to preprints…” etc. Limitations and constraints. We have also added a new section in the Methods section on “Limitations and constraints”. The main limitation with qualitative research is lack of generalisability. Whilst results may be transferable to other contexts, generalisability is not possible from qualitative research alone. Qualitative research is designed to explore specific instances of phenomena and generate theory and hypotheses which might then be tested using other methods, including quantitative methods where findings might be generalised. Our new section reads:“Limitations and constraints. Like many kinds of qualitative research, this study was designed to be exploratory; in this case, to map out key aspects of the preprints space and suggest policy responses. Our conclusions are tentative. Many of interviewees were selected because of their knowledge of the issues under investigation, and although our findings based on their views may be transferable to other contexts, they cannot be generalised without further testing, as with most qualitative research. There was a bias in our sampling of participants aware of and engaged with preprints. Further research, using other methods, will be needed in future in order to generalise across communities as a whole, including non-engaged researchers. Furthermore, some stakeholder groups, such as publishers (who only have very limited representation in this study), and other groups (such as non-academic users of the research literature) could usefully be included in future studies. Our coding was undertaken using agreed protocols and involved a process of validation provided by three different members of the authorial team but necessarily involves interpretation and judgement on the part of the researchers.” Semi-structured interviews. Semi-structured interviews have ‘spine’ of common questions running through the interview shared by all participants (or all participants in a particular group), but allow room for the interviewer to pursue areas of interest arising from participant responses, including probing for greater clarity, where needed. It is probably the most common interviewing approach for qualitative research. We have added a brief explanation to the paper and also a reference to a more detailed explanation should the reader be interested to follow it up:“Interviews were conducted using a semi-structured approach – incorporating a ‘spine’ of common questions for all participants, and some questions specific to different actor groups – allowing room for the interviewer pursue areas of interest arising from participant responses, including probing for greater clarity, where needed (Bryman, 2015).” Sampling approach. We have described purposive heterogeneous sampling above in our introductory explanation. We have expanded the explanation in the paper itself to clarify this.“The study adopted a heterogeneous purposive sampling approach, aiming to include a wide range of perspectives from actors in the area, selected in a “strategic way” in order to address the objectives of the study (Bryman, 2015). The sample was heterogeneous in a number of respects: firstly, it contained representatives of different roles in the scholarly communication system; secondly, it included participants from different countries (and therefore policy environments); thirdly, it comprised interviewees and with different views and levels of experience of using preprints. Participants comprised senior representatives from research funders, research-conducting organisations (universities and research institutes), preprint services, other related service providers (such as infrastructure providers), as well as researchers, both researchers demonstrably engaged with preprints (they had themselves posted a preprint) and non-engaged (there was no evidence of them having posted a preprint)…” etc.Sample size. Sample size is addressed in our introductory explanation. 38 is a relatively large number for a study of this kind, enabling the study to achieve the desired heterogeneity and theoretical saturation. We have added a sentence explaining this in the Method: “The sample allowed the study to achieve the desired heterogeneity of actors and perspectives.” Reviewers’ comments: Please provide more information about the subjects to help readers assess relevance: What is their geographical context? How knowledgeable were subjects, and do they bring bias for or against open science into their statements? How did interviewees arrive at their opinions? How much close experience do they have with preprints? Were participants asked to answer only within their own expertise? Authors’ response:All of our participants and their affiliations are named and so readers can draw judgements from this – this information is available in the extended data. As already discussed, we have an ethical commitment not to associate individuals with specific quotations, which is conventional for research of this kind as explained in the introductory explanation above. Reviewers’ comments: There are quoted statements that reflect inaccurate understandings and are not always qualified by the author: for example, “relating to whether preprints are seen as an appropriate object for evaluation in exercises such as the UK’s Research Excellence Framework, about which there remains ambiguity. One representative of a university stated: “they’re not acceptable for REF, so they’re not even part of the equation. So it’s the author’s accepted manuscript is the currency we deal with.” (Research performing organisation)” This is inaccurate and misleading (see https://www.ref.ac.uk/publications/guidance-on-submissions-201901/) but there is no further commentary from the authors. Please add clarification that the interviewee responses are not necessarily factual. (It would be interesting to compare interviewee opinions with known facts to understand which topics are well-understood.) Authors’ response:On the specific issue of the REF and preprints, the guidance was been updated at the beginning of 2019 whilst we were still undertaking our interviews. We have added a comment in the paper clarifying the situation, but acknowledging there is still ambiguity in this area in terms of practice: the official rules of the REF and actual practice may often be in tension:“The concept of “standing” (Neylon et al., 2017) is relevant here, with, as an example, standing relating to whether preprints are seen as an appropriate object for evaluation in exercises such as the UK’s Research Excellence Framework, about which there had apparently been uncertainty. One representative of a university stated:“they’re not acceptable for REF, so they’re not even part of the equation. So it’s the author’s accepted manuscript is the currency we deal with.” (Research performing organisation) The REF guidance was updated in January 2019 (whilst our interviews were still ongoing) and now states that preprints can be included in REF submissions, but expresses a preference for “final versions” of papers rather than preprints (Research England, 2019). It is, however, also possible that institutions have developed local conventions for REF submission which in fact discourage submission of preprints, as might be inferred from this quotation.”In some ways, there is a balance to be achieved here. Qualitative research aims to find out about perceptions and this can include misunderstandings. It is important to report these, as misunderstandings often shape behaviours. The extent to which the researcher should correct the participants in their reporting is a moot point in general. That being as it may, in this case, we do recognise there was a need to comment. Reviewers’ comments: Clarity of presentation: Please provide more quantitative context to the opinions expressed. For example, for phrases such as “Some researchers” it would be useful to be more specific. Please also be more quantitative in assessment of the themes — it is useful to know if a sentiment was expressed by a majority or minority, as shown clearly in tables of benefits and challenges. Please provide aggregate data of thematic analyses, not just quotes, and use specific numbers and proportionality (Two of Five researchers mentioned X as a benefit). Authors’ response:We have addressed this comment by amending wording to be as precise as possible within the constraints of a qualitative study. Once again, there is a balance to struck here. On the one hand, as a general principle, it is essential to recognise that qualitative research should not be analysed quantitatively. Inferences cannot be drawn from the data in any statistically valid way from quantitative analysis of a dataset like this, and it is important not to give the impression that they can. Certainly, including percentages would not be appropriate. In any case, the data is not sufficiently structured to allow for reliable identification of a type of response which could be reported quantitatively. Every datum in a qualitative dataset is actually unique and although it is possible to identify commonalties, it is not normally possible to say that a certain proportion of participants said one thing or another. This likely to lead inappropriate homogenisation of views.Furthermore, it is important not to confuse prevalence with significance. In a qualitative dataset, some ideas may be mentioned by a large number of participants, but this does not necessarily mean they are significant. Significance may not, of course, be easy to judge (and it is a matter of judgement based on careful analysis) but it is not a simple issue of numbers of times an issue is mentioned in the dataset. We have however made some attempts to identify prevalence of issues raised in discussing pros and cons of preprints, something which is possible (simply identifying topics mentioned), with the relevant caveats.On the other hand, it is perfectly reasonable to mention some quantitative terms, “the majority”, or “a minority” or if an issue was only mentioned by one or was mentioned by all participants. That is what we have tried to do within the bounds of what is defensible. Reviewers’ comments: The paper combines elements of a literature review, white paper/policy piece, and a qualitative study. It would be helpful to clarify the origin of ideas by separating the work clearly into Results and Discussion sections. (As one example, under the heading “Financial sustainability and business models,” it is unclear whether the proposed taxonomy was expressed by interviewees or generated by the authors. Similarly, it is unclear whether use of the term “seminal” comes from interviewees or authors.) Authors’ response:This point is well made. The systematisation of the different models or scenarios of delivery of preprint services is ours based on a composite analysis of our data. Many of our participants talked about ownership and sustainability issues but none of them organised their remarks in this way. We had thought the systematisation of views helped to contextualise the reporting of the data but it does create the potential for confusion, as the reviewers point out. So we have, therefore, pulled this section out of the findings and added it into the discussion in order to make it clear that it is our analysis arising from the data not a direct reporting of the data. Reviewers’ comments: Readability: Please use active voice and concise language to improve readability. Authors’ response:We are conscious that conventions vary in this area across disciplines or even journals. Many life sciences researchers have moved over recent years to using the active voice. However, we were not aware of there being a house style for F1000. The discursive style we have adopted using a combination of active and passive voice is conventional for the reporting of the research we have carried out. We have, however, made some adjustments to improve clarity. Reviewers’ comments: Bring interview questions and list of participants into this report — it is hard to interpret findings while having to continuously refer back to other sources, e.g. the 2019b interview structure paper. Authors’ response:We believe the comments from the two sets of peer reviewers are somewhat in tension with regard to the level of detail that should be included, with the other reviewers stating:The reviewers wonder if the results section as a whole, which is relatively long in comparison to the rest of the paper, should be limited to Table 5 and its description and avoid the unending narrative of anecdotal specific responses. Given the insertion of the interview questions and list of participants would make the paper still longer, we believe it remains appropriate to make them available as separate, citable objects which are referenced by the paper.Finally, sharing interview questions and participants via ‘extended data’ is required by the F1000Research Author Guidelines, which state that: “F1000Research does not accept supplementary material. Additional materials that support the key claims in the paper but are not absolutely required to follow the study design and analysis of the results, e.g. questionnaires, or supporting images or tables, can be included as extended data.” Reviewers’ comments: Claims: The abstract sets the reader up to expect a review of preprint servers that have emerged since 2013. However, the methods section discloses that coverage of the study is limited to biology, chemistry, and psychology. It would be helpful to readers to state this up front. It is also unclear how this scope was applied in the selection of study subjects. It is difficult to determine if the scope influenced the selection of participants as listed in https://zenodo.org/record/2654832#.XTeUB5NKjs0 – the questions listed at https://zenodo.org/record/3240426 do not seem specific to these three disciplines and the quoted answers in this report suggest interviewees’ responses were not limited to these three disciplines. Please clarify how the scope of the interviews was constrained to the stated subjects, or if not, please revise the stated scope to more accurately reflect the scope employed. Authors’ response:We have changed the text at the beginning of the methods section to provide greater clarity about our approach, as follows:“We chose to carry out detailed interviews of key actors in this space who could explain in depth their perceptions, attitudes and practices in relation to preprints. Participants were asked about their perspectives on preprints in general, but we intentionally recruited interviewees (where they had disciplinary affiliations) particularly from disciplines where preprint services are relatively new and rapidly growing. These were biology, chemistry and psychology, corresponding to preprint servers, bioRxiv, ChemRxiv and PsyArXiv. Focusing on these areas helped us to gauge the impact that preprints are having in areas where they are more innovative, and since many of our participants were able to speak more generally about preprints, we were able to draw comparisons with disciplines where preprints are established and which are better represented in the literature (e.g. physics, computer science, and economics).”It is important to note that our study was not designed to elicit comments about specific servers in particular but rather about participants views on preprints in general. Their own experiences, based as some of them were in particular disciplinary communities, will, of course, be coloured by their familiarity with particular servers, but we wanted to explore their views on preprints more generally than that. Reviewers’ comments: The abstract does not reflect the content. The interview responses are summarised in general terms, not related to defined scope or the newer servers. A substantial proportion of this article is review material: either in the explicit literature review section, or as author-contributed content interspersed with summaries of interviewee responses. For example, conclusions listed in the abstract appear to be drawn from the literature review. Authors’ response:We have made changes to the abstract that we hope correct this, including ensuring findings reported relate closely to the data (although for the most part these do correlate with the literature). Reviewers’ comments: “Our study is the first using empirical data to understand the new wave of preprint servers” — this is not supported by the study content: no results are specifically related to the newer servers identified in the introduction; quoted excerpts indicate that interviewees responded in general about all servers and disciplines. Furthermore, prior surveys and bibliometric analyses have looked at a subset of these servers, either individually or in groups (eg https://osf.io/5fk6c/).1 Authors’ response:We have made changes to the abstract that we hope correct this.Minor commentsReviewers’ comments:We recommend making the following amendments to improve the clarity and accuracy of the report.In the Introduction and literature review: The summary of the longer history of preprint efforts across disciplines, would benefit from incorporating and citing ‘The Brief Prehistory of Preprints” (Cobb, 2017)2 Authors’ response:Added. Reviewers’ comments: Zuckerberg Foundation should be corrected to Chan Zuckerberg Initiative. Authors’ response:Corrected.Reviewers’ comments: Discussion of new servers would be improved with a note as to their current size, and with awareness that not all new ‘preprint’ servers are exclusively for preprint content (many COS servers include postprints that are not possible to easily distinguish from preprint versions). Authors’ response:We have added figures for bioRxiv, ChemRxiv and PsyArXiv. Reviewers’ comments: “This paper aims to explore…by investigating current practices, drivers and barriers to [preprint] use.” The interviews indicate perceptions and attitudes but do not reveal actual behaviours or practices. Please clarify how this study examines current practices. Authors’ response:The data covers behaviours and practices in a number of ways. Most prominently, the data indicates differences in uptake of preprints by researchers, with some being engaged and talking about their experiences and the positive outcomes that have accrued in their view. Others were sceptical and spoke about their lack of engagement based on their views of the some of the challenges presented by preprints. The thematic area covering perceived benefits and challenges has experience of adoption (or non-adoption) running through it. The theme of “Disciplines, cultures and practices” also has behaviours and practices throughout. In it, we refer to differing levels of awareness and “adoption”, “a willingness to experiment” etc. and present illustrative quotations from interviews. The earlier section on definitions also has some elements of behaviour, with one participant, for example, talking about posting a preprint when it was “submission ready for a journal”. The behaviours and practices are then closely linked to and shaped by the perceptions and attitudes, as might be expected. We have tried to make this clear at the beginning of the sub-section on benefits and challenges: “Participants highlighted a number of (potential) benefits and challenges of preprints which were seen to relate to particular practices around adoption or non-adoption of preprints”. Reviewers’ comments:Figures and tables: Fig 1: To increase accessibility, please provide as raw text with headings or tags to indicate which stakeholder group you believe is most relevant Authors’ responseFigure 1 is now available in csv format (text only) via Zenodo. This is referenced in the text as Chiarelli et al 2019c - http://doi.org/10.5281/zenodo.3539032. Reviewers’ comments:Literature review: The authors focus on four main issues — how were these arrived at? Was the literature review systematic in any way? Authors’ responseWe did not perform a formal systematic literature review but our reading of the literature is that these issues emerge. We have added a new category to cover the more empirical studies now emerging, mostly in 2019. Reviewers’ comments: Given the emergent phase of preprint infrastructure in biology, chemistry and psychology, and as the authors note that empirical data is lacking, it may be equally valuable to include more grey literature (blogposts, webpages) in this review, given that several of the cited articles are opinion pieces, albeit as editorials or peer-reviewed review articles. Of note, we find personal blogs are a useful source of stories that detail benefits and drawbacks of preprints from personal experiences. Authors’ responseWe have included some of these more informal sources, including information form the ASAPbio web site, Scholarly Kitchen blog, SciELO blog and numerous news sections of journals. We have added an example of an exchange on Twitter. We do not claim to be exhaustive but have been intentionally selective to represent the main strands of debates. Including many more would make an already long article even longer when we would prefer the main focus of the article to on the data we present. Reviewers’ comments: \"Dis-benefits:\" is not a common term, perhaps use “drawbacks,” “concerns,”, “disadvantages” or “potential risks”. Authors’ response:We have changed the term to “challenges”. Reviewers’ comments: When discussing the lack of quality assurance, the authors seem to make an implicit assumption here that journal-led peer review assures quality. It would be fair to contextualise this section with some literature on peer review to understand variety of peer review outcomes on quality of paper and validity of findings. Authors’ response:We have added reference (Lee, et al., 2013) in the literature review on problems with peer review. Concerns about quality were raised by our participants, which we report, although as we also say, many were eager to report the imperfections of peer review as well. Reviewers’ comments:Results: In the discussion of preprints in the medical field, please note that the concerns raised are being addressed by medRxiv. https://www.medrxiv.org/ Authors’ response:medRxiv had not been released when our data was collected. Our data are inevitably a snapshot from the time it was collected. However, we note that the quotation from this area in the paper covers many of the checks now being carried out by medRxiv. Reviewers’ comments: In the table of business models: Note that bioRxiv uses 3rd party technology, Highwire. Authors’ response:Corrected Reviewers’ comments: “Publisher driven” models are not new; PeerJ is older than bioRxiv, Nature had Nature Preceedings in the early 2000s, and bioRxiv has had J2B preprints since its inception. Authors’ response:Noted and details about the closure of PeerJ preprints also now added. Reviewers’ comments: How does the outsourcing of infrastructure to a third party enable sustainability? Arguably, depending on the third party, this leaves the service vulnerable to changes in service entirely outside its control. Authors’ response:We have added clarification on this, as follows:“Model 2 is a version of 1 in which some infrastructure is outsourced to a third party, something which might help to enable sustainability by creating efficiencies through economies of scale (a common benefits of outsourcing). The infrastructure provided by the Center for Open Science is an example of such benefit, with multiple preprint servers being run by the same organisation and on the same infrastructure, avoiding duplication, and therefore creating efficiency.” Reviewers’ comments: Individual authors can still submit to PeerJ. Authors’ response:We have updated references to PeerJ following the announcement of their closure. Reviewers’ comments: Most publisher-mediated preprint submissions are voluntary for authors (a notable exception being F1000). Authors’ response:We have added a note to this effect in the Introduction. Reviewers’ comments: Regarding the statement of subject preferences: we have conducted a survey on these preferences that may provide additional context: https://asapbio.org/asapbio-funders-workshop Authors’ response:We have reviewed the survey results at the URL cited and agree they are useful and interesting. However, we cannot see substantial information about “subject preferences” in the data. We have not therefore made any changes in response since we are unsure what the recommendation of the reviewers refers to. The following suggestions may also help improve the paper but are not essential. Reviewers’ comments:In the Introduction and literature review: Open Research platforms could be discussed in context of F1000Research, since this is the model they are derived from (in collaboration with F1000). Authors’ response:The Open Research Central services are further examples of the model developed by F1000. Since we are not attempting to be exhaustive in listing of services but are rather giving examples, and because Open Research Central uses the same model as is “powered by” F1000, we would prefer to keep our introduction at the length it is without giving more examples, which would further extend it. Reviewers’ comments: Please clarify the age of PeerJ and Preprints.org in comparison with bioRxiv. Authors’ response:We have done this, adding a note of the launch (and closure) of PeerJ in the opening paragraph. Reviewers’ comments: PLOS is not the only journal that deposits preprints to biorxiv using the J2B mechanism — several do, please refer to the list of journals offering direct transfers to bioRxiv (https://www.biorxiv.org/about-biorxiv). Authors’ response:We have done this, adding a reference in the opening paragraph. Reviewers’ comments: Regarding trust in preprints, it would be relevant to mention or cite the current effort by COS to better understand indicators of trust in preprints: https://cos.io/about/news/center-open-science-receives-grant-alfred-p-sloan-foundation-study-trust-and-credibility-preprints/. Reviewers’ comments:Figures and tables: Fig 1 introduces some interesting questions, however many of these are related to topics that are brought up for the first time in this figure. Authors’ response:Yes, ours was an exploratory study which had as one of its aims to identify areas requiring further work. Our data pointed to a wide range of questions which now need to be pursued in order to understand more about the (potential) future of preprints. What we have done is to help to provide clarity around what the they key questions are and how they might begin to be addressed (e.g. which actors should be involved in addressing them). This was designed to be helpful to policy makers in particular. Reviewers’ comments: Table 6: The concern of reputational damage is indeed in the literature; please see point 5 here: https://smallpondscience.com/2017/07/24/whats-up-with-preprints/ Authors’ response:Updated Reviewers’ comments:Literature: “However, Bourne et al. (2017) controversially extend the definition to include “a paper that has been peer reviewed and…was rejected, but the authors are willing to make the content public”.” — Please provide reasoning or citation as to why this is controversial. Authors’ response:The context as a whole is important: “For (3) versioning, the relationship of a preprint to peer review is central. Desjardins-Proulx et al.’s (2013, p. 1) observation is typical in stating that preprints are made available, “before, or in parallel to, submitting them to journals for traditional peer review”. Suber (2012, p. 102) points out that this is not to “bypass peer review”, but that it applies to “works destined for peer review but not yet peer reviewed”. However, Bourne et al. (2017) controversially extend the definition to include “a paper that has been peer reviewed and…was rejected, but the authors are willing to make the content public”.The point is that if preprints are defined as papers “destined for peer review” then including papers was rejected in a peer review process extends the definition and does so in a controversial way because it might imply a by-passing (or ignoring) of the peer review process. We note that in our data some participants questioned whether an output can be considered a preprint if it is not ultimately formally published. Reviewers’ comments: Accessibility — note that preprints are not necessarily open access, where they are not provided with permissive licensing, and this is certainly the case for bioRxiv.3 Authors’ response:Point noted. We have changed the description in Table 1 to, “Preprints are openly available online”. In the associated discussion we talking about availability of content but do not use the term ‘open access’. This was intentional, as it allows us to side step the question of definitions of open access, which is a controversial issue (gratis, libre etc). We appreciate the point about licensing and this was one issue mentioned in our dataset and to which we make reference. Reviewers’ comments: One potential benefit to ECRs that is not mentioned here is being able to demonstrate productivity to funders and hirers. This would be useful to append to the current paragraph, given it is raised in the interview questions. Authors’ response:Good point, and as this is a somewhat different point than achieving visibility, we have added it to the paragraph:“Early dissemination is seen by some as especially useful to a number of members of the scholarly community in particular, with early career researchers (ECRs) commonly identified as specific potential beneficiaries, as preprints can allow them to rapidly achieve “visibility” and demonstrate productivity in job and grant applications (Desjardins-Proulx et al., 2013; Sarabipour et al., 2018; Tennant et al., 2019).” Reviewers’ comments: “There also appears to be little acknowledgement that the claim stands in tension to the one cited earlier that preprints often differ little from final published versions.” — There is only tension if both claims concern all preprints. It is possible for there to be preprints that improve through a lot of feedback as well as preprints that undergo no/very little change. Authors’ response:We have updated this sentence to read “There also appears to be little acknowledgement that the claim, when used to make the case for preprints in general terms, stands in tension to the one cited earlier that preprints often differ little from final published versions.” Reviewers’ comments: Please use unshared or unpublished instead of “homeless”. Authors’ response:“Homeless” has been used elsewhere in the context of preprints (e.g. Berlin, 2018) and more generally in the context of scholarly communication. Mega-journals have been described as “allowing for the publication of research that might not find a home in traditionally more selective journals” (Wakeling, et al. 2017). That definition helps to make the point that “homeless” does not just mean outputs that are unshared or unpublished but ones that might not otherwise find a venue for publication. We therefore propose to retain the term.Berlin, S. (2018). If the papers don’t come to the journal…. EMBO Reports, 19(4). https://doi.org/10.15252/embr.201845911Wakeling, S., Spezi, V., Creaser, C., Fry, J., Pinfield, S., & Willett, P. (2017). Open access megajournals: The publisher perspective (Part 1: Motivations). Learned Publishing, 30(4), 313–322. https://doi.org/10.1002/leap.1118Reviewers’ comments: We recommend avoiding the term “policy stack,” as there is no dependent relationship among these different groups/stakeholders. Authors’ response:We have removed the term from the paper. Reviewers’ comments:Methods: This section ignores the efforts of researchers as change agents. Authors’ response:We have now acknowledged the potential for change agents to affect community norms within our description of the ‘nature of the social system’. Reviewers’ comments:Results: Please provide evidence for this statement: “we note that adjacency between disciplines appears to be playing an important role.” Authors’ response:We have adjusted the wording of this to make sure it is clearly defensible:“Finally, we note that adjacency between disciplines may play an important role. For example, disciplines close to those which posted preprints (e.g. those close to some areas of computer science or physics, which use arXiv consistently) may be more favourably disposed to the practice compared to those from other disciplines.”The extension of arXiv to other adjacent disciplines is evidence of our point and we see hints of this elsewhere – but at early points. Reviewers’ comments: “Many of the participants agreed that preprints servers could be a useful outlet for otherwise ‘homeless’ research outputs, even though this goes counter to the emphasis of many of preprints as early versions of outputs later formally published elsewhere.” — this attitude may reflect a lack of publication venues for such content, not a lack of desire to publish these other outputs in a peer-reviewed destination. Authors’ response:Yes, agreed (see our remarks on “homeless” above). It does shift the model of preprints as being early versions of papers that are later formally published elsewhere, however." } ] } ]	1	https://f1000research.com/articles/8-971
https://f1000research.com/articles/6-1873/v1	23 Oct 17	{ "type": "Research Article", "title": "Patient predictors of poor drug sensitive tuberculosis treatment outcomes in Kyiv Oblast, Ukraine", "authors": [ "Omowunmi Aibana", "Andrej Slavuckij", "Mariya Bachmaha", "Viatcheslav Krasiuk", "Natasha Rybak", "Timothy P. Flanigan", "Vasyl Petrenko", "Megan B. Murray", "Andrej Slavuckij", "Mariya Bachmaha", "Viatcheslav Krasiuk", "Natasha Rybak", "Timothy P. Flanigan", "Vasyl Petrenko", "Megan B. Murray" ], "abstract": "Background: Ukraine has high rates of poor treatment outcomes among drug sensitive tuberculosis (DSTB) patients, while global treatment success rates for DSTB remain high.\n\nWe evaluated baseline patient factors as predictors of poor DSTB treatment outcomes. Methods: We conducted a retrospective analysis of new drug sensitive pulmonary TB patients treated in Kyiv Oblast, Ukraine between November 2012 and October 2014. We defined good treatment outcomes as cure or completion and poor outcomes as death, default or treatment failure. We performed logistic regression analyses, using routine program data, to identify baseline patient factors associated with poor outcomes. Results: Among 302 patients, 193 (63.9%) experienced good treatment outcomes while 39 (12.9%) failed treatment, 34 (11.3%) died, and 30 (9.9%) defaulted. In the multivariate analysis, HIV positive patients on anti-retroviral therapy (ART) [OR 3.50; 95% CI 1.46 – 8.42; p 0.005] or without ART (OR 4.12; 95% CI 1.36 – 12.43; p 0.01) were at increased risk of poor outcomes. Alcohol abuse (OR 1.81; 95% CI 0.93 - 3.55; p 0.08) and smear positivity (OR 1.75; 95% CI 1.03 - 2.97; p 0.04) were also associated with poor treatment outcomes. Conclusions: High rates of poor outcomes among patients with newly diagnosed drug sensitive TB in Kyiv Oblast, Ukraine highlight the urgent need for programmatic interventions, especially aimed at patients with the highest risk of poor outcomes.", "keywords": [ "tuberculosis", "drug sensitive", "treatment outcomes", "patient predictors", "HIV", "HIV-TB coinfection", "Ukraine", "Eastern Europe" ], "content": "Introduction\n\nTuberculosis (TB) control remains challenging worldwide, with approximately 10.4 million new cases diagnosed and 1.8 million TB deaths in 20151. Although incidence rates have declined in parts of Eastern Europe, TB continues to be a significant public health problem in many former Soviet Union countries including Ukraine, which currently has the second highest burden of multi-drug resistant TB (MDR-TB) in the WHO European Region after Russia1. National TB control measures in Ukraine include annual screening with chest radiographs for at risk groups i.e. immunosuppressed patients, diabetics, homeless patients, migrants, incarcerated individuals, and all medical staff in primary healthcare facilities2. In addition, surveillance for resistant TB includes routine drug susceptibility testing (DST) for all culture positive isolates; and in 2007 the country adopted WHO recommended directly observed therapy short course (DOTS). However, despite these efforts, Ukraine’s National TB Program (UNTP) still has low treatment success rates. According to the most recent WHO data available for new smear and/or culture positive TB cases in Ukraine, the treatment success rate among these cases was 58% in 2011 in contrast to a global success rate of 83%3.\n\nTB treatment outcomes vary depending on the distribution of risk factors within a treatment cohort, as well as on the quality and nature of TB health services. Patient-related predictors of poor outcome among patients with drug-sensitive TB (DSTB) include HIV, diabetes mellitus (DM), alcohol or substance use, and homelessness4–10. Health system factors that influence TB outcomes include ease of access to services, diagnostic capabilities, drug availability, social support for patients, and collaboration of TB/HIV services11,12. Currently, no published research addresses predictors of poor DSTB outcomes in the context of the UNTP, despite the frequency of this outcome. Here, we used routinely collected program data in the Kyiv Oblast of Ukraine to examine the association between baseline patient risk factors and DSTB treatment outcomes. These findings can help develop targeted interventions to address patient populations at the greatest risk of poor outcomes.\n\n\nMaterials and methods\n\nThe study was approved by the Institutional Review Board at The Miriam Hospital, Lifespan, Providence; RI (215014 45CFR 46.110[5]) and the Research Ethics Committee at Bogomolets Medical University in Kyiv, Ukraine. Informed consent was not required because the data were analyzed anonymously, and written informed consent was waived by the Institutional Review Boards.\n\nWe conducted a retrospective chart review to identify baseline risk factors for poor treatment outcomes among drug-sensitive pulmonary TB patients in the Kyiv Oblast of Ukraine, where the notification rate for new pulmonary TB in 2014 was approximately 62 per 100 000 persons. TB diagnosis and management in Kyiv Oblast is provided free of charge and according to Ukraine’s NTP2. National guidelines specify that all TB suspects undergo sputum smear microscopy and culture, molecular testing with Xpert® MTB/RIF and chest X-ray to confirm diagnosis. TB suspects include patients evaluated in primary care settings with complaints of cough, fever, night sweats, weight loss, chest pain, and dyspnea or patients that providers consider at risk for TB based on clinical history. General practitioners then refer suspects to TB specialists for diagnosis and further management. The 2014 UNTP specify the following: baseline susceptibility testing to rifampin (R), isoniazid (H), ethambutol (E), pyrazinamide (Z) and streptomycin (S) on all culture positive TB isolates; baseline screening for pre-specified risk factors including alcohol abuse, intravenous drug use (IVDU), and homelessness (notably, screening for alcohol and substance use relies on patient self-report); baseline HIV testing and provision of anti-retroviral therapy (ART) to those that are positive as soon as possible after initiation of TB treatment; and repeat DST among DSTB patients who are culture positive at three months or at the end of treatment. In Kyiv Oblast, treatment for alcohol or substance abuse is not provided for patients during TB treatment.\n\nUNTP guidelines also specify that DSTB patients receive treatment with two months of RHZE and four months of RH. The previous UNTP guidelines specified inpatient treatment during the intensive phase in specialized TB hospitals, while the subsequent continuation phase occurs in an ambulatory setting. Dedicated adult TB hospitals exist in each administrative region of Ukraine where patients receive testing and treatment. Although latest national guidelines in 2014 now recommend outpatient management of DSTB2, many regions in Ukraine, including Kyiv Oblast, have yet to implement this practice and continue to hospitalize patients during the intensive phase.\n\nFor newly diagnosed patients with HIV, ART is initiated during hospitalization and after discharge, HIV-related care occurs at HIV programs that are distinct from the TB clinics, which provide outpatient TB care. Standard ART regimen for co-infected patients include Tenofovir, Lamivudine, and Efavirenz. In Kyiv Oblast, outpatients may receive daily directly observed therapy or receive a supply of medication at 7 – 10 day intervals. Clinicians can continue the inpatient care of individuals at high risk for default (e.g. homeless patients) for the entire treatment duration, although compliance is not enforced; and patients are free to leave the hospital any time. UNTP guidelines also recommend follow up for DSTB patients at yearly intervals for three years after treatment completion.\n\nWe analyzed routinely collected clinical and programmatic data from the three TB hospitals in Kyiv Oblast, which together admit approximately 1100 patients annually for pulmonary TB. We included all adult patients (≥ 16 years) treated for newly diagnosed drug-sensitive pulmonary TB between November 2012 and October 2014. We excluded patients who did not yet have a treatment outcome assigned because they were undergoing the initial course of TB treatment at the time of data extraction in November 2014, and those with previous TB history.\n\nWe extracted the following information routinely collected by the TB program in an electronic database: age, gender, residence, employment status, history of TB contact, homelessness, immigration status, previous incarceration, HIV status with ART initiation dates, history of alcohol abuse and intravenous (IV) drug use, as well as mode of case finding (active or passive); passive TB case finding refers to the diagnosis of TB among patients who self-initiate contact with healthcare providers for management of TB symptoms. We also recorded baseline sputum smear, culture and DST results. The NTP provides standardized paper forms used by TB providers to record all baseline demographic and clinical information for routine program monitoring. In Kyiv Oblast TB hospitals, all data are subsequently entered in an electronic database by the statistics department.\n\nTreatment outcomes for DSTB are classified in Kyiv Oblast according to WHO guidelines13. Good treatment outcomes include cure and treatment completion, while poor outcomes include deaths, default and treatment failure. The WHO considers a DSTB patient cured if he or she remains smear or culture negative in the last month of treatment and on at least one previous occasion. A patient is considered to have completed treatment if he or she has received a full course of therapy but has not received smear or culture in the last month of treatment. Any deaths during TB treatment are considered TB related. A patient is considered to have defaulted if he or she interrupts treatment for two or more consecutive months. Patients who remain smear or culture positive at month 5 or later during treatment are considered treatment failures; and in Kyiv Oblast patients who acquire resistance are also categorized as treatment failures. The exact dates of treatment outcomes or last follow up visits were not captured in the database.\n\nWe analyzed only patients with confirmed drug sensitive pulmonary TB, and we excluded from the analysis patients who transferred out or had missing outcomes. We did not follow up with patients in the community to ascertain treatment outcome among those with missing data on final outcome. We compared categorical variables with Fisher’s exact test and continuous variables with the Wilcoxon rank sum test. We performed univariate and multivariate logistic regression analyses to identify baseline predictors of combined poor treatment outcomes. For the multivariate model, we included baseline variables previously known to be associated with poor outcomes (age, sex, HIV, alcohol abuse, homelessness) and any variable associated with poor outcomes at p value less than 0.2 in the univariate analysis. We further evaluated baseline predictors for the outcomes of death and treatment failure separately. We used complete case analysis in the regression models. We used the regression coefficients specified by the final multivariate model to predict probability of combined poor outcomes. Data were analyzed using SAS v9.4 (SAS Institute, Cary, NC 2013).\n\n\nResults\n\nWe identified 561 patients treated for new DSTB between November 2012 and October 2014. Among them, we excluded 99 (17.6%) patients who did not yet have a treatment outcome because they were still undergoing TB treatment at the time of analysis (Figure 1). Table 1 lists baseline characteristics of the remaining 462 patients; among them, 122 (26.4%) patients had no drug susceptibility testing performed. 340 patients (73.6%) had a baseline DST to confirm drug sensitive pulmonary TB, and 181 (39.2%) underwent Xpert/Rif testing at baseline. 75 (16.2%) patients tested HIV positive, while HIV status was not recorded for 8 (1.7%) patients. Among the HIV positive patients, 34 (45.3%) were initiated on ART during TB treatment. Median time to ART initiation from TB treatment start date was 43.5 days (IQR 34.0 – 59.5).\n\nIQR: Interquartile Range.\n\na N = 418\n\nb N = 461\n\nc N = 460\n\nOf the 340 patients with DST results, 38 (11.2%) had missing outcome data. Among the remaining 302 patients, 104 (34.4%) experienced treatment cure and 89 (29.5%) completed treatment, while 39 (12.9%) failed treatment, 34 (11.3%) died, 30 (9.9%) defaulted, and 6 (2.0%) transferred out.\n\nIn the univariate analysis, significant baseline predictors of poor treatment outcomes included alcohol abuse (OR 1.95; 95% CI 1.05 - 3.61; p 0.03), and smear positive disease (OR 1.70; 95% CI 1.04 - 2.75; p 0.03) (Table 2). Compared to HIV negative, HIV patients were also at increased risk of poor outcomes; those who were not initiated on ART were four times as likely to experience poor outcomes (OR 4.07; 95% CI 1.45 – 11.39; p 0.01), while patients on ART were more than twice as likely to have a poor treatment outcome (OR 2.58; 95% CI 1.14 – 5.85; p 0.02). Homeless patients were also at increased risk of poor outcomes, although this association was not significant at the .05 level (OR 7.76; 95% CI 0.86 – 70.32). Unemployment (OR 1.59; 95% CI 0.97 – 2.61; p 0.06) and passive case finding (OR 1.78; 95% CI 0.94 – 3.39; p 0.07) also conferred borderline significantly increased risk of poor treatment outcomes in the univariate analysis. Time to ART initiation was not associated with poor outcomes (OR 1.02; 95% CI 0.98 – 1.06; p 0.33) (Table 2).\n\nOR: Odds Ratio.\n\na Patients with confirmed drug sensitive TB and outcomes of cure, completion, death, treatment failure and default.\n\nb Adjusted for age, gender, HIV, alcohol abuse, homelessness, baseline smear status, unemployment and passive case finding.\n\nc N = 294\n\nd N = 26\n\ne N = 269\n\nf Excluded from multivariate analysis because only 4 patients had known TB contact.\n\nWhen we adjusted for other risk factors, we found that smear positivity (OR 1.75; 95% CI 1.03 - 2.97; p 0.04) and HIV positivity (on ART [OR 3.50; 95% CI 1.46 – 8.42; p 0.005] and without ART [OR 4.12; 95% CI 1.36 – 12.43; p 0.01]) all remained significant predictors of poor outcome. Patients with alcohol abuse also had a modest increase in risk of poor outcomes (OR 1.81; 95% CI 0.93 – 3.55; p 0.08) and the odds of poor outcomes among the homeless continued to be high but not statistically significant at 6.38 (95% CI 0.69 – 59.40) (Table 2). Unemployment (OR 1.26; 95% CI 0.72 – 2.20; p 0.43) and passive case finding (OR 1.18; 95% CI 0.60 – 2.35; p 0.63) were no longer associated with increased risk of poor outcomes in the adjusted analysis (Table 2). Our multivariate model predicted that a 40-year-old male who is HIV positive but not on ART, with alcohol abuse and smear positive disease, has a 75.8% probability of poor treatment outcome.\n\nWhen we separately evaluated risk factors for death during DSTB treatment, in the adjusted analysis, we found age (OR 1.03; 95% CI 1.00 – 1.06; p 0.03), HIV positivity (OR 4.21; 95% CI 1.44 – 12.30; p 0.01) and alcohol abuse (OR 2.54; 95% CI 1.00 – 6.42; p 0.05) were associated with statistically significant increased risk of death (Table 3). HIV positivity (OR 7.42; 95% CI 2.56 – 21.54; p < 0.001) and smear positive disease at baseline (OR 4.99; 95% CI 2.00 – 12.45; p 0.001) were the strongest predictors of DSTB treatment failure (Table 4).\n\nOR: Odds Ratio.\n\na Patients with confirmed drug sensitive TB and outcomes of cure, completion and death.\n\nb Adjusted for age, gender, HIV, alcohol abuse, baseline smear status, and passive case finding.\n\nc N = 225\n\nd Excluded from multivariate analysis because there were only 3 homeless patients.\n\ne N = 226\n\nf N = 206\n\nOR: Odds Ratio.\n\na Patients with confirmed drug sensitive TB and outcomes of cure, completion and treatment failure.\n\nb Adjusted for age, gender, HIV, alcohol abuse, baseline smear status, and passive case finding.\n\nc N = 230\n\nd Excluded from multivariate analysis because there were only 3 homeless patients and 3 patients with known TB contact.\n\ne N = 211\n\n\nDiscussion\n\nWe found that only 64% of patients treated for drug-sensitive TB in Kyiv Oblast achieved treatment cure or completion, and this is far below global treatment success rates of 83%1. We also identified alcohol abuse and HIV as patient determinants of failure, death or default in this setting. Our findings support the idea that TB control efforts in this setting should urgently prioritize interventions aimed at the patient populations identified as at risk.\n\nWe show that routinely collected baseline programmatic data in Ukraine’s NTP reasonably predicts patients at high risk of poor DSTB treatment outcome at the beginning of treatment. Notably, this routine program data did not include other known predictors of poor TB treatment outcomes (e.g. DM, smoking, socioeconomic status, and poor nutritional status)4,9,10,14–17, therefore, we could not evaluate relative contributions of these unmeasured patient factors. Furthermore, the UNTP does not employ validated screening tools for harmful alcohol or substance use but instead relies on patient self-report; stigma associated with alcohol abuse and IVDU likely limits patients’ willingness to accurately report this information. Hence, rates of reported alcohol and IVDU were likely underestimated. Studies from other settings have demonstrated that incorporating dedicated treatment for alcohol abuse within TB programs is feasible18 and access to treatment for substance use improves TB outcomes19,20. Nevertheless, despite the limitations of routine program data, our findings demonstrate that within the current operations of Ukraine’s TB program, there is sufficient data to identify patients who can be targeted for early intervention to mitigate their risk of poor outcome. Improved screening for additional co-morbidities will also help identify other populations at higher risk for poor TB outcomes in this setting.\n\nIt is also important to note that health system factors influence patient-predictors and limit treatment success rates among patients with and without known risk factors at baseline. Hence, TB control efforts in this setting should also address how the current TB care delivery system in Ukraine adversely affects treatment outcomes for all patients. For instance, while the TB program in Kyiv Oblast tested most patients for HIV, less than half of HIV/TB co-infected patients were initiated on ART. We do not have data on the specific reasons why many HIV-positive patients were not initiated on ART during TB treatment. Anecdotal reports indicate TB providers sometimes defer ART for patients with high CD4 counts despite national guidelines for ART initiation regardless of CD4 count. Even among those treated, ART did not significantly mitigate the risk of poor TB treatment outcomes. Timing of ART initiation also did not predict outcome. Health system factors such as quality of ART and inconsistent availability may explain these findings. Provision of ART to all HIV/TB patients, as recommended by the WHO21, will likely improve treatment success rates among co-infected patients. However, additional system interventions such as integrated HIV and TB care during the entire phase of TB treatment may also be required to sufficiently address the excess risk of poor TB outcomes among HIV patients in this setting.\n\nPrevious evaluations of Ukraine’s TB program have already enumerated specific health system factors that hamper successful treatment outcomes, including: unnecessarily prolonged hospital-based care; interruptions in drug supply; protocol deviations; limited social support for patients; and suboptimal infection prevention that increases nosocomial TB transmission22,23. Our findings have been presented to policy makers in Ukraine including during a National Round Table discussion in Kyiv (November 2015) in preparation for updates to UNTP guidelines, which will focus on scaling up ambulatory-based care, targeted interventions for populations at risk of poor outcomes and patient-oriented approaches to improve treatment adherence. New policy changes create the possibility of further analyzing health system contributions to poor outcomes, and assessing how systems improvements will influence success rates among patients with baseline increased risk of poor outcomes. Future research can also evaluate providers’ understanding of and compliance with guidelines.\n\n\nConclusion\n\nWe found extremely low rates of treatment cure and completion for new drug sensitive TB in the Kyiv Oblast of Ukraine. In addition to specific interventions targeted at vulnerable patients, there is also a need to address and mitigate the impact of health system factors on Ukraine’s TB treatment success rates.\n\n\nData availability\n\nData have been de-identified for ethical, data protection and security reasons. Permission for use and publication of the anonymized data granted by the Institutional Review Board of Bogomolets Medical University.\n\nDataset 1. Data for patients analyzed in the retrospective cohort study. DOI, 10.5256/f1000research.12687.d17951324.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding support to OA was provided by the National Institute on Drug Abuse training grant T32 DA013911 and the NIH-NIMH grant R25MH83620.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWorld Health Organization: Global Tuberculosis Report. 2016. Accessed 10 August 2017. Reference Source\n\nMinistry of Health of Ukraine: Unified Clinical Protocol for Primary, Secondary (Specialized) and Tertiary (Highly Specialized) Medical Care for Adults with Tuberculosis. 2014. Accessed 10 August 2017. Reference Source\n\nWorld Health Organization: Global Tuberculosis Report. 2013. Accessed 10 August 2017. Reference Source\n\nMuture BN, Keraka MN, Kimuu PK, et al.: Factors associated with default from treatment among tuberculosis patients in Nairobi province, Kenya: a case control study. BMC Public Health. 2011; 11(1): 696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenegar C, Behets F, Vanden Driessche K, et al.: Mortality among tuberculosis patients in the Democratic Republic of Congo. Int J Tuberc Lung Dis. 2012; 16(9): 1199–1204. PubMed Abstract\n\nGelmanova IY, Keshavjee S, Golubchikova VT, et al.: Barriers to successful tuberculosis treatment in Tomsk, Russian Federation: non-adherence, default and the acquisition of multidrug resistance. Bull World Health Organ. 2007; 85(9): 703–711. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKliiman K, Altraja A: Predictors and mortality associated with treatment default in pulmonary tuberculosis. Int J Tuberc Lung Dis. 2010; 14(4): 454–463. PubMed Abstract\n\nJakubowiak WM, Bogorodskaya EM, Borisov SE, et al.: Risk factors associated with default among new pulmonary TB patients and social support in six Russian regions. Int J Tuberc Lung Dis. 2007; 11(1): 46–53. PubMed Abstract\n\nBaker MA, Harries AD, Jeon CY, et al.: The impact of diabetes on tuberculosis treatment outcomes: a systematic review. BMC Med. 2011; 9: 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaitt CJ, Squire SB: A systematic review of risk factors for death in adults during and after tuberculosis treatment. Int J Tuberc Lung Dis. 2011; 5(7): 871–885. PubMed Abstract \| Publisher Full Text\n\nGarner P, Smith H, Munro S, et al.: Promoting adherence to tuberculosis treatment. Bull World Health Organ. 2007; 85(5): 404–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Global strategy and targets for tuberculosis prevention, care and control after 2015. Accessed 10 August 2017. Reference Source\n\nWorld Health Organization: Definitions and reporting framework for tuberculosis – 2013 revision. Updated December 2014, Accessed 11 August 2017. Reference Source\n\nLin HH, Ezzati M, Murray M: Tobacco smoke, indoor air pollution and tuberculosis: a systematic review and meta-analysis. PLoS Med. 2007; 4(1): e20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMishra P, Hansen EH, Sabroe S, et al.: Socio-economic status and adherence to tuberculosis treatment: a case-control study in a district of Nepal. Int J Tuberc Lung Dis. 2005; 9(10): 1134–1139. PubMed Abstract\n\nAnanthakrishnan R, Kumar K, Ganesh M, et al.: The profile and treatment outcomes of the older (aged 60 years and above) tuberculosis patients in Tamilnadu, South India. PLoS One. 2013; 8(7): e67288. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurton NT, Forson A, Lurie MN, et al.: Factors associated with mortality and default among patients with tuberculosis attending a teaching hospital clinic in Accra, Ghana. Trans R Soc Trop Med Hyg. 2011; 105(12): 675–682. PubMed Abstract \| Publisher Full Text\n\nShin S, Livchits V, Connery HS, et al.: Effectiveness of alcohol treatment interventions integrated into routine tuberculosis care in Tomsk, Russia. Addiction. 2013; 108(8): 1387–1396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGelmanova IY, Taran DV, Mishustin SP, et al.: 'Sputnik': a programmatic approach to improve tuberculosis treatment adherence and outcome among defaulters. Int J Tuberc Lung Dis. 2011; 15(10): 1373–1379. PubMed Abstract \| Publisher Full Text\n\nMorozova O, Dvoryak S, Altice FL: Methadone treatment improves tuberculosis treatment among hospitalized opioid dependent patients in Ukraine. Int J Drug Policy. 2013; 24(6): e91–e98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: WHO policy on collaborative TB/HIV activities: guidelines for national programmes and other stakeholders. Geneva, Switzerland: World Health Organization; 2012. Accessed 10 August 2017. Reference Source\n\nWorld Health Organization, Regional Office for Europe: Review of the National Tuberculosis Programme in Ukraine. 10-22 October 2010. Accessed 10 August 2017. Reference Source\n\nVassall A, Chechulin Y, Raykhert I, et al.: Reforming tuberculosis control in Ukraine: results of pilot projects and implications for the national scale-up of DOTS. Health Policy Plan. 2009; 24(1): 55–62. PubMed Abstract \| Publisher Full Text\n\nAibana O, Slavuckij A, Bachmaha M, et al.: Dataset 1 in: Patient predictors of poor drug sensitive tuberculosis treatment outcomes in Kyiv Oblast, Ukraine. F1000Research. 2017. Data Source" }	[ { "id": "30175", "date": "01 Feb 2018", "name": "Bernt Lindtjorn", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors address an important issue. I have the following comments:\nMost of the literature is old, and the authors should use more up-to-date literature. This also includes the methods and how to calculate default rates. The authors chose a method whereby they exclude patients who were lost to follow-up. However, this is a too simplistic approach. The authors should read the latest WHO recommendations on doing such calculations.\n\nThe biggest problem with this paper is how they handle missing information. They state that: \"We analysed only patients with confirmed drug sensitive pulmonary TB, and we excluded from the analysis patients who transferred out or had missing outcomes.\" Also, as there is a high prevalence of drug resistance in their country, the exclusions of these patients can affect the result of this paper. Even if they wish to examine only patients with bacteria sensitive to antituberculosis drugs, the numbers should be mentioned so the reader would understand the total picture.\n\nFrom the paper, I understand there are about 1200 new patients with tuberculosis in their Oblast. This calculation is based on the population in the province and the incidents estimate the authors provide. They find only 562, and of those, 380 were expected to complete the treatment. The authors should explain the discrepancy between what was expected and what was found.\nPatients who were lost to follow-up were excluded from their analysis. This is not how it should be done according to the latest WHO recommendations. Also, the authors should do some sensitivity analysis on data. So, if we include those who were missing and those who were transferred out, the treatment complete rate is only 56%.\n\nIn the discussion, the authors do not discuss these major weaknesses and how they analysed the data. In addition, they need to explain the seeming discrepancy between incident cases during the year and the numbers they analysed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3488", "date": "13 Mar 2018", "name": "Omowunmi Aibana", "role": "Author Response", "response": "Comment 1: Most of the literature is old, and the authors should use more up-to-date literature. This also includes the methods and how to calculate default rates. The authors chose a method whereby they exclude patients who were lost to follow-up. However, this is a too simplistic approach. The authors should read the latest WHO recommendations on doing such calculations. Response 1: We have included updated references (refs 1, 4 - 14) in our background discussion of known patient predictors of poor drug sensitive TB treatment outcomes. The data we provided in the introduction on treatment outcomes for new DSTB patients in Ukraine is the most recent WHO reported information available for the country. The WHO now reports treatment outcomes for new and relapsed cases combined but we did not evaluate TB relapses in this study for comparison. Please note that treatment outcomes for DSTB in this study were categorized according to the latest WHO recommendations, which includes considering a patient to be lost to follow up (previously defaulted) if he or she interrupts treatment for two or more consecutive months. Therefore, we did not exclude patients lost to follow up (per WHO definitions) in our main analysis. As indicated below, we excluded patients whose final treatment outcomes were not assessed either because they transferred out and had missing outcome data. We have addressed this issue of handling missing outcome data in comment 2 below.We have further replaced “default” with “lost to follow up” throughout the document to clarify that patients lost to follow up according to WHO definitions were not excluded from our analysisComment 2: The biggest problem with this paper is how they handle missing information. They state that: \"We analysed only patients with confirmed drug sensitive pulmonary TB, and we excluded from the analysis patients who transferred out or had missing outcomes.\" Also, as there is a high prevalence of drug resistance in their country, the exclusions of these patients can affect the result of this paper. Even if they wish to examine only patients with bacteria sensitive to antituberculosis drugs, the numbers should be mentioned so the reader would understand the total picture. Response 2: A survival analysis approach would have allowed us to evaluate the relative contributions of all patients (including those whose outcomes were not assessed) in our assessment of determinants of treatment outcomes. However, as noted in paragraph 3 of the statistical analysis section, the exact dates of treatment outcomes or last follow up visits were not captured in the electronic database, therefore we were unable to perform such analysis. As an alternative, we have conducted a sensitivity analysis where we considered patients whose treatment outcomes were not assessed (i.e. transferred out and missing outcome data) as having a poor outcome. The results in this analysis do not differ from our main analysis; we found that patients with HIV and smear positive disease at baseline remained at increased risk of poor outcomes while high alcohol consumption conferred a modest risk of poor outcomes. We have added Table 5 to depict these results and we included discussion of this sensitivity analysis in the methods and results section as follows: Methods (paragraph 4 of statistical analysis): “In a sensitivity analysis, we categorized patients whose treatment outcomes were not assessed (transferred out and missing final outcome data) as having poor outcomes.”Results (paragraph 6): “When we categorized patients with missing outcome data andpatients who transferred out as having poor outcomes, the results did not differ from the findings in our main analysis of predictors of poor treatment outcomes (Table 5).”Please note that we have separately published results from a cohort of drug resistant TB patients in Kyiv Oblast [1]. Therefore, our primary aim in this manuscript was to report on predictors of poor outcomes among patients with confirmed drug sensitive pulmonary TB. We have now included in the methods section (paragraph 1 of statistical analysis) the following: “We have previously reported on a cohort of approximately 600 patients initiated on treatment for drug resistant TB in Kyiv Oblast between 2012 and 2015”.1. Aibana O, Bachmaha M, Krasiuk V, Rybak N, Flanigan TP, Petrenko V, et al. Risk factors for poor multidrug-resistant tuberculosis treatment outcomes in Kyiv Oblast, Ukraine. BMC Infect Dis. 2017;17(1): 129. Comment 3: From the paper, I understand there are about 1200 new patients with tuberculosis in their Oblast. This calculation is based on the population in the province and the incidents estimate the authors provide. They find only 562, and of those, 380 were expected to complete the treatment. The authors should explain the discrepancy between what was expected and what was found.Response 3: Please note that the TB notification rates we reported for Kyiv Oblast is for all pulmonary TB diagnosed and therefore includes both sensitive and resistant TB cases. We have now also reported the number of MDR-TB patients initiated on treatment in Kyiv Oblast as indicated above in response 2. Any remaining discrepancy in the expected number of TB cases might be related to lack of active screening. In addition to providing MDR-TB numbers for reference, we have now included a discussion (paragraph 4) about lack of active case finding in this setting, which may contribute to under-notification of TB. “We found that majority of TB patients were identified through passive case finding, which may also contribute to poor treatment outcomes. The WHO recommends systematic screening for active TB as a complement to passive case finding [1]. Studies have also shown that active TB case finding results in early detection and reduces risk of extensive disease at diagnosis [2-6], which may potentially decrease risk of poor outcomes. Active case finding also reduces risk of TB transmission [6-9] and may contribute to reducing TB prevalence. Lack of active case TB finding may further result in under notification of TB in this setting. TB control efforts in Ukraine will likely benefit from strengthening and improving health systems capacity for active case finding.”1. World Health Organization. Systematic screening for active tuberculosis. Principles and Recommendations. 2013. Available: http://apps.who.int/iris/bitstream/10665/84971/1/9789241548601_eng.pdf?ua=1 Accessed 20 February 2018. 2. den Boon S, Verver S, Lombard CJ, Bateman ED, Irusen EM, Enarson DA, et al. Comparison of symptoms and treatment outcomes between actively and passively detected tuberculosis cases: the additional value of active case finding. Epidemiol Infect. 2008;136(10): 1342-1349. 3. Eang MT, Satha P, Yadav RP, Morishita F, Nishikiori N, van-Maaren P, et al. Early detection of tuberculosis through community-based active case finding in Cambodia. BMC Public Health. 2012;12: 469.4. Datiko DG, Yassin MA, Theobald SJ, Blok L, Suvanand S, Creswell J, Cuevas LE. Health extension workers improve tuberculosis case finding and treatment outcome in Ethiopia: a large-scale implementation study. BMJ Glob Health. 2017;2(4): e000390.5. Kranzer K, Afnan-Holmes H, Tomlin K, Golub JE, Shapiro AE, Schaap A, Corbett EL, Lönnroth K, Glynn JR. The benefits to communities and individuals of screening for active tuberculosis disease: a systematic review. Int J Tuberc Lung Dis. 2013 Apr;17(4):432-46.6. Cheng S, Chen W, Yang Y, Chu P, Liu X, Zhao M, et al. Effect of diagnostic and treatment delay on the risk of tuberculosis transmission in Shenzhen, China: an observational cohort study, 1993-2010. PLoS One. 2013;8(6): e67516.7. Lin X, Chongsuvivatwong V, Lin L, Geater A, Lijuan R. Dose-response relationship between treatment delay of smear-positive tuberculosis patients and intra-household transmission: a cross-sectional study. Trans R Soc Trop Med Hyg. 2008;102(8): 797-804.8. Fox GJ, Barry SE, Britton WJ, Marks GB. Contact investigation for tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2013;41(1): 140–156.9. Jia ZCS, Ma Y, Zhang Y, Bai L, Xu W, He X, et al. Tuberculosis burden in China: a high prevalence of pulmonary tuberculosis in household contacts with and without symptoms. BMC Infect Dis. 2014;14: 64.Comment 4: Patients who were lost to follow-up were excluded from their analysis. This is not how it should be done according to the latest WHO recommendations. Also, the authors should do some sensitivity analysis on data. So, if we include those who were missing and those who were transferred out, the treatment complete rate is only 56%. Response 4: Please see responses 1 and 2 above where we clarified the definition of lost to follow up and presented the suggested sensitivity analysis. Comment 5: In the discussion, the authors do not discuss these major weaknesses and how they analysed the data. In addition, they need to explain the seeming discrepancy between incident cases during the year and the numbers they analysed.Response 5: We have addressed the analysis of missing data and the discrepancy between expected and analyzed cases as indicated above. We have expanded the discussion on limitations to include: “We also found a high proportion of patients did not have their treatment outcome assessed, which further reduces the rate of successful treatment outcomes in this setting. However, when we included patients with missing data on final outcomes in the analysis, our results did not change; we identified the same patient predictors of poor treatment outcomes.”" } ] }, { "id": "29231", "date": "05 Feb 2018", "name": "Erica Lessem", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important article, highlighting the previously overlooked problem of poor outcomes in nearly half of people with drug-susceptible TB, which is usually considered \"low hanging fruit\" and relatively easy to treat (as opposed to more complicated drug-resistant TB). The article is generally well-written and provides important specifics to help readers understand what was done, the context, and what conclusions can be fairly drawn from the data. It is good that the authors highlight the importance of ART initiation and earlier ART, ambulatory care, screening for other known correlates of poor outcomes (DM, smoking, etc.), as well as employing validated screening tools for potential risk behaviors (alcohol and drug use)\nThere are a few areas that could be improved:\nI was surprised that a major recommendation was not to conduct more active case finding. The article mentions briefly that there is some active case finding in this setting, but it is not analyzed that 82% of cases were identified through passive case finding. While not identified as a risk factor for poor DS-TB per this analysis, it could be just that there were so few cases for active case finding that the N in the comparison was too small for significance. The fact that the vast majority of cases are found through passive case finding indicates a weak system for active finding, which leads to late detection, higher probability of transmission to more people, and greater chance of extensive disease (which could contribute to poor outcomes).\n\nRelatedly, cavitation (which is a marker of extent of disease) was not explored as a risk factor for poor outcomes and could likely be easily included as part of a baseline analysis of risk if Ukraine is already routinely performing radiography on all patients.\n\nAside from individual patient risk factors, the fact that 11% of cases are missing data and the high rate of loss to follow up indicate need for patient support across the board to ensure patients are able to stay in care through cure.\n\nThe authors do not describe what kind of interventions could help improve outcomes for those at high risk, despite a large body of evidence on useful elements of patient support.\n\nAvoid the use of stigmatizing language, in line with recommended language: http://www.stoptb.org/assets/documents/resources/publications/acsm/LanguageGuide_ForWeb20131110.pdf. For example, use \"lost to follow up\" as opposed to \"defaulted\"; \"persons in need of evaluation for TB\" instead of \"suspects\" and define what is considered \"alcohol abuse\" and consider using a less stigmatizing term (which is indeed avoided in certain parts of the document) such as \"frequent alcohol use\" or \"high alcohol consumption\").\n\nWhile not the main focus of the article, the authors refer to but do not make notes about several areas where the UNTP guidance is inappropriate; it is worth calling these out as well. For example:\n\nThe authors note that routine DST is done for all culture positive cases but do not specify using what method-- liquid or solid culture phenotypic testing? Are line probe assays available? The availability (or lack thereof) of rapid methods of DST (in addition to Xpert, which only identifies rifampicin resistance) could be another recommendation that could help improve treatment outcomes by identifying need for regimen changes earlier on.\n\nChest radiography is done for high risk groups annually --this is potentially a lot of exposure to radiation—why not use symptom screen and Xpert?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3487", "date": "13 Mar 2018", "name": "Omowunmi Aibana", "role": "Author Response", "response": "Comment 1: I was surprised that a major recommendation was not to conduct more active case finding. The article mentions briefly that there is some active case finding in this setting, but it is not analyzed that 82% of cases were identified through passive case finding. While not identified as a risk factor for poor DS-TB per this analysis, it could be just that there were so few cases for active case finding that the N in the comparison was too small for significance. The fact that the vast majority of cases are found through passive case finding indicates a weak system for active finding, which leads to late detection, higher probability of transmission to more people, and greater chance of extensive disease (which could contribute to poor outcomes).Response 1: We have added the following to our discussion (paragraph 4):“We found that majority of TB patients were identified through passive case finding, which may also contribute to poor treatment outcomes. The WHO recommends systematic screening for active TB as a complement to passive case finding [1]. Studies have also shown that active TB case finding results in early detection and reduces risk of extensive disease at diagnosis [2-6], which may potentially decrease risk of poor outcomes. Active case finding also reduces risk of TB transmission [6-9] and may contribute to reducing TB prevalence. Lack of active case TB finding may further result in under notification of TB in this setting. TB control efforts in Ukraine will likely benefit from strengthening and improving health systems capacity for active case finding.”1. World Health Organization. Systematic screening for active tuberculosis. Principles and Recommendations. 2013. Available: http://apps.who.int/iris/bitstream/10665/84971/1/9789241548601_eng.pdf?ua=1 Accessed 20 February 2018. 2. den Boon S, Verver S, Lombard CJ, Bateman ED, Irusen EM, Enarson DA, et al. Comparison of symptoms and treatment outcomes between actively and passively detected tuberculosis cases: the additional value of active case finding. Epidemiol Infect. 2008;136(10): 1342-1349. 3. Eang MT, Satha P, Yadav RP, Morishita F, Nishikiori N, van-Maaren P, et al. Early detection of tuberculosis through community-based active case finding in Cambodia. BMC Public Health. 2012;12: 469.4. Datiko DG, Yassin MA, Theobald SJ, Blok L, Suvanand S, Creswell J, Cuevas LE. Health extension workers improve tuberculosis case finding and treatment outcome in Ethiopia: a large-scale implementation study. BMJ Glob Health. 2017;2(4): e000390.5. Kranzer K, Afnan-Holmes H, Tomlin K, Golub JE, Shapiro AE, Schaap A, Corbett EL, Lönnroth K, Glynn JR. The benefits to communities and individuals of screening for active tuberculosis disease: a systematic review. Int J Tuberc Lung Dis. 2013 Apr;17(4):432-46.6. Cheng S, Chen W, Yang Y, Chu P, Liu X, Zhao M, et al. Effect of diagnostic and treatment delay on the risk of tuberculosis transmission in Shenzhen, China: an observational cohort study, 1993-2010. PLoS One. 2013;8(6): e67516.7. Lin X, Chongsuvivatwong V, Lin L, Geater A, Lijuan R. Dose-response relationship between treatment delay of smear-positive tuberculosis patients and intra-household transmission: a cross-sectional study. Trans R Soc Trop Med Hyg. 2008;102(8): 797-804.8. Fox GJ, Barry SE, Britton WJ, Marks GB. Contact investigation for tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2013;41(1): 140–156.9. Jia ZCS, Ma Y, Zhang Y, Bai L, Xu W, He X, et al. Tuberculosis burden in China: a high prevalence of pulmonary tuberculosis in household contacts with and without symptoms. BMC Infect Dis. 2014;14: 64. Comment 2: Relatedly, cavitation (which is a marker of extent of disease) was not explored as a risk factor for poor outcomes and could likely be easily included as part of a baseline analysis of risk if Ukraine is already routinely performing radiography on all patients.Response 2: Our analysis was based on information captured in the electronic database, which in Kyiv Oblast did not include an adequate level of detail regarding cavitary lesions. Therefore, we were unable to analyze cavitation as a predictor of poor outcomes in this study. Comment 3: Aside from individual patient risk factors, the fact that 11% of cases are missing data and the high rate of loss to follow up indicate need for patient support across the board to ensure patients are able to stay in care through cure.The authors do not describe what kind of interventions could help improve outcomes for those at high risk, despite a large body of evidence on useful elements of patient support.Response 3: We have expanded our discussion (paragraphs 2 and 5) as follows:“Studies from other settings have demonstrated that incorporating dedicated treatment for high alcohol use within TB programs is feasible [1] and access to treatment for substance use improves TB outcomes [2,3]. For instance, one study in Ukraine showed methadone treatment for TB patients with IVDU led to improved retention in care and medication adherence [3].” “Our finding that 10% of patients are lost to follow up also highlights the importance of providing patient support to all TB patients during treatment, not just individuals identified at baseline as having high risk for poor outcomes. Prior studies have shown incentives and other enablers of treatment adherence are an effective strategy for improving TB treatment outcomes [4-8]. Such enablers include use of community health workers, food and transportation assistance, reminder systems, education and counseling geared towards adherence as well as enhanced access to social services. Although Ukraine’s newest TB guidelines encourage additional social support for TB patients, currently there is limited funding dedicated to incentivizing treatment adherence.” 1. Shin S, Livchits V, Connery H S, Shields A, Yanov S, Yanova G, et al. Effectiveness of alcohol treatment interventions integrated into routine tuberculosis care in Tomsk, Russia. Addiction. 2013;108(8): 1387-1396.2. Gelmanova IY, Taran DV, Mishustin SP, Golubkov AA, Solovyova AV, Keshavjee S. 'Sputnik':a programmatic approach to improve tuberculosis treatment adherence and outcome among defaulters. Int J Tuberc Lung Dis. 2011;15(10): 1373-1379.3. Morozova, Dvoryak S, Altice FL. Methadone treatment improves tuberculosis treatment among hospitalized opioid dependent patients in Ukraine. Int J Drug Policy. 2013;24(6): e91-e98.4. Schluger N, Ciotoli C, Cohen D, Johnson H, Rom WN. Comprehensive tuberculosis control for patients at high risk for noncompliance. Am J Respir Crit Care Med. 1995;151(5): 1486-90. 5. Volmink J, Garner P. Systematic review of randomised controlled trials of strategies to promote adherence to tuberculosis treatment. BMJ. 1997; 315: 1403-1406. 6. Davidson H, Schluger NW, Feldman PH, Valentine DP, Telzak EE, Laufer F N. The effects of increasing incentives on adherence to tuberculosis directly observed therapy. Int J Tuberc Lung Dis. 2000;4: 860-865. 7. Liu Q, Abba K, Alejandria MM, Balanag VM, Berba RP, Lansang MA. Reminder systems and late patient tracers in the diagnosis and management of tuberculosis. Cochrane Database Syst Rev. 2008;4: CD006594.8. M'Imunya MJ, Kredo T, Volmink J. Patient education and counseling for promoting adherence to treatment for tuberculosis. Cochrane Database Syst Rev. 2012;5: CD006591. Comment 4: Avoid the use of stigmatizing language, in line with recommended language: http://www.stoptb.org/assets/documents/resources/publications/acsm/LanguageGuide_ForWeb20131110.pdf. For example, use \"lost to follow up\" as opposed to \"defaulted\"; \"persons in need of evaluation for TB\" instead of \"suspects\" and define what is considered \"alcohol abuse\" and consider using a less stigmatizing term (which is indeed avoided in certain parts of the document) such as \"frequent alcohol use\" or \"high alcohol consumption\").Response 4: We have made changes throughout the manuscript to avoid stigmatizing language as suggested. We changed “default” to “lost to follow up,” replaced “alcohol abuse” with “frequent alcohol use” and now refer to TB suspects as individuals in need of evaluation for TB. We have also clarified in the description of study setting (paragraph 1) that “screening for frequent alcohol use and substance use relies on patient self-report without specific definitions about what is considered high or harmful alcohol consumption.” Comment 5: While not the main focus of the article, the authors refer to but do not make notes about several areas where the UNTP guidance is inappropriate; it is worth calling these out as well. For example:The authors note that routine DST is done for all culture positive cases but do not specify using what method-- liquid or solid culture phenotypic testing? Are line probe assays available? The availability (or lack thereof) of rapid methods of DST (in addition to Xpert, which only identifies rifampicin resistance) could be another recommendation that could help improve treatment outcomes by identifying need for regimen changes earlier on. Chest radiography is done for high risk groups annually -- this is potentially a lot of exposure to radiation—why not use symptom screen and Xpert?Response 5: We have now noted in the methods section that: “In the Kyiv Oblast laboratory, solid and liquid culture DST for first line drugs are performed using LJ medium and the M960 system.”Please note that our analysis also revealed that available technology such as DST and Xpert that are currently mandated per UNTP guidelines remain under-utilized in Kyiv Oblast. For instance, less than half of patients in this cohort had a baseline Xpert test. Therefore, introduction of additional technology such as line probe assay, with its requisite costs in materials and training, may not necessarily have an immediate and beneficial impact on treatment outcomes in this setting. Paragraph 4 in the discussion section now states: “The use of newer technologies and approaches to optimize early identification of TB patients and prompt diagnosis of resistant TB may also lead to improved treatment outcomes in this setting.”" } ] } ]	1	https://f1000research.com/articles/6-1873
https://f1000research.com/articles/8-1955/v1	22 Nov 19	{ "type": "Systematic Review", "title": "Dental stem cells in tooth repair: A systematic review", "authors": [ "Mary Sabry Tawfik Tadros", "Maha Abd-El Salam El-Baz", "Mohamed Adel Ezzat Khairy Khairy" ], "abstract": "Background: Dental stem cells (DSCs) are self-renewable teeth cells, which help maintain or develop oral tissues. These cells can differentiate into odontoblasts, adipocytes, cementoblast-like cells, osteoblasts, or chondroblasts and form dentin/pulp. This systematic review aimed to summarize the current evidence regarding the role of these cells in dental pulp regeneration. Methods: We searched the following databases: PubMed, Cochrane Library, MEDLINE, SCOPUS, ScienceDirect, and Web of Science using relevant keywords. Case reports and non-English studies were excluded. We included all studies using dental stem cells in tooth repair whether in vivo or in vitro studies. Results: Dental pulp stem cell (DPSCs) is the most common type of cell. Most stem cells are incorporated and implanted into the root canals in different scaffold forms. Some experiments combine growth factors such as TDM, BMP, and G-CSF with stem cells to improve the results. The transplant of DPSCs and stem cells from apical papilla (SCAPs) was found to be associated with pulp-like recovery, efficient revascularization, enhanced chondrogenesis, and direct vascular supply of regenerated tissue. Conclusion: The current evidence suggests that DPSCs, stem cells from human exfoliated deciduous teeth, and SCAPs are capable of providing sufficient pulp regeneration and vascularization. For the development of the dental repair field, it is important to screen for more effective stem cells, dentine releasing therapies, good biomimicry scaffolds, and good histological markers.", "keywords": [ "tooth repair", "dental stem cell", "pulp regeneration", "SCAPs", "SHEDs" ], "content": "Introduction\n\nRegenerative dentistry is designed to recover dental anatomy and function. Regenerative endodontics procedures (REPs) of a damaged tooth are a series of biological processes aimed to restore the dental pulp's physiological functions, cure periapical lesions, and substitute pulp-dentin complex cells and dentin1,2. Three components are involved in these techniques: stem cells, growth and bio-materials, which are often known as scaffolds or templates3\n\nThe dental pulp consists of nerves, blood vessels and connective tissue to maintain teeth's integrity. The nerves of the pulp can mediate pain, blood flow control, recruit immunocompetent cells, and act as a mesenchymal stem cells (MSCs) niche4. Loss of tooth pulp stops the development of permanent root teeth that can weaken the periodontal connection and lead to teeth loss. Recent animal studies indicate that vascular dental pulp can be regenerated by cell-based therapy5.\n\nDental stem cells (DSCs) are self-renewable teeth cells, which help maintain or develop oral tissues6. In the literature, there are various types of dental adult stem cells, such as dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells or stem cells of the developing root apical papilla (SCAPs), dental follicle stem cells (DFSCs), and dental MSCs (DMSCs)7,8. Such cells can differentiate into dentine/pulp, odontoblast, adipocyte, cementblast-like, osteoblast, and chondroblast cells. DSC regenerative potential is explained through both natural and experimental conditions9,10. Differentiated dentinoblasts, also called secondary odontoblasts, produce new dentine in response to dental cell injury. This regenerative process is called reparative tertiary dentinogenesis11. This process of dentinogenesis was suggested to be used in the recruitment of endogenous DSCs. Most recent animal studies have investigated the role of DSCs in the regeneration of dental pulp tissues.\n\nBakhtiar et al.12 conducted a systematic review on 47 studies that investigate the role of stem cell therapy in regeneration of dentine‑pulp complex; the current systematic review aimed to update this previous systematic review, presenting 57 articles, and summarizes the current evidence regarding the efficacy of dental stem cells in dental pulp regeneration in animal models.\n\n\nMethods\n\nWe report this manuscript following the preferred reporting items systematic reviews and meta-analysis (PRISMA statement) guideline13. All methods used in this review were conducted in strict accordance with the Cochrane Handbook for Systematic Reviews of Interventions14.\n\nWe searched the following databases from January 2000 to June 2019: PubMed, Cochrane Library, MEDLINE, SCOPUS, ScienceDirect, and Web of Science using the following keywords (((Dental pulp stem cells OR DPSCs OR stem cells OR human exfoliated deciduous teeth OR SHEDs OR Periodontal ligament stem cells OR developing root apical papilla OR SCAPs OR dental follicle stem cells OR DFSCs OR dental mesenchymal stem cells OR DMSCs) AND (pulp OR pulpal tissue OR pulp treatment OR pulpal therapy) AND (endodontic treatment OR deciduous teeth OR permanent teeth OR primary teeth OR dentition))) to identify the relevant studies.\n\nTwo authors screened the titles and abstracts of retrieved literature records. For titles and abstracts that deemed relevant to the research question, the full-text articles of these records were obtained and screened for eligibility according to the following criteria:\n\nWe included studies that meet the following PICOS criteria:\n\n1) Population: Both in vitro and in vivo studies that investigate the endodontic regeneration following treatment with dental pulp stem cells.\n\n2) Intervention/Comparator: studies that use all of the following types of stem cell in the regeneration of dental pulp tissue: DPSCs, SHEDs, SCAPs or DMSCs.\n\n3) Outcomes: pulpal regeneration or repair.\n\n4) Study design: All in vivo, in vitro, animal, or human studies.\n\nWe excluded all of the following studies: 1) Case reports and case series; and 2) non-English studies. In the case of multiple reports for the same study population, we analyzed data from the most updated dataset. Any discrepancies were resolved by discussion and consensus between reviewers.\n\nData extraction was performed manually and data were entered into a structured Microsoft Excel sheet (For Windows, Professional Plus version 2016). We extracted data of the following domains: 1) Characteristics of study design; 2) Baseline criteria of the included population; and 3) Study outcomes. There was not sufficient data for meta-analysis.\n\n\nResults\n\nThe electronic search retrieved 4433 unique articles. After removing duplications, 2780 articles were enrolled in the title/abstract screening. This led to the retrieval and screening of 327 full-text articles for eligibility. Studies that were not eligible with our criteria were excluded. In total 57 articles were included in the qualitative synthesis. A flow diagram of the selection process is shown in Figure 1. A summary of characteristics, models, and populations of the included studies and their key outcomes are shown in Table 1. Variation of the extracted data is reported in Table 2.\n\nThis table was adapted from a previous systematic review with written permission from the authors and under a Creative Commons Attribution 4.0 International License12.\n\nBC, blood clot; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BMSC, bone marrow mesenchyme stem cell; CC, costal chondrocyte; CDHA, calcium carbonate hydroxyapatite; CNCC, cranial neural crest cell; CXCL14, chemokine (CXC motif) ligand-14; DFSC, dental follicle stem cell; DPSC, dental pulp stem cell; FGF, fibroblast growth factor; GCSF, granulocyte colony-stimulating factor; GF, growth factors; HA, hydroxyapatite; iPS, induced pluripotent stem cell; MCP1, monocyte chemoattractant protein-1; MSC, mesenchymal stem cell; MTA, mineral trioxide aggregate; NA, not exogenously added; ND, not determined; NGF, nerve growth factor; PCL, polycaprolactone; PGA, polyglycolic acid; PGDF, platelet-derived growth factor; PLGA, polylactic-co-glycolic acid; PLLA, poly-L-lactic acid; PRF, platelet-rich fibrin; PRP, platelet-rich plasma; REP, regenerative endodontic procedure; SCAP, stem cell of the apical papilla; SHED, stem cell from human exfoliated deciduous teeth; TCP, tricalcium phosphate; TDM, treated dentin matrix; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; DBCs, dental bud cells\n\n*The number of included studies is 57 studies; however, the number of trials is 67 trials. Therefore, each study may include one or more types of cells.\n\nBC, blood clot; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BMSC, bone marrow mesenchyme stem cell; CC, costal chondrocyte; CDHA, calcium carbonate hydroxyapatite; CNCC, cranial neural crest cell; CXCL14, chemokine (CXC motif) ligand-14; DFSC, dental follicle stem cell; DPSC, dental pulp stem cell; FGF, fibroblast growth factor; GCSF, granulocyte colony-stimulating factor; GF, growth factors; HA, hydroxyapatite; iPS, induced pluripotent stem cell; MCP1, monocyte chemoattractant protein-1; MSC, mesenchymal stem cell; MTA, mineral trioxide aggregate; NA, not exogenously added; ND, not determined; NGF, nerve growth factor; PCL, polycaprolactone; PGA, polyglycolic acid; PGDF, platelet-derived growth factor; PLGA, polylactic-co-glycolic acid; PLLA, poly-L-lactic acid; PRF, platelet-rich fibrin; PRP, platelet-rich plasma; REP, regenerative endodontic procedure; SCAP, stem cell of the apical papilla; SHED, stem cell from human exfoliated deciduous teeth; TCP, tricalcium phosphate; TDM, treated dentin matrix; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.\n\nIn this systematic review, we reviewed multiple types of stem cells, such as dental follicle stem cell (DFSC), bone marrow mesenchyme stem cell (BMSC), periodontal ligament stem cell (PDLSC), dental pulp extracellular matrix (DPEM), adipose-derived stem cell (ADSC), DPSC, SCAP, and SHED. The majority of the studies (n=31; 46%) used DPSCs for regeneration of dentine-pulp complexes15–45. Two out of eight studies, in which stem cells were transplanted into the renal capsule, used DPSCs15,16. Moreover, DPSCs were used by 19 out of 40 studies on subcutaneous transplantation17–21,27,31–34,37–45 , three out of four studies on intra-canal transplantation25,30,35, and two studies about allogeneic direct implantation into socket22,24.\n\nSCAP was reported in eight studies15,46–52, six studies with subcutaneous implantation46–48,50–52 and two studies on renal capsule transplantation15,49. There are no experiments utilizing transplanted SCAP into root canal. DFSC is reported in six studies42,53–57; five subcutaneous implantation studies42,54–57 and one into-socket transplantation53. Across four experiments that were all on subcutaneous transplants used SHEDs43,58–60. Four experiments used BMSCs; two were transplanted to the renal capsule61,62, one to the subcutaneous63, and the other to the root canal64. Three trials tried to regenerate periodontal ligament (PDL) tissue using PDLSCs; two subcutaneously transplanted41,55 and one transplanted into a socket for extraction22.\n\nAll included experiments utilizing DPSCs were isolated from human healthy pulp tissues, usually orthodontics, to be used in an animal model. Stem cells from exposed pulp have also been reported to be more likely to differentiate into osteoblastic cells than dentinogenic ones. In this review, 20 articles used DPSCs in mice models16–21,27,31–34,37–45, three in rat models15,25,36, four in dogs26,28–30, and three in pigs15,22,23. It was observed that DPSC transplantation was associated with regeneration of pulp-like tissue22,29,30,32, successful revascularization36,38, enhanced chondrogenesis31,37, and tissue regeneration with direct vascular supply. However, two studies reported that DPSCs formed an inflamed pulp-like tissue15,20.\n\nSCAPs were commonly isolated from immature third molars. Wang et al.15 reported that SCAPs have greater generation of mineralized tissue than those with DPSCs and higher differentiation of osteo/odontoblast in the supplemental medium khpo4. Furthermore, SCAPs have been reported to have re-vascularizing properties, heterotopic dental pulp/dentin complex formation, faster proliferation and mineralization, and more efficient migration and telomerase than DPSCs46,65.\n\nPDLSCs demonstrated a significant role in maintenance of MSC characteristics after implantation41. In addition, the dentin tissue structure produced by dental follicle cell (DFC) was more complete. In the included experiments, Gao et al.22 used PDL for regeneration of a fresh bio-root. They developed an effective bio-root of PDL tissue, using a mixture of DPSC-hydroxyapatite wrapped in a layer of PDLSCs. These freshly produced miniature pig roots, both in mineral components and biomechanical characteristics, had comparable characteristics to natural teeth, but only 20% of the samples attained success, whilst titanium implants were 100% effective.\n\nMurakami et al.64 showed that BMSCs generated a potential pulp, but with less volume. Zhang et al.63 proposed applying endogenous BM-MSC to a subcutaneous root canal tooth to a regenerative tissue after the systemic homing in the root canal, driven by the use of the stromal cell-derived factor-1 (SDF-1). The use of dentine-matrix-scaffolding cells was associated to the differentiation of the stem cells in a dentinal tubule in polarized odontoblast-like cells62.\n\nSHEDs, which rare derived from extracted deciduous teeth, were used in mice models in four studies43,58–60. It was observed that the capability of mineralization of SHEDs was higher than DPSCs43. Casagrande et al.60 reported that SHEDs express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE).\n\n\nDiscussion\n\nThis is the largest and most updated systematic review aiming to investigate the role of DSCs in tooth repair. We found that multiple DSCs have a potent role in tissue regeneration and vascularization of dental pulp-like tissues54,56,58,61,66,70,72. Most of the included research assessed the 4–8 week dentine-pulp regeneration following transplant57,67,69,70. These studies used the ectopic models of dentine pulp-complex. A few studies used long-term evaluation of up to 36 weeks after surgery71. However, there are some studies that had multiple time points for evaluation.\n\nLike other tissues, three primary components are required to regenerate a necrotic pulp: 1) vital root canal cells, which can distinguish into normal pulp cells, 2) morphogenic and growth factors to activate and encourage cell distinction, and 3) a matrix that ensures an environment that maintains their vitality and growth and supports cells in a mechanical way58,59,61,62.\n\nGrowth factors, drugs, bioactive products, glycosaminoglycans and other small molecules and motifs of peptide are considered promotive healing factors that can be used for stem cells and matrix to improve the effectiveness of stem cell therapy for dentine-pulp regeneration and biodegradation. Growth factors have a short half-life; therefore, degradable materials are required to control their release47,48,73.\n\nRecent studies for dentine pulp regeneration have been done in various types of stem cells from various sources in body. DPSCs are the preferred cells in the majority of these studies and have demonstrated their capacity to regenerate the complex dentine15,22,25,46,65. Although the great tendency for dentine-pulp complex regeneration, SCAP and SHED were rarely administered44,45. DPSCs and SHEDs were evaluated with adequate or partly successful histological outcomes in various REP research. DPSC, collagen or polylactic/glycol and scarce factors with or without growth factors are optimized when compared to REPs with growth factors but without amplified stem cells19,20. Several dentin therapies demonstrate further excellent outcomes, which should be followed by platelet-rich plasma/platelet-rich fibrin (PRP/PRF) or collagen gels in REPs and improved biomimicry to maintain various levels of the variables that release oral stem cell niche formation. Recently, cell survival of stem cells is much easier than in the past due to the appropriate interaction with dentine-released factors21,23. Thus, screening for more appropriate stem cells, dentine releasing treatments, scaffolds with good biomimicry and good histological markers is an exciting activity for future REP improvements.\n\nBesides dental sources, non-dental cells, such as the MSCs derived from bone marrow and adipose stem cells, are able to regenerate the pulp tissue.\n\nGenerally, our study showed that adult stem cells appear to be able to regenerate dentine-pulp complexes; therefore, the criteria of selection should be considered the most cost-effective and cheapest, particularly when the main obstacle is the expense28,32,35. Moreover, our findings demonstrated that the human body is a wealthy source of stem cells; therefore, the third molars or any orthodontic tooth originated from a human body are excellent sources of stem cells. As regards cells circulating, these cells migrate to sites and engage in a recovery process in the presence of chemotactic gradients, as their capacity for root canal migration was shown33.\n\nThis study showed two limitations; 1) we could not conduct a meta-analysis due to insufficient data; and 2) we failed to find a suitable tool to assess the quality of included studies and risk of bias.\n\nIn conclusion, the current evidence suggests that the DPSCs, SHEDs, and SCAPs are capable of providing a sufficient pulp regeneration and vascularization. Nevertheless, the efficacy of stem cell transplantation in therapy locations and their cost may be obstacles to their clinical use. Scaffolds and biomaterials provide a useful strategy for stronger incorporation of stem cells and development factors together with monitored regeneration rates. Hence, we suggest future studies to concentrate on offering definite guidance on appropriate and preferable biomaterial characteristics for use in regenerative endodontics.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Dental stem cells in tooth repair: A systematic review’, https://doi.org/10.6084/m9.figshare.10315835.v174.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAngelova Volponi A, Zaugg LK, Neves V, et al.: Tooth Repair and Regeneration. Curr Oral Health Rep. 2018; 5(4): 295–303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJung C, Kim S, Sun T, et al.: Pulp-dentin regeneration: current approaches and challenges. J Tissue Eng. 2019; 10: 204173141881926. 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Biomaterials. 2013; 34(8): 1888–97. PubMed Abstract \| Publisher Full Text\n\nIohara K, Imabayashi K, Ishizaka R, et al.: Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue Eng Part A. 2011; 17(15–16): 1911–20. PubMed Abstract \| Publisher Full Text\n\nSrisuwan T, Tilkorn DJ, Al-Benna S, et al.: Revascularization and tissue regeneration of an empty root canal space is enhanced by a direct blood supply and stem cells. Dent Traumatol. 2013; 29(2): 84–91. PubMed Abstract \| Publisher Full Text\n\nSchmalz G, Widbiller M, Galler KM: Material Tissue Interaction--From Toxicity to Tissue Regeneration. Oper Dent. 2016; 41(2): 117–31. PubMed Abstract \| Publisher Full Text\n\nDissanayaka WL, Zhu L, Hargreaves KM, et al.: Scaffold-free Prevascularized Microtissue Spheroids for Pulp Regeneration. J Dent Res. 2014; 93(12): 1296–303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurakami M, Horibe H, Iohara K, et al.: The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential. Biomaterials. 2013; 34(36): 9036–47. PubMed Abstract \| Publisher Full Text\n\nWang X, He H, Wu X, et al.: Promotion of dentin regeneration via CCN3 modulation on Notch and BMP signaling pathways. Biomaterials. 2014; 35(9): 2720–9. PubMed Abstract \| Publisher Full Text\n\nLei M, Li K, Li B, et al.: Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation. Biomaterials. 2014; 35(24): 6332–43. PubMed Abstract \| Publisher Full Text\n\nYang X, Han G, Pang X, et al.: Chitosan/collagen scaffold containing bone morphogenetic protein-7 DNA supports dental pulp stem cell differentiation in vitro and in vivo. J Biomed Mater Res Part A. 2012. PubMed Abstract \| Publisher Full Text\n\nWang X, Sha XJ, Li GH, et al.: Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol. 2012; 57(9): 1231–40. PubMed Abstract \| Publisher Full Text\n\nChen B, Sun HH, Wang HG, et al.: The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars. Biomaterials. 2012; 33(20): 5023–35. PubMed Abstract \| Publisher Full Text\n\nWang YX, Ma ZF, Huo N, et al.: Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells. J Tissue Eng Regen Med. 2011; 5(5): 354–62. PubMed Abstract \| Publisher Full Text\n\nHuang GT, Yamaza T, Shea LD, et al.: Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A. 2010; 16(2): 605–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang W, Dang M, Zhang Z, et al.: Dentin regeneration by stem cells of apical papilla on injectable nanofibrous microspheres and stimulated by controlled BMP-2 release. Acta Biomater. 2016; 36: 63–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J, Wang Z, Jiang Y, et al.: Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro. Exp Cell Res. 2015; 332(2): 259–66. PubMed Abstract \| Publisher Full Text\n\nYan M, Wu J, Yu Y, et al.: Mineral trioxide aggregate promotes the odonto/Osteogenic differentiation and dentinogenesis of stem cells from apical papilla via nuclear factor kappa B signaling pathway. J Endod. 2014; 40(5): 640–7. PubMed Abstract \| Publisher Full Text\n\nYadlapati M, Biguetti C, Cavalla F, et al.: Characterization of a Vascular Endothelial Growth Factor-loaded Bioresorbable Delivery System for Pulp Regeneration. J Endod. 2017; 43(1): 77–83. PubMed Abstract \| Publisher Full Text\n\nJin B, Choung PH: Recombinant Human Plasminogen Activator Inhibitor-1 Accelerates Odontoblastic Differentiation of Human Stem Cells from Apical Papilla. Tissue Eng Part A. 2016; 22(9–10): 721–32. PubMed Abstract \| Publisher Full Text\n\nHilkens P, Bronckaers A, Ratajczak J, et al.: The Angiogenic Potential of DPSCs and SCAPs in an In Vivo Model of Dental Pulp Regeneration. Stem Cells Int. 2017; 2017: 2582080. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen G, Chen J, Yang B, et al.: Combination of aligned PLGA/Gelatin electrospun sheets, native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root regeneration. Biomaterials. 2015; 52: 56–70. PubMed Abstract \| Publisher Full Text\n\nChen G, Sun Q, Xie L, et al.: Comparison of the Odontogenic Differentiation Potential of Dental Follicle, Dental Papilla, and Cranial Neural Crest Cells. J Endod. 2015; 41(7): 1091–9. PubMed Abstract \| Publisher Full Text\n\nTian Y, Bai D, Guo W, et al.: Comparison of human dental follicle cells and human periodontal ligament cells for dentin tissue regeneration. Regen Med. 2015; 10(4): 461–79. PubMed Abstract \| Publisher Full Text\n\nJiao L, Xie L, Yang B, et al.: Cryopreserved dentin matrix as a scaffold material for dentin-pulp tissue regeneration. Biomaterials. 2014; 35(18): 4929–39. PubMed Abstract \| Publisher Full Text\n\nGuo L, Li J, Qiao X, et al.: Comparison of odontogenic differentiation of human dental follicle cells and human dental papilla cells. PLoS One. 2013; 8(4): e62332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa L, Makino Y, Yamaza H, et al.: Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem cell resource for regenerative medicine. Câmara NOS, editor. PLoS One. 2012; 7(12): e51777. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeon M, Song JS, Choi BJ, et al.: In vitro and in vivo characteristics of stem cells from human exfoliated deciduous teeth obtained by enzymatic disaggregation and outgrowth. Arch Oral Biol. 2014; 59(10): 1013–23. PubMed Abstract \| Publisher Full Text\n\nCasagrande L, Demarco FF, Zhang Z, et al.: Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res. 2010; 89(6): 603–8. PubMed Abstract \| Publisher Full Text\n\nHashmi B, Mammoto T, Weaver J, et al.: Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds. Stem Cell Res. 2017; 24: 55–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLei G, Yu Y, Jiang Y, et al.: Differentiation of BMMSCs into odontoblast-like cells induced by natural dentine matrix. Arch Oral Biol. 2013; 58(7): 862–70. PubMed Abstract \| Publisher Full Text\n\nZhang LX, Shen LL, Ge SH, et al.: Systemic BMSC homing in the regeneration of pulp-like tissue and the enhancing effect of stromal cell-derived factor-1 on BMSC homing. Int J Clin Exp Pathol. 2015; 8(9): 10261–71. PubMed Abstract \| Free Full Text\n\nMurakami M, Hayashi Y, Iohara K, et al.: Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells from Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration. Cell Transplant. 2015; 24(9): 1753–65. PubMed Abstract \| Publisher Full Text\n\nNa S, Zhang H, Huang F, et al.: Regeneration of dental pulp/dentine complex with a three-dimensional and scaffold-free stem-cell sheet-derived pellet. J Tissue Eng Regen Med. 2016; 10(3): 261–70. PubMed Abstract \| Publisher Full Text\n\nLei G, Yan M, Wang Z, et al.: Dentinogenic capacity: immature root papilla stem cells versus mature root pulp stem cells. Biol Cell. 2011; 103(4): 185–96. PubMed Abstract \| Publisher Full Text\n\nJiang N, Zhou J, Chen M, et al.: Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis. Biomaterials. 2014; 35(7): 2172–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Yu Y, Chen L, et al.: Human Umbilical Cord Mesenchymal Stem Cells: A New Therapeutic Option for Tooth Regeneration. Stem Cells Int. 2015; 2015: 549432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPan S, Dangaria S, Gopinathan G, et al.: SCF promotes dental pulp progenitor migration, neovascularization, and collagen remodeling - potential applications as a homing factor in dental pulp regeneration. Stem Cell Rev Reports. 2013; 9(5): 655–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuo N, Tang L, Yang Z, et al.: Differentiation of Dermal Multipotent Cells Into Odontogenic Lineage Induced by Embryonic and Neonatal Tooth Germ Cell–Conditioned Medium. Stem Cells Dev. 2010; 19(1): 93–104. PubMed Abstract \| Publisher Full Text\n\nKuo TF, Huang AT, Chang HH, et al.: Regeneration of dentin-pulp complex with cementum and periodontal ligament formation using dental bud cells in gelatin-chondroitin-hyaluronan tri-copolymer scaffold in swine. J Biomed Mater Res Part A. 2008; 86(4): 1062–8. PubMed Abstract \| Publisher Full Text\n\nYang J, Zhang Y, Sun Z, et al.: Dental pulp tissue engineering with bFGF-incorporated silk fibroin scaffolds. J Biomater Appl. 2015; 30(2): 221–9. PubMed Abstract \| Publisher Full Text\n\nEbada MA, Alkanj S, Ebada M, et al.: Safety and Efficacy of Levetiracetam for the Management of Levodopa- Induced Dyskinesia in Patients with Parkinson's Disease: A Systematic Review. CNS Neurol Disord Drug Targets. 2019; 18(4): 317–325. PubMed Abstract \| Publisher Full Text\n\nTadros MST: PRISMA checklist for ‘Dental stem cells in tooth repair A systematic review’. pdf. figshare. Online resource. http://www.doi.org/10.6084/m9.figshare.10315835.v1" }	[ { "id": "56928", "date": "21 Jan 2020", "name": "Sesha Hanson-Drury, D.D.S", "expertise": [ "Reviewer Expertise stem cells and regeneration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript “Dental stem cells in tooth repair: A systematic review” is a review by Tadros et al. on a very timely topic, tooth regeneration. However, this paper does not read like a review but rather as a catalogue of papers. This review requires conceptual work towards synthesizing the great findings in the field. For example, many notable, recent papers are excluded from this review. In addition, the following comments need to be addressed.\nProvide citations for every statement (e.g. “Loss of tooth pulp stops the development of permanent root teeth that can weaken the periodontal connection and lead to teeth loss” Page 3, Paragraph 2.)\n\nSpelling and grammar review. Multiple incidences of unclear sentences (e.g. “Although the great tendency for dentine-pulp complex regeneration, SCAP and SHED were rarely administered.” Page 10, Paragraph 5) or incorrect word used (“SHEDs, which rare derived from extracted deciduous teeth” Page 10, Paragraph 1).\n\nDevelop appropriate tool to assess the quality of included studies and risk of bias such as that described in Cochrane Handbook for Systematic Reviews of Interventions.\nHow titles and abstracts were “deemed relevant” to the research question. Why were studies excluded/removed? What databases/sources were used? What search terms were used? Studies included:\nReviews are not listed though there are reviews included. Humans are not listed in populations studied though human studies are cited (e.g. “All included experiments utilizing DPSCs were isolated from human healthy pulp tissues…” Page 4, Paragraph 3).\n\nWhy there was insufficient data for meta-analysis. Meaning of “usually orthodontics” Page 4, Paragraph 3. Does this mean teeth were extracted due to orthodontic treatment plans? “Fresh bio-root” and “success” when discussing PDLSCs and “potential pulp” on Page 4, Paragraph 4 and 5. TERM approach. “Scarce factors” and “excellent outcomes” discussed on Page 10, Paragraph 5.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "58864", "date": "23 Jun 2020", "name": "Weidong Tian", "expertise": [ "Reviewer Expertise Dental regeneration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this review, the authors summarized recent studies of dental pulp regeneration using stem cells. In general, it is a timely and detailed review of stem cells used in dental regeneration. But this manuscript needs to be carefully modified.\n\nComments:\nIn the abstract and Table 2, the authors described “TDM (treated dentin matrix)” as a growth factor. In fact, TDM is a matrix containing growth factors. “Growth factors derived from TDM” is more suitable.\n\nThe language of this manuscript should be checked and polished by a native English speaker.\n\nAccording to the methods, the authors searched the databases from January 2000 to June 2019: Since numbers of studies of dental pulp regeneration have published after June 2019, it is recommended for the authors to update their data to June 2020.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1955
https://f1000research.com/articles/8-176/v1	11 Feb 19	{ "type": "Clinical Practice Article", "title": "Allopurinol reverses mercaptopurine-induced hypoglycemia in patients with acute lymphoblastic leukemia", "authors": [ "Melissa Zhang", "Bruce Bostrom", "Melissa Zhang" ], "abstract": "Fasting hypoglycemia is a known complication of mercaptopurine (6MP) maintenance therapy for acute lymphoblastic leukemia (ALL). It is associated with high levels of the methylated metabolite 6-methyl-mercaptopurine (6MMP). Symptoms of hypoglycemia include morning tremulousness, nausea and vomiting. We have previously shown that switching 6MP dosing from evening to morning resolved hypoglycemia by reducing 6MMP; however, the reduction of 6MMP was only transient, potentially resulting in return of hypoglycemia. In children and adults with Crohn’s disease, co-prescribing allopurinol with 6MP blocks the activity of thiopurine methytransferase (TPMT), reducing 6MMP and improving its tolerance. As a consequence of inhibiting TPMT, 6MP is shunted toward the production of 6-thioguanine nucleotide (6TGN), which will result in pancytopenia if the dose of 6MP is not reduced. We demonstrate that allopurinol with a reduced dose of 6MP in two patients with ALL and 6MMP-associated hypoglycemia resulted in a complete and sustained suppression of 6MMP and rapid reversal of hypoglycemia and its symptoms.", "keywords": [ "mercaptopurine", "allopurinol", "hypoglycemia", "morning nausea", "thiopurine methyltransferase" ], "content": "Introduction\n\nMercaptopurine (6MP) maintenance therapy is critical for the cure of ALL. There is no acceptable alternative. In general, 6MP is well tolerated with minimal side-effects such as facial or generalized rash and asymptomatic elevations of hepatic transaminases. Occasionally, more serious side effects, such as direct hyperbilirubinemia, pancreatitis, and fasting hypoglycemia, may occur, requiring discontinuation or reduction in the dose of 6MP1.\n\nPreviously, we had shown that 6MP induced fasting hypoglycemia is related to elevated levels of red cell 6-methyl-mercaptopurine (6MMP)2. We also showed that altering the administration time of 6MP from evening to morning or splitting the dose to twice a day results in lower 6MMP concentrations and resolution of hypoglycemia symptoms. However, in some of the patients there was a rebound in 6MMP, which may result in recurrence of symptomatic hypoglycemia.\n\nAllopurinol has been used by gastroenterologists for many years in patients with inflammatory bowel disease who have elevations of alanine aminotransferase (ALT) or gastrointestinal symptoms from the use of 6MP or azathioprine3–6. Recently, pediatric patients with ALL have been treated with allopurinol to reduce elevated 6MMP levels resulting in pancreatitis, hepatotoxicity, or inability to get absolute neutrophil count in target range despite increasing the dose of 6MP7–9.\n\n\nCase series\n\nThe electronic medical records of two children with ALL who developed symptomatic hypoglycemia on maintenance therapy were reviewed. After an extensive risk-benefit discussion with parents, they were started on allopurinol with a reduced dose of 6MP.\n\nThiopurine metabolites were measured with a CLIA-approved test (www.prometheuslabs.com). The reference values for this assay only apply to patients with inflammatory bowel disease on azathioprine or 6MP and not for ALL patients on 6MP. Unpublished data on 200 patients with ALL from day 85 of the first maintenance cycle on Children’s Oncology Group COG1922 demonstrated the 5th, 50th, and 95th percentiles for 6MMP are 320, 4900, and 19,000 pmol/ 8×108 RBC, respectively. The 5th, 50th, and 95th percentiles for 6TGN are 75, 260, and 690 pmol/ 8×108 RBC, respectively10. This two-patient case report was reviewed by Children’s Minnesota Institutional Review Board and deemed not research allowing publication.\n\nPatient UPN 1 is an African-American girl who was diagnosed with B-lineage ALL at age 10 years. She was enrolled on high-risk protocol Children’s Oncology Group (COG) AALL1131 (ClinicalTrials.gov Identifier: NCT02883049), but was taken off protocol after induction due to desire to use triple intrathecal therapy for blasts in diagnostic cytospin (CNS-2 status). Germline testing for methylene tetrahydrofolate reductase (MTHFR C667T) and thiopurine methyltransferase (TPMT) were homozygous normal.\n\nMaintenance therapy doses were started at 6MP (62 mg/m2/day) and MTX (15 mg/m2/week). On day 57 of maintenance, the dose of 6MP was increased to 75 mg/m2/day with no change in the methotrexate (MTX) dose. On day 73, she presented to the emergency department with shaking. Upon questioning she disclosed having episodes of morning shaking, nausea and vomiting for about a month. Serum glucose was 3.18 mmol/L (53 mg/dL). The hemoglobin A1C level was 4.9%. Thiopurine metabolites showed an extremely elevated 6MMP level of 41,000 pmol/8×108 RBC and 6TGN level of 456 pmol/ 8×108 RBC. She was neutropenic with absolute neutrophil count (ANC) of 0.462 × 109 cells/L so oral chemotherapy was halted.\n\nOn day 98, 6MP was restarted at 30 mg/m2/day along with 50 mg of allopurinol given with each dose of 6MP. MTX was also restarted at the previous dose. The episodes of morning nausea, vomiting, and shakes resolved. No further episodes of hypoglycemia were seen. On day 142, the ANC was 0.29 ×109 cells/L, so oral chemotherapy was halted. Subsequently the hemoglobin level fell to 57 g/L and platelets to 82,000/µl. On day 163, 6MP was restarted at 15 mg/m2/day with 50 mg allopurinol and the previous MTX dose, which continued to the end of therapy without interruption. MTX dose remained unchanged at 75 mg/m2/week and 6MP was increased to 18 mg/m2/day to keep ANC within the target range (0.5-2 x108/L). The patient remains in remission 24 months off therapy. Figure 1 contains details of oral chemotherapy doses and laboratory values.\n\nOn the horizontal axis is the day of maintenance therapy from the start to completion. On the vertical axis are the red cell thiopurine metabolite values and drug doses. Interruption in drug doses is noted by a break in the line. For graph clarity, 6-methyl-mercaptopurine (6MMP) values were divided by 100 and 6-thioguanine nucleotide (6TGN) values by 10. After introduction of allopurinol, the 6MMP levels rapidly fell to undetectable levels with stable 6TGN. Following the initial introduction of allopurinol in patient 1 the mercaptopurine (6MP) and methotrexate (MTX) doses required interruption due to neutropenia which did not recur with a dose reduction of 6MP. As expected the myelosuppression was associated with a very elevated 6TGN level.\n\nPatient UPN 2 is a Caucasian girl diagnosed with B-lineage ALL at 3 years of age. Genotyping for TPMT was normal and MTHFR C677T was heterozygous. She was enrolled on the standard risk protocol COG AALL0932 (ClinicalTrials.gov Identifier: NCT01190930) and removed from the protocol when allopurinol was started.\n\nAround day 124 of maintenance, she had problems with morning vomiting daily. She had been on full dose 6MP (75 mg/m2/day) and MTX (20 mg/m2/week) since the start of maintenance with no interruptions. On day 229, she was diagnosed steroid-induced hyperglycemia with rebound hypoglycemia. Hemoglobin A1C was normal. Home glucose monitoring was started. Glucose levels were noted to be elevated after completion of a 5-day dexamethasone pulse. Metformin 500 mg extended release every morning was started with a subsequent dexamethasone pulse on day 255, with the resolution of steroid induced hyperglycemia. However symptomatic hypoglycemia continued.\n\nThiopurine metabolite levels were drawn, which showed an extremely high 6MMP level (32,718 pmol/8×108 RBC) with a 6TGN level of 182 pmol/8×108 RBC. She then was switched to morning dosing of 6MP, based on prior publication2. She continued to have symptoms of morning hypoglycemia, which was confirmed on five low serum glucose values over a 40-day period (values of 46, 44, 42, 37, and 36 mg/dL = 2.8, 2.7, 2.6, 2.5, 2.2, and 2.2 mmol/L).\n\nAfter extensive discussion with the parents concerning the risks and benefits of the treatment, she was taken off COG 0932 protocol per physician preference and started on allopurinol 50 mg daily with reduced dose 6MP (12 mg/m2/day) and MTX (11 mg/m2/day) on day 316 of maintenance. Within 2 weeks she had no hypoglycemia symptoms and no low glucose values on home testing. On day 392 the doses were increased to 6MP (20 mg/m2/day and MTX (17 mg/m2/week) to keep ANC in the target range (0.5-2 × 108/L). Metformin was continued for 5 months during dexamethasone pulses. Metformin was omitted the last 5 months of maintenance, without rebound hyperglycemia, which was completed on day 547. She remains in remission 24 months off therapy. Figure 2 contains details of oral chemotherapy doses and laboratory values.\n\nOn the horizontal axis is the day of maintenance therapy from the start to completion. On the vertical axis are the red cell thiopurine metabolite values and drug doses. For graph clarity, 6-methyl-mercaptopurine (6MMP) values were divided by 100 and 6-thioguanine nucleotide (6TGN) values by 10. After introduction of allopurinol, the 6MMP levels rapidly fell to undetectable levels with stable 6TGN. Of note the high glucose values during dexamethasone pulses resolved after introduction of metformin on day 255.\n\n\nDiscussion\n\n6MP is a pro-drug that is metabolized in nucleated cells to form 6TGN, which is felt to be the active anti-leukemic metabolite. Alternatively, 6MP can also undergo oxidation through a pathway catalyzed by xanthine oxidase/dehydrogenase and aldehyde oxidase to form thiouric acid, which is excreted in urine. It is also metabolized in nucleated cells by TPMT, producing 6MMP. In patients with inflammatory bowel disease, high levels of 6MMP (>5300 pmol/8×108 RBCs) have been associated with hepatotoxicity, decreased therapeutic efficacy, and symptoms of hypoglycemia11.\n\nPreviously we showed that switching 6MP from evening to morning administration reduced elevated 6MMP levels and resolved symptomatic hypoglycemia2. The mechanism of this effect is not fully known, but the most reasonable interpretation is inhibition of TPMT activity. The current study is an extension of that observation, showing that co-administration of a low dose allopurinol (50 mg) once a day with a reduced dose of 6MP (~20 mg/m2/day) also resolves 6MMP-induced symptomatic hypoglycemia without any rebound of 6MMP levels, as we saw with switching the administration time of 6MP. A reduction in the dose of 6MP is needed because on average the 6MMP to 6TGN ratio during ALL maintenance is approximately 25 to 1, per unpublished data from the COG9506 study12.\n\nApproximately 90% of people, including the patients in this study, have wild-type TPMT, the genotype responsible for high levels of TPMT activity. These patients require higher doses of 6MP to reach therapeutic levels of 6TGN. It is unknown what proportion of these patients will develop the symptoms related to elevated 6MMP8.\n\nAllopurinol was first developed by Gertrude Elion to potentiate the therapeutic index of oral 6MP for treatment of leukemia. Albeit allopurinol stimulated the anti-tumor activity of 6MP, it was associated with increased hematologic toxicity. Allopurinol use with 6MP was abandoned in the 1960s and fell into the niche of managing gout. However, our data shows success of co-prescription of allopurinol to reverse hypoglycemia in children with ALL by reducing 6MMP13.\n\nSeinen et al. demonstrated that allopurinol inhibited xanthine oxidase/dehydrogenase and increased hypoxanthine guanine phosphoribosal transferase in blood samples from patients taking 6MP who were started on allopurinol14. Notably, they did not show change in TPMT activity but did show a slight increase in 6TGN and significant decrease in 6MMP. However, Blaker et al. demonstrated inhibition of TPMT by a metabolite of allopurinol thioxanthine (2-hydroxymercaptopurine) in vitro11. Our data shows that the same mechanism of allopurinol to inhibit TPMT to treat hepatotoxicity in IBD can be applied to reverse symptoms of hypoglycemia by lowering 6MMP levels. Both patients exhibited a reduction of 6MMP to undetectable levels following co-prescription of allopurinol with 6MP.\n\nOf concern is the theoretical possibility that reducing production of 6MMP may have a negative effect on leukemia therapy. An in vitro study with MOLT-4 ALL cells showed that knocking down TPMT expression did not affect sensitivity to 6MP, and that increasing the 6MMP to 6TGN ratio in the MOLT-4 ALL cell line by adding S-adenosylmethionine (SAM) decreases cytotoxicity of 6MP15,16. These two prior studies suggest 6MMP is not an active metabolite in the treatment of leukemia. However, another in vitro study showed that transfection with TPMT gene increased sensitivity to 6MP in human CCRF-CEM cell lines, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotide17. To our knowledge, no studies on animals or humans, including currently unpublished results from the COG1922/B925 study, have demonstrated a correlation of intracellular levels of 6MMP with a decrease in ALL relapse. Thus, we are left with conflicting in vitro data and no patient data suggesting that 6MMP is necessary to cure ALL. Indeed, when prescribed by itself, allopurinol is also known to inhibit de novo purine synthesis, similar to the effect of 6MMP, suggesting it may have anti-leukemic effects. Following the submission of our manuscript the senior author reviewed for publication a two patient report further confirming our observations18. In our opinion, the benefit of preventing symptomatic 6MMP-induced hypoglycemia, and the likely reduction or omission of 6MP, outweighs the unproven theoretical possibility of interfering with ALL therapy.\n\n\nData availability\n\nDeidentified clinical values for each patient by day are available on figshare. DOI: https://doi.org/10.6084/m9.figshare.766640919.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the parents of the patients.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSchmiegelow K, Nielsen SN, Frandsen TL, et al.: Mercaptopurine/Methotrexate maintenance therapy of childhood acute lymphoblastic leukemia: clinical facts and fiction. J Pediatr Hematol Oncol. 2014; 36(7): 503–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMelachuri S, Gandrud L, Bostrom B: The association between fasting hypoglycemia and methylated mercaptopurine metabolites in children with acute lymphoblastic leukemia. Pediatr Blood Cancer. 2014; 61(6): 1003–1006. PubMed Abstract \| Publisher Full Text\n\nSparrow MP, Hande SA, Friedman S, et al.: Allopurinol safely and effectively optimizes tioguanine metabolites in inflammatory bowel disease patients not responding to azathioprine and mercaptopurine. Aliment Pharmacol Ther. 2005; 22(5): 441–446. PubMed Abstract \| Publisher Full Text\n\nSmith MA, Blaker P, Marinaki AM, et al.: Optimising outcome on thiopurines in inflammatory bowel disease by co-prescription of allopurinol. J Crohns Colitis. 2012; 6(9): 905–912. PubMed Abstract \| Publisher Full Text\n\nMin MX, Weinberg DI, McCabe RP: Allopurinol enhanced thiopurine treatment for inflammatory bowel disease: safety considerations and guidelines for use. J Clin Pharm Ther. 2014; 39(2): 107–111. PubMed Abstract \| Publisher Full Text\n\nIhekweazu FD, Kellermayer R: Allopurinol: a useful adjunct to thiopurine therapy for pediatric ulcerative colitis in the biologic era. J Pediatr Gastroenterol Nutr. 2014; 59(1): 22–24. PubMed Abstract \| Publisher Full Text\n\nZerra P, Bergsagel J, Keller FG, et al.: Maintenance Treatment With Low-Dose Mercaptopurine in Combination With Allopurinol in Children With Acute Lymphoblastic Leukemia and Mercaptopurine-Induced Pancreatitis. Pediatr Blood Cancer. 2016; 63(4): 712–715. PubMed Abstract \| Publisher Full Text\n\nGiamanco NM, Cunningham BS, Klein LS, et al.: Allopurinol Use During Maintenance Therapy for Acute Lymphoblastic Leukemia Avoids Mercaptopurine-related Hepatotoxicity. J Pediatr Hematol Oncol. 2016; 38(2): 147–151. PubMed Abstract \| Publisher Full Text\n\nBrackett J, Schafer ES, Leung DH, et al.: Use of allopurinol in children with acute lymphoblastic leukemia to reduce skewed thiopurine metabolism. Pediatr Blood Cancer. 2014; 61(6): 1114–1117. PubMed Abstract \| Publisher Full Text\n\nBostrom BC, Sensel MR, Sather HN, et al.: Dexamethasone versus prednisone and daily oral versus weekly intravenous mercaptopurine for patients with standard-risk acute lymphoblastic leukemia: a report from the Children's Cancer Group. Blood. 2003; 101(10): 3809–17. PubMed Abstract \| Publisher Full Text\n\nBlaker PA, Arenas-Hernandez M, Smith MA, et al.: Mechanism of allopurinol induced TPMT inhibition. Biochem Pharmacol. 2013; 86(4): 539–547. PubMed Abstract \| Publisher Full Text\n\nBell BA, Brockway GN, Shuster JJ, et al.: A comparison of red blood cell thiopurine metabolites in children with acute lymphoblastic leukemia who received oral mercaptopurine twice daily or once daily: a Pediatric Oncology Group study (now The Children's Oncology Group). Pediatr Blood Cancer. 2004; 43(2): 105–9. PubMed Abstract \| Publisher Full Text\n\nElion GB: Nobel lecture in physiology or medicine--1988. The purine path to chemotherapy. In Vitro Cell Dev Biol. 1989; 25(4): 321–30. PubMed Abstract\n\nSeinen ML, van Asseldonk DP, de Boer NK, et al.: The effect of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes: results from a prospective pharmacological study. J Crohns Colitis. 2013; 7(10): 812–819. PubMed Abstract \| Publisher Full Text\n\nKarim H, Ghalali A, Lafolie P, et al.: Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells. Biochem Biophys Res Commun. 2013; 437(2): 280–6. PubMed Abstract \| Publisher Full Text\n\nMilek M, Karas Kuzelicki N, Smid A, et al.: S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts. Biochem Pharmacol. 2009; 77(12): 1845–1853. PubMed Abstract \| Publisher Full Text\n\nDervieux T, Blanco JG, Krynetski EY, et al.: Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells. Cancer Res. 2001; 61(15): 5810–5816. PubMed Abstract\n\nMiller MB, Brackett J, Schafer ES, et al.: Prevention of mercaptopurine-induced hypoglycemia using allopurinol to reduce methylated thiopurine metabolites. Pediatr Blood Cancer. 2018; e27577. PubMed Abstract \| Publisher Full Text\n\nBostrom B: 6MP Allopurinol hypoglycemia patient data SPSS. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7666409.v1" }	[ { "id": "44316", "date": "20 Feb 2019", "name": "Maureen O'Brien", "expertise": [ "Reviewer Expertise Pediatric oncology", "leukemia" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide an interesting report of the use of allopurinol to decrease 6-MMP levels and ameliorate 6MP-induced hypoglycemia. Their experience in these two cases is supported by other recent case reports with similar results. This article provides good background and discussion of the mechanism, as well as guidance regarding the risks of myelosuppression with this therapy and the need for 6MP dose reduction and close monitoring.\n\nThe authors should review the therapy details closely, particularly related to dosing. For patient #1 they state that the methotrexate dose was 75mg/m2 which is clearly a typo, but draws into question if other doses provided are correct.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "44317", "date": "26 Feb 2019", "name": "Michael E. Rytting", "expertise": [ "Reviewer Expertise Pediatric leukemia and lymphoma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report on two patients with hypoglycemia and increased 6MMP provides an interesting discussion of management of this unusual but significant problem. The description of the use of allopurinol along with the biochemical rationale for using the drug is very helpful, and should be of value to pediatric hematologists. The discussion, including the possibility of altering the effectiveness of chemotherapy, is fair and balanced, as well.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-176
https://f1000research.com/articles/8-1939/v1	20 Nov 19	{ "type": "Case Report", "title": "Case Report: Intussepting mucocele appendix", "authors": [ "Tom Crawley-Smith" ], "abstract": "Background: A case study of a presentation of a mucocele appendix, a rare condition accounting for 0.2% of appendicectomies. The case and operative management are discussed along with the possible progression to pseudomyxoma peritoneii and its differing management. Case: A 15-year-old girl had two presentations with atypical Right Iliac Fossa pain over 2 months. This was investigated with ultrasound and CT which revealed a calcified, intussusepting mucocele of the appendix. This was surgically resected with partial Right Hemicolectomy. The patient was discharged on day 3 with no complications. Discussion: The presentation, malignant potential, investigation and management of the mucocele appendix are discussed. The rare presentation of a mucocele appendix necessitates care to eliminate the risk of pseudomyxoma peritoneii. The operative management should minimise disturbance of the peritoneum in this presentation. In this case, due to an intersussepting nature a limited Right Hemicolectomy had to be performed. This is compared to the literature.", "keywords": [ "Mucocele", "Appendix", "Pseudomyxoma peritoneii" ], "content": "Introduction\n\nMucinous or mucocele appendix is a rare but clinically significant presentation, presenting as 0.2–0.3% of appendicectomies1. While uncommon, it has potential to cause great morbidity if not recognised. It may be detected incidentally or in the setting of appendicitis. The landmark study by Higa et al.2 advocated describing mucocele appendix as a spectrum encompassing both benign and malignant cystadenoma, as both have potential to progress to pseudomyxoma peritoneii. In 25–50% of presentations the condition is not suspected prior to operation1,2. There may however be prior suspicion if the nature of the presenting complaint is chronic from intusseption. Classically 50%1 of cases will also present with a palpable mass in the right iliac fossa (RIF). In the case presented below, there was suspicion raised due to the chronicity of the pain, and negative markers on investigation of infection or inflammation. A ‘volcano’ sign may be seen at the appendiceal orifice at colonoscopy3.\n\nThe condition may also present late with development of pseudomyxoma peritoneii. This condition, also known as ‘jelly belly’ has a poor prognosis, with a 5-year survival of 25%4,5. It typically presents with nonspecific abdominal pain and painless increasing abdominal girth. On CT, intraperitoneal mucus is seen encasing small bowel and having the same density as ascites. There will be a calcified cyst and frequently a perforated appendix. The suspicion or operative findings of pseudomyxoma can drastically change the management and prognosis of the patient. Clinical and intraoperative suspicion allows for this potent pathology to be managed safely.\n\n\nCase\n\nA timeline of the case is shown in Table 1.\n\nThe patient, a 15-year-old-girl, had been experiencing 2 months of intermittent right iliac fossa pain. She presented to Toowoomba Hospital, a public facility in regional SE Queensland. It was an atypical presentation, with normal inflammatory markers, βHCG and white cell count. Initial ultrasonography eliminated gynaecological pathology but failed to visualise the appendix. The patient was initially observed overnight and discharged before presenting the next day with ongoing pain. A repeat ultrasound showed an ‘aparistatic’ segment of distal ileum (Figure 1). This was followed up with CT that revealed a calcified mucocele appendix which was intussepted into the caecum (Figure 2).\n\nThe patient was taken to theatre for resection (Figure 3 and Figure 4). The plan was for laparoscopic resection, with low threshold for open resection due to the risk of pseudomxyoma peritoneii. The caecum was able to be safely mobilised during the case, however due to the intussepted mucocele in situ it was not felt safe to do a limited ileocolic resection with a linear stapler without risk of spillage. The decision was made to proceed to a lap-assisted limited ileocolic resection. The specimen was safely removed and sent for histopathology (Figure 5).\n\nThe patient recovered quickly from the surgery, being discharged on day three post op. She was not given post-operative antibiotics, only prophylactic 1g cefazolin intraoperatively. She required only paracetamol and ibuprofen as required for pain relief. She was seen in the outpatient clinic at 2 weeks for wound review and check of histopathology. Her wounds were well healed and there were no malignant cells seen in the histopathology.\n\n\nDiscussion\n\nThe decision to perform a CT scan of the abdomen and pelvis facilitated early diagnosis and the opportunity to plan out the operative procedure. This avoided putting the patient at risk of rupture of the mucocele itself and inadvertent iatrogenic seeding of the peritoneum. Classically it has been advocated that a mucocele appendix be removed via an open approach to facilitate dissection of the caecum and minimise risk of rupture3,4. In this case, it was possible to mobilise the caecum to facilitate a limited Right Hemicolectomy.\n\nThe presentation of this case allowed for the early detection and operative planning of the mucocele resection. In the event of finding an unexpected mucocele appendix, there is controversy as to whether an initial right hemicolectomy should be completed. A study by Fernado et al. recommend a typlectomy if there is a base larger than 2 centimetres. If the base was less than this an appendicectomy with mesoappendiceal resection could be undertaken. Frozen section, if available, could allow the elimination and confirm whether a further colectomy would be required on table for oncological resection. If this were not available they recommend returning for further right hemicolectomy if necessary. In this case however, due to an intersussepting mucocele, it was not safe or possible to perform a typhlectomy with a linear stapler without risking spillage of mucus. The caecum was safely mobilised laparoscopically, and the specimen was safely delivered intact.\n\nIn the event of finding a ruptured mucocele with free mucus the question arises whether to perform an immediate Right Hemicolectomy or proceed to a more limited appendicectomy, with the option for more major surgery later. A study by Gonzalez-Mareno and Sugarbaker5 found that there was increased risk of seeding of the retroperitoneum with an initial Right Hemicolectomy. In their prospective study of 501 patients treated with proven peritoneal seeding requiring peritoneal cytoreductive surgery and intraperitoneal chemotherapy, it was found that the initial surgery itself was more predictive of mortality and morbidity than nodal status.\n\nThe rare nature of this presentation makes the clinical suspicion of this condition prior to operation difficult. The morbidity and mortality of pseudomxyoma peritoneii, risk of progression either in benign or malignant mucoceles and the young presenting age of this patient, emphasise a careful approach, avoiding handling of the mucocele itself and minimal disruption to the peritoneum. In this case the chronic nature of the pain on the history, lack of inflammatory signs on the bloodwork and detection on imaging facilitated this approach.\n\n\nConsent\n\nInformed written consent from the patient’s guardian was obtained for publication of this case.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nDachman AH, Lichtenstien JE, Friedman AC: Mucocele of the appendix and pseudomyxoma peritonei. AJR Am J Roentgenol. 1985; 144(5): 923–929. PubMed Abstract \| Publisher Full Text\n\nHiga E, Rosai J, Pizzimbono CA, et al.: Mucosal hyperplasia, mucinous cystadenoma, and mucinous cystadenocarcinoma of the appendix. A re-evaluation of appendiceal “mucocele”. Cancer. 1973; 32(6): 1525–41. PubMed Abstract \| Publisher Full Text\n\nFilho J, De Lira E: Mucocele of the appendix: appendectomy or colectomy? J Coloproctol (Rio J). 2011; 31(3): 276–84. Publisher Full Text\n\nForano R, Frascio M, Sticchi C, et al.: Appendectomy or right hemicolectomy in the treatment of appendiceal carcinoid tumors? Tumori. 2007; 93(6): 587–590. PubMed Abstract \| Publisher Full Text\n\nGonzález-Moreno S, Sugarbaker PH: Right hemicolectomy does not confer a survival advantage in patients with mucinous carcinoma of the appendix and peritoneal seeding. Br J Surg. 2004; 91(3): 304–311. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66543", "date": "21 Jul 2020", "name": "Antonio Manenti", "expertise": [ "Reviewer Expertise General surgery", "portal hypertension", "rectal cancer." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reported case refers to a mucocele of appendix: the subsequent hemicolectomy seems an overtreatment, mainly today when intra-operative histology is possible. Please,discuss it and explain the reason for absence of an intra-operative histological diagnosis. In this way the text could be clearer and the scientific impact of the paper greater.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5756", "date": "22 Jul 2020", "name": "Tom Crawley-Smith", "role": "Author Response", "response": "Thank you for your report. Unfortunately in the regional hospital in Queensland Australia intraoperative pathology was not readily available. It could possibly have made a difference to the operative outcome. However the reason we had to proceed was that the mucocele was intussepted into the Caecum and we did not want to risk rupturing it by reducing it as discussed." } ] }, { "id": "123538", "date": "18 Mar 2022", "name": "Bora Barut", "expertise": [ "Reviewer Expertise Liver transplantation and Emergency Surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that this article, which I refereed, will contribute to the literature. The background of the case’s history and progression is described in sufficient detail. The picture and case presentation content used is quite good. The discussion section is rich enough. The resources section is suitable for the content of the article. I think it is appropriate for the article to be indexed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1939
https://f1000research.com/articles/8-101/v1	25 Jan 19	{ "type": "Research Article", "title": "Anaemia in solitary acyanotic ventricular septal defect in comorbid with pneumonia or pulmonary hypertension: A retrospective study of 75 paediatric cases", "authors": [ "Geoffrey Joseph Changwe", "Haizhou Zhang", "Hongxin Li", "Zeeshan Farhaj", "Marlvin Anemey Tewara", "Wenlong Zhang", "Chengwei Zou", "Geoffrey Joseph Changwe", "Hongxin Li", "Zeeshan Farhaj", "Marlvin Anemey Tewara", "Wenlong Zhang", "Chengwei Zou" ], "abstract": "Background: Ventricular septal defects (VSD) are the second commonest congenital heart defects after bicuspid aortic valve. When left unrepaired, they can undergo spontaneous closure or elicit a spectrum of complications including pneumonia (PNA) or pulmonary hypertension (PH) with subsequent anaemia. In this retrospective study, we aim to establish and compare the prevalence of anaemia in patients with solitary acyanotic VSD in comorbid with PNA or PH. Methods: A total of 75 case files of patients with solitary acyanotic VSD, who underwent surgical closure or device occlusion had haemoglobin level analysed prior to the procedure. The cohort included patients with (history of) PNA and PH, and asymptomatic. The cohort included 27 females and 48 males with mean age and weight of 8.3±5.72 (3-24) months and 5.9±3.9 (2.7-17.8) kilograms, respectively. Depending on associated complication and age, the cohort was divided: PNA (A), PH (B) and Control (C); and (I) young children (≥3-6≤) and (II) older children (>6-≤24) months. We used 95 and 105 grams per litre as haemoglobin lower threshold level for (I) and (II), respectively. Results: According to data analysis 27 patients (36%) in total had anaemia. Of the anaemia cohort 16 (59.3%) had PNA, 9 (33.3%) PH and 2 (7.4%) were asymptomatic. Of the cohort, 42 were young children, with anaemia prevalence of 19/42 (45.2%), while 24.2% of the older children had anaemia. Intergroup ANOVA independent sample t-test was significant (p<0.05). In addition, intergroup Tukey HSD test for haemoglobin: A/B (p>0.05), A/C (p<0.01), B/C (p<0.01). Conclusion: Paediatric patients with acyanotic VSD in comorbid with PNA or PH are 8 and 4 times more susceptible to develop anaemia compared to asymptomatic counterparts. Susceptibility is even higher among young children (3-6months). However, a prospective study is needed to validate our findings.", "keywords": [ "Ventricular septal defect", "Pneumonia", "Pulmonary hypertension", "Anaemia" ], "content": "Introduction\n\nAnaemia is a common complication of a myriad medical conditions often met in general ward. Its aetiology is complex and multifactorial, encompassing intrinsic and extrinsic factors1–3. Cyanotic congenital heart defects are known to severely derail smooth blood flow with subsequent iron homeostasis dysregulation. Prevalence of anaemia amongst patients with cyanotic VSD is known and well documented in a variety of world literature2. On the contrary, solitary acyanotic ventricular septal defect (VSD) exhibit a relatively smooth blood with unremarkable iron homeostasis dysregulation. However, depending on size and duration, solitary VSD can lead to pulmonary hyper-circulation with subsequent complications, including recurrent lower respiratory tract infection (pneumonia; PNA), pulmonary hypertension (PH), pulmonary vascular disease, heart failure and death4,5.\n\nPNA is known to cause local hypoxia with subsequent upregulation of erythropoiesis. In addition, certain causatives of PNA are known to compete for iron with the host, while others induce haemolysis, both leading to anaemia6. Equally, PH has been associated with severe forms of haemolytic anaemia in patients with haemoglobin disorder and erythrocyte membranopathies2,7. In addition, free haemoglobin produced as a result of haemolysis depletes nitric oxide with subsequent vessel constriction, local hypoxia and exacerbation of PH8,9. Both PNA and PH exhibit tendencies of inducing local hypoxia, leading to upregulation of erythrocyte synthesis. Adults and older children with enough iron store can tolerate such a condition without developing anaemia. However, infants and young children have inadequate iron stores. In addition, children below the age of 6-months fed exclusively on breast milk have limited source of iron replacement. For the reasons mentioned above, we hypothesize that paediatric patients with solitary acyanotic VSD coexisting with PNA or PH retain a risk of developing anaemia. In this retrospective study, we aim to establish the prevalence of anaemia in patients with solitary acyanotic VSD in comorbid with PNA or PH.\n\n\nMethods\n\nBetween February 2014 and September 2018, 90 case files of patients with solitary acyanotic-VSD, who underwent either surgical or minimal invasive closure in our Department of Cardiac Surgery, Shandong Provincial Hospital Affiliate of Shandong University were primarily selected for this study.\n\nHowever, only 75 case files met study criteria, which included patients with a history of proven PNA by chest radiography with positive bacterial culture of trans-tracheal aspirate or polymerize chain reaction from nasopharyngeal swab. Pulmonary hypertension diagnosis was echocardiography based, except in 5 patients from PNA group, who presented in heart failure state. Excluded from this study were 15 files of patients: 7, sickle cell; 4, β-Thalassemia; 4, blood transfusion.\n\nAmong the 75 files were 48 males (75.64%) and 27 females with mean age and weight of 8.3±5.7 (3–24) months and 3.8±3.0 kilograms, respectively. Depending on the associated complication, the cohort was then divided into three groups: A, PNA (n=30); B, PH (n=25); and C, control (n=20). Based on age, the cohort was further split into two groups: I, young children (≥3-≤6); II, older children (>6-≤24) months. The patient demographic and clinic characteristics (Table 1) and hematologic profile (Table 2) reflects pre-procedure state.\n\nStatistical analysis. Data was analysed using IBM SPSS-software (one-way-ANOVA) and all statistics expressed as mean ± standard deviation. Intergroup haemoglobin level was compared using independent samples student’s t-test. Statistical comparison of proportions was analysed using Tukey HSD Test, and the probability value of less than 0.05 was considered significant. Patient proportions are expressed in number and percentage (n, %).\n\n\nResults\n\nIn this study, the haemoglobin reference range for groups: I and II were (95–135g/L) and (105–135g/L) respectively. This thus, conforms with local protocol.\n\nAccording to data analysis in Table 3, 27 patients (36%) in total had anaemia. Of the anaemia cohort, 16 (59.3%) had PNA, 9 (33.3%) had PH, and 2 (7.4%) were asymptomatic. These results can be expressed as ratios 16:9:2=8:4.5:1; therefore, patients with PNA and PH are 8 and 4 times more susceptible to developing anaemia than asymptomatic patients.\n\nOf the cohort, 42 were young children, and of those 19 had anaemia (45.2%), while 24.2% of the older children had anaemia. Haemoglobin intergroup (ANOVA) independent sample t-test was significant (p<0.05). In addition, intergroup Tukey HSD test for haemoglobin: A/B (p>0.05), A/C (p<0.01), B/C (p<0.01). The mean white blood cells in patients with PNA was higher and intergroup p-value was significant (p<0.05).\n\n\nDiscussion\n\nAnaemia, defined as haemoglobin (Hb) concentration below the 5th percentile for age at sea-level, is a common complication of a myriad medical conditions often met in the general ward1. Its aetiology is complex and multifactorial, encompassing intrinsic and extrinsic factors. Both pneumonia (PNA) and pulmonary hypertension (PH) due to cyanotic congenital heart defect (CHD) have been implicated in the occurrence of anaemia2,3,7. In addition, sporadic reports linking anaemia to PNA or PH amongst patients with acyanotic ventricular septal defect (VSD) have been publish.\n\nVSD is the second commonest CHD after bicuspid aortic valve4,10, and solitary cases account for almost 20%. One of the most common defects associated with elevated pulmonary artery pressure is a large VSD. Elevated pulmonary artery pressure in CHD can be due to pulmonary hyper-circulation, pulmonary vasoconstriction, and pulmonary vascular disease, either alone or in combination. In an infant, despite pulmonary pressure being at systemic level, pulmonary vascular resistance is low; therefore, minor shunt easily elicits hyper-circulation2,4,7,11.\n\nPH, defined as mean pulmonary artery pressure of ≥25mmHg at rest as measured by cardiac catheterization in children aged ≥3months, is a serious disorder with a high morbidity and mortality rate2. Blood shunt may cause haemolysis due to shear stress and produce free haemoglobin, which in turn depletes nitric oxide leading to endothelial dysfunction, vasoconstriction, pulmonary oedema and hypoxia. Furthermore, haemolysis produces arginase, which converts L-arginine to ornithine; therefore, bypassing nitric oxide production2,9,12.\n\nPNA, defined as an inflammation of alveoli (with or without pus) due to an infection is a serious morbidity and mortality reason for children (≤2years) with hemodynamic significant VSD5,13. Similar to PH, inflammatory state in PNA causes pulmonary oedema, homeostasis and hypoxia, with subsequent upregulation of erythrocyte synthesis8,14. Prolonged upregulated erythropoiesis in young children with low iron store and limited iron source (exclusive breast milk) can develop low haemoglobin levels1. In addition, microangiopathic haemolytic anaemia in CHD and PH has been reported3, a complication commonly observed in primary PH. Unlike PH, a number of PNA causatives are known to cause haemolytic and iron deficient anaemia. Mycoplasma pneumonia has been reported to cause severe haemolytic anaemia15,16. In addition, Klebsiella is known to compete for iron with host via siderophores6.\n\nOur study shows that patients with acyanotic VSD within mean sizes 1.2±0.3 and 0.89±0.2 cm were prone to develop recurrent lower respiratory tract infection and pulmonary vascular disorder. The two pathological conditions primarily share common features including pulmonary oedema, haemostasis and hypoxia, with an immediate non-hemodynamic response in form of increased erythropoiesis. However, unlike PH, toxins from certain causatives (bacteria) in PNA induce haemolysis, while other pathogens compete for iron with the host. Owing to LDH and WBC elevation in group A, it’s evident that there was some haemolysis of a certain degree as a result of an infection. Other potential causes include microangiopathic haemolysis in CHD with PH, feeding difficulties and malabsorption. Given the high proportion of anaemia in the ‘young children’ group, it is logical to assume they were fed exclusively on breast (artificial) milk, possibly limiting the source of iron. From our study, it is evident that diagnosis of hemodynamic significant acyanotic VSD in comorbid with PNA or PH should heighten the suspicion index of anaemia. However, we acknowledge that aetiology of anaemia is multifactorial, and so many intrinsic and extrinsic factors are at play, hence, every case requires a holistic approach.\n\nTo the best of our knowledge this comparative concept is new; therefore, a larger cohort in a prospective model would validate our findings.\n\n\nConclusion\n\nPaediatric patients without hematologic disorders, diagnosed with hemodynamic significant acyanotic VSD in comorbid with PNA or PH are 8 and 4 times susceptible to develop anaemia compared to asymptomatic counterparts. Susceptibility is even high amongst young children (3–6months).\n\n\nEthical considerations\n\nThe Shandong Provincial Hospital Ethics Committee approved this study, and waived individual patient consent as the study was based on archived data.\n\n\nData availability\n\nHarvard Dataverse: Anaemia in solitary acyanotic ventricular septal defect in comorbid with pneumonia or pulmonary hypertension; a retrospective study of 75 paediatric cases, https://doi.org/10.7910/DVN/2B328D17.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nFunding for this project was provided by Shandong University PR China; and National Key R & D program of P.R. China (Grant no. 2017YFC1308000).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank the management and staff of the Department of Cardiovascular Surgery and Imaging of Shandong Provincial Hospital, affiliated to Shandong University.\n\n\nReferences\n\nJanus J, Moerschel SK: Evaluation of anemia in children. Am Fam Physician. 2010; 81(12): 1462–71. PubMed Abstract\n\nMathew R, Huang J, Wu JM, et al.: Hematological disorders and pulmonary hypertension. World J Cardiol. 2016; 8(12): 703–718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuzuki H, Nakasato M, Sato S, et al.: Microangiopathic Hemolytic Anemia and Thrombocytopenia in a Child with Atrial Septal Defect and Pulmonary Hypertension. Tohoku J Exp Med. 1997; 181(3): 379–84. PubMed Abstract \| Publisher Full Text\n\nBerger J: Pulmonary Hypertension in Congenital Heart Disease. 8.\n\nÖzdemir Şahan Y, Kılıçoğlu E, Ülger Tutar Z: Evaluation of Children with Congenital Heart Disease Hospitalized with the Diagnosis of Lower Respiratory Tract Infection. J Pediatr Res. 2018; 5(1): 32–6. Publisher Full Text\n\nHolden VI, Breen P, Houle S, et al.: Klebsiella pneumoniae Siderophores Induce Inflammation, Bacterial Dissemination, and HIF-1α Stabilization during Pneumonia. mBio. 2016; 7(5): pii: e01397-16 PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWahl S, Vichinsky E: Pulmonary hypertension in hemolytic anemias. F1000 Med Rep. 2010; 2: pii: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThachil J: The enigma of pulmonary hypertension after splenectomy--does the megakaryocyte provide a clue? QJM. 2009; 102(10): 743–5. PubMed Abstract \| Publisher Full Text\n\nIvy D: Pulmonary Hypertension in Children. Cardiol Clin. 2016; 34(3): 451–72. PubMed Abstract \| Free Full Text\n\nSimonneau G, Galiè N, Rubin LJ, et al.: Clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2004; 43(12 Suppl S): S5–12. PubMed Abstract \| Publisher Full Text\n\nMetivier F, Marchais SJ, Guerin AP, et al.: Pathophysiology of anaemia: focus on the heart and blood vessels. Nephrol Dial Transplant. 2000; 15 Suppl 3: 14–8. PubMed Abstract \| Publisher Full Text\n\nLuisada AA, Cardi L: Acute pulmonary edema; pathology, physiology and clinical management. Circulation. 1956; 13(1): 113–35. PubMed Abstract \| Publisher Full Text\n\nReade MC, Weissfeld L, Angus DC, et al.: The prevalence of anemia and its association with 90-day mortality in hospitalized community-acquired pneumonia. BMC Pulm Med. 2010; 10(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZijlstra WM, Douwes JM, Ploegstra MJ, et al.: Clinical classification in pediatric pulmonary arterial hypertension associated with congenital heart disease. Pulm Circ. 2016; 6(3): 302–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhoury T, Abu Rmeileh A, Kornspan JD, et al.: Mycoplasma pneumoniae Pneumonia Associated With Methemoglobinemia and Anemia: An Overlooked Association? Open Forum Infect Dis. 2015; 2(1): ofv022. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurugol Z, Onen SS, Koturoglu G: Severe Hemolytic Anemia Associated with Mild Pneumonia Caused by Mycoplasma pneumonia. Case Rep Med. 2012; 2012: 649850. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChangwe GJ: Anaemia in solitary acyanotic ventricular septal defect in comorbid with pneumonia or pulmonary hypertension; a retrospective study of 75 paediatric cases. Harvard Dataverse, V1, UNF:6:4K4wd5s1QrnBqf+D3UuXYA== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/2B328D" }	[ { "id": "44745", "date": "27 Mar 2019", "name": "Shengli Li", "expertise": [ "Reviewer Expertise Fetal medicine", "prenatal diagnosis of fetal malformations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is a small-size retrospective study about the prevalence of anaemia in patients with solitary acyanotic VSD in comorbid with PNA or PH; they find that patients with acyanotic VSD in comorbid with PNA or PH were 8 and 4 times more susceptible to develop anaemia compared to asymptomatic counterparts.\n\nFor the VSD patients with PNA or PH, does the increased incidence of anaemia have any influence on the therapy or clinical management?\n\nAs the aetiology of anaemia is multifactorial, any intrinsic and extrinsic factors should be analysed in this article. This is crucial.\n\nAs mentioned above, it suggests that this article needs to be revised before accepting for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "54739", "date": "15 Oct 2019", "name": "Raymond N. Haddad", "expertise": [ "Reviewer Expertise Pediatrics", "Pediatric cardiology", "Pediatric interventionnal cardiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirstly, I want to thank The Editorial Team of F1000Research for this opportunity to review the following manuscript.\nI want to also thank the authors for recommending as a reviewer for their manuscript. Although this comparative concept is extremely new and interesting, I will generate candid comments which at times may seem overly critical. Please accept these criticisms in the positive spirit in which they are intended. I believe that the manuscript would be suitable for indexing if the comments/questions are addressed and appropriate changes made.\n\nThe authors in their Introduction largely reviewed the anemia and the contributing factors of PNA and PH which in large part are repeated in the discussion section. For that, I recommend that authors should summarize their introduction while briefly reviewing the role of VSD closure in the anemia (especially when all their patients had repaired defects either surgically or with minimal invasive approach).\n\nAuthors are encouraged to detail the method of defect repair. When is was performed? Is it early on diagnosis or later in time? The reason for closure especially in small defects? What do they actually mean by minimal invasive closure? Is it percutaneously or using a hybrid approach? Moreover, authors should explain why did they consider PNA as the only lower respiratory tract infection? And diagnosis of PHT should be more clearly detailed and if international guidelines were used reference must be cited. Additionally, where all patients screened for other possible hematological diseases to be excluded from the study? Table 1 : What does the patients mean by VSD size? is it the LV entry or the RV exit? I advice the authors to mention minimum and maximum values especially in each of the 3 groups for the PHT row as it may seems that some of Group A patients had PHT.\n\nStatistics: SPSS version must reported.\n\nTable 1 and 2 should be briefly described in the results sections.\n\nTable 3 must be just reported in the text.\n\nThe first 4 paragraphs of the discussion are pure literature review without any relevant discussion with the study results. In fact excessive review could impose more stratified results: should authors report the etiology on the PNA since the described the possible implications of infectious organisms in anemia. Therefore, discussion should focus in some part on the prevalence of anemia in repaired VSD (and if possible discussing the timing of closure and its possible effect of the results) while highlighting what is has been previously written by the authors.\n\nNo changes are needed for the limitations and conclusions section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-101
https://f1000research.com/articles/8-795/v1	06 Jun 19	{ "type": "Research Article", "title": "Non-communicable diseases in the Western Area District, Sierra Leone, following the Ebola outbreak", "authors": [ "Ibrahim Baimba Koroma", "Dena Javadi", "Katrina Hann", "Anthony D. Harries", "Francis Smart", "Thomas Samba", "Ibrahim Baimba Koroma", "Katrina Hann", "Anthony D. Harries", "Francis Smart", "Thomas Samba" ], "abstract": "Background: Non-communicable diseases (NCDs) are the leading causes of morbidity and mortality in the world. During infectious disease outbreaks, such as the Ebola virus disease outbreak in West Africa from 2014-2015, the health system is often strained, and diagnosis, management and care of NCDs may be compromised. This study assessed numbers and distribution of NCDs in all health facilities in the Western-Area District, Sierra Leone, in the post-Ebola period (June–December 2015) comparing findings with the pre-Ebola (June–December 2013) and Ebola outbreak (June–December 2014) periods. Methods: This was a cross-sectional study using secondary data from routine records of aggregate monthly NCD reports. Data were analysed using Open EPI and comparisons were made between the post-Ebola and pre-Ebola/Ebola periods using the chi square test. Results: There were 10,011 people reported with NCDs during the three six-month periods, with 6194 (62%) presenting at peripheral health units (PHU). Reported NCDs decreased during Ebola and increased post-Ebola, but did not recover to pre-Ebola levels. Hypertension cases remained fairly constant throughout being mainly managed at PHU. Numbers with diabetes mellitus generally stayed the same except for a significant post-Ebola increase in tertiary hospitals. Small numbers were reported with mental health disorders across all facilities in all time periods. Conclusion: NCD reporting is recovering in the immediate post-Ebola period. Decentralization of NCD care is welcome and is an effective strategy for management as evidenced by hypertension. To be successful, this must be supported by strengthening other elements of the health system such as training of health workers, robust information and referral systems and reliable medicine supply chains.", "keywords": [ "SORT IT", "operational research", "Sustainable Development Goals", "Universal Health Coverage", "health systems" ], "content": "Introduction\n\nNon-communicable diseases (NCDs) are the leading causes of morbidity and account for 70% of worldwide deaths, with the major burden felt in low- and middle-income countries (LMICs)1. The problem is highlighted in the UN Sustainable Development Goals (SDGs), with SDG target 3.4 aiming to reduce premature mortality from NCDs by one-third between 2015 and 20302. Progress is slow with poor health system responsiveness and weak monitoring capacity being important obstacles3.\n\nIn 2014–2015, Sierra Leone was hit by a devastating Ebola virus disease outbreak that highlighted the need for resilient health systems4,5. At that time, diagnosis, care and management of NCDs were in the process of decentralization from hospital to primary healthcare settings. In 2017, we reported on the burden and distributions of selected NCDs in a sample of health facilities in the Western-Area District, Sierra Leone, before and during the Ebola outbreak6. There was a marked decline in reported NCD numbers in the Ebola compared with the pre-Ebola periods, and this was especially observed for hypertension (HTN) and diabetes mellitus (DM) and in secondary and tertiary hospitals compared with peripheral health units (PHUs). Several recommendations were made that included strengthening the diagnosis, management and monitoring of NCDs in all district health facilities.\n\nWe now have comprehensive reports from all the district facilities for six-month periods before Ebola (June–December 2013), during Ebola (June–December 2014) and post-Ebola (June–December 2015) and have taken the opportunity to assess what has happened to NCDs during this time. The study objectives were to assess reported NCD numbers, the distribution of NCDs and their stratification by health facility level in the Western-Area District in the post-Ebola period and compare findings with Ebola and pre-Ebola periods outlined abov\n\n\nMethods\n\nThis was a cross-sectional study using secondary data from routine records. The setting and district monitoring system have been previously described6. In the current study, however, all public health facilities in the Western-Area District, Sierra Leone, were included: 110 PHUs, 9 secondary and 4 tertiary hospitals. The study population included all people reported with cardiovascular disease (CVD), HTN, DM, mental health disorder (MHD) and tumours/cancer in June–December 2013, June–December 2014 and June–December 2015. Data sources were the aggregate monthly NCD reports available at the Directorate of Policy, Planning and Information, Ministry of Health and Sanitation.\n\nData were analysed using Open EPI version 3.03 (updated 2014/09/22), with comparisons made between the post-Ebola and pre-Ebola/Ebola periods using the chi square test. P<0.05 was considered to indicate a significant difference.\n\nEthics approval was obtained from the Sierra Leone Ethics and Scientific Committee (dated 14 December 2018) and the Ethics Advisory Group, International Union Against Tuberculosis and Lung Disease, Paris, France (EAG number 63/18). As aggregate data were used with no identifiers, the need for informed consent was waived by the ethics committees.\n\n\nResults\n\nAltogether there were 10,011 people reported with NCDs during the three six-month periods, of whom 6194 (62%) were at PHU, 720 (7%) at secondary and 3097 (31%) at tertiary hospitals. Numbers with the five selected NCDs in the three different time periods are shown in Table 1. Key findings were: i) a decrease followed by an increase in NCDs in Ebola and post-Ebola periods with numbers not reaching pre-Ebola levels and this distribution was mirrored for HTN; ii) a large increase in DM in the post-Ebola period; iii) a decrease in CVD and tumours/cancer in the Ebola outbreak which continued to decline or stay unchanged in the post-Ebola period; and iv) small numbers of MHD in all three periods. These changes between the different periods were similar for males and females.\n\nPre-Ebola: June to December 2013; Ebola: June to December 2014; Post-Ebola: June to December 2015.\n\nNCD distributions in the three health facility levels are shown in Table 2. Key findings were: i) 72% of reported HTN and 94% of reported MHD cases were at PHU, while 83% of reported CVD, 64% of reported DM and 94% of reported tumour/cancer cases were at tertiary hospitals; ii) the proportion of reported HTN cases was maintained over the three six-month periods at PHU in contrast to the hospital settings; iii) numbers with DM generally stayed the same except for a significant post-Ebola increase in tertiary hospitals; iv) those with CVD decreased significantly in the post-Ebola period in PHU and tertiary hospitals.\n\nPre-Ebola: June to December 2013; Ebola: June to December 2014; Post-Ebola: June to December 2015.\n\nThe chi-square test was used to compare results of categorical variables in the post-Ebola period with the pre-Ebola and Ebola periods: P< 0.05; *<P<0.001\n\n\nDiscussion\n\nThe three most common NCDs during the study period were HTN, DM and CVD, with DM assuming greater importance in the post-Ebola period. Reasons are unclear, although DM has been predicted to increase dramatically in the next 10–20 years throughout sub-Saharan Africa7. In all three six-month periods it was encouraging to see that HTN was predominately managed at the PHU as opposed to hospitals, suggesting that the process of decentralisation in this area is well underway. The management of DM could benefit from the same approach. The small number of individuals reported with MHD is of concern, especially given the high prevalence of anxiety, depression and post-traumatic stress disorder following the Ebola outbreak8.\n\nThe strengths of the study are the inclusion of all public health facilities, making this study representative of the district. Limitations include use of routine aggregate data (the accuracy of which could not be verified), the lack of data from the private sector and missing hospital data for 2016 and 2017, which prevented further assessment of the post-Ebola recovery period.\n\nThere are important policy recommendations from this study. It is clear that the majority of NCDs in the district are managed at the PHU; this move for decentralisation is in line with Sierra Leone’s 2017–2021 National Health Sector Strategic Plan9. To ensure quality of care, however, the availability of medicines, diagnostic tools, a trained workforce, use of standardised NCD protocols and robust information systems must also be decentralised10. Health facility reporting forms and processes must also be reassessed to improve consistency of case definitions and ensure complete and accurate records, without which resource management to strengthen NCD care is not possible.\n\nIn conclusion, this study shows that NCD reporting is recovering in the immediate post-Ebola period. Decentralisation could be an effective and resilient strategy for management as evidenced by HTN. This encouraging momentum must be accompanied by health system strengthening—particularly for information systems—which will help move the country to universal health coverage and achievement of SDG3.4.\n\n\nData availability\n\nOpen Science Framework: Kamara_IbrahimK_SORTIT2_NCD_data 2019. https://doi.org/10.17605/OSF.IO/5Q3AX11.\n\nThis project contains the number of cases of each disease for each time period, alongside a data dictionary.\n\nThe Sierra Leone Health Management Information Systems, the District Health Information System 2 (DHIS2), is accessible with a Ministry of Health and Sanitation login through https://sl.dhis2.org/. The Directorate of Policy, Planning, and Information (DPPI) can be contacted through Dr. Francis Smart (drfsmart@gmail.com), Director, DPPI, MOHS, with an information request detailing the specific data request and purpose of use. Applicants will be asked to provide details of the reason for the request and details pertaining data request (such as data points, disaggregation, time period). In this case, data access wolud be granted to persons who request data for research purposes if they can provide appropriate ethical approval documentation.", "appendix": "Grant information\n\nThe programme was funded by the Special Programme for Research and Training in Tropical Diseases hosted at the World Health Organization (TDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) and implemented with partners. The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: WHO/TDR, the Sierra Leone Ministry of Health, WHO Sierra Leone, the Centre for Operational Research, The Union, Paris, France; the Alliance for Public Health, Ukraine; the Institute of Tropical Medicine, Antwerp, Belgium; and Sustainable Health Systems, Freetown, Sierra Leone.\n\n\nReferences\n\nNCD Countdown 2030 collaborators: NCD Countdown 2030: worldwide trends in non-communicable disease mortality and progress towards Sustainable Development Goal target 3.4. Lancet. 2018; 392(10152): 1072–1088. PubMed Abstract \| Publisher Full Text\n\nUnited Nations: Transforming our world: the 2030 agenda for sustainable development. 2015; (Accessed 20th February 2019). Reference Source\n\nNishtar S, Niinistö S, Sirisena M, et al.: Time to deliver: report of the WHO Independent High-Level Commission on NCDs. Lancet. 2018; 392(10143): 245–252. PubMed Abstract \| Publisher Full Text\n\nGostin LO, Friedman EA: A retrospective and prospective analysis of the west African Ebola virus disease epidemic: robust national health systems at the foundation and an empowered WHO at the apex. Lancet. 2015; 385(9980): 1902–1909. PubMed Abstract \| Publisher Full Text\n\nDecroo T, Fitzpatrick G, Amone J: What was the effect of the West African Ebola outbreak on health programme performance, and did programmes recover? Public Health Action. 2017; 7(Suppl 1): S1–S2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamba T, Bhat P, Owiti P, et al.: Non-communicable diseases in the Western Area District, Sierra Leone, before and during the Ebola outbreak. Public Heath Action. 2017; 7(Suppl 1): S16–S21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMbanya JC, Motala AA, Sobngwi E, et al.: Diabetes in sub-Saharan Africa. Lancet. 2010; 375(9733): 2254–2266. PubMed Abstract \| Publisher Full Text\n\nJalloh MF, Li W, Bunnell RE, et al.: Impact of Ebola experiences and risk perceptions on mental health in Sierra Leone, July 2015. BMJ Glob Health. 2018; 3(2): e000471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health and Sanitation: National Health Sector Strategic Plan 2017-2021. Freetown, Sierra Leone. 2017. Reference Source\n\nKane J, Landes M, Carroll C, et al.: A systematic review of primary care models for non-communicable disease interventions in Sub-Saharan Africa. BMC Fam Pract. 2017; 18(1): 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHann K, Koroma IB, Samba T: Kamara_IbrahimK_SORTIT2_NCD_data. 2019. http://www.doi.org/10.17605/OSF.IO/5Q3AX" }	[ { "id": "49596", "date": "19 Jul 2019", "name": "Jeffery Edwards", "expertise": [ "Reviewer Expertise Public health", "NCDs", "Ebola", "HIV/TB", "infectious diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the opportunity to review this well written and timely manuscript submitted by Koroma et al. It is certainly telling of the impact of the Ebola outbreak and the effects on the health system within Sierra Leone. Although it only uses aggregate data, the analysis is straight forward and highlights the struggles of providing/reporting on healthcare within a developing country.\n\nI believe this manuscript is very much worth of indexing and will add significantly to the growing knowledge base surrounding healthcare system management in developing countries. I would suggest one minor correction and an area which could be further discussed, where there is a gap in services being at least reported, if not provided.\nFirst, the last sentence in the 3rd paragraph is incomplete and needs to be corrected.\nSecondly, it is extremely interesting the lack of reporting of mental health visits - across all three levels of care. Additionally, I am curious, as with other African countries, do mental health visits include epilepsy as well? If so, this is an interesting perspective that should be mentioned.\n\nThe authors mention that \"the small number of individuals reported with MHD is of concern...especially... post Ebola...\" I think this could be another key finding, especially if it includes those with epilepsy. The prevalence of MHD and epilepsy in developing countries is unknown and regardless of the Ebola outbreak, the tremendous gap in reporting found in this study suggests that they are under-recognized and under-treated. The authors might want to point this out and provide some recommendations for Sierra Leone and other countries with similar contexts.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4993", "date": "19 Nov 2019", "name": "Katrina Hann", "role": "Author Response", "response": "Zena NyakoojoSenior Managing EditorF1000 Research Ltd.Middlesex House34-42 Cleveland StreetLondon W1T 4LBUK Dear Zena Nyakoojo, RE: Non-communicable diseases in the Western Area District, Sierra Leone, following the Ebola outbreak [version 1; peer review: 2 approved]We have carefully read through the peer review and have revised our manuscript accordingly. We see the manuscript as improved as a result of this peer review process, and thank you for your support in taking the process to this stage.Please find for your consideration the following: A point-by-point response to the comments and suggestions of the reviewers A new revised version of the manuscript, in tracked changes A new, revised version of the manuscript, no tracked changes. We hope that these modifications meet with your favourable review. Please do not hesitate to request any further changes. Kind regards, Ibrahim Baimba KoromaDirectorate of Policy, Planning and Information (DPPI)Ministry of Health and Sanitation (MoHS)FreetownSierra Leone Author Response to Reviewer Report for Version 1 from Jeffery Edwards 19 Jul 2019 Thank you for your thoughtful review of the manuscript and your insightful suggestions. We have responded as below. 1. Reviewer comment:I appreciate the opportunity to review this well written and timely manuscript submitted by Koroma et al. It is certainly telling of the impact of the Ebola outbreak and the effects on the health system within Sierra Leone. Although it only uses aggregate data, the analysis is straight forward and highlights the struggles of providing/reporting on healthcare within a developing country. I believe this manuscript is very much worth of indexing and will add significantly to the growing knowledge base surrounding healthcare system management in developing countries. Response:Thank you very much. 2. Reviewer comment:I would suggest one minor correction and an area which could be further discussed, where there is a gap in services being at least reported, if not provided. First, the last sentence in the 3rd paragraph is incomplete and needs to be corrected.Response:Thank you for this direction. We have amended the sentence based on this feedback. 3. Reviewer comment:Secondly, it is extremely interesting the lack of reporting of mental health visits - across all three levels of care. Additionally, I am curious, as with other African countries, do mental health visits include epilepsy as well? If so, this is an interesting perspective that should be mentioned. Response:In Sierra Leone, epilepsy is considered as a neurological condition, but it is documented using a separate data capture form and reported into a different programme. Therefore, our study does not capture epilepsy results, and thus, we are limited in our ability to make conclusions on epilepsy specifically.We have made a clarification on this point in the description of the NCD reporting form in the methods section. 4. Reviewer comment:The authors mention that \"the small number of individuals reported with MHD is of concern...especially... post Ebola...\" I think this could be another key finding, especially if it includes those with epilepsy. The prevalence of MHD and epilepsy in developing countries is unknown and regardless of the Ebola outbreak, the tremendous gap in reporting found in this study suggests that they are under-recognized and under-treated. The authors might want to point this out and provide some recommendations for Sierra Leone and other countries with similar contexts.Response:Thank you for this insight. We have added an additional point on underreporting and the treatment gap in the discussion." } ] }, { "id": "49599", "date": "22 Jul 2019", "name": "Priyakanta Nayak", "expertise": [ "Reviewer Expertise Infectious Disease Epidemiology", "Emergency Outbreak Preparedness & Response", "Tuberculosis", "HIV", "Malaria", "Neglected Tropical Disease (Leishmaniasis and Lymphatic Filariasis)", "NCD", "Workforce and Institutional Development", "Operational Research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good study, a kind of situational analysis of NCD in Sierra Leone during Ebola episodes. The methodology is good and the investigator has done a good job in evaluating the NCD reports across the PHUs. The only concerned thing here is the conclusion. This is very generalized and did not necessarily explain about the artifacts. Its obvious that it would be increased awareness and increased surveillance/screening activities as a result of the endemic but the same can not be generalized to the overall scenario. I recommend more clarity in the conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4994", "date": "19 Nov 2019", "name": "Katrina Hann", "role": "Author Response", "response": "Zena Nyakoojo Senior Managing Editor F1000 Research Ltd. Middlesex House 34-42 Cleveland Street London W1T 4LB UK Dear Zena Nyakoojo, RE: Non-communicable diseases in the Western Area District, Sierra Leone, following the Ebola outbreak [version 1; peer review: 2 approved] We have carefully read through the peer review and have revised our manuscript accordingly. We see the manuscript as improved as a result of this peer review process, and thank you for your support in taking the process to this stage. Please find for your consideration the following: A point-by-point response to the comments and suggestions of the reviewers A new revised version of the manuscript, in tracked changes A new, revised version of the manuscript, no tracked changes. We hope that these modifications meet with your favourable review. Please do not hesitate to request any further changes. Kind regards, Ibrahim Baimba Koroma Directorate of Policy, Planning and Information (DPPI) Ministry of Health and Sanitation (MoHS) Freetown Sierra Leone Point-by-point response to the reviewer suggestions Author Response to Reviewer Report for Version 1 from Priyankanta Nayak 22 Jul 2019 Thank you for your thoughtful review of the manuscript and your suggestions. We have responded as below. 1. Reviewer comment:This is a good study, a kind of situational analysis of NCD in Sierra Leone during Ebola episodes. The methodology is good and the investigator has done a good job in evaluating the NCD reports across the PHUs. Response: Thank you very much. 2. Reviewer comment:The only concerned thing here is the conclusion. This is very generalized and did not necessarily explain about the artifacts. Its obvious that it would be increased awareness and increased surveillance/screening activities as a result of the endemic but the same can not be generalized to the overall scenario. I recommend more clarity in the conclusion. Response: Thank you for this thoughtful reflection. The conclusion only gave a snap shot of the latest developments of NCD reporting during the post-Ebola period and suggested strategies for the health sector to strengthen NCD reporting." } ] } ]	1	https://f1000research.com/articles/8-795
https://f1000research.com/articles/8-1925/v1	18 Nov 19	{ "type": "Research Article", "title": "Analysis of the Arabidopsis organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130", "authors": [ "Kenton Ko", "Jeremy Guenther", "Nicholas Ostan", "Joshua Powles", "Jeremy Guenther", "Nicholas Ostan", "Joshua Powles" ], "abstract": "Background: Four distinct rhomboid genes appear to function in Arabidopsis plastids, two “active” types from the secretases and presenilin-like associated rhomboid-like (PARL) categories (At1g25290 and At5g25752) and two “inactive” rhomboid forms (At1g74130 and At1g74140). The number of working rhomboids is further increased by alternative splicing, two reported for At1g25290 and three for At1g74130. Since At1g25290 and At1g74130 exist as alternative splice variants, it would be necessary to assess the splicing patterns of the other two plastid rhomboid genes, At5g25752 and At1g74140, before studying the Arabidopsis plastid rhomboid system as a whole.\n\nMethods: This study thus specifically focused on an analysis of the At1g74140 transcript population using various RT-PCR strategies.\n\nResults: The exon mapping results indicate splicing patterns different from the close relative At1g74130, despite similarity between the exonic sequences. The splicing patterns indicate a high level of sequence “discontinuity” in the At1g74140 transcript population with a significant portion of the discontinuity being generated by two regions of the gene.\n\nConclusion: The overall discontinuous splicing pattern of At1g74140 may be reflective of its mode of involvement in activities like controlling gene expression.", "keywords": [ "Arabidopsis", "plastid rhomboid proteins", "alternative splicing", "short RNA transcript production" ], "content": "Introduction\n\nThe rhomboid protein gene system in Arabidopsis is complex with 13 transcribed genes encoding both “active” and “inactive” rhomboid protein types (Knopf & Adam, 2012; Koonin et al., 2003; Tripathi & Sowdhamini, 2006). A recent survey of the databases indicates at least 22 entries for Arabidopsis (Powles & Ko, 2018). These entries encompass the same range of rhomboid types as that observed in other organisms (Lemberg & Freeman, 2007). One intriguing aspect about these different rhomboids is that they appear to operate simultaneously, such as in the Arabidopsis plastid compartment. In Arabidopsis, there are four distinct plastid rhomboid genes predicted - two encoding “active” types (At1g25290 and At5g25752) and two encoding “inactive” forms (At1g74130 and At1g74140). This number is further increased by alternative splicing, as reported for At1g25290 and At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012). Alternative splicing of At1g25290 transcripts introduced variant forms with and without a putative cyclin-binding RVL motif (Sedivy-Haley et al., 2012). For At1g74130, alternative splicing generated three variants with different carboxyl segments, changing from a predicted seven to a six transmembrane structure (Powles et al., 2013). These three At1g74130 variants exhibited different functionalities, such as their interactions with the Tic40 substrate and the yeast mitochondrial rhomboid protease Rbd1 and Mgm1 in live cell contexts, and their abilities in sensitizing cells to antimicrobial drugs (Powles et al., 2013; Powles & Ko, 2018). These different functionalities are especially interesting since one potential role of At1g74130 appears to be in the early stages of tissue development and plastid biogenesis (Sedivy-Haley et al., 2011).\n\nThe picture emerging for the Arabidopsis plastid rhomboids suggests that alternative splice variants are involved in regulatory type activities. Such a phenomenon is observed in other organisms, such as for the human rhomboid gene RHBDD2, as an example (Abba et al., 2009). The levels of the two alternatively spliced RHBDD2 mRNAs were elevated in breast cancer cell lines (Abba et al., 2009). In terms of other regulatory roles, rhomboid proteins themselves are often associated with regulatory activities in different organisms and in a range of cellular pathways, such as development, mitochondrial membrane remodeling, protein transport, quorum sensing, and stress response (see examples in Adrian & Freeman, 2012; Jeyaraju et al., 2013; Lemberg, 2013; McQuibban et al., 2003; Sedivy-Haley et al., 2011; Sekine et al., 2012; Stevenson et al., 2007; Thompson et al., 2012; Urban, 2006; Yan et al., 2008). The possibility of alternative splicing contributing further to these different types of regulatory activities is thus intriguing and warrants investigation.\n\nIt is now clear that to understand how the plastid rhomboid system works, it is necessary to identify all potential rhomboid variants in play. This study was thus designed to examine the splicing activities of At1g74140, a close relative to At1g74130 and located tandemly next to At1g74130. An analysis of the At1g74140 transcript population would help uncover potential modes of operation for this gene and its possible role in Arabidopsis plastids.\n\n\nMethods\n\nGrowth chambers were used to propagate the plants employed in this study. The cultivation conditions for the Arabidopsis line CS60000 (“wild-type”) were derived from information posted on the Arabidopsis Biological Resource Center (Ohio State University) website (http://abrc.osu.edu) (Arabidopsis Biological Resource Center, RRID:SCR_008136) (Alonso et al., 2003). The growth chamber parameters used were: 21°C; 70% humidity; and a 16:8 hour light:dark photoperiod using 150–200 μmol·m-2·s-1 of fluorescent and incandescent lighting. Seeds were cold stratified for 3 days before transfer to growth chambers. All plants were subjected to a regime of daily watering and weekly fertilization.\n\nThe genomic DNA entries used for At1g74130 and At1g74140 are of the tandem genes located on chromosome 1 (Chromosome 1 NC_003070.9). The accession numbers used for the At1g74140 cDNA work were: NM_001124127.1, NM_001084350.2, NM_106074.2, NM_001036203.1, and NM_001084351.2. The accession numbers used for the At1g74130 cDNA entries were NM_106073.5 and NM_202412.1 (NCBI, RRID:SCR_006472; NCBI BLAST, RRID:SCR_004870).\n\nProcedures and optimization details related to the analysis of transcript structure were the same as those described by Sedivy-Haley et al. (2012). Briefly, all plant tissues destined for RNA extraction were collected between 11:00 and 13:00 h during the light period to equalize the circadian effects. Total RNA was isolated and treated with DNase using Qiagen kits (RNeasy kit and RNase-free DNase set, Qiagen, Hilden, Germany) (QIAGEN, RRID:SCR_008539). Transcript structure was studied using Qiagen PCR kits (One-Step RT-PCR kit, Qiagen, Hilden, Germany) (QIAGEN, RRID:SCR_008539) and a set of exon-exon specific DNA primers. A summary of the DNA primers used is provided in the Extended data: Table S1. The RT-PCR steps and cycle timings used were as recommended by the manufacturer, Qiagen, without modifications. The RT-PCR assays were all conducted with 20 ng of total RNA with cycles between 35 and 40 to visualize lower level products. The temperature used was 55°C. Prior optimization RT-PCR assays were conducted with varying temperatures, cycles, and total RNA amounts. All assays were conducted using an Eppendorf Mastercycler for microplates. PCR products were resolved by running samples in standard 4% (w/v) Tris-borate – EDTA polyacrylamide gels. The DNA markers used were purchased from Frogabbio (Toronto, Ontario, Canada).\n\n\nResults\n\nThere are currently four genes predicted for plastidial rhomboid proteins in Arabidopsis (Kanaoka et al., 2005; Knopf & Adam, 2012; Koonin et al., 2003; Tripathi & Sowdhamini, 2006). Three are located in chromosome 1 and one in chromosome 5. Our previous work on the transcript structures of At1g25290 and At1g74130 revealed alternatively spliced variants, two for At1g25290 and three for At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012). The other rhomboid gene in chromosome 1, At1g74140, is highly similar to At1g74130 and located downstream from At1g74130. At1g74130 is located between nucleotide positions 27873611 and 27876033, and At1g74140 occupies 27876905 to 27879780. The overall exon-intron structures of At1g74130 and At1g74140 are similar, each with 8 exons and 7 introns (Figure 1A). Although the exon lengths are similar, there appear to be substantial differences in the lengths of the middle introns 3, 4, and 5 (Figure 1A). A comparison of the translated products from the available At1g74130 and At1g74140 cDNA entries or sequences (five and three, respectively) also indicated a high degree of similarity with a few gaps (Figure 1B). The differences in intronic composition may thus be reflective of the roles of the two genes and this notion continues to be the case when examining the outcomes of the At1g74140 transcript structure analysis below.\n\n(A) A schematic comparison of the related gene structures (using information derived from the entry Chromosome 1 NC_003070.9). Exons are in depicted black and numbered sequentially. Introns are depicted in gray. (B) A comparison of the amino acid sequences derived from five At1g74140 variants and three At1g74130 variants (The accession numbers for the At1g74140 variants were: NM_001124127.1, NM_001084350.2, NM_106074.2, NM_001036203.1, and NM_001084351.2. The accession numbers for the At1g74130 variants were NM_106073.5 and NM_202412.1, and from Powles et al., 2013). Gaps are denoted by hyphens. The yellow highlighted amino acids indicate the residue locations for part of the active site.\n\nFor the analysis of At1g74140 transcripts, the overall RT-PCR strategy and primer pairs used were designed to map alternative splicing activities in the context of transcript populations (Table S1 summarizes the details of the exon-exon specific primers used). These primers should allow detection of the many differently spliced exonic structures in the transcript population, along with changes to levels of neighboring exons represented in the resulting products. Such modulations in RT-PCR product levels should be indicative of different transcript splicing activities. This approach was employed to characterize alternatively spliced variants in our previous studies on At1g25290 and At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012).\n\nA combined RT-PCR strategy, stepped and laddered primer pairs, was used to characterize the different structures that existed in the transcript population. The outcomes were sorted and summarized schematically in Figure 2–Figure 4 along with the corresponding primer strategies. The various RT-PCR results used for the construction of Figure 2–Figure 4 are presented in Figure 5–Figure 10. The presence or absence of each exon and their relative levels were surveyed and compiled. These exon specific RT-PCR outcomes are sorted and organized schematically as black lines in Figure 2 (below the gene map). The weight of the black lines, for example, thick versus thin, represents relative RT-PCR product levels. From this summation map of the stepped RT-PCR strategy, we were able to observe missing exon-exon products as well as unbalances in the relative levels of neighboring exon-exon products. Products primed from the start region of exon 1 and forward region of exon 8 were not detected. Products primed from the latter end of exon 1 to exon 7 were present, but exhibited changes to their relative levels, even if the neighboring RT-PCR products were expected to be comparable with respect to size and RT-PCR assay parameters.\n\nThe results summarized in this figure are presented in Figure 5 through Figure 10 and Figures S1 to S3. Primer pairs focused on exon-exon borders are summarized as black solid lines. Primer pairs focused on 5’UTR and START are summarized separately as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product. The At1g74140 gene structure and map of priming sites are provided schematically.\n\nThe results summarized are presented in Figure 5 through Figure 10 and Figures S1 to S3. The primer pair focused on exons 3 to 5 is represented as a black solid line. Primer pairs focused on the exon 2 region are summarized separately as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product.\n\nThe results summarized are presented in Figure 5 through Figure 10 and Figures S1 to S3. Primer pairs focused on the exon 7 region are summarized as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product.\n\nThe forward 1F primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe forward primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe forward primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe forward primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe forward primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe forward primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.\n\nThe outcomes were next sorted and organized as sequential sets of laddered primers in the 5’ to 3’ direction. Different splicing activities were detected by the appearance or disappearance of different-sized RT-PCR splice products, and by changes in the levels of RT-PCR products derived from neighboring exons (the red lines in Figure 2–Figure 4). The overall patterns generated indicate that At1g74140 transcripts were not processed as predicted. Primers derived from the 5’UTR, or the start of exon 1, or the end of exon 8, or 3’UTR, did not consistently result in RT-PCR products that spanned the entire open reading frame. Products that did arise with the exon 8 or 3’UTR primers were lower than the levels observed for many of the products generated by the other primer pairs. This pattern is different from the closely related At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012). The overall pattern displayed for At1g74140 indicates a high level of alternative splicing activity with extensive discontinuity in the resulting products.\n\nThe high level of discontinuity appears to originate mainly from two regions, from sequences spanning exon 2 and exon 7 (Figure 3 and Figure 4). Both regions appear to separately and in combination contribute to the observed discontinuity phenomenon. Signs indicating splicing discontinuity manifest as disappearances of predicted RT-PCR products and lower levels of predicted products as a result of re-distribution into differently spliced transcript sub-populations. These occurrences are represented as red lines in Figure 3 and Figure 4 (hatched for missing, solid for present, and thickness for lower relative levels). The patterns, when considered together, suggest that the sequence spanning the 5’end of exon 1 to approximately exon 6 is frequently separated from the latter section spanning approximately from exon 6 to exon 8. The resulting products from the two regions appear to be distributed into different transcript sub-populations.\n\n\nDiscussion and conclusions\n\nThe Arabidopsis rhomboid system consists of at least 13 expressed full-length genes for active and inactive types that work in different cellular locations (Knopf & Adam, 2012; Koonin et al., 2003; Lemberg & Freeman, 2007; Tripathi & Sowdhamini, 2006). Of these 13 genes, two are for active plastidial types (At1g25290 and At5g25752) and two are for inactive plastidial types (At1g74130 and At1g74140) (Knopf et al., 2012; Sedivy-Haley et al., 2011; Sedivy-Haley et al., 2012; Thompson et al., 2012). The number of plastid rhomboid variants are further increased by alternative splicing, such as that reported for At1g25290 (Sedivy-Haley et al., 2012) and At1g74130 (Powles et al., 2013). It is thus important to characterize the status of all rhomboid forms before studying the plastid rhomboid system. This particular study focuses on a close relative of At1g74130, At1g74140.\n\nThe various RT-PCR outcomes obtained in this study, together, uncovered a complex, atypical alternative splicing pattern for At1g74140. In contrast to the alternative splicing patterns of At1g25290 and At1g74130 where each generated defined variants, the splicing activities of At1g74140 produced an array of shorter, spliced transcripts derived from different exonic stretches of the gene sequence. Unlike its close relative At1g74130, spliced At1g74140 transcripts that span the entire gene sequence were not produced at our level of detection and are thus not represented as a significant pool in the transcript population. This outcome suggests that the function of At1g74140 was likely not based on the full length of the gene as for At1g74130, but on many shorter stretches. It is not known at this juncture if the different shorter, spliced transcripts are functional at the translational level. The shorter, spliced transcripts themselves are more likely to represent functional entities, such as microRNAs or RNA interference.\n\nThe complex splicing pattern of At1g74140 appears to be triggered predominantly by two regions, the stretch spanning exon 2 and the stretch spanning exon 7. Many of the spliced products predicted to span exon 2 and/or 7 were absent or produced in lower than expected relative levels. The lowering of relative levels is also due to the splitting of the one predicted pool into several pools with different spliced transcript variants. A preliminary analysis of sequences and RT-PCR products from the two regions revealed microRNA possibilities, but this is being further investigated in depth at this point.\n\nDespite our limited understanding of the roles played by splice variants, we have witnessed the employment of alternative splicing as a mechanism for diversifying plastid rhomboid protein function in Arabidopsis. Alternative splicing is used to create protein variants with changes to functionality for At1g25290 and At1g74130. Here in this report, the data provide evidence that another Arabidopsis plastid rhomboid gene, At1g74140, carries the potential to function through its alternatively spliced short transcript products. This function could be in the form of shorter translational products and/or short RNAs. This potential is quite different from its close relative At1g74130. Preliminary studies with different developmental tissues indicate that some of the alternatively spliced short transcript products fluctuate between mature and young tissues, hence the short transcripts appear to bear functional significance (see Underlying data: Figures S1-S3). For now, the role(s) played by the short transcripts of At1g74140, although intriguing they may be, awaits elucidation.\n\n\nData availability\n\nZenodo: Splicing patterns of the Arabidopsis organellar rhomboid At1g74140 change during development from young to mature leaf tissues, https://doi.org/10.5281/zenodo.3533891 (Ko et al., 2019a).\n\nThis project contains the following underlying data:\n\nFigure S1. RT-PCR results for various primer combination set 1 and the two developmental stages analyzed.\n\nFigure S2. RT-PCR results for various primer combination set 2 and the two developmental stages analyzed.\n\nFigure S3. RT-PCR results for various primer combination set 3 and the two developmental stages analyzed.\n\nZenodo: Splicing patterns of the Arabidopsis organellar rhomboid At1g74140 - Table S1 for primers used in the RT-PCR assays, https://doi.org/10.5281/zenodo.3534013 (Ko et al., 2019b).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbba MC, Lacunza E, Nunez MI, et al.: Rhomboid domain containing 2 (RHBDD2): a novel cancer-related gene over-expressed in breast cancer. Biochim Biophys Acta. 2009; 1792(10): 988–997. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdrian C, Freeman M: New lives for old: evolution of pseudoenzyme function illustrated by iRhoms. Nat Rev Mol Cell Biol. 2012; 13(8): 489–498. PubMed Abstract \| Publisher Full Text\n\nAlonso JM, Stepanova AN, Leisse TJ, et al.: Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003; 301(5633): 653–657. PubMed Abstract \| Publisher Full Text\n\nJeyaraju DV, Sood A, Laforce-Lavoie A, et al.: Rhomboid proteases in mitochondria and plastids: keeping organelles in shape. Biochim Biophys Acta. 2013; 1833(2): 371–380. PubMed Abstract \| Publisher Full Text\n\nKanaoka MM, Urban S, Freeman M, et al.: An Arabidopsis rhomboid homolog is an intramembrane protease in plants. FEBS Lett. 2005; 579(25): 5723–5728. PubMed Abstract \| Publisher Full Text\n\nKnopf RR, Adam Z: Rhomboid proteases in plants - still in square one? Physiol Plant. 2012; 145(1): 41–51. PubMed Abstract \| Publisher Full Text\n\nKnopf RR, Feder A, Mayer K, et al.: Rhomboid proteins in the chloroplast envelope affect the level of allene oxide synthase in Arabidopsis thaliana. Plant J. 2012; 72(4): 559–571. PubMed Abstract \| Publisher Full Text\n\nKo K, Guether J, Ostan N, et al.: Splicing patterns of the Arabidopsis organellar rhomboid At1g74140 change during development from young to mature leaf tissues. Zenodo. 2019a. http://www.doi.org/10.5281/zenodo.3533891\n\nKo K, Guenther J, Ostan N, et al.: Splicing patterns of the Arabidopsis organellar rhomboid At1g74140 - Table S1 for primers used in the RT-PCR assays [Data set]. Zenodo. 2019b. http://www.doi.org/10.5281/zenodo.3534013\n\nKoonin EV, Makarova KS, Rogozin IB, et al.: The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers. Genome Biol. 2003; 4(3): R19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLemberg MK: Sampling the membrane: function of rhomboid-family proteins. Trends Cell Biol. 2013; 23(5): 210–217. PubMed Abstract \| Publisher Full Text\n\nLemberg MK, Freeman M: Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases. Genome Res. 2007; 17(11): 1634–1646. 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Publisher Full Text\n\nSedivy-Haley K, Powles J, Ko K: Characterization of two alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g25290. Botany. 2012; 90(12): 1252–1262. Publisher Full Text\n\nSekine S, Kanamaru Y, Koikem M, et al.: Rhomboid protease PARL mediates the mitochondrial membrane potential loss-induced cleavage of PGAM5. J Biol Chem. 2012; 287(41): 34635–34645. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevenson LG, Strisovsky K, Clemmer KM, et al.: Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc Natl Acad Sci USA. 2007; 104(3): 1003–1008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson EP, Smith SG, Glover BJ: An Arabidopsis rhomboid protease has roles in the chloroplast and in flower development. J Exp Bot. 2012; 63(10): 3559–3570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTripathi LP, Sowdhamini R: Cross genome comparisons of serine proteases in Arabidopsis and rice. BMC Genomics. 2006; 7: 200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUrban S: Rhomboid proteins: conserved membrane proteases with divergent biological functions. Genes Dev. 2006; 20(22): 3054–3068. PubMed Abstract \| Publisher Full Text\n\nYan Z, Zou H, Tian F, et al.: Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth. Mol Cancer Ther. 2008; 7(6): 1355–1364. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "59597", "date": "12 Feb 2020", "name": "Ezequiel Petrillo", "expertise": [ "Reviewer Expertise Gene expression", "alternative splicing regulation in plants." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “Analysis of the Arabidopsis organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130” by Ko and colleagues is an attempt to characterize the alternative splicing patterns of At1g74140 in comparison to the previously characterized one, At1g74130. After carefully reading the paper and carefully analyzing the methods, I have to sadly say that I do not think this manuscript represents an advance in the knowledge of these genes (or At1g74140 in particular), since the alternative splicing isoforms are already annotated in JBrowse, Araport and TAIR, and I do not think the methods used are appropriate to search for (or validate) alternative splicing isoforms. The use of different overlapped/stepped PCR amplicons to check for “different splicing activities” is not a reliable method. PCR is extremely sensitive and with enough cycles could amplify even noise. Primer design is wrong in many cases. When the authors use primers that should span exon junctions they have far too many bases on the 3’ end of the primers. This means that these oligos could partially bind (and with partially I mean 13-16 bases) to a target and give a PCR product that would be misleading. When generating a primer for a junction the authors must limit the binding to the 3’ exon/region to only 3-5 bases. Moreover, these 2 genes do not show any expression at all in the RNA-seq data on JBrowse in any condition there. In addition, I was analyzing other RNA-seq data, publicly available, to see if I could find if these genes are expressed at all, and I wasn’t able to find trustable reads for these loci in several conditions tested. From my point of view, the expression of these genes, and of At1g74140 in particular, is marginal (in several conditions). Hence, any conclusion based on the expression of a few, or just some partial, RNA molecules wouldn’t be reliable. As I was saying before, by using PCR you can get a signal even from the-almost-non-existent expression of a gene. Since there are no amplification controls, for example using genomic DNA to test the primers, when there is no amplification we cannot say that a particular cDNA (corresponding to an isoform) is absent or that a particular combination of exons is not in the sample. The lack of amplification of the longer amplicons (“full length RNA”) should be taken as a signal of a problem. Since, as expressed before, there are no amplification controls and some primers do work in some combinations or in shorter amplicons, I would guess that the expression of the “full length RNA” is so low that the authors are just able to get some partial RNAs that are most likely expression noise. The authors do not inform the amount of cycles they used; hence, I do not know for sure whether the expression is low or not. However, I am concluding this from the intensity of the bands in the gels, the lack of amplification of the longer amplicons, and the fact that there are no reads at all for this gene in different RNA-seq experiments. The authors should provide this information, so the reader knows how many cycles are needed to get a signal from the different amplicons. In addition, if the authors have evidence that expression is not marginal in their conditions, they should add that into this manuscript. Moreover, showing different conditions where splicing patterns are affected would also increase the impact of this work. Besides these main issues I would also like to add that the conclusion that the shorter, spliced transcripts, could be functional entities such as miRNAs is far far beyond the data presented. Furthermore, the authors introduce the terms “active”/”inactive” but they never explain the meaning of these in the context of these proteins. In addition, the authors discuss about splicing patterns but they do not see that in their data, and they introduce the concept (?) of ‘sequence “discontinuity”’ that it is not further explained and seems to be, actually, they are talking about alternative splicing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "60928", "date": "23 Mar 2020", "name": "Han Xiao", "expertise": [ "Reviewer Expertise transcriptome" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlternative splicing (AS) definitely contributes to the complexity of gene regulation at post-transcriptional level. To understand if and how AS is involved in the regulation of gene expression, for a particular multiexon gene, it will be necessary to determine the pattern of alternative splicing and to identify functional splice variants. In this study by Ko et al. report their alternative splicing analysis of an Arabidopsis gene At1g74140. They applied RT-PCR approach with a variety of primer sets to detect alternative spliced variants of At1g74130. Main results obtained in this study are that alternative splicing of At1g74140 was mainly attributed to two regions of this gene. Based on the RT-PCR results, a conclusion was reached by the authors that the overall discontinuous splicing pattern of the At1g74140 gene may reflect its mode of involvement in activities like controlling gene expression.\nComments: Since the study just verified the splice variants annotated in public available database, no new variant was detected. RT-PCR can be no doubt used to detect particular type of splicing events, but this method is prone to false positives, especially when too many PCR cycles were used. Given the PCR cycles used in this study were 35-40, it is difficult to conclude that some of the faint bands saw in the gel were come from mRNA rather than contaminations. At least, no RT samples should be included as negative controls in these PCR experiments, which was missing. Further, the conclusion that the discontinuous splicing pattern of the At1g74140 gene is associated with gene expression does not have any support from these RT-PCR analysis; no gene expression analysis was conducted to show any of these splice variant contribute to gene expression levels.\n\nSuggestions:\nFigures 2-4 may be combined with Figures 5-10 and Figures S1-S3, which will help readers to better understand how the experiments were performed and what these figures are showing. Essentially, these figures summarized results from RT-PCR validation of alternative splicing events of At1g74140 in the tissues sampled.\n\nThe gel images in Figure 5-10 are of low quality. The unnecessary portions of the gel images in the six figures can also be cropped off, they are just distracting.\n\nWill the authors be able to show which variant(s) was detected predominantly in particular tissues or under specific growth conditions? That information may link AS events with physiological processes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/8-1925
https://f1000research.com/articles/8-155/v1	05 Feb 19	{ "type": "Case Report", "title": "Case Report: Myocarditis along with fulminant hepatic failure secondary to acute hepatitis E infection", "authors": [ "Lajpat Rai", "Osama Salam", "Uzair Yaqoob", "Ujala Zubair", "Lajpat Rai", "Osama Salam", "Ujala Zubair" ], "abstract": "Myocarditis, defined as the inflammation of myocardial tissue has many causes which may be viral, metabolic or bacterial in origin. In this case we report a patient aged 22 years who was admitted with presenting complains of loss of consciousness, generalized muscle rigidity and yellowish discoloration of skin. During the course of his hospital stay, patient developed signs of myocarditis and later died of hypotensive shock. Viral serology was positive for the presence of hepatitis E virus (HEV), a rare cause of myocarditis. HEV infection can range from asymptomatic disease course to fulminant hepatitis but in rare cases it has been found to be a cause of myocarditis. This is so far the sixth case of hepatitis E induced myocarditis.", "keywords": [ "Myocarditis", "Hepatitis", "hepatic failure", "Hepatitis E", "Acute hepatitis" ], "content": "Introduction\n\nHepatitis E (HEV) virus is an important cause of morbidity and mortality and constitutes a significant public health problem1. Hepatitis E transmission includes fecal-oral, vertical and transfusion routes2. Symptoms of HEV infection ranges from asymptomatic to fulminant hepatitis, which is most common in pregnant women. Other extra-hepatic manifestations seen in HEV infection includes Guillian Barre syndrome, neuralgic amyotrophy, glomerulonephritis, cryoglobulinemia, pancreatitis, leukemia, thrombocytopenia, meningitis, thyroiditis, neuro-myopathy, vestibular neuritis and arrhythmias3,4. This extra-hepatic involvement seen in an association of HEV is documented in various articles but needs further clinical studies and research3. Here we report a rare case of HEV virus associated myocarditis in Karachi, Pakistan. The other four reported cases are documented in India5,6. and one case was documented in a western hemisphere traveler7.\n\n\nCase presentation\n\nA 22 year old male student, from the Sindh province of Pakistan, unmarried, presented to the emergency department of Jinnah Postgraduate Medical Centre, Pakistan, in April 2018 with loss of consciousness, convulsions, and generalized rigidity. He was admitted to the gastroenterology high dependency unit (HDU). On physical examination his temperature was 100°F, pulse was 136 bpm, blood pressure (BP) of 110/70 mm/Hg, and respiratory rate of 28 breaths per minute. The patient was unresponsive to pain with bilateral reactive pupil, and there was a slight yellow skin pigmentation of palms and sole with yellow sclera. The patient was not maintaining oxygen saturation hence intubation and ventilatory support was required and so patient was shifted to the intensive care unit. Abdominal exam was normal without hepatosplenomegaly or ascites, respiratory and heart sounds were audible and normal. His past medical history was negative and was non-contributory. Laboratory parameters at that time revealed, altered liver function tests (LFTs) with serum glutamic pyruvic transaminase (SGPT) 1160 u/L (reference range, 7–56 u/L); aspartate aminotransferase (AST) 225 u/L (reference range, 10–40 u/L); gamma-glutamyltransferase (GGT) 51 u/L (reference range, 9–50 u/L); alkaline phosphatase (ALP) 372 u/L (reference range, 150–480 u/L); serum albumin 4.2 g/dL (reference range, 3.5–5.5 g/dL); total bilirubin 4 mg/dL (reference range, 0.1–1.2 mg/dL); platelet time (PT) 13 seconds (reference range, 11–14 seconds); and international normalized ratio (INR) 13 seconds (reference range, 0.9–1.2 seconds). His blood urea was 61 mg/dl (Reference range, 7–20 mg/dL), serum creatinine of 1.64 (reference range, 0.6–1.2 mg/dL), with a normal serum electrolyte panel. A viral screen of the patients blood was ordered and HEV IgM and IgG was positive, with negative serology for Hepatitis B, Hepatitis C, Hepatitis D, Dengue, Typhoid, Cox-B, Epstein-Barr virus (EBV), Leptospira, Herpes simplex virus, Adenovirus and HIV. Blood cultures were negative, rapid malaria test was negative, labs for antinuclear antibody, anti-mitochondrial antibody and anti-smooth muscle antibody was also negative. Sonographic investigation showed normal abdominal structures. Patient’s blood urea and creatinine returned to normal on the second day of admission and LFT’s started recovering by the fourth day. On the seventh day of hospitalization, the patient had normal total bilirubin with slightly raised LFT’s of ALP 120 u/l, SGPT 115 u/l, AST 62 u/l, GGT 68 u/l and platelet of 201×109, leukocytes 27×109 with high-grade fever. On the seventh day of admission the patient became hypotensive with BP of 92/68 mm/Hg and a pulse of 148. Physical examination revealed distended external jugular veins, with bilateral pedal edema and bilateral chest congestion. Chest examination showed decreased respiratory sounds from the right and left lung base with muffled heart sounds. On further investigations and workups a chest X-Ray showed bilateral pulmonary congestion at the base of the lung with cephalization of vessels. An electrocardiography (ECG) showed nonspecific ST segment and T-wave abnormalities with supraventricular tachycardia (Figure 1). Creatinephosphokinase (CPK) came out to be 8414 u/l (the patient had no history of seizures). Coronary angiography was performed to exclude acute coronary syndrome, which revealed no obstructive lesion in the coronary artery. Echocardiography was performed which demonstrated impaired left ventricular function with diffuse hypokinesia without any pericardial effusion and ejection fraction of 30 %. The sign and symptoms together with workups and labs were highly suggestive of myocarditis. The patient was treated accordingly an injection of Risek (40 mg once daily), 25% dextrose (as per needed), colomycin (2 million IU thrice daily) and moxifloxacin (400 mg once daily), acyclovir (500 mg thrice daily); syp duphalac (30 ml 6 hourly) was given nasogastrically; and patient was kept sedated with propofol. Despite aggressive medical support, the patient abruptly developed cardiopulmonary arrest on the eighth day of admission, resuscitation was performed but the patient didn’t recover and died of cardiopulmonary arrest. More specific investigations were planned but could not be performed post-mortem.\n\n\nDiscussion\n\nAccording to the World Health Organization, the annual incidence of HEV infection is 20 million around the globe, with the majority of cases from East and South Asia8. It is common in resource-limited countries with limited access to essential water, sanitation and health services. Our patient contracted an acute HEV infection causing acute fulminant hepatitis leading to hepatic encephalopathy, coma and loss of consciousness. He was intubated and managed appropriately in intensive medical care, patients LFT’s were recovering but unfortunately, he developed myocarditis and died of cardiopulmonary arrest. Myocarditis is the inflammation of the myocardium with inflammatory infiltrate, necrosis and degeneration of myocytes in the myocardium8. Among all other causes of myocarditis, viral etiology appears to be the most common in developed countries, whereas in developing countries its rheumatic carditis, Chagas disease and HIV associated diseases are more common9,10. As there is so much clinical variability, diagnosing myocarditis is a challenge. Diagnostic guidelines such as those of American college of cardiology/American heart association and Dallas criteria provide help with early diagnosis4. On eighth hospitalization day, our patient developed hypotension and distended jugular venous distension, pedal edema, bilateral pulmonary edema and muffled heart sounds. On initial workup, cardiac enzymes, ECG, CPK tests were planned. Echocardiography is recommended as an initial diagnostic tool for suspected myocarditis11. According to small observational studies, it was suggested that magnetic resonance imaging (MRI) can identify morphological and anatomical changes such as inflammation, edema and myocyte injury in the myocardium. Due to myocardial inflammation T1 and T2 relaxation times and spin densities gives accurate tissue characterization12. Endomyocardial biopsy is also an important diagnostic tool in myocarditis and sometimes is the only means of diagnosing myocarditis. Endomyocardial biopsy may be used for patients whose condition does not respond to conventional supportive therapy, a patient with acute dilated cardiomyopathy associated with hemodynamic compromise, refractory cases, patients with life-threatening arrhythmias and high-grade AV block13. Histological evaluation is also diagnostic for myocarditis, however, because of variable observer interpretations and focal inflammation sampling error can be high14. In our case our diagnosis was limited as these confirmatory tests just mentioned i.e. echocardiography, biopsy and MRI, could not be performed due to the early death of the patient.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the relatives of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nEuropean Association for the Study of the Liver: EASL Clinical Practice Guidelines: management of hepatitis C virus infection. J Hepatol. 2011; 55(2): 245–64. PubMed Abstract \| Publisher Full Text\n\nKhati C: Hepatitis E-not so benign after all! J Med Res. 2016; 2(4): 118–20. Reference Source\n\nPischke S, Hartl J, Pas SD, et al.: Hepatitis E virus: Infection beyond the liver? J Hepatol. 2017; 66(5): 1082–95. PubMed Abstract \| Publisher Full Text\n\nWoolson KL, Forbes A, Vine L, et al.: Extra-hepatic manifestations of autochthonous hepatitis E infection. Aliment Pharmacol Ther. 2014; 40(11–12): 1282–91. PubMed Abstract \| Publisher Full Text\n\nPremkumar M, Rangegowda D, Vashishtha C, et al.: Acute viral hepatitis e is associated with the development of myocarditis. Case Reports Hepatol. 2015; 2015: 458056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoyal B, Mishra DK, Kawar R, et al.: Hepatitis E associated myocarditis: an unusual entity. Bombay Hosp J. 2009; 51(3): 361–2. Reference Source\n\nDougherty T, Showkat B, Adam MK, et al.: Acute myopericarditis due to Hepatitis E virus infection: the first reported case in the western hemisphere. J Gastrointest Dig Syst. 2016; 6: 386. Publisher Full Text\n\nMaisch B, Portig I, Ristic A, et al.: Definition of inflammatory cardiomyopathy (myocarditis): on the way to consensus. A status report. Herz. 2000; 25(3): 200–9. PubMed Abstract \| Publisher Full Text\n\nCooper LT Jr: Myocarditis. N Engl J Med. 2009; 360(15): 1526–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahrholdt H, Wagner A, Deluigi CC, et al.: Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation. 2006; 114(15): 1581–90. PubMed Abstract \| Publisher Full Text\n\nPinamonti B, Alberti E, Cigalotto A, et al.: Echocardiographic findings in myocarditis. Am J Cardiol. 1988; 62(4): 285–91. PubMed Abstract \| Publisher Full Text\n\nLaissy JP, Messin B, Varenne O, et al.: MRI of acute myocarditis: a comprehensive approach based on various imaging sequences. Chest. 2002; 122(5): 1638–48. PubMed Abstract \| Publisher Full Text\n\nSchultz JC, Hilliard AA, Cooper LT Jr, et al.: Diagnosis and treatment of viral myocarditis. In: Mayo Clin Proc. Elsevier. 2009; 84(11): 1001–1009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaughman KL: Diagnosis of myocarditis: death of Dallas criteria. Circulation. 2006; 113(4): 593–5. PubMed Abstract \| Publisher Full Text" }	[ { "id": "50664", "date": "15 Jul 2019", "name": "Jun Inoue", "expertise": [ "Reviewer Expertise Hepatology", "Viral hepatitis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, Rai et al. described a patient with acute hepatitis E virus infection who developed acute heart failure. Some cases with hepatitis E virus-associated myocarditis have been reported previously and the authors considered the development of myocarditis also in this case, but the diagnosis was not based on enough tests.\nThe authors diagnosed him as having myocarditis, but the evidence is not enough for the diagnosis.The finding of electrocardiogram is not typical. As discussed in the manuscript, endomyocardial biopsy or other supportive tests are required.\n\nIn Figure 1, why are the same 3 pictures of electrocardiogram (only lead I, II, and III) shown? 12-lead electrocardiogram should be shown.\n\nIn Introduction, HEV is an abbreviation of ‘hepatitis E virus’, not ‘hepatitis E’.\n\nPlease show general names of drugs that were used for this patient.\n\nA result of HAV test is not shown.\n\nUnits of platelet and leukocyte counts are not shown.\n\nThe characteristics of reported cases with hepatitis E virus-associated myocarditis should be described in the discussion section.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [ { "c_id": "5007", "date": "05 Nov 2019", "name": "Uzair Yaqoob", "role": "Author Response", "response": "Thank you so much for taking your time out and reviewing our work. we have worked and updated our article according to your comments and it will soon be online. Can you please elaborate Number \"7\" of your comments a little more?" }, { "c_id": "5011", "date": "18 Nov 2019", "name": "Uzair Yaqoob", "role": "Author Response", "response": "(Response to rest of the comments) 1.as patient was on mechanical ventilation, he had dyspnoea, became tachycardic with raised JVP, ECG got done showed ST-T wave abnormalities along with SVT, and Echocardiogram showed Ejection fraction of 30%. So on basis of ECG, Echocardiogram and clinical findings patient diagnosed as myocarditis. 2. added in the updated version, with apologies for the mistake earlier 3. Corrected 4.done 5. Added, it was non-reactive 6. Added" } ] }, { "id": "51461", "date": "08 Aug 2019", "name": "Madhumita Premkumar", "expertise": [ "Reviewer Expertise Cirrhotic cardiomyopathy", "critical care in cirrhosis", "coagulation in cirrhosis." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report describes a 22- year old patient who had acute viral Hepatitis E who presented with altered sensorium possibly hepatic encephalopathy, but there are several unexplained details which alter the diagnostic specificity:\nWhy did the patient have generalized muscle rigidity?\n\nClearly, he was intubated for poor sensorium and it is apparent from the clinical examination that he had no heart failure signs at presentation, with normal heart sounds and chest examination.\n\nWhy are the authors mentioning SGPT on one hand and AST on the other? Surely it will be wiser to use AST and ALT, as is the norm.\n\nThe authors write ‘platelet time (PT) 13 seconds (reference range, 11–14 seconds); and international normalized ratio (INR) 13 seconds (reference range, 0.9–1.2 seconds)’. There is no test called ‘platelet time’ I believe that they intend to write ‘Prothrombin time’. The INR is a ratio. It could not be 13 seconds with the reference range being 0.9-1.2.\n\nThe authors write ‘platelet of 201×109, leukocytes 27×109’ No units are mentioned.\n\nThe sign and symptoms together with workups and labs were highly suggestive of myocarditis. The criteria for myocarditis need to be met. It is likely the patient was in sepsis. The levels of biomarkers like Troponin I and T is not mentioned. Also, the MB fraction of creatine kinase needs to be done in serial tests. Echocardiographic images would be required. The ECG supplied shows sinus tachycardia with secondary ST-T changes.\n\nThe patient was treated accordingly an injection of Risek (40 mg once daily), 25% dextrose (as per needed), colomycin (2 million IU thrice daily) and moxifloxacin (400 mg once daily), acyclovir (500 mg thrice daily); syp duphalac (30 ml 6 hourly) was given nasogastrically; and patient was kept sedated with propofol. Please avoid use tradenames of drugs used in the treatment. The indication for acyclovir is unclear.\n\nWhen was the coronary angiogram done? Are images available? Was there a suspicion of acute coronary syndrome in this patient?\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [ { "c_id": "5010", "date": "18 Nov 2019", "name": "Uzair Yaqoob", "role": "Author Response", "response": "(Responses numbered acc to comments) 1.Patient was in hepatic encephalopathy and was showing signs of raised ICP due to which muscle tone was increased and muscle rigidity was present. 2. Exactly, there were no signs of myocarditis on presentation. After patient was intubated and kept under support, he became hemodynamically unstable and arrhythmic. 3. done in the next update 4. Corrections made in the next update 5. Mentioned in the next update 6. Agreed, all these investigations were planned but due to the unfortunate demise of the patient, these could not be done. Portable echocardiogram was since the patient was on mechanical ventilation, where just the important findings are given by an experienced radiologist on a wriiten note, so due to the limited resources in our setup images could not be provided 7. Names corrected, since viral myocarditis was suspected, we started acyclovir 8. It was planned, but as mentioned, could not be done" } ] } ]	1	https://f1000research.com/articles/8-155
https://f1000research.com/articles/8-1922/v1	15 Nov 19	{ "type": "Study Protocol", "title": "Impact of interventions including vaccination against Neisseria meningitidis on the frequency of meningitis in the African meningitis belt: a scoping review protocol", "authors": [ "Niurka Molina", "Greissi Justiniani", "Lisset Urquiza", "Maria Eugenia Toledo", "Chukwuemeka Onwuchekwa", "Kristien Verdonck", "Ermias Diro", "Nivaldo Linares-Pérez", "Niurka Molina", "Greissi Justiniani", "Lisset Urquiza", "Maria Eugenia Toledo", "Chukwuemeka Onwuchekwa", "Ermias Diro", "Nivaldo Linares-Pérez" ], "abstract": "In the African meningitis belt (region from Senegal to Ethiopia), there are around 30,000 reported cases of meningococcal disease per year. The main aetiological agent is Neisseria meningitidis of serogroup A. Since 2010, vaccination efforts have increased and hundreds of millions of people have been vaccinated. There are indications that the epidemiology of meningococcal disease is changing. This is the protocol of a scoping review, the objective of which is to describe the extent and nature of the research evidence about the impact of vaccination on meningitis frequency. Primary studies and reviews are eligible for inclusion in the review if they assess the impact of interventions that include N. meningitidis vaccination in countries of the African meningitis belt, report meningitis frequencies, and include an element of comparison. The sources of records are electronic databases (MEDLINE, Cochrane register of clinical trials, African Index Medicus, and clinicaltrials.gov), surveillance reports at country level, online resources of large stakeholders involved in vaccination, reference lists of included records, and experts in the field. The search strategy is based on the combination of the condition of interest, the intervention, and the geographical region. The findings of this review will be presented using figures, tables, and thematic narrative synthesis. This review will not produce a pooled estimate of what the impact of vaccination is, but will give insight in how the authors of the included records assessed the impact.", "keywords": [ "Neisseria meningitidis", "vaccination", "health impact assessment", "scoping review" ], "content": "Introduction\n\nNeisseria meningitidis is a Gram-negative bacterium that is found in the mucous membrane of the nasopharynx and tonsils of about 10% of the human population1. Most N. meningitidis strains are harmless, but some encapsulated clones are virulent and can cause meningococcaemia, meningitis, and septic shock1. Historically, the highest incidence of meningococcal disease has been described in sub-Saharan Africa, in the so-called African meningitis belt, which stretches from the west of Senegal to the east of Ethiopia2. In this region, endemic rates are high, and large-scale epidemics have occurred every 8–12 years for more than a century, typically in dry seasons1–4. The number of cases of meningococcal meningitis reported from the African meningitis belt is around 30,000 per year4.\n\nThere are at least 13 serogroups of N. meningitidis (A, B, C, D, E, H, I, K, L, W135, X, Y, and Z) that are classified based on differences in capsule polysaccharides1. Serogroup A used to be the main causative agent of epidemics in Africa, but massive vaccination campaigns are changing the epidemiology3,5. Validated and licensed conjugate vaccines are available for serogroups A (MenAfriVac®) and C, and there is also a tetravalent vaccine for serogroups A, C, Y, and W1354,6. These vaccines can be used in routine settings (part of routine immunisation scheme) and in response to outbreaks (reactive vaccination)4. Vaccination efforts intensified in 2010 and since then, hundreds of millions of Africans have received a dose of MenAfriVac®3. As a consequence, the incidence of meningococcal meningitis due to serogroup A has decreased, but outbreaks of new clones have been reported3,5.\n\nOur main objective is to evaluate the impact of vaccination on morbidity and mortality due to meningococcal disease in countries of the African meningitis belt. Before engaging in a systematic review, we will assess the size and scope of the body of literature6. The aim at this stage is not to produce a pooled estimate of what the impact of vaccination is, but to evaluate how the authors of the included records have assessed the impact.\n\n\nObjectives\n\nThe central question of this scoping review is: what is the extent and the nature of the research evidence about the impact of interventions including vaccination against Neisseria meningitidis on the frequency of meningitis in the African meningitis belt? The review question is formulated using the SPICE (setting, perspective, intervention, comparison, evaluation) framework and the key elements are summarised in Table 17.\n\n\nMethods\n\nRecords will be included in the review if they meet all the following criteria:\n\n- Reports of primary studies or review articles (not opinion papers); and\n\n- About people living in one of 27 countries corresponding to the African meningitis belt (any population group, any age); and\n\n- Assessing the impact of interventions that include N. meningitidis vaccination; and\n\n- Including an element of comparison (populations with versus without vaccination, or before versus after vaccination); and\n\n- Reporting meningitis frequency. The reported condition can be meningitis due to N. meningitidis, meningitis in general, or death due to meningitis. Disease frequency can be expressed as absolute number of cases, prevalence, or incidence. The denominator can be the general population or a subgroup (e.g. meningitis patients).\n\nRecords reporting the impact of mixed interventions (vaccination for N. meningitidis + other interventions such as chemoprophylaxis to prevent meningococcal disease among contacts or vaccination for other pathogens) will be included. If we find a record that reports findings both from countries inside and outside the African meningitis belt, we will include that record and extract only that part of the data that comes from one of the 27 target countries for this review.\n\nA sheet with detailed eligibility criteria will be used for record screening (based on titles and abstracts) and selection (based on full-text papers). A preliminary version of this sheet is available as extended data8. The detailed selection criteria will be pilot-tested on 50 titles and abstracts and refined if necessary.\n\nWe will search the following electronic databases: MEDLINE, the Cochrane register of clinical trials, African Index Medicus, and clinicaltrials.gov. Other sources of information will be surveillance reports at country level and online resources of the World Health Organization and other large stakeholders involved in vaccination campaigns (to be identified via the included records). Finally, we also intend to screen the reference lists of included records (especially review papers) and contact experts in the field to check if we have missed any potentially relevant records. There will be no restrictions regarding language, publication date, or study design.\n\nThe search strategy is based on the combination of three concepts: the condition of interest, the intervention, and the geographical region (Figure 1). The Boolean operators “AND” and “OR” are used to combine search terms. The planned search syntax for PubMed is given in Table 2. The same general strategy will be used to search the other databases, but small adjustments will be made such as the translation of key words to French, and the adaptation of truncation symbols and parentheses to different search engines.\n\nData management. Retrieved records will be automatically exported to Microsoft Excel if possible (e.g. from PubMed) and manually added otherwise. All records will get a unique identifier. Information extracted from the included records will be stored in the Excel file. Records that remain after title and abstract screening will also be kept in an EndNote file.\n\nSelection process. Record screening and selection will be done in duplicate by two independent members of the review team (LU and/or GJ and/or NM). Any discordances during screening of titles and abstracts or full-text papers will be solved through discussion with a third member of the review team (KV). For each full-text record that we exclude, the main reason for exclusion will be recorded. The search and selection process will be documented in a PRISMA flowchart9.\n\nData collection process. Two members of the review team (LU and/or GJ and/or NM) will independently extract the information from the included records using a standard form (preliminary version available as extended data8). This data extraction form will be piloted on at least three full-text records and refined if necessary. The extracted information will first be filled out on the data extraction form (one form per reviewer and per record) and then passed to the Excel file. In case the information is unclear or incomplete, we will describe it as such; we do not intend to contact investigators. Any discordances between the two reviewers will be discussed with a third reviewer (KV).\n\nWe will collect information about the record itself and about the study described in the record. Table 3 gives an overview of the data items. The complete preliminary data extraction form is available as extended data8.\n\nRather than focusing on one or a few specific outcomes, this review focuses on the size and scope of the available research literature and on the nature and extent of the research evidence. The approach will be descriptive; we do not foresee outcome prioritization.\n\nThe assessment of risk of bias will be done at study level and independently by two people of our review team. As we are using broad eligibility criteria for this scoping review, we expect to include information in heterogeneous formats and coming from studies following different designs. For randomised trials of interventions, we plan to use the risk-of-bias assessment tool of the Cochrane Collaboration, and for non-randomised studies the ROBINS-I tool. For studies following other designs, we will only describe the study design and the ways impact of vaccination was described and assessed. We plan to describe and discuss the findings of the assessment of risk of bias and will not use them in any other way in data synthesis.\n\nThe findings of this review will be presented using figures, tables, and thematic narrative synthesis. Data from the included studies will not be pooled and the synthesis will not lead to recommendations on vaccination for N. meningitidis.\n\nA preliminary structure of the results section is given below, but this may slightly change depending on the content of the included papers:\n\n- Search and selection (with PRISMA flowchart)\n\n- Characteristics of included records: publication type, year, journal, author affiliations\n\n- Study populations: country, setting, size, general population or subgroups\n\n- Interventions: vaccination alone or in combination, rationale, in outbreak, routine or research settings, by government or others\n\n- Vaccines used: type, brand, provider\n\n- Approaches to assess impact: overview of definitions and operationalisation of impact\n\n- Study design: according to the study authors and according to the review team\n\n- Risk of bias assessment\n\n\nReporting and registration\n\nThe present review protocol was developed following the Preferred Reporting Items for Systematic review and Meta-Analysis (PRISMA) guidelines, more specifically the checklist for review protocols (PRISMA-P 2015) and the extension for scoping reviews (PRISMA-ScR 2018)9–11.\n\nThe review protocol will be published so that it is publicly available before the actual reviewing activities start. PROSPERO is a specialized platform for review protocols but does not accept scoping reviews. We therefore publish the current protocol on F1000Research, an open access scientific publishing platform. Any changes in the reviewing activities after protocol registration will be listed in the final review paper.\n\n\nPlanning\n\n- Protocol publication: November 2019\n\n- Search, selection, data extraction and synthesis: November 2019 – February 2020\n\n- Writing of review paper: February 2020 – April 2020\n\n\nReview team and roles\n\nThe review team is presented in Table 4.\n\n\nStudy status\n\nWhile preparing the present protocol, we tried out preliminary searches to get an idea of the size of the available literature. At the time of submission, formal reviewing activities had not started yet.\n\n\nData availability\n\nNo underlying data are associated with this study.\n\nFigshare: SupplementaryInformation_Eligibility_DataExtraction. https://doi.org/10.6084/m9.figshare.10078928.v18.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBrandtzaeg P: Oxford textbook of medicine: infection. 5th ed. Oxford (United Kingdom): Oxford University Press; Chapter 6.5, Meningococcal infections; 2012; 318–332. Reference Source\n\nMustapha MM, Harrison LH: Vaccine prevention of meningococcal disease in Africa: major advances, remaining challenges. Hum Vaccin Immunother. 2018; 14(5): 1107–1115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammed I, Iliyasu G, Habib AG: Emergence and control of epidemic meningococcal meningitis in sub-Saharan Africa. Pathog Glob Health. 2017; 111(1): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Meningococcal meningitis key facts. (accessed 24 October 2019). Reference Source\n\nBorrow R, Caugant DA, Ceyhan M, et al.: Meningococcal disease in the Middle East and Africa: Findings and updates from the Global Meningococcal Initiative. J Infect. 2017; 75(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nGrant MJ, Booth A: A typology of reviews: an analysis of 14 review types and associated methodologies. Health Info Libr J. 2009; 26(2): 91–108. PubMed Abstract \| Publisher Full Text\n\nBooth A: Clear and present questions: Formulating questions for evidence based practice. Library Hi Tech. 2006; 24(3): 355–368. Publisher Full Text\n\nVerdonck K: SupplementaryInformation_Eligibility_DataExtraction. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10078928.v1\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015; 350: g7647. PubMed Abstract \| Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018; 169(7): 467–473. PubMed Abstract \| Publisher Full Text" }	[ { "id": "63667", "date": "03 Jun 2020", "name": "Clare French", "expertise": [ "Reviewer Expertise Epidemiology", "meningococcal disease", "research synthesis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments This is a clearly written protocol for a scoping review to describe the extent and nature of the research evidence about the impact of vaccination on meningitis frequency. We note that this is being conducted before the research team embark on a full systematic review on the topic.\n\nIntroduction Importantly the numbers of cases (30,000 a year) quoted refer to suspected cases due to any cause, not specifically meningococcal meningitis. I note that the WHO reference to key facts is misleading on this point. Most reported cases are not laboratory confirmed. The majority of confirmed cases are meningococcal, but a substantial proportion are pneumococcal meningitis1.\nThe authors should emphasize this in the protocol and clarify how/that they will take account of information on laboratory confirmation of meningococcal and other pathogens when interpreting the data obtained. This is clearly important in assessing the impact of meningococcal vaccines and the validity of the review.\nMinor: There are currently 12 (not 13) serogroups, and W135 is no longer used, it has been replaced by W2. In the abstract, not in the main text, it is stated that Serogroup A is the main aetiological agent of meningitis in the belt. This was true until 2010, but not since. There are 26 (not 27) countries in the meningitis belt according to WHO https://www.who.int/gho/epidemic_diseases/meningitis/en/.\n\nTrotter, C.L., et al., Impact of MenAfriVac in nine countries of the African meningitis belt, 2010-15: an analysis of surveillance data. Lancet Infect Dis, 2017. 17(8): p. 867-872.1. Harrison, O.B., et al., Description and nomenclature of Neisseria meningitidis capsule locus. Emerg Infect Dis, 2013.# 19(4): p. 566-73.2.\n\nMethods The authors plan to search various sources in addition to electronic databases but they don’t mention searching conference abstracts. Though this is not essential for a scoping review it could be another way of identifying work in progress – such work may well have been published by the time the authors conduct their planned systematic review on the topic so it might be helpful to include it at this stage.\nThe search strategy looks fine for the purpose of a scoping review. The authors might consider adding the following Mesh term: ‘Meningococcal Infections’. The authors state that they will include both primary studies and review articles. Some clarification is required as to how they will avoid reporting on individual studies more than once (i.e. where an eligible individual study is identified and that study is also included in an eligible systematic review) - will reviews be used as a means of identifying individual studies to report on or will they be reported on at the review level?\nIt sounds as though the search hits will be screened for eligibility in Excel. This is fine if it is the authors’ preference but an alternative could be to use free screening software such as Rayyan which can help organise and speed up the screening process (https://rayyan.qcri.org/welcome).\nThe authors state that they will assess the risk of bias for individual studies included in their review. Although this will be an important element of the full systematic review planned by the authors it is not necessarily required for a scoping review e.g. see the PRISMA extension for scoping reviews guidance on this: https://www.acpjournals.org/doi/10.7326/M18-0850. It is not clear that an assessment of risk of bias is required here. We suggest the authors give careful consideration as to whether this step is necessary, particularly given that it can be quite time consuming, and what it will add to the scoping review. If the authors do feel that risk of bias assessment is required, they should provide clear reasoning for this in relation to the review objectives.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "121240", "date": "14 Mar 2022", "name": "Charlene M Kahler", "expertise": [ "Reviewer Expertise Meningococcal disease", "meningococcal genomic epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments The scoping review is well written and should achieve the outcomes stated.\nMinor comments Serogroup B disease is an issue in Africa and is increasing in frequency by causing local sporadic clusters, so this should be added to your search terms. Meningococcal disease is only one of many agents causing meningitis, so please be sure that your search terms can make this distinction or try to find studies that use follow up steps to confirm the causative agent. All cases of meningitis are treated the same - with ceftriaxone or penicillin, so you cannot really dissect useful information using this as a term to extract this information.\nOverall, I look forward to the final outcome of this study as a review on this topic has not been done for some time.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1922
https://f1000research.com/articles/8-1734/v1	09 Oct 19	{ "type": "Case Report", "title": "Case Report: Case report: A rare case of middle-ear Rhabdomyosarcoma in a 4-year-old boy", "authors": [ "Richard Menzies-Wilson", "Gentle Wong", "Prodip Das", "Gentle Wong", "Prodip Das" ], "abstract": "We present a rare case of a four-year-old boy with a botyroid embryonal rhabdomyosarcoma of the right middle ear. Rhabdomyosarcoma is a soft tissue malignancy which is thought to originate from embryonic mesenchymal cells of striated skeletal muscle. It is a disease primarily of children and is exceptionally rare in parameningeal regions. The diagnosis is often delayed and easily misdiagnosed as aural polyp. Therefore, advanced disease is common at the time of diagnosis. A four-year-old boy presented with a four-month history of recurrent left ear blood and pus discharge, otalgia and fevers. He attended his GP three times and paediatric A&E 13 times where he received antibiotics for presumed otitis media and externa. He was eventually referred to the otolaryngology department and underwent an examination under anaesthesia of ear and excisional biopsy of a suspicious aural polyp. Staging chest CT and PET scan showed no loco-regional spread or distal metastasis. Magnetic resonance imaging demonstrated absence of invasion into adjacent organs. Histology confirmed embryonal rhabdomyosarcoma, botryoid subtype. Subsequent to the initial excision of the polyp, he was started on an ifosfamine, vincristine and actinomycin (IVA) chemotherapy regime in three weekly cycles for nine cycles with concomitant radiotherapy. Two weeks subsequent to his first chemotherapy dose he presented with a House-Brackmann II-III facial nerve palsy but no other middle ear complications. He was started on intravenous antibiotics and dexamethasone. The facial nerve palsy incompletely resolved with treatment.", "keywords": [ "Rhabdomyosarcoma", "middle ear" ], "content": "Introduction\n\nRhabdomyosarcoma is a rare soft tissue malignancy with unknown aetiology. It is thought to originate from embryonic mesenchymal cells of striated skeletal muscle. It is a disease primarily of children, with two-thirds of patients presenting under the age of six years1. The commonest areas of the body to be affected are the head and neck, the bladder and reproductive organs. They are rare elsewhere and are exceptionally rare in parameningeal regions, including the ear and mastoid.\n\nSymptoms depend on the location of the tumour. Aural involvement often has non-specific symptoms such as fever and otalgia. If the tumour is significant in size and protrudes through the tympanic membrane, then otorrhoea and hearing loss may result. Rarely, facial nerve palsy may occur. The diagnosis is often delayed and easily misdiagnosed as aural polyp. Therefore, advanced disease is common at the time of diagnosis.\n\nWe present a rare case of a four-year-old boy with a botyroid embryonal rhabdomyosarcoma of the right middle ear and review the literature.\n\n\nCase report\n\nA four-year-old boy presented with a four-month history of recurrent left ear blood and pus discharge, otalgia and fevers. He attended his general practitioner (GP) three times and Paediatric Accident & Emergency 13 times where he received oral, topical and intravenous antibiotics for presumed otitis media and externa. He had no relevant family history. He was eventually referred to the otolaryngology department after 19 months and was listed for an examination under anaesthesia of and excisional biopsy of a suspicious aural polyp 2 weeks after presentation to the otolaryngology department team. Intra-operatively, a large lesion extending from the middle ear through the tympanic membrane and into the external auditory canal was noted which was resected. The lesion was found to be highly vascular.\n\nHistopathological findings were in keeping with embryonal rhabdomyosarcoma, botryoid subtype. This rare diagnosis required a second opinion from the Histology Department at the Royal Marsden Hospital. Immunochemical staining revealed sheets of rounded tumour cells with scant cytoplasm and inconspicuous nucleoli, which were distinguished by the formation of polypoid and grapelike tumour masses. Malignant cells in an abundant myxoid stroma were observed. Staining was positive for desmin and muscle-specific actin.\n\nMagnetic resonance imaging (MRI) of the head was performed within two weeks of resection of the tumour, which demonstrated absence of invasion into adjacent organs. Staging chest MRI showed no evidence of metastases and PET scan showed no loco-regional spread or distal metastasis. Bone marrow trephine and CSF were negative. The patient’s haemoglobin levels were borderline anaemic but all routine blood tests were otherwise within normal limits.\n\nAt 5 weeks after initial presentation he was started on nine cycles of IVA chemotherapy: ifosfamine (3 g/m2 on days 1 and 2); vincristine (1.5 mg/m2 weekly during the first 7 weeks, then only on day 1 of each cycle); and Dactinomycin (at 1.5 mg/m2 on day 1) in three weekly cycles with concomitant radiotherapy.\n\nTwo weeks subsequent to his first chemotherapy dose he was admitted with neutropenic sepsis and also presented with a House-Brackmann II-III facial nerve palsy but no other middle ear complications. He was started on paediatric doses of intravenous Piperacillin / tazobactam and gentamicin for 5 days as an inpatient prior to being discharged on a paediatric oral dose of ciprofloxacin for the remainder of his chemotherapy course. The facial nerve palsy incompletely resolved with a 2-week course of 3 mg dexamethasone twice daily. He is being followed up on the following schedule: 3–4 months in the first 2–3 years, then twice a year up to the fifth year,\n\n\nLiterature review\n\nWe searched EMBASE, MEDLINE, UpToDate, CINAHL, TRIP Database, BMJ Best Practice, Google Scholar, Cochrane Database of Systematic Reviews and NICE Evidence Search with the following search terms: rhabdomyosarcoma (RMS); ear; aural; middle ear; inner ear; ear canal, parameningeal; ear neoplasm in July 2018.\n\nInclusion criteria were: diagnosis of rhabdomyosarcoma of the middle ear; published in a peer reviewed journal; any geographical location or language; any publication date; any age of patient.\n\nIn total 11 case reports were found. Of these, there was an equal male-female split. The clinical presentations were similar throughout the case reports, mimicking recurrent otitis media/externa. The average survival across the case reports was 7.2 months. We summarise the case reports in Table 1.\n\n\nDiscussion\n\nRhabdomyosarcoma of the middle ear and mastoid is rare2. Around 30% of cases of paediatric rhabdomyosarcoma occur in the head and neck involving the orbits, base of skull, nasal cavity and nasopharynx3 but only 3% occur in the ear or mastoid4. In total, 63% of rhabdomyosarcomas occur in children under 10 years old, with a peak between 2 and 5 years old6.\n\nPresentation often involves purulent or blood-stained discharge, hearing loss, aural polyps and granulation tissue. Clinical diagnosis is often delayed since the presentation is similar to that of chronic suppurative otitis media7. By the time of diagnosis, a facial nerve palsy is usually present and there is involvement of both the middle and external ear and petrous bone8,9. Metastases commonly spread to the lungs, liver, bones and extremities, and are present in approximately 30% of cases3.\n\nAccording to the International Classification of Rhabdomyosarcoma, these tumours are histopathologically classified into five categories: botryoid, spindle cell, embryonal, alveolar, and undifferentiated. Embryonal rhabdomyosarcoma is the most common, accounting for 70–80%, occurring mostly in the orbit and parameninges3. Alveolar is the second most common, usually presenting in the deep soft tissues of the extremities4.\n\nRisk factors for rhabdomyosarcomas are poorly understood. Epidemiological studies have suggested a link with radiation and recreational drug in utero, lower socioeconomic status and use of antibiotics soon after birth10. Several genetic syndromes, including some commonly known to otolaryngologists such as Rubinstein-Taybi syndrome and Beckwith-Wiedemann syndrome, are associated with increased prevalence of rhabdomyosarcoma.\n\nHistorically, rhabdomyosarcomas were invariably fatal. Now, 5-year survival rates of around 80% have been reported11; however, surgical removal is often difficult in head and neck cases given the stage of the neoplasm and anatomy. Consequently, the mainstay of treatment is often chemoradiotherapy. Parameningeal tumours, especially of the middle ear and mastoid, have a poor prognosis because of the proximity to the brain7. Of the categories, embryonal have the best prognostic factor and alveolar the worst and children have a better prognosis than adults4.\n\n\nConclusion\n\nRhabdomyosarcoma of the ear is a rare presentation but one which should be considered in young children with recurrent otitis media. If missed, facial nerve involvement is a common as well as local meningeal and distant metastasis. Multi-disciplinary involvement and patient education is mandatory in ensuring the highest survival rates. Treatment consists of a combination of surgery, chemotherapy and/or radiotherapy.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details as obtained from the parent of the patient study.", "appendix": "References\n\nRies L, Harkins D, Krapcho M: SEER Cancer Statistics Review. National Cancer Institute, Bethesda. 2003; Accessed 14 December 2017. Reference Source\n\nAgarwal NM, Popat VC, Traviad C, et al.: Clinical and histopathological study of mass in ear: a study of fifty cases. Indian J Otolaryngol Head Neck Surg. 2013; 65(Suppl 3): 520–525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerraz-filho J, Felipe LF, Floriano VH, et al.: Clinical and radiological findings of the middle ear' embryonal rhabdomyosarcoma and temporal bone in the children. Int Arch Otorhinolaryngol. 2010; 14(1): 123–126. Reference Source\n\nShirani S, Alizadeh F, Kashani S: Rhabdomyosarcoma of the Middle and Inner Ear. Iran J Radiol. 2003; 1: 157–160. Reference Source\n\nDurve DV, Kanegaonkar RG, Albert D, et al.: Paediatric rhabdomyosarcoma of the ear and temporal bone. Clin Otolaryngol Allied Sci. 2004; 29(1): 32–37. PubMed Abstract \| Publisher Full Text\n\nCotton R, Rothschild M, Zwerdling T: Tumors of the head and neck in children. In: Thawley SE, Panje WR, Batsakis JG, Lindberg RD, editors. Comprehensive Management of Head and Neck Tumors 2nd ed. Philadelphia: W.B. Saunders; 1999; 1846–1902.\n\nAkbar A, Sohail A: Rhabdomyosarcoma of the middle ear and mastoid: a case report and review of the literature. Ear Nose Throat J. 2005; 84(12): 780–784. Publisher Full Text\n\nWeiss S, Goldblum J: Soft Tissue Tumurs. 4th ed. St Louis: Mosby; 2001; 785–835.\n\nCastillo M, Pillsburg HC 3rd: Rhabdomyosarcoma of the middle ear: imaging features in two children. AJNR Am J Neuroradiol. 1993; 14(3): 730–733. PubMed Abstract\n\nFatih M, Hicks J: Rhabdomyosarcoma in childhood and adolescence: Epidemiology, pathology, and molecular pathogenesis. Uptodate. 2017; Accessed 14 December 2017. Reference Source\n\nDevaney KO, Boschman CR, Willard SC, et al.: Tumours of the external ear and temporal bone. Lancet Oncol. 2005; 6(6): 411–420. PubMed Abstract \| Publisher Full Text" }	[ { "id": "54913", "date": "16 Oct 2019", "name": "Max Cunnane", "expertise": [ "Reviewer Expertise Working as specialist registrar in otolaryngology. Particular interest include paediatric ENT and laryngology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting, concise and well-constructed case report and review of the relevant literature surrounding rhabdomyosarcoma affecting the middle ear. This is a rare entity and as such additional case reports are valuable in adding to the existing literature.\n\nThe case report contains adequate description of the patient and detail of the presentation and initial investigations, which would be in line with the reviewers own practice.\n\nThe case highlights the need for high clinical suspicion when treating non-resolving recurrent ear infections in children which is a useful message for clinicians.\n\nThe literature review results are presented clearly and contain all relevant information regarding the case’s reported. However, there is a lack of detail surrounding which chemotherapy/radiotherapy regimes were used. It is also unclear if they were adjuvant or primary treatment modalities for the other cases described.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "54912", "date": "31 Oct 2019", "name": "Craig Hickson", "expertise": [ "Reviewer Expertise Otology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a generally well written case report detailing a rare disease process in an unusual anatomical location. It covers pathogenesis, presentation, problems with diagnosis, treatment and prognosis.\nIt has multiple grammatical errors which need review and correcting:\n'Recreational drugs in utero.'\nThe diagnosis is often delayed and easily misdiagnosed as AN aural polyp\n'A four-year-old boy presented with a four-month history of recurrent left ear blood and pus discharge' - please revise to A four-year-old boy presented with a four-month history of recurrent haemo-purulent discharge from the left ear.\nIn addition I have the following comments/suggestions:\nIntra- operatively, a large lesion extending from the middle ear through the tympanic membrane and into the external auditory canal was noted which was resected. The lesion was found to be highly vascular.\n\nWas it totally resected? Via a tympanotomy? Or was the tissue lateral to the TM resected. If a tympanotomy was performed what was the status of the ossicles?\nYou say that the facial nerve palsy incompletely resolved. Please give details using House-Brackmann or other suitable classification.\nHow do you arrive at '3% of paediatric rhabdomyosarcoma occurring in the head and neck involving the …………..'? There are only 11 cases reported. You would need 100/3 = 30 cases or more to be able to state this?\nCould intra operation images and high res CT image be included?\nPlease state the disease free followup to date of your patient.\nPlease comment on hearing outcome.\nThere is no mention specific mention of MDT discussion? You state a second opinion was sought but surely this case went through an MDT?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5020", "date": "15 Nov 2019", "name": "Richard Menzies-Wilson", "role": "Author Response", "response": "Many Thanks for your comments. We have adjusted the manuscript to reflect your feedback." } ] }, { "id": "54909", "date": "05 Nov 2019", "name": "Adam Haymes", "expertise": [ "Reviewer Expertise Otolaryngology", "Rhinology", "Head & Neck surgery", "Tissue engineering", "regenerative medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a case of botyroid embryonal rhabdomyosarcoma of the right middle ear in a four year old child. It covers the clinical presentation, workup and subsequent management of this rare entity and reviews the pertinent literature of other case descriptions.\nThe case report is very well written, informative, covers an interesting and unusual topic, and adequately covers all of the objectives set out in the guidelines.\n\nThe background of the case’s history and progression are described in excellent detail.\nDescription of the physical examination and workup of the patient are detailed and methodical, as are the descriptions of diagnostic tests, treatments and outcomes.\nEmphasis is placed on the rarity of this clinical entity and thus it's contribution to the literature is a useful one. There were no novel treatments in this case report, however the description of the chemotherpay regimen will be useful for other clinicians. Description of the recurrent presentations to the GP and A&E departments are a sobering reminder of the need for these clinicians to recognise unusual clinical patterns, not rely on previous diagnoses and seek help from specialists early in such scenarios.\n\nImages of the histology would have been a nice to see given the rarity of this clinical entity.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1734
https://f1000research.com/articles/8-1919/v1	13 Nov 19	{ "type": "Research Article", "title": "Analysis of rural egg production to improve the economy of the Andean communities in Ecuador", "authors": [ "María Belén Bravo Avalos", "Maritza Vaca Cárdenas", "José Luis López Salazar", "María Fernanda Herrera Chico", "Jenny Margoth Villamarín Padilla", "Maritza Vaca Cárdenas", "José Luis López Salazar", "María Fernanda Herrera Chico", "Jenny Margoth Villamarín Padilla" ], "abstract": "Background: An economically active population refers to people involved in any economic activity, such as agriculture, livestock, or industry. This activity can supply benefits to its owners and may generate the growth of small businesses. Our study presents a qualitative analysis of the egg production in Ecuadorian Andean communities (Tzimbuto, Bayushig \"La Liberad,\" and La Victoria \"Pusuca.\") Methods: Analysis was performed using a random sample of 81 hens and 29 eggs obtained from 65 producers, belonging to 50 farming families at the Utopia Community Fair. Zoometric characterization of the hens was carried out according to FAO protocols, and egg yolk quality, egg width and weight, and egg shell weight and thickness were calculated. The 65 producers were interviewed to answer questions pertaining to selling eggs, costs of production, and realized profit. The profit/cost indicator was calculated from the ratio of expenditure (egress) and total revenue of egg sales. Results: 84% of surveyed participants indicated that they purchase eggs for their nutritional value, 12% because the animals are raised with organic food, and 4% showed preference for the eggs’ special flavor. The quality analysis of the eggs sold at the Fair demonstrated that the average weight of the eggs was 47.24 g; the length was 55.24 mm; and the width was 41.66 mm; weight and thickness of the shell were 4.90 g and 0.42 mm, respectively. The color of the yolk had a brightness of 57.62 L, 5.53 a for red/green tones and 47.15 b* for yellow/blue tones. The costs to produce the eggs for the Fair was US$0.21 with a profit/cost of US$0.04 per egg; the unit sale price is US$0.25. Conclusions: 84% of surveyed participants indicated that they purchase eggs for their nutritional value, revealing the need to train the communities toward more efficient egg production.", "keywords": [ "domestic economies", "rural development", "consumer protection", "rural egg productionn" ], "content": "Introduction\n\nAn economically active population (EAP) attempts to grow small businesses, allowing significantly more employment opportunities. Jiménez (2012) states that the purpose of any economic activity, whether agricultural, livestock or industrial, is to supply a benefit to its owners. The main global activity carried out in rural areas is animal raising for commercial purposes (Ordoñez & Lasso, 2010). Poultry farming is a key activity that can provide several benefits especially for the rural economy. However, it is important that poultry farmers become aware of the importance and value of poultry in the creation of companies, more than just meeting basic needs; when the obtained products are marketed, an EAP is formed.\n\nIn this study, we examine egg production in Ecuadorian Andean communities (Tzimbuto, Bayushig \"La Liberad,\" and La Victoria \"Pusuca.\") A quality analysis of the eggs and an analysis regarding the demand for egg consumption was carried out at the Utopia Community Fair, Riobamba, Ecuador.\n\n\nMethods\n\nThe research was performed over 120 days between February 2019 and <May 2019. Zoometric measures and egg quality analysis were carried out in the Escuela Superior Politécnica de Chimborazo, Riobamba. Egg consumption rates and motivation for selling eggs were obtained from producers at the Utopia Community Fair located at Av. Juan Félix Proaño, Riobamba municipality, Chimborazo Province (GPS coordinates: -1.685193, -78.644049).\n\n50 families from the communities of Tzimbuto, Bayushig \"La Libertad\" and La Victoria \"Pusuca\" were randomly selected from the main producers of the Utopia Community Fair. 81 hens owned by the selected families were chosen, having undefined age and weight. In addition, 29 eggs were analyzed and 65 consumers/producers from the 50 families were recruited for the study. The methodology related to egg/hen analyses followed procedures described in Martínez, 2016.\n\nTable 1 shows all variables considered in the present study.\n\nHen data. Zoometric characterization was performed for each hen (n = 81) according to FAO (1981); Lázaro et al. (2012) and Pérez & Polanco (2003). In brief, the birds were weighed manually by Chick Scale (model: 103; Agrologic Ltd., Israel), and direct observation of the animals was used to obtain color of feathers, types of feather, types of crest, ear color, beak color, skin color, tarsus color, and possible presence of feathers on the legs (Espinosa, 1991; SOCPA, 2007).\n\nEgg data. In total, 29 eggs from three communities were analyzed. The width of the egg was measured with a digital Vernier caliper (Dongguan Kuaijie Ltd., China); thickness of the eggshell was measured by Egg Shell Thickness Gauge (Orka Food Technology, LLC., USA); and the weight of the egg and the eggshell without the egg was determined by means of digital balance (model galaxy SF-107R; Ravi Scientific Industries, Delhi). Konica Minolta (CR 400) Colorimeter was used for three-dimensional interpretation of the yolk's color. Color of the egg yolk was obtained on three scales L, a, b: L, luminosity, ranging from 0 (absolute black) to 100 (absolute white); a, red and green tones, ranging from – 60 to +60, with green tones indicated by negative values and red tones assessed by positive values; b, yellow and blue tones, ranging from – 60 to + 100, with blue tones shown by negative values and yellow tones corresponding to positive values (González et al., 2015; Ochoa, 2014).\n\nHuman participant data. A total of 65 registered members (demographics: age, gender, marital status, number of family members) were subjected to a descriptive statistical analysis according to Rustom (2012). To understand the demand for eggs, we analyzed the type of consumed eggs, frequency of consumption, quantity of consumed eggs, amount of money spent on buying eggs, preferred characteristics of eggs and place of purchase (Underlying data (Bravo Avalos, 2019d)) using central trend measures (mean, media and mode) and deviation (variance and standard deviation). For the economic analysis, the profit/cost indicator was established from the ratio of expenditure (egress) to total revenue corresponding to the sale of eggs as follows: total revenues (US$) divided by expenditure (US$) (Anzola, 2002; Díaz, 2005; Hammershoj, 2015). See Extended data for the survey used in the study.\n\nSummary percentages were calculated for survey responses from participants, and hen and egg parameters. Egg quality analysis, production costs, and demand analysis for egg consumption were analyzed according to Bruni (2000); FAO (2014); Jeréz (2014); Juarez, (1999); Juarez, (2000); Quintana (2011); Villanueva (2015), and World Visión (2008).\n\nAll statistical analyses were performed with SPSS (version 22.0).\n\nThe Committee Fundación UTOPIA approved this study. Participants provided informed oral consent to participate in the study prior to its start. Oral consent was obtained from participants instead of written consent because of time constraints and illiteracy of some participants.\n\n\nResults and discussion\n\nFrom farming family surveys, it can be observed that 100% of producers own Creole birds. The majority of producers (72 %) own 0–10 birds and 28 % own 10–20 birds.\n\nAnalysis of the types of egg consumed found that 49% of families consume laying eggs, 47% consume rural eggs, while only 4% consume quail eggs.\n\nIn total, 84% of producers buy eggs because of their nutritional value, 12% because the animals are fed with organic food, and 4% because the eggs have a special flavor. Therefore, it is necessary to inform producers about the requirements of hens to produce eggs rich in essential nutrients, and to convey an added value to the product by disseminating the nutritional composition of the egg being commercialized, based on nutritional information.\n\nRegarding the commercialization of eggs, 57% of producers sell their products at the Utopia Community Fair, 30% sell them in stores, and 6–7% go to markets or deliver them directly to consumers’ homes. This shows that there is great support for direct community association with producers. Therefore, producers understand that direct association with customers results in beneficial sales channels, as they avoid intermediate vendors, who usually obtain higher profits without investing in or producing the product.\n\nAccording to the producers, 96% of them sell 1–2 egg cartons, each containing 30 eggs, and only 4% sell 3–4 cartons. Using the latter figure, this means that 90–120 eggs are sold at a cost of US$0.25/egg. These statistics represent that the egg production is intended for a retail scale; however, an increase in production will yield greater profits.\n\nThis study demonstrated that production cost of eggs contains several expenses: cost of poultry, chick feed, natural alternative medicines for hens, labor cost, and transportation (Table 2). Poultry purchase according to Andrade (2011) indicates that production cost of the chicken at the end of the breeding phase is US$3.78. When all incurred costs are considered, the total cost of rearing the 81 birds included in this study summed to US$306.18/hen.\n\nFor the purchase and rearing of animals (including food and medicine), labor and transportation, the total costs are US$1263.77. Sales of eggs amounted to US$1,500.00. The profit/cost indicator for egg production is US$1.19, indicating that producers have a profit margin of 0.19 cents for each dollar invested, value that can vary according to the age, production phase, and feeding of birds, complying with very strict food principles, the quality of production, and necessary marketing (Table 2).\n\nCalculation explanation: Total cost for feeding 81 birds over a time period of four months was valued at US$769.07. For this calculation, one considered the type of diet supplied to hens, which consisted of maize, barley, grass, and harvest waste. The value of harvest waste represented 5% of total diet. The cost of medicinal plants for the 81 birds was of US$3.00 per month, and a total cost of US$12.00 was incurred, since it is a common practice to use medicinal plants for improving birds’ health conditions. One day’s wage of a producer is US$15.00. Time needed for feeding 81 hens and collecting the eggs is 30 minutes a day (estimated cost, US$0.94/day). This item is multiplied by four months for a total cost of US$112.50. The fortnightly commuting cost (round trip) for moving products from communities to the Utopia Community fair is US$4.00. During this study, nine trips were made for nine fairs, leading to a total expenditure of US$36.00.\n\nOut of the surveyed producers, 75.4% were women and 24.6% were men; 61% have a family composed of 4 to 5 members, followed by 28% with 2 to 3 members, and the remaining 11% with 6 to 7 members. In total, 98.46% of producers surveyed include eggs in their diet, while only 1.54% do not consume eggs.\n\nThe results show that 72.31% of producers purchase both types of egg (processed and rural), 16.92% prefer processed eggs, 9.23% like for rural eggs, while as few as 1.54% do not consume eggs at all. Exploration of consumer behavior revealed that 48.44% of the producers surveyed buy eggs daily, 37.50% purchase eggs fortnightly, 12.50% acquire eggs weekly, and 1.56% buy them monthly. The average number of eggs that families purchase is 13 units daily, 33 weekly, 30 fortnightly, and 60 per month. The average amount that producers pay to buy eggs is US$7.50 for rural eggs and US$3.25 for commercial eggs; producers said they allocate US$8.00 to buy any type of eggs per week.\n\nA total of 60% of the producers go shopping in stores located in their neighborhood, 18.46% go to Utopia Community Fair, 13.85% choose public markets, while 4.62% prefer to buy them directly from producers.\n\nThe main characteristics of the eggs sold at the Utopia Community Fair show that the average weight of the eggs was 47.24 g; the length 55.24mm; width 41.66mm; weight and thickness of the shell 4.90 g and 0.42 mm, respectively. The color of the yolk obtained a brightness of 57.62 L, 5.53 a for red/green tones and 47.15 b* for yellow/blue tones. The measurement of color and brightness of the yolks reveals high quality of the overall egg, which can be rated as a top product offered by the Ecuadorian market.\n\n\nConclusions\n\nThe costs to produce a rural egg for the Utopia Community Fair is US$0.21 with a profit/cost of US$0.04 per egg (the unit sale price being US$0.25).\n\nStatistical data of our study reveal that 18.46% of producers purchase rural eggs, their average consumption is 30 units, and they spend US$7.50 fortnightly. It can be noticed that all producers possess Creole birds; 72% own 0-10 birds and 28% own 10-20 birds. Therefore, it was considered that poultry production is not widespread even though it is considered a very profitable business. Training those who farm Creole hens can be provided in the future so that they provide greater livestock and demand in the market. Analysis of the type of eggs consumed found that 50% of produces consume Creole hen eggs, 44% consume eggs from laying birds, and only 1% prefer quail eggs, which demonstrates high acceptance of Creole eggs by families living in the area of the Utopia Community Fair.\n\n\nData availability\n\nFigshare: Zoometric characterization.xlsx, https://doi.org/10.6084/m9.figshare.9978890.v1 (Bravo Avalos, 2019a).\n\nFigshare: Egg quality.xlsx, https://doi.org/10.6084/m9.figshare.9978896.v1 (Bravo Avalos, 2019b).\n\nFigshare: Egg colour (3 repetitions).xlsx, https://doi.org/10.6084/m9.figshare.9978899.v1 (Bravo Avalos, 2019c).\n\nFigshare: Individual survey results for the economic analysis, https://doi.org/10.6084/m9.figshare.9980033.v1 (Bravo Avalos, 2019d).\n\nFigshare: Survey, https://doi.org/10.6084/m9.figshare.9979025.v2 Fig share: Survey, https://doi.org/10.6084/m9.figshare.9979025.v2 (Bravo Avalos, 2019e).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAndrade C: Determinación de parámetros reproductivos y productivos de gallinas criollas para huevo verde, desde la recolección de huevos hasta la etapa inicial. (Tesis de grado. Ingeniero Zootecnista). Escuela Superior Politécnica de Chimborazo. Riobamba - Ecuador. 2011.\n\nAnzola R: Administración de pequeñas empresas. La pequeña empresa típicamente familiar. (2ª ed). México: McGraw-Hill. 2002; 22–65.\n\nBarroeta A, Izquierdo D, Pérez J: Manual de avicultura; breve manual de aproximación a la empresa avícola para estudiantes de veterinaria. Recuperado el 10 de febrero del 2017, de UAB. 2009.\n\nBravo Avalos MB: Zoometric characterization.xlsx. figshare. Dataset. 2019a. http://www.doi.org/10.6084/m9.figshare.9978890.v1\n\nBravo Avalos MB: Egg quality.xlsx. figshare. Dataset. 2019b. http://www.doi.org/10.6084/m9.figshare.9978896.v1\n\nBravo Avalos MB: Egg colour (3 repetitions).xlsx. figshare. Dataset. 2019c. http://www.doi.org/10.6084/m9.figshare.9978899.v1\n\nBravo Avalos MB: Individual survey results for the economic analysis. figshare. Dataset. 2019d. http://www.doi.org/10.6084/m9.figshare.9980033.v1\n\nBravo Avalos MB: Survey. figshare. Dataset. 2019e. http://www.doi.org/10.6084/m9.figshare.9979025.v2\n\nBruni M, Hamelin C, Hernández J, et al.: Calidad del huevo. Expectativas de los consumidores europeos. Madrid: Roche Vitamins Europe Ltd. 2000.\n\nCardenas E, Moreira J, Vera E: Manejo sanitario, infraestructura técnica y alimentación en la cría de las gallinas criollas (Gallusgallus) en las comunidades norte, sur y este del cantón Olmedo. (Tesis de grado. Médico Veterinario). Universidad Técnica de Manabí. Facultad de Ciencias Veterinarias. Portoviejo - Ecuador. 2006. Reference Source\n\nCisneros M: Aves de traspatio modernas en el Ecuador. Recuperado el 19 de febrero del 2017. 2002. Reference Source\n\nCuca J, Gómez G, López E, et al.: Producción y manejo de aves domésticas. Universidad Autónoma de Chapingo. 2003; 114–133.\n\nDíaz I: Indicadores de calidad del huevo que se comercializa en la ciudad de Morelia Michoacán. (Tesis de grado. Médico Veterinario). Facultad de Medicina Veterinaria y Zootecnia. Universidad Michoacana de San Nicolás de Hidalgo. Morelia - México. Recuperado el 14 de febrero del 2017. 2005.\n\nEspinosa R: Caracterización morfológica de la gallina mestiza del Estado de Chiapas. (Tesis de grado. Médico Veterinario). Universidad Autónoma de Chiapas. Tuxtla Gutiérrez - México. 1991.\n\nFood and Agriculture Organization (FAO): Descriptores de especies avícolas. En banco de datos de recursos genéticos animales. Roma - Italia. 1981; 13–15.\n\nFood and Agriculture Organization (FAO): Genética y cría de aves de corral en los países en desarrollo. 2014. Reference Source\n\nFood and Agriculture Organization (FAO): Historia de la producción avícola doméstica. 2014a. Reference Source\n\nGonzález E, Moscoso C, Oliva A, et al.: Manual de producción y manejo de aves de patio. (1° ed) Turrialba - Costa Rica: CATIE. (Serie técnica. Manual técnico/CATIE; no.128). 2015; 64.\n\nHammershøj M, Steenfeldt S: Organic egg production. II: the quality of organic eggs is influenced by hen genotype, diet and forage material analyzed by physical parameters, functional properties and sensory evaluation. Anim Feed Sci Technol. 2015; 208: 182–197. Recuperado el 10 de febrero del 2017. Publisher Full Text\n\nHernández Z, Lázaro G, Martínez L, et al.: Use of morphometric characters in the classification of local chickens. Actas Iberoamericanas de Conservación Animal. 2012; 2: 109–114. Reference Source\n\nHerrera H, Jerez S, Vásquez D: La gallina criolla en los Valles Centrales de Oaxaca. Instituto Tecnológico Agropecuario No 23 de Oaxaca/ Colegio de Postgraduados Montecillos - Texcoco. 2002.\n\nJeréz S, Herrera H, Vásquez D: La gallina criolla en los Valles Centrales de Oaxaca. Instituto Tecnológico Agropecuario No 23 de Oaxaca/ Colegio de Postgraduados. Montecillos. Texcoco - México. 2014; 80.\n\nJiménez M: Políticas públicas sobre ganadería de traspatio y seguridad alimentaria en México. III Foro internacional sobre ganadería de traspatio y seguridad alimentaria. Colegio de Posgraduados. Campus Veracruz. 2012.\n\nJuárez C, Manríquez A, Segura C: Rasgos de apariencia fenotípica en la avicultura rural de los municipios de la Ribera del Lago de Patzcuaro. Michoacán - México. Recuperado el 14 de febrero del 2017. 1999.\n\nJuárez C, Manríquez A, Segura C: Rasgos de apariencia fenotípica en la avicultura rural de los municipios de la Ribera del Lago de Patzcuaro, Michoacan, Mexico. (Tesis de grado. Médico Veterinario). Universidad Michoacana de San Nicolás de Hidalgo. Livestock Research for Rural Development. Recuperado el 22 de febrero del 2017. 2000. Reference Source\n\nMartínez J: Evaluación productiva de gallinas de campo de la región sierra del Ecuador. (Tesis de grado. Ingeniero Zootecnista). Escuela Superior Politécnica de Chimborazo. Riobamba - Ecuador. 2016. Reference Source\n\nMelgar I: Mujeres en el campo, la otra revolución. México D.F. México: El Norte. 2014.\n\nMolina M: Comparación de dos sistemas de producción y manejo sanitario de las aves criollas de traspatio en los municipios de Ignacio de la Llave y Teocelo, Veracruz. (Tesis de grado. Médico Veterinario). Universidad Veracruzana. Veracruz - México. 2013. Reference Source\n\nNorth O: Manual de Producción Avícola. (3ª. ed). Editorial El Manual Moderno. México. Recuperado el 18 de febrero del 2017. 2010; 829.\n\nOrdoñez A, Lasso E: Implementación del programa de gallinas ponedoras criollas en la Universidad Nacional de Loja. (Tesis de grado. Médico Veterinario). Loja - Ecuador. 2010.\n\nPérez A, Polanco G: La avicultura de traspatio en zonas campesinas de la provincia de Villa Clara - Cuba. Livestock Research for Rural Development. Recuperado el 10 de febrero del 2017. 2003; 15(2).\n\nQuintana J: Contribución al estudio de la dieta de las gallinas criollas de traspatio. (Tesis de pregrado. Médico Veterinario). Universidad Michoacana de San Nicolás de Hidalgo. Morella - Michoacán. 2011.\n\nTorres E: Evaluación de los parámetros productivos del pollo criollo vs pollo comercial. (Tesis de grado. Médico Veterinario). Universidad Veracruzana - México. 2010. Reference Source\n\nVaca L: Producción avícola, razas. 1 a. Bogotá, Colombia. Edit Pearson education. 2013; 45–56.\n\nVillanueva Najarro C, Oliva A, Torres Á, et al.: Manual de producción y manejo de aves de patio, Serie técnica. Manual técnico No. 128. (1° ed). Recuperado el 10 de febrero del 2017. 2015. Reference Source\n\nWorld Visión: Manual de crianza de gallinas ponedoras. Lima - Perú. Recuperado el 10 de marzo del 2017. 2008. Reference Source" }	[ { "id": "62000", "date": "29 May 2020", "name": "János Felföldi", "expertise": [ "Reviewer Expertise Agribusiness", "Farm business management", "Agri-food industry", "Agri-food supply chains" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general, the article partially meets the standards of a scientific research paper.\n1. Abstract:\nThe abstract gives the reader a general and clear idea about the article. It could be good if the author highlights the used statistical techniques.\n\n2. Jel classification could be added.\n3. Introduction:\nThe introduction is not enough as an introduction to a scientific research paper. It should be longer to give the reader an idea about the considered variables.\n\n4. Literature review:\nIt is missed in this article, and that is not accepted. Any research article should contain in addition to the introduction, a good theoretical part to show the author's level of literary knowledge about the topic. Otherwise, in case there are not enough pages, the author can write a comprehensive and coherent introduction to express the theoretical background of the article, in addition, to mention the objectives of the article at the end of the introduction.\n\n5. Methods:\nThe methodology part was discussed well.\n\nResearch design: The time horizon and measurements have been discussed.\n\nSamples and sampling techniques have been discussed. It is much better to mention clearly if the producers are the consumers themselves.\n\nData collection and data sources have been mentioned.\n\nData analysis, the author can provide some tables to show the outcomes of the analysis especially for descriptive analysis by SPSS.\n\n6. Results and conclusion:\nAs a paragraph, it had discussed well, the author can compare his results to other studies which had done before by other authors and consider the same geographic areas or compare them to other areas in the same country, to show if the consumption trends are the same or changed by time.\n\n7. References:\nThe list of references should reorganize again. Some references used in the text of the article but not mentioned in the references list, on the other hand, some references mentioned in the final list but not used in the text of the article.\n\nSummary:\nAccepted after doing major corrections. It should send back to the reviewer again after doing the recommended modifications.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "68841", "date": "19 Aug 2020", "name": "Terence Zimazile Sibanda", "expertise": [ "Reviewer Expertise Poultry science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis brief research reports a cost-benefit analysis of egg production in three communities of Ecuador. The element of cost-benefit analysis is clearly articulated in the study that shows the potential of the egg production Ecuador and one would want to see further analysis of the challenges in egg poultry production. Egg quality, zoometric measurements, and producer survey data are presented are collected although the zoometric data is completely missing in the results and discussion section. In general, it is not clear why the study was carried out. What the problem was? Correct terminology should be used. The introduction is too short and doesn`t cover variables that are going to be studied. The sample size of 29 eggs is not a good representative of the 3 communities? How many hens are in total in these communities? Justify your sample sizes.\nAreas of improvement:\nThe purpose and background of the study should be clearly articulated.\n\nThe author should include subsections on the results to improve structure. A clear structure of the data collected is presented nicely in Table two and this should be maintained in presenting the results.\n\nThe zoometric measurements and tables of descriptive data of the three communities should be included in the results.\n\nFurther discussion of the egg quality and zoometric measurements is required.\n\nMore comparison of the results to other studies is highly warranted in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1919
https://f1000research.com/articles/8-1916/v1	13 Nov 19	{ "type": "Research Article", "title": "Knowledge of pastoralists on livestock diseases and exposure assessment to brucellosis within rural and peri-urban areas in Kajiado, Kenya", "authors": [ "Joshua Onono", "Penina Mutua", "Philip Kitala", "Peter Gathura", "Penina Mutua", "Philip Kitala", "Peter Gathura" ], "abstract": "Background: Livestock diseases impact the livelihoods of pastoralists. Brucellosis, a neglected zoonotic disease is highly prevalent in this system with an estimated 16% of livestock population in sub-Saharan Africa infected with the disease. The objective of this study was to assess knowledge of livestock diseases and the risk of exposure to brucellosis among pastoralists living in Kajiado County of Kenya. Methods: The study sites included pastoralist communities living in rural and peri-urban areas within the County. Both primary and secondary data were collected using participatory methods including pairwise ranking, proportional piling and probing and a review of the published literature. Exposure risk assessment was conducted according to the CODEX Alimentarius framework: Hazard identification, hazard characterization, exposure assessment and risk estimation. Results: According to pastoralists, livestock diseases that frequently occurred in their flocks and herds were contagious caprine pleuropneumonia, lumpy skin disease and foot and mouth disease; but zoonoses, including anthrax and brucellosis, were also mentioned during focus group discussions. Potential pathways of exposure to brucellosis and other zoonoses included consumption of unpasteurized milk, handling infected aborted materials without protective measures and consumption of raw meat and raw blood. Consumption of unpasteurized milk and handling infected aborted materials without protectives were linked with high risk of exposure to household members living in rural areas, with the risk level within the peri-urban areas ranked very low to low for most of these risk practices. Conclusions: The results call for enhanced public education targeting vulnerable groups to mitigate risks of disease spread and other impacts of brucellosis within the affected pastoralist production systems.", "keywords": [ "Qualitative risk assessment", "Zoonoses", "Pastoralism", "Livestock", "Kenya" ], "content": "Introduction\n\nBrucellosis is a neglected bacterial zoonosis with global distribution, and it has been reported in 56 countries with more than 500,000 new human cases reported annually (Christou, 2011; Seimenis et al., 2006). The disease is prevalent in pastoralist systems within developing countries and it has been associated with increased public health concerns, especially in the Mediterranean region, western Asia, Latin America and other parts of Africa (Corbel, 2006; Gwida et al., 2010). In sub-Saharan Africa, about 16% of all livestock raised within the region are estimated to be infected with brucellosis (Kiambi, 2012). Similarly, several studies have identified Brucella species including Brucella abortus, Brucella melitensis and Brucella suis in different parts of Africa, with isolates which are closely related to strains circulating in the Mediterranean basin (Ducrotoy et al., 2017; Lounes et al., 2014).\n\nIn Kenya, brucellosis was first reported in 1916 with the first case confirmed in a laboratory in 1931 (Njeru et al., 2016). To date, animal and human brucellosis is reported annually especially from pastoralist production systems (Kiambi, 2012; Maichomo et al., 2000; Richards et al., 2010). Furthermore, Brucella species have been isolated from both wild and domestic animals (Muendo et al., 2012; Oomen, 1976). B. melitensis and B. abortus have also been isolated in human patients from different parts of the country (Njeru et al., 2016). According to results of a systematic review of brucellosis in Kenya, the estimated national sero-prevalence was about 3.0%, (Njeru et al., 2016). This high sero-prevalence in humans is reported to occur in several counties including Kajiado, Marsabit, Turkana, Machakos and Garissa, which are counties mainly inhabited by pastoralists (Ogola et al., 2014). However, a low sero-prevalence has been reported in Kiambu, Naivasha, Busia and Nairobi counties, which are counties where the predominant production system is intensive and semi-intensive livestock production systems (Njeru et al., 2016; Osoro et al., 2015).\n\nThe pastoralist communities are highly dependent on livestock for supply of their household nutritional needs, besides their economic and social utilization (Bechir et al., 2012; Zinsstag et al., 2006). However, some cultural practices inherent in these communities such as consumption of unpasteurized milk, raw meat and blood, contribute to increased risk of transmission of brucellosis from livestock to humans (Adesokan et al., 2013; John et al., 2010). Indeed, other practises such as increased mobility of pastoralist communities with their livestock in search for pasture and communal watering grounds also contributes to the spread of infectious diseases in animal populations.\n\nVarious studies have shown varying degrees of knowledge about zoonoses, especially brucellosis, amongst populations. This level of awareness is thought to significantly contribute to the control of spread of such infections (Lindahl et al., 2015). Likewise, lack of awareness on such infections may delay patients from seeking health care in time (Kansiime et al., 2014), thus enhancing spread of infection amongst populations. Some studies have shown higher level of awareness about brucellosis among the educated when compared to the non-educated populations (Lindahl et al., 2015). In their study, increased awareness on brucellosis was also reported among the agro-pastoralist population when compared to the group of nomadic pastoralists and farmers who engaged with veterinarians on service delivery for their animal health problems (Kansiime et al., 2014; Lindahl et al., 2015). A number of studies have also shown that while the majority of the population have heard about brucellosis, only a small percentage of the farming population have knowledge of how the disease is transmitted and its manifestation both in man and animals (Adesokan et al., 2013; Ljung, 2013).\n\nIn Kenya, previous studies conducted have focused on determining prevalence and risk factors associated with spread of brucellosis both in livestock and human populations (Kang’ethe et al., 2007; Osoro et al., 2015). However, there is paucity of studies that reports associations of these risk practices by farming communities to exposure of household members to infections with brucella from the livestock they keep. This study examined the role of pastoralist practices and behaviour in disease management that increases their risk of exposure to brucellosis from their livestock. The findings of this report are useful in understanding why brucellosis continues to persist within pastoralist communities despite the increasing knowledge amongst both livestock and human health professionals on the available control strategies, including vaccinations and improved breeding management through artificial insemination, which have been used to control the disease in other parts of the World.\n\n\nMethods\n\nThis was a descriptive study that examined knowledge, attitudes and practices of the pastoralist community living within selected rural areas in Kajiado and another group who were living within the peri-urban areas. The study was carried out in August 2015. These communities were selected to compare the risk practices of exposure to zoonoses between those who still practiced pastoralist livelihoods system within the county, and another who had transitioned to peri-urban livelihoods systems, but were still bound by cultural ties based on the community customs and traditions. Kajiado County is predominantly inhabited by the Maasai community, which still have members who practices nomadic pastoralism. However, there are immigrants from other communities in the county, with different cultural orientations from those of the Maasai. These have mostly inhabited the peri-urban areas within the county.\n\nKajiado County has two main livestock production systems: nomadic pastoralism and mixed livestock and crop farming systems (agro-pastoralism) practiced around up-coming urban centers. The county was therefore stratified in two strata, based on these livelihood systems. The study sites were selected through a two-stage sampling process. The sub-counties where the study was carried out were first selected purposively due to available logistical arrangements for accessing the pastoralists, and administrative wards where interviews were carried out were selected from these sub-counties through a random selection process using a lottery method where all wards in each sub county were listed in different pieces of paper, which were then rolled up and placed in a box. From each box two pieces of paper were drawn sequentially, and names of the selected wards recorded. For each of the selected sub-counties, all administrative wards had an equal opportunity of being selected to be included in the study through a process of sampling without replacement. The process was guided by the research team with assistance from a local government extension agent in Kajiado who was familiar with geography of the area and Maasai culture and language. Two sub-counties were selected from this process: Kajiado Central representing the nomadic pastoralism system and Kajiado East representing the peri-urban communities (Figure 1). In both sub-counties, the number of administrative wards were five. The wards included Purko, Ildamat, Matapato South, Matapato North and Dalalekutuk in Kajiado Central sub county, while for Kajiado East, these were Poka, Imaroro, Kaputei North, Kitengela and Oloosirkon/sholinke wards. Two wards were selected from each of the two sub counties. For Kajiado Central, the selected wards were Matapato North and Matapato South, while in Kajiado East; Kitengela and Kaputei North wards were selected.\n\nAdministration officers (village elders) from each of the selected wards were recruited to help with mobilization of study participants to attend focus group discussions. The participants who were invited to the group discussions were drawn from different parts of the wards where the village elders represented. Each group comprised of between 8 to 11 participants who were identified by village elders to have lived in the area for several years, and who were either of male or female gender were recruited for the study, while anyone who was non-Maasai speaking were not invited for the discussion. This was considered to comply with group composition description of 6–12 participants for a group discussion as was described by Dilshad & Latif (2013), where individuals with a certain characteristics are brought together by a researcher to explore attitudes, perceptions, feelings and ideas about a topic. The local veterinary personnel were also recruited to the study to serve as key informants as well as in assisting during the focus group discussion sessions to triangulate the information on livestock diseases that participants identified. A total of six groups comprising both male and female participants and two groups comprising each gender separately were conducted to explore any differences in household and farm practices with regard to handling livestock and their products which could be gender specific, especially for a patriarchal society like the Maasai.\n\nTwo focus group discussions were conducted in each selected ward led by a member of the research team with support from local government extension personnel who was conversant with the Maasai culture and language. The group discussions were held in homesteads of local village elders where community leaders would always meet to resolve communal problems or under makeshift structures which were also used as places of worship by the community. Secondary data on the other hand, was obtained through a review of surveillance data from previous published studies and grey literature sources. Published participatory methods including proportional piling, pair wise ranking and disease matrix scoring were used for data collection during the focus group discussions (Catley, 2006; Catley et al., 2012). Briefly, pairwise ranking is a structured method of prioritizing a list of factors for decision making through consensus, where each box on a matrix represent an intersection of two constraints. For each pair of listed diseases, participants determined which of the two was most important through consensus and the important disease was marked in the appropriate box. The procedure was repeated until all boxes in the matrix were filled. Diseases were then ranked according to number of times they appeared in the matrix. Proportional piling involve allocating percentage scores to identified list of factors that have been identified by a group of people through consensus. Disease impact matrix scoring employed proportional piling method to estimate effects of livestock diseases on production parameters as perceived by pastoralists. For this case a total of 100 beads representing different weights for impact of livestock diseases were applied. Production parameters assessed included weight loss, mortality, loss in milk yield, and abortion rate. This was based on how participants perceived the disease to impact on these production parameters. The production parameters were entered on the left column of the matrix, while diseases were entered on top of the matrix. The next step involved allocating scores to diseases according to how pastoralist perceived them to impact on each production parameter. This was done sequentially focusing on one production parameter each time by allocating scores across the disease columns until all the rows of the matrix were filled. The scores allocated for each production parameter were then summed to give an overall score. The results obtained were probed to obtain reasons for the observed pattern.\n\nThe guiding questions during these sessions included: list of livestock diseases in order of their perceived importance, and what is the effect of listed diseases on livestock production parameters including milk yield, mortality rates, morbidity rates, and abortion rates. For the risk assessment, both primary and secondary data were collected through use of the questionnaire guide during the focus group discussions, and through a review of published and grey literature sources (see extended data (Onono et al., 2019)). Primary data were collected on participants’ knowledge on brucellosis in animals and humans, modes of transmission of brucellosis from animals to man and the potential exposure factors contributing to spread of brucellosis in man. The data were recorded in flip charts, while the research assistant would take additional notes that participants provided to support their argument on the types of choices they made during discussions. The participants were asked whether they knew about brucellosis in animals and in man, what were the symptoms of brucellosis in animals, the ways through which people could acquire brucellosis, factors that contributed to spread of brucellosis in man, disease management in animals and what were their perceptions on the impact of human brucellosis. The factors identified were useful in exposure assessment and estimation of risk of brucellosis infection among the population under study. The literature review was based on studies that had reported presence of the disease (hazard identification) through detection with either immunological or molecular methods within pastoral herds and flocks in Kajiado and neighboring pastoral areas. The search was done in electronic databases including PubMed, Medline and CABI direct, and the search terms included “brucellosis, Brucella & melitensis, Brucella & abortus, livestock, Kenya”. Data were also obtained from grey literature sources including published thesis and reports. The inclusion criteria for studies were those which were conducted across all farming systems in Kenya, while exclusion criteria was any other study on brucellosis which was not done in Kenya. Hazard characterization was based on the livestock species from which the hazard was detected in biological samples through either immunological or molecular methods. Before the focus group discussions were conducted, participants were asked if they accepted to participate in the study through a written consent statement which was read to them, and those who accepted to participate were asked to provide their written consent by signing an attendance form. Further ethical clearance for the study was obtained from the University of Nairobi, Faculty of Veterinary Medicine ethics and biosafety committee (FVM: BAUEC/2015/138).\n\nData on scores and ranks obtained during focus group discussions were entered in Microsoft Excel software version 2010. Data for various questions which were obtained from the focus group discussions were analyzed using descriptive statistical methods and results presented as frequencies, while scores were analyzed through non-parametric methods using GenStat Statistical package 13th edition (VSN International, 2011). For the scores, the analysis aimed at measuring the level of agreement amongst groups of the FGDs using Kendall’s coefficient of concordance, while Kruskal Wallis One Way Anova was also used to test whether the average scores obtained for each disease as ranked by participants were significantly different from zero. The impact of the listed diseases on the various production parameters: herd/flock milk yield, abortion rates, growth rates (weight gain), and mortality rates were presented as averages.\n\nQualitative risk assessment was done according to the CODEX Alimentarius guidelines (FAO & WHO, 2009). The exposure assessment to infection with brucellosis through the various pathways was determined based on the scores allocated for the likelihood of occurrence of these events in the community by the participants. A high risk score of 5 was allocated when the event occurred very often in the surveyed community; medium risk score of 4 was allocated if the event occurred regularly; low risk score of 3 was allocated if the event ever occurred but was very rare; very low risk score of 2 was allocated if the event was very rare but could not be excluded and a negligible risk score of 1 was allocated if the event was so rare and it did not merit consideration. Risk estimation was achieved by summation of the scores obtained for each exposure factor across the groups for both peri-urban and rural areas. If the summed scores for a risk practice was between 1 and 4, then the risk was considered negligible, between 5 and 8 very low risk, between 9 and 12 low risk; between 13 and 16 medium risk and between 17 and 20 high risk.\n\n\nResults\n\nAmongst the rural pastoralist category, a total of 38 respondents participated, and out of this number, 32 % were female. Of these respondents, 47 % of participants were between 25–30 years of age, 34 % were between 31–40 years and 19 % were above 40 years. With regard to the level of education, 45 % of the respondents had attained primary school education, 21 % had obtained secondary school level of education, and 3 % had tertiary education, while 31 % had no formal education. The peri-urban group of agro-pastoralists attracted 33 respondents, of which 46 % were female. Of these respondents from peri-urban areas, 15 % were between 25–30 years of age, 52 % between 31–40 years and 33 % were above 40 years. About 18 % of the respondents had primary school education, 49 % had secondary school level of education while 33 % had obtained tertiary level of education (see underlying data (Onono et al., 2019)).\n\nA total of 11 livestock diseases were listed from all the eight groups of participants. There were more similarities on the livestock diseases suggested by the groups of rural pastoralists when compared to those within the peri-urban groups. However, only two of the peri-urban groups mentioned brucellosis among livestock diseases that affected their flocks and herds and none from rural pastoralists ever mentioned brucellosis. According to these pastoralists, livestock diseases which were most prevalent included contagious caprine pleuropneumonia, lumpy skin disease, and foot and mouth disease and these diseases were described to have a high impact on livestock production (Z > 1.96) (Table 1). Amongst all listed livestock diseases, zoonoses (Anthrax and brucellosis) were ranked low, which could be due to the fact that these groups of pastoralist lack knowledge and awareness of the impact of these livestock diseases on their wellbeing and health. The level of agreement across these groups of pastoralists on rank orders for the livestock diseases based on their impact of wellbeing and health was considered weak, with the calculated Kendall’s coefficient of concordance ‘W’ estimated at 0.007. The high impact of these livestock diseases on mortality was reported for East Coast fever and contagious caprine pleuropneumonia, while milk yield was mostly affected by foot and mouth disease, lumpy skin disease, heart water, East Coast fever and diarrhea. Increased incidences of abortions were due to infection with foot and mouth disease, lumpy skin disease, contagious caprine pleuropneumonia and East Coast fever. The pastoralists from peri-urban areas associated brucellosis with an increased level of abortions in herds and flocks while anthrax was associated with high morbidity rates in livestock herds. Rank orders for livestock diseases according to pastoralists are presented as underlying data (Onono et al., 2019).\n\nHazard identification was achieved through review of published literature under the different farming systems in Kenya (see underlying data (Onono et al., 2019)). Various Brucella species had been detected in livestock raised in different farming systems in Kenya, and also in humans through serological tests (Table 2). Antibodies of B. abortus had been detected in bovine milk through milk ring tests and Elisa test (Kang’ethe et al., 2004). According to a recent review by Njeru et al. (2016), B. abortus and B. melitensis had been isolated in cattle and humans and B. suis in rodents. Pastoralists listed various practices which may act as possible exposure factors for transmission of brucellosis from livestock to members of the community. These included handling birth products without protective gear while assisting livestock during birth process and consumption of unpasteurized milk, which were associated with high risk of exposure by household members to brucellosis among the rural pastoralists. These practices would increase exposure to Brucella pathogens and therefore increasing the risk of its transmission from livestock to household members. Within the rural community, young children were often drinking fresh raw milk and colostrum which were perceived to enhance their immunity against infections, which is also a potential risk factor for infection.\n\nKey: number of study units or samples which were included for specific studies which were reviewed (n); Masters of Science (MSc); milk ring test (MRT); and enzyme linked immunosorbent assay (ELISA)\n\nWithin the peri-urban group, the risk associated with exposure to brucellosis through handling of birth products without protective gear and drinking of raw fresh milk was considered to be low. These peri-urban pastoralists kept only a few livestock and they did not keep mixed livestock species. Moreover, the majority also relied on professional veterinary services during delivery or treatment of sick livestock, hence they were less exposed to brucellosis infection through handling of infected birth products. Besides, these pastoralists often used pasteurized milk, while others would always boil the raw milk from their livestock but for those households who did not own livestock, they often boiled the milk they would purchase from local shops or neighboring farms before using it within the household.\n\nConsumption of raw meat and blood were associated with medium risk of brucellosis infection among the rural pastoralists. Consumption of raw blood was often practiced during traditional ceremonies like marriage and rites of passage when young Maasai boys are initiated to adulthood, during which time livestock were slaughtered at home. Pastoralists reported that kidneys from livestock and occasionally the liver would be consumed in their raw state during slaughtering process. This practice of home slaughter was prevalent amongst rural pastoralists due to a lack of established slaughter premises where they lived, and the long distances they would have to walk to get to the nearest marketplaces where slaughter facilities were established. The risk of exposure to brucellosis was, however, ranked low because only a few people within households were engaged in drinking the raw blood or eating the raw meat from kidneys and the liver.\n\nBased on the risk estimation criteria, the risk of exposure to brucellosis was categorized as high amongst the rural pastoralists when compared to peri-urban pastoralists. This was due to cultural practices around consumption of fresh raw milk, raw blood and meat, and handling of birth products without protection while assisting livestock during delivery. Based on the criteria for risk estimation, consumption of fresh raw milk had a score of 19 for the rural pastoralists group and only 10 for the peri-urban group; eating of raw meat had a score of 14 for rural pastoralists and 7 for the peri-urban group; drinking of raw blood was scored 15 for the rural pastoralists and only 6 for the peri-urban group; and finally, handling of birth products without gloves while assisting livestock delivery was scored 20 for the rural pastoralists and 11 for the peri-urban group (Table 3). Based on this criterion, risk of brucellosis infection in the rural pastoralists was high for handling birth products without protectives and drinking of fresh raw milk; while the drinking of blood and eating raw meat were rated to be of medium risk. For pastoralist within the peri-urban areas, handling of birth products and consumption of raw milk were ranked as low risk, while for consumption of raw meat and blood the risk was ranked as very low.\n\nKey: +++++ = high risk score; ++++ = medium risk score; +++ = low risk score; ++ = very low risk score; + = negligible risk score\n\n\nDiscussion\n\nThis study identified livestock diseases which negatively impacted on livelihoods of pastoralists within Kajiado County, through reduction in milk yield, increased mortality rates, increased morbidity rates and abortions in herds and flocks. In addition, zoonoses including brucellosis and anthrax were identified by communities as a threat both to their wellbeing and health. According to the pastoralists, livestock diseases that were significantly impacting on their livelihoods included contagious caprine pleuropneumonia, Lumpy Skin disease and foot and mouth disease, which were ranked high. These results agree with findings of a previous report which was done in Narok County, Kenya, where foot and mouth disease and lumpy skin disease were identified amongst other diseases to be most prevalent and also had negative impacts on family incomes (Onono et al., 2013). In addition to occurrence of these livestock diseases which affected levels of productivity, their knowledge on occurrence of brucellosis had previously been corroborated by findings from studies that have reported the disease in herds and flocks through serological testing and microbiological methods. According to a review of the literature on studies that were carried out in similar pastoralist systems in Kenya, brucellosis had been reported to occur either due to B. melitensis or B. abortus (Kang’ethe et al., 2007; Muendo et al., 2012; Ogola et al., 2014). However, the pastoralists had only ranked brucellosis 7th out of the 11 livestock diseases that were identified to affect their livelihoods. Another zoonosis identified by the pastoralist was anthrax and it was ranked 5th amongst all the livestock diseases affecting their wellbeing and health. These results reveal the lack of knowledge on impact of zoonoses to their livelihoods and health. Indeed the findings are not surprising based on the findings of a previous study which had reported the nature of inconsistencies in level of knowledge of zoonoses among pastoralists living in the Northern parts of Cameroon (Moritz et al., 2013). Similarly, the knowledge of the community on brucellosis in animals with regard to its clinical signs, transmission patterns and control was very poor when compared to that in humans. This finding is similar to that by Adesokan et al. (2013), who reported poor knowledge of brucellosis amongst a pastoralist community in Nigeria. But another study by Buhari et al. (2015), had reported a high level of knowledge on brucellosis in cattle when compared to infection in man, amongst a specific group of pastoralists in northern Nigeria. No explanation was provided for the cause of this apparent difference in knowledge amongst pastoralists in northern Nigeria, but it can be hypothesized that the community had obtained some form of training through extension agents on the importance and significance of brucellosis control in livestock. Indeed, the pastoralists from northern Nigeria are reportedly reliant on media teachings on improved livestock production which could explain the rise in levels of awareness on this disease. In the current study, only one group of pastoralists reported abortions as a clinical sign associated with occurrence of brucellosis in animals. This finding is consistent with reports from other studies that have documented lack of knowledge by pastoralists in northern Uganda on the clinical manifestation of brucellosis in livestock through abortions (Kansiime et al., 2014). The occurrence of brucellosis in herds and flocks is a serious public health threat to the people who are in contact with these livestock. Indeed, clinical disease in human have been reported in various parts of Kenya (Njeru et al., 2016). Furthermore, the practice by pastoralists of raising mixed livestock species per farm poses a greater risk of cross infection since the Brucella species are multi-host. It has been reported that when Brucella melitensis establish itself in cattle herds, it poses greater health risk since localization of infection in the udder result in shedding of large quantities of the bacteria which would contaminate the environment and therefore a greater public health hazard to humans (Díaz Aparicio, 2013; Ducrotoy et al., 2017).\n\nWith regard to exposure factors for human infection, participants identified consumption of raw unpasteurized milk, blood and raw meat from infected animals as common practices by pastoralists. These were factors which were identified to highly increase the risk of exposure to people. Indeed, according to Kansiime et al. (2014), consumption of raw fresh milk had been associated with increased occurrence of brucellosis in Uganda, and often the health professionals in that country would educate pastoralists to avoid consumption of raw fresh milk as a measure to control the disease. Furthermore, a study performed in Ethiopia also corroborates this finding where cases of human brucellosis was reported in up to 86% of patients who were consuming raw milk (Regassa et al., 2009). The consumption of raw milk is therefore a major exposure factor for brucellosis to humans, and based on a review that has reported that raw milk consumption is rampant within sub Saharan Africa context, the general population would be at a greater risk of infection with brucellosis (Mfinanga et al., 2003). This practice also increases the risk of transmission of other zoonoses as was argued in the publication by John et al. (2010). Most of the pastoralists in the rural area would feed raw fresh unpasteurized milk products and sometimes colostrum directly to their young children, which they believed would boost the immunity of these children. This is a practice which has also been reported amongst the Fulani pastoralists community of northern Nigeria, who suckle milk directly from the udder of animals (Adesokan et al., 2013). These practices are generally a major risk to pastoralist community and their children for transmission and acquiring of brucellosis.\n\nOther factors such as consumption of raw blood, kidneys and liver during traditional ceremonies and handling birth materials without protective gear were also often practised by pastoralists. This finding is consistent with results from a study which was conducted in Ethiopia, where pastoralists would often consume raw liver (Desta, 2015; Desta, 2016a). Indeed, the consumption of uncooked meat is reportedly a common practice amongst African communities according to previous reviews (Corbel, 2006; John et al., 2010). In addition to the exposure factors discussed above, the pastoralists would often assist livestock especially cattle during deliveries without proper protective gear which increases their risk of exposure to brucellosis and other zoonoses. Indeed, in a study which was done in Jordan, less than 6% of the study participants believed that livestock owners would use protective gear while assisting animals during deliveries (Musallam et al., 2015), while other livestock owners have also been reported to handle aborted foetal materials without protective gear (Desta, 2016b; Lindahl et al., 2015). With regard to the disposal of aborted birth materials, six groups reported that they would give the aborted foetuses to dogs and another two groups would bury them. This agrees with results obtained from a study done in Jordan where about 55% of the participants would feed the aborted material to dogs and less than 10% would bury or burn these aborted materials (Musallam et al., 2015). Brucella abortus has been isolated in sick dogs, which demonstrate that feeding dogs with aborted foetal parts can act as reservoirs for future occurrence of disease in both livestock and man due to the close interactions between dogs, livestock and man in the pastoralist set ups (Díaz Aparicio, 2013).\n\nThe estimated risk of exposure to brucellosis infection was high in rural areas where it was associated with handling birth products without protective gear and drinking unpasteurized milk. The pastoralists would keep large herds of livestock of mixed species and they also practiced seasonal animal breeding, therefore parturition would occur during a specific time period. In such instances, if brucellosis was present in flocks or herds, many pastoralists can get exposed in a shorter time interval given that majority of them often engaged in assisting animal deliveries and handling birth contents without protective gear, which is a possibility as was discussed by Musallam et al. (2015) in a study in Jordan where only 6% of respondents reportedly were using some form of protective gear while assisting on livestock delivery. Feeding of raw fresh milk and colostrum to young children to boost their immunity is a practice which requires a lot of attention, since the consequences of this practices exposes them to increased risk of contracting brucellosis, and indeed Adesokan et al. (2013), in their study also described the rampant use of raw fresh milk by these pastoralist communities.\n\nConsumption of raw meat and blood were associated with medium risk of exposure amongst pastoralist living in the rural set-up. However, they still predispose communities to increased risk of exposure because they are practices that are often practiced during traditional ceremonies where many people are involved. During these traditional ceremonies, pastoralists would often slaughter animals at home without any meat inspection services. This has been reported as a common practice among African countries, which is associated with occurrence of zoonotic diseases (Corbel, 2006; John et al., 2010).\n\nThe level of risk of exposure to brucellosis within the peri-urban areas was, however, categorized as low from practices of drinking raw fresh milk, handling birth products without protective gears, and consuming raw meat and blood. This could be due to the fact that cultural practices described within rural set up are gradually being replaced by the influence of increased information on public health for the communities who have moved closer to urban set ups. Indeed, the consumption of raw fresh milk was low amongst this group since majority would boil milk before use. In addition, most households in peri-urban areas would purchase pasteurized milk. This group of pastoralists was not consuming raw meat and blood, and therefore was at a very low or negligible risk of getting exposed to brucellosis through their consumption practices.\n\nIn conclusion, the level of risk of exposure to brucellosis for pastoralists living in rural areas was high when compared to pastoralists living within peri-urban areas. This is linked to their cultural practices and how they interact with livestock products and by-products including consumption of raw fresh milk, raw blood and meat and handling birth products without protectives. Action planning to mitigate risk from these practices through public education regarding brucellosis and its exposure factors to the vulnerable communities should be established and, this will enable them know the disease and how it is transmitted. Likewise, there is need for enhanced collaboration between the Departments of Medical and Veterinary services in zoonotic disease surveillance and its control through the established government departments like the Zoonotic Disease Unit, a body created by both the Ministry of Health and Veterinary Services to respond to occurrence of zoonoses within the country.\n\n\nData availability\n\nFigshare: KNOWLEDGE OF PASTORALISTS ON LIVESTOCK DISEASES AND EXPOSURE ASSESSMENT TO BRUCELLOSIS WITHIN RURAL AND PERI-URBAN AREAS IN KAJIADO, KENYA. https://doi.org/10.6084/m9.figshare.9848168.v5 (Onono et al., 2019)\n\nRaw data.xls (Excel file containing focus group responses, pastoralists rankings of livestock diseases and estimates of brucellosis exposure)\n\nFigshare: KNOWLEDGE OF PASTORALISTS ON LIVESTOCK DISEASES AND EXPOSURE ASSESSMENT TO BRUCELLOSIS WITHIN RURAL AND PERI-URBAN AREAS IN KAJIADO, KENYA. https://doi.org/10.6084/m9.figshare.9848168.v5 (Onono et al., 2019)\n\nFocus group questionnaire guide.docx (Word document containing questions used in focus groups)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe acknowledge the support from the office of the County Director of Veterinary Services in Kajiado who allowed me to conduct research and pastoralist communities from Kajiado County who participated in the study.\n\n\nReferences\n\nAdesokan HK, Alabi PI, Stack JA, et al.: Knowledge and practices related to bovine brucellosis transmission amongst livestock workers in Yewa, south-western Nigeria. 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PubMed Abstract \| Publisher Full Text\n\nDilshad RM, Latif MI: Focus Group Interview as a Tool for Qualitative Research: An Analysis. Pakistan Journal of Social Sciences (PJSS). 2013; 33(1): 191–198. Reference Source\n\nDucrotoy M, Bertu WJ, Matope G, et al.: Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control. Acta Tropica. 2017; 165: 179–193. PubMed Abstract \| Publisher Full Text\n\nFAO and WHO: Food safety: Principles and methods for the risk assessment of chemicals in food. Environmental Health Criteria 240. 2009; ISBN: 978 92 41572408. Reference Source\n\nGwida M, Al Dahouk S, Melzer F, et al.: Brucellosis - regionally emerging zoonotic disease? Croat Med J. 2010; 51(4): 289–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohn K, Fitzpatrick J, French N, et al.: Quantifying risk factors for human brucellosis in rural northern Tanzania. PLoS One. 2010; 5(4): e9968. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiambi SG: Prevalence and factors associated with brucellosis among febrile patients attending Ijara District Hospital, Kenya. Abstracts of Postgraduate Thesis, 2012. Reference Source\n\nLindahl E, Sattorov N, Boqvist S, et al.: A study of knowledge, attitudes and practices relating to brucellosis among small-scale dairy farmers in an urban and peri-urban area of Tajikistan. PLoS One. 2015; 10(2): e0117318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLjung I: Brucellosis in small ruminants – a seroprevalence study in peri-urban farming around the region of Dushanbe, Tajikistan. Uppsala. 2013; ISSN 1652-8697. Reference Source\n\nLounes N, Cherfa MA, Le Carrou G, et al.: Human brucellosis in Maghreb: existence of a lineage related to socio-historical connections with Europe. PLoS One. 2014; 9(12): e115319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaichomo MW, McDermott JJ, Arimi SM, et al.: Study of brucellosis in a pastoral community and evaluation of the usefulness of clinical signs and symptoms in differentiating it from other flu-like diseases. Afr J Health Sci. 2000; 7(3–4): 114–9. PubMed Abstract\n\nMfinanga SG, Mørkve O, Kazwala RR, et al.: Tribal differences in perception of tuberculosis: a possible role in tuberculosis control in Arusha, Tanzania. Int J Tuberc Lung Dis. 2003; 7(10): 933–941. PubMed Abstract\n\nMoritz M, Ewing D, Garabed RB: On Not Knowing Zoonotic Diseases: Pastoralists' Ethnoveterinary Knowledge in the Far North Region of Cameroon. Hum Organ. 2013; 72(1): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuendo EN, Mbatha PM, Macharia J, et al.: Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes. Trop Anim Health Prod. 2012; 44(1): 17–20. PubMed Abstract \| Publisher Full Text\n\nMusallam II, Abo-Shehada MN, Guitian J: Knowledge, Attitudes, and Practices Associated with Brucellosis in Livestock Owners in Jordan. Am J Trop Med Hyg. 2015; 93(6): 1148–1155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNanyende DW: Estimation of the prevalence of brucellosis in humans and livestock in Northern Turkana district, Kenya. (Unpublished Msc Thesis, University of Nairobi) 2007. Reference Source\n\nNjeru J, Wareth G, Melzer F, et al.: Systematic review of brucellosis in Kenya: disease frequency in humans and animals and risk factors for human infection. BMC Public Health. 2016; 16(1): 853. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgola E, Thumbi S, Osoro E: Sero-prevalence of Brucellosis in Humans and their Animals: A Linked Cross-sectional Study in Two Selected Counties in Kenya. Online J Public Health Inform. 2014; 6(1): e67. Publisher Full Text \| Free Full Text\n\nOnono J, Mutua P, Kitala P, et al.: knowledge of pastoralists on livestock diseases and exposure assessment to brucellosis within rural and peri-urban areas in Kajiado, Kenya. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9848168.v5\n\nOnono JO, Wieland B, Rushton J: Constraints to cattle production in a semiarid pastoral system in Kenya. Trop Anim Health Prod. 2013; 45(6): 1415–1422. PubMed Abstract \| Publisher Full Text\n\nOomen LJ: Human brucellosis in Kenya. Trop Geogr Med. 1976; 28(1): 45–33. PubMed Abstract\n\nOsoro EM, Munyua P, Omulo S, et al.: Strong Association between Human and Animal Brucella Seropositivity in a Linked Study in Kenya, 2012–2013. Am J Trop Med Hyg. 2015; 93(2): 224–231. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRegassa G, Mekonnen D, Yamuah L, et al.: Human Brucellosis in Traditional Pastoral Communities in Ethiopia. Int J Trop Med. 2009; 4(2): 59–64. Reference Source\n\nRichards AL, Jiang J, Omulo S, et al.: Human Infection with Rickettsia felis, Kenya. Emerg Infect Dis. 2010; 16(7): 1081–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichards AL, Jiang J, Omulo S, et al.: Human Infection with Rickettsia felis, Kenya. Emerg Infect Dis. 2010; 16(7): 1081–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeimenis A, Morelli D, Mantovani A: Zoonoses in the Mediterranean region. Ann Ist Super Sanita. 2006; 42(4): 437–445. PubMed Abstract\n\nVSN International: GenStat for Windows 14th Edition. VSN International, Hemel Hempstead, UK. Web page: GenStat.co.uk. 2011. Reference Source\n\nZinsstag J, Ould Taleb M, Craig PS: Editorial: health of nomadic pastoralists: new approaches towards equity effectiveness. Trop Med Int Health. 2006; 11(5): 565–568. PubMed Abstract \| Publisher Full Text" }	[ { "id": "60300", "date": "11 Mar 2020", "name": "Marta Guerra", "expertise": [ "Reviewer Expertise Epidemiology", "One Health", "veterinary medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods:\nSelection of study subjects: Unclear how participants were selected for focus groups e.g. one per household? Were participants related, which would increase similarities?\nData collection:\nUnclear what, if any, information was gathered per participants or was all the data obtained through the focus groups?\nResults:\nQualitative risk assessment: Unclear in the text what data were from the study and which were from the articles in Table 2.\nDiscussion:\nDid not see where differences in the demographics of the focus groups (sex, age) which could influence the results were discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "73118", "date": "04 Nov 2020", "name": "Gezahegne Mamo", "expertise": [ "Reviewer Expertise Zoonotic diseases (Tuberculosis", "brucellosis", "Anthrax)", "Emerging infectious diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods:\nTechnically, the study lacks clarity as it is quite difficult to say whether the participants have knowledge on livestock diseases or not because the clinical signs alone are not enough to define a diseases like brucellosis.\n\nDetail of the methods used, including the major parameters used to assess the knowledge are not described. The authors should describe briefly what criteria used to say a participating group has knowledge or not, for that supplementary file of the questionnaire should be added to the manuscript\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1916
https://f1000research.com/articles/8-1903/v1	11 Nov 19	{ "type": "Research Article", "title": "Knowledge and practices about breastfeeding in rural areas of Rajshahi District, Bangladesh: A cross-sectional study", "authors": [ "Ruhani Mat Min", "Md Mosharaf Hossain", "Ruhani Mat Min" ], "abstract": "Background: Breastfeeding is an important indicator for child health and mortality. The aim of this study was to determine the level of knowledge and practices regarding EBF and its relation to various socio-economic and demographic factors among mothers with at least one child age (6-12 years) in the rural areas of the Rajshahi district in Bangladesh. Methods: A study based at village hospitals was conducted and a semi-structured questionnaire was used. A total of 513 mothers who had at least one child's age (6-12) months from 32 different village hospitals in rural areas of the Rajshahi District, Bangladesh from September to December 2015. The composite index, chi-square test and binary logistic regression model were used in this study. Results: The incidence of EBF good knowledge and practices was 32.0% and 27.9% among mothers with at least one child age (6-12) months. The analysis shows that the age of mothers ≥ 31 years have less knowledge and practice about EBF compared to mothers aged ≤ 30 years. Mothers who are housewives had a higher probability of good knowledge and practice than mothers who were service providers. Nursing mothers at home have less knowledge and practices about EBF than mothers who gave birth in the hospital. Mothers that had a monthly family income of ≤ 6 699 BDT had less knowledge and practices about EBF compared to mothers with a family income of >6 699 BDT. Conclusions: This study showed a huge gap in EBF knowledge and practices among mothers who have at least one child age (6-12) months. This study suggests that EBF education and interventions can play an important role in increasing EBF good knowledge and practices among mothers with at least one-to-one (6-12) months of age children. Malnutrition will be reduced if the EBF is widely established in Bangladesh.", "keywords": [ "Exclusive Breastfeeding", "Knowledge and Practice", "Composite Index", "Chi-square Test", "Binary Logistic Regression." ], "content": "Introduction\n\nExclusive breastfeeding (EBF) is one of the best nutrition practices for child health, growth and nutrition and is an optimal strategy for feeding newborn and young infants1. According to WHO and UNICEF, EBF should start within less than one hour of delivery and should continue for up to 6 months of infants’ age as it is the only diet and source of fluids for babies at that age2.\n\nChildren, especially new born babies, are in high danger of malnutrition during the first six months of life when breast milk alone is necessary to meet all nutritious supplies and breastfeeding needs to continue during this time3. Good practice of EBF can prevent 13.8% and 11.6% of all deaths among infants aged <2 years and those under 5-years, respectively4; however, a report estimated that in 2012 only 35% of infants were exclusively breastfed globally5. EBF, due to its various recognized health welfare for babies, children and their mothers, is a crucial plan to improve public health6. Low breastfeeding rates have been found in Canada, as well as other industrialized countries7, and EBF for at least 6 months is not a general practice in developed nations, and is even less in developing nations8. Usually infant development is measured by nutritional level9.\n\nNearly all Bangladeshi children are breastfed to some extent in the first year of life and many mothers continue to breastfeed up to the second year of a baby’s life (91%)10. Bangladesh has the highest prevalence of malnourishment in South East Asia with a high percentage of children aged 59 months being underweight10. To determine knowledge and practices of newborn nourishment is imperative11.\n\nSeveral studies have been performed to assess the knowledge, perception and practices of breastfeeding among women and to assess the global trends of EBF12,13. For instance, previous studies have been conducted in Nigeria about knowledge, attitude and techniques of breastfeeding mothers of under five children14,15. In Ethiopia, special concern has been paid to the association between schoolgirls’ perception and knowledge about breastfeeding, and knowledge and practice of mothers towards EBF16.\n\nOnly a few studies have been carried out on EBF, and most of these studies were carried out in developing countries17,18. Furthermore, methodological concerns associated with the measurement of knowledge and practices about EBF have not been adequately addressed in earlier studies. The difficulty of judging knowledge lies in its multidimensional aspects; most of studies have been focused on a few indicators. To the best of our knowledge, in Bangladesh this type of study has not been conducted. Therefore, the aim of the present study was to assess the knowledge and practices of EBF among mothers who have at least one child aged 6–12 months in Rajshahi District, Bangladesh.\n\n\nMethods\n\nA village hospital based study was conducted in the rural area of Rajshahi district, Bangladesh. There are several reasons why we selected mothers who have at least one child aged 6–12 months from different village hospitals in Rajshahi district. Firstly, to the best of our knowledge in this area no studies have been conducted on EBF; secondly, this area is situated in the remote areas of Rajshahi19. Most of the sample population included all participants that were living near different village hospitals in Rajshahi district, Bangladesh.\n\nThe following formula has been used for calculating sample size: n= N/ (1+Nd2), where n = required sample size, N= population size (5,123), d = marginal error (0.05)20. The formula provided that the minimum sample size was estimated to be 366 for this study. For a better result, we collected data from 513 participants.\n\nBefore sampling, lists of children aged 6–12 months were gathered from the Charghat upazila (sub-district) Health Complex, Rajshahi, from lists used in expanded programmes on immunization. A two-stage purposive sampling approach was chosen to enrol mothers that have at least one child aged 6–12 months from Rajshahi district. In the first stage, out of nine upazila of Rajshahi District, one upazila was purposively selected. In the second stage, purposive sampling was used for the selected sample size. The inclusion criteria of the participants was mothers who have at one child aged 6–12 months and those with no psychological disorders. Exclusion criteria was male parents. The participants asked to be interviewed during routine check-ups. The interviews took place at the participants homes.\n\nFrom September to December 2015, we collected the following data from the mothers for the study: (i) socio-demographic characteristics and (ii) knowledge about EBF using a semi-structured questionnaire by face-to-face interviews from the villages in Rajshahi District. The survey questionnaires were drafted in Bangla, the national and mother tongue of Bangladesh and was then for research purpose translated into English (Extended data). Five fully trained and experienced enumerators conducted the interviews.\n\nThe dependent variable in our study is the level of good knowledge about EBF, which was calculated through nine different questions, namely: i) Do you know what is meant by EBF?; ii) Do you know when EBF should be started?; iii) Do you know when supplementary feeding is needed?; iv) Do you know if water is allowed in the EBF period?; v) Do you know if honey is allowed in EBF period?; vi) Do you know what the appropriate duration of EBF is?; vii) Do you know what the benefits of EBF are?; viii) Do you know what happens if EBF is not done?; ix) Do you know if any additional feed is essential during the EBF period?\n\nOther outcome variables in this study were good practices about EBF, which was measured through two different questions, namely: i) Did you feed anything else to your last baby during EBF?; ii) What type of feed did you allow during your EBF period for your last baby?\n\nThe respondent’s knowledge and practice were scored using a system adopted from earlier studies. A correct reply was given 1 point, while incorrect replies received 0 points [34].\n\nSocio-economic and demographic factors were included as independent variables. Age was classified into two groups: ≤ 30 years and ≥ 31years. Place of delivery was divided into two groups (hospital and home) and occupation was classified into two groups (housewife and service holder). Education was classified based on the formal learning system in Bangladesh: Illiterate and literate. Size of family was categorized as joint (both parents) or single family. Respondent’s monthly income was categorized as yes or no to the question: do you earn ≤6,999 Bangladeshi Taka (BDT)? – (≤6,999 BDT = yes; ≥7,000 BDT = no).\n\nStatistical Package for Social Science (SPSS) version 22 IBM was used to analyse the data. Descriptive analyses were conducted to ascertain the socio-economic and demographic variables, and the good knowledge and practice scores. Demographic differences regarding good knowledge and practices of EBF were assessed by χ2 analysis significance, and all analyses was set at p<0.05. Completely adjusted models were used to analyse each binary outcome variable. All variables were inputted into binary logistics regression models. The adjusted odds ratio (AOR) was observed to assess the strength of the associations, and 95% confidence intervals (Cis) for significance test were used.\n\nThe knowledge index was calculated through the sums of binary input variables, where the highest and lowest values were selected for each underlying pointer. To determine the knowledge pertaining to breastfeeding, ten questions about the knowledge of breastfeeding were provided. The question was answered CORRECT or INCORRECT. A score of 1 was given for a correct answer and 0 for the incorrect answer. The scores varied from 0–9 points and were classified into two levels, as follows: Bloom’s cut off point, 60%-80%. The items were all assessed using a zero-one indicator (dummy variables). These variables were given a value of zero (low knowledge less than 6 points) for ‘No’ (Bloom’s cut off point less than 59%), and a value of one (high knowledge more than or equal to 6 points) for ‘Yes’ (Bloom’s cut off point 60% – 80% or high). The enactment of individually pointer was articulated using a unit-free index between 0 and 1 in accordance with the structure technique of the Human Development Index21.\n\nKnowledge index = (Actual value - Minimum value) / (Maximum value - Minimum value)\n\nThe scores were categorized as the following groups [35, 36]: knowledge - poor = 0-2, moderate = 3-4, and good = 5-9; practice - poor = 0-<1, and good = ≥1.\n\nThis study was approved by the Department of Population Science and HRD, University of Rajshahi, Bangladesh (Ref: 2658/89, Date: 22/12/2014). Written informed consent was obtained from participants before data collection.\n\n\nResults\n\nA total of 513 mothers were involved in this study. From the total sample population, approximately 61% were ≤30 years of age, 60% of deliveries were at hospital and 61% respondents were housewives. Regarding education, 27.5% were illiterate, 19.1% were primary educated and the remaining 53.4% had secondary or higher level of education. A total of 79.5% were from a joint family, and a major portion of respondent’s (59.8 %) had a monthly family income <6,999 BDT.\n\nThere was a good level of knowledge and practice of EBF among the mothers that participated in this study. Table 1 and Table 2 show the socio-economic and demographic factors associated with good knowledge and practice. From the total sample population, 32% mothers had a good level of knowledge and 27.9% mothers had a good level of practice about EBF, which was statistically significant (p<0.05) for all variables apart from education.\n\nRegression analysis of the factors associated with good knowledge and practices among mothers on EBF showed (Table 3) that mothers aged ≤30 years (adjusted odds ratio (AOR) = 0.040; 95% CI: 0.021-0.079), gave birth in a hospital (AOR = 0.039; 95% CI: 0.017-0.095) and had a ≤6,999 BDT monthly family income (AOR = 0.197; 95% CI: 0.088-0.442) were less likely to have good knowledge of EBF compared to their counterparts (p<0.05). Mothers that were housewives (AOR = 21.352; 95% CI: 5.170-88.174) and had joint families (AOR = 27.445;95% CI: 11.494-65.537) were more likely to have good knowledge of EBF compared to their counterparts (p<0.05).\n\nRreference category\n\nIn Table 4, Mothers aged ≤30 years (AOR = 0.084; 95% CI: 0.050-0.143), gave birth at home (AOR = 0.208; 95% CI: 0.111-0.389), had a ≤6,999 BDT monthly family income (AOR = 0.092; 95% CI: 0.050-0.168), and had a joint family (AOR = 0.024; 95% CI: 0.010-0.057) were less likely to have good practice of EBF compared to their counterparts (p<0.05). Mothers that were housewives (AOR = 9.992; 95% CI: 4.485-22.260) were more likely to have good practice of EBF compared with their counterparts (p<0.05).\n\nRreference category\n\n\nDiscussion\n\nThis study surveyed the knowledge and practice of EBF among mothers in rural area of Rajshahi district, Bangladesh. There are two major findings for this study. First, poor knowledge and practice of EBF was seen in 32.0% and 27.9% of mothers. Second, mothers that had good knowledge and practice about EBF were aged ≤30 years, were housewives, had a hospital delivery, were joint family members and had ≤6,999 BDT monthly family income.\n\nThe study assumed that most of the mothers would have good knowledge and practice of EBF; however, the study demonstrated that a small percentage of mothers in this area were assessed as having a good level of knowledge and practice of EBF. This study therefore highlights the need for EBF health education programs to educate mothers.\n\nUntil now, according to the best of our knowledge this type of study has not been performed in Bangladesh, but similar studies have been conducted in different populations22. The study found that, middle aged mothers (≤30 years) had low knowledge and practices as compared with older respondents (>31 years) and similar results have been found in other countries23. The present study found that hospital delivery respondents had low knowledge and practices as compared with their counterpart, which is consistent with a previous study in Ethiopia24. An extra assumption was that most of the service holder mothers, and those with secondary and higher level of education would have a better knowledge and practice than housewives or those who did not have a high level of education; however, we found that housewives had good knowledge and practices compared with those that were service holders. This study result is consistent with previous other studies25,26.\n\nThose mothers that had joint families had a good of knowledge and practice compared with single mothers. This may be because those mothers in joint families can share their knowledge with other family members. The study also found that mothers from families with ≤6,999 BDT monthly income had good knowledge and practice.\n\nAs a final point, the idea of good knowledge and practices of EBF had various definitions. Therefore it is challenging to measure, particularly using the questionnaire used in the present study. However, this study measures knowledge and practice through a lot of indicators, which were seen in a previous study27.\n\nThis study had a few limitations. Firstly, it was a village based study and people are busy. Secondly, there are 64 districts and 491 sub-districts (upazilas) in Bangladesh, and in this study we considered only one district and one upazila; therefore, more upazilas should be looked.\n\n\nConclusions\n\nThis study found that there are huge knowledge and practice gaps regarding EBF among mothers that have at least one child aged 6–12 months. As malnutrition will be decreased if EBF is widely established, this study suggests that EPF related education and interventions could play an important role to increase the level of knowledge and practice concerning EBF among this population of mothers. Health policy makers of Bangladesh should consider performing a study with a larger sample size so that further information can be obtained regarding knowledge and practice of EBF in Bangladesh.\n\n\nData availability\n\nThe underlying data for this study cannot be openly shared since the consent to participate obtained from the mothers explicitly stated that their data would remain confidential and only be reported in an aggregated manner. Anyone wishing to access the underlying data should first contact the corresponding author (md.hossain@umt.edu.my) who will facilitate contact with the ethical review board who approved the study. Data will be provided to all applicants that apply to access the data.\n\nFigshare: Knowledge and practices about breastfeeding in rural areas of Rajshahi District, Bangladesh: A cross-sectional study, https://doi.org/10.6084/m9.figshare.9975704.v128.\n\nThis project contains the following extended data:\n\n- Questionnaire in Bangla and English.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors gratefully acknowledge the authority of the village hospitals of Rajshahi District, Bangladesh, for giving us permission to use from their catchment area and University Malaysia Terengganu.\n\n\nReferences\n\nImdad A, Yakoob MY, Bhutta ZA: Effect of breastfeeding promotion interventions on breastfeeding rates, with special focus on developing countries. BMC Public Health. 2011; 11(3): S24. 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National center for chronic disease prevention and Health Promotion. Centers for Disease Control and Prevention. 2016. Reference Source\n\nvon Kries R, Koletzko B, Sauerwald T, et al.: Breast feeding and obesity: cross sectional study. BMJ. 1999; 319(7203): 147–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuigley MA, Carson C, Sacker A, et al.: Exclusive breastfeeding duration and infant infection. Eur J Clin Nutr. 2016; 70(12): 1420–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsman H, El Zein L, Wick L: Cultural beliefs that may discourage breastfeeding among Lebanese women: a qualitative analysis. Int Breastfeed J. 2009; 4(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNIPORT: Bangladesh Demographic and Health Survey 2007. National Institute of Population and Training, Dhaka. 2009. Reference Source\n\nDGHS: National Strategy for Infant and Young Child Feeding. Directorate General of Health Services (DGHS) Ministry of Health and Family Welfare (MOHFW) Institute of Public Health Nutrition Dhaka-1212. 2007.\n\nAlFaleh KM: Perception and knowledge of breast feeding among females in Saudi Arabia. J Taibah Univ Med Sci. 2014; 9(2): 139–42. Publisher Full Text\n\nMbada CE, Olowookere AE, Faronbi JO, et al.: Knowledge, attitude and techniques of breastfeeding among Nigerian mothers from a semi-urban community. BMC Res Notes. 2013; 6(1): 552. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOche MO, Umar AS, Ahmed H: Knowledge and practice of exclusive breastfeeding in Kware, Nigeria. Afr Health Sci. 2011; 11(3): 518–523. PubMed Abstract \| Free Full Text\n\nAsfaw MM, Argaw MD, Kefene ZK: Factors associated with exclusive breastfeeding practices in Debre Berhan District, Central Ethiopia: a cross sectional community based study. Int Breastfeed J. 2015; 10(1): 23. Publisher Full Text\n\nZenebu BB, Belayneh KG, Alayou G, et al.: Knowledge and practice of mothers towards exclusive breastfeeding and its associated factors in Ambo Woreda West Shoa Zone Oromia Region, Ethiopia. Epidemiology: Open Access. 2015; 5(1).\n\nRahman MS, Basar A, Karmaker H, et al.: Body Mass Index of University Students and Gender Differential: Survey in Rajshahi University, Bangladesh. South Asian Anthropologist. 2016; 16(1): 27–33. Reference Source\n\nDGHS: Health Bulletin 2015 \| Rajshahi Civil Surgeon Office. Directorate General of Health Services (DGHS) Ministry of Health and Family Welfare (MOHFW) Institute of Public Health Nutrition Dhaka-1212. 2015. Reference Source\n\nHaque SE, Rahman M, Mostofa MG, et al.: Reproductive health care utilization among young mothers in Bangladesh: does autonomy matter? Womens Health Issues. 2012; 22(2): e171–e80. PubMed Abstract \| Publisher Full Text\n\nLing Oh A, Hassali MA, Al-Haddad MS, et al.: Public knowledge and attitudes towards antibiotic usage: a cross-sectional study among the general public in the state of Penang, Malaysia. J Infect Dev Ctries. 2011; 5(5): 338–47. PubMed Abstract \| Publisher Full Text\n\nMoya EM, Biswas A, Chávez Baray SM, et al.: Assessment of stigma associated with tuberculosis in Mexico. Public Health Action. 2014; 4(4): 226–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVelusamy V, Premkumar PS, Kang G: Exclusive breastfeeding practices among mothers in urban slum settlements: pooled analysis from three prospective birth cohort studies in South India. Int Breastfeed J. 2017; 12(1): 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKasahun AW, Wako WG, Gebere MW, et al.: Predictors of exclusive breastfeeding duration among 6-12 month aged children in gurage zone, South Ethiopia: a survival analysis. Int Breastfeed J. 2017; 12(1): 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMekuria G, Edris M: Exclusive breastfeeding and associated factors among mothers in Debre Markos, Northwest Ethiopia: a cross-sectional study. Int Breastfeed J. 2015; 10(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRana M, Sayem A, Karim R, et al.: Assessment of knowledge regarding tuberculosis among non-medical university students in Bangladesh: a cross-sectional study. BMC Public Health. 2015; 15(1): 716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDerso T, Biks GA, Tariku A, et al.: Correlates of early neonatal feeding practice in Dabat HDSS site, northwest Ethiopia. Int Breastfeed J. 2017; 12(1): 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHossain, Mat Min R: Knowledge and practices about breastfeeding in rural areas of Rajshahi District, Bangladesh: A cross-sectional study. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9975704.v1" }	[ { "id": "56807", "date": "04 Dec 2019", "name": "Felix Emeka Anyiam", "expertise": [ "Reviewer Expertise Public Health", "Population Health", "Global Health", "Epidemiological Research", "Biostatistics", "Open Research Data", "Open Science", "Data Science", "Non-communicable diseases", "Maternal and Child Health", "Vulnerable Populations", "Population at Risk", "Reproductive Health", "Data Management", "Community-Based Research", "HIV/AIDS Prevention", "TB Prevention", "Health Management and Health Promotion" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article titled: Knowledge and practices about breastfeeding in rural areas of Rajshahi District, Bangladesh, is a cross-sectional study focused on mothers with at least one child age 6-12 months residing in a rural community. The study is relevant, especially as it is carried out in a rural area where majority of mothers may not have adequate access to Antenatal Care services which is a vital contact for enhancing knowledge and practice relating to EBP. Poor Knowledge and Practice about EBF could be a pointer towards strengthening the Antenatal care services in rural communities. As \"Infant development is measured by nutritional level\", it becomes appropriate to create more awareness towards EBF practices,especially at the rural level.\n\nBelow are my comments and recommendations:\nAbstract\nAt the Background section, the authors stated that the study was among mothers with at least one child age (6-12 years). But in the Methods section they mentioned (6-12) months. Of course it can’t be both. Please revisit and use the correct age.\n\nIn the Result section, the authors mentioned: “The incidence of EBF good knowledge and practices was 32.0% and 27.9% among mothers with at least one child age (6-12) months.” I believe this value here is prevalence and not incidence as this is a cross sectional study, as incidence is a measure of the occurrence of new cases of disease (or some other outcome) during a span of time.\n\nIn the Result section, the authors mentioned: “Mothers that had a monthly family income of ≤ 6 699 BDT had less knowledge and practices about EBF compared to mothers with a family income of >6 699 BDT.” I think from the results in Tables 1 & 2, it’s more (or good which is the right term to use) Knowledge and Practice and not less for both.\n\nKeywords: Knowledge should be separated from Practice (Knowledge, Practice), Chi-square Test and Binary Logistic Regression shouldn’t be key words as this is not a Statistical methodology paper. Other key words are recommended.\nIntroduction\nIn text Referencing: The information in Reference 8, “EBF for at least 6 months is not a general practice in developed nations, and is even less in developing nations” is too old (1999) and may not be the true state of things in the present time. A reference not older than 10 years should be sought and is recommended.\n\nIn text Referencing: The information in Reference 10, “Nearly all Bangladeshi children are breastfed to some extent in the first year of life and many mothers continue to breastfeed up to the second year of a baby’s life (91%)” is from one study and not suitable for this sort of generalization. A systematic review would have been more appropriate.\n\nIn text Referencing: Also the same reference 10 stated something contrary from the previous sentence above: “Bangladesh has the highest prevalence of malnourished in South East Asia with a high percentage of children aged 59 months being underweight.” I am not sure which to consider appropriate.\n\nThe authors stated in the last paragraph of the introduction: “Only a few studies have been carried out on EBF, and most of these studies were carried out in developing countries.” Although my own personal search showed several studies. Authors should use a more rigorous search strategy.\n\nThe authors stated in the last paragraph of the introduction: “Furthermore, methodological concerns associated with the measurement of knowledge and practices about EBF have not been adequately addressed in earlier studies.” If this is so, then the studies applicable should be stated.\n\nThe authors stated in the last paragraph of the introduction: “The difficulty of judging knowledge lies in its multidimensional aspects; most of studies have been focused on a few indicators.” I understand accessing knowledge of any kind requires a multidimensional approach but I do not agree that it’s a difficult process. Please rephrase sentence.\n\nOn the last paragraph of the introduction, the authors stated, “…most of studies have been focused on a few indicators.” Should read “most of the studies have been focused on a few indicators.”\n\nThe statement: “To the best of our knowledge, in Bangladesh this type of study has not been conducted” is not completely accurate as my personal search found several studies.\n\nMethodology\nStudy design\nStudy design was not mentioned. Although from the title of the study, it was clear that this is a cross-sectional study but it was not mentioned in the right place. What was said here was: “A village hospital based study…” which is not a study design. Please include the study design in this section.\n\nThe authors stated: “There are several reasons why we selected mothers who have at least one child aged 6–12 months” and I saw only two reasons. Maybe it should be stated two reasons and not several reasons.\n\nThe authors stated, “Firstly, to the best of our knowledge in this area no studies have been conducted on EBF” and personally for me, I found several similar studies in my search.\nSimple size determination\nThe authors stated, “The following formula has been used for calculating sample size: n= N/ (1+Nd2)…” and they gave a reference from Haque et al., (2012). The original author for this formula is Taro Yomane, and the Reference link is:[Yamane, Taro. 1967. Statistics, An Introductory Analysis, 2nd Ed., New York: Harper and Row.1]\n\nUsing this formula, the minimum sample size should have been 371 when calculated and not 366 as stated.\n\nParticipants\n“A two-stage purposive sampling approach was chosen to enrol mothers..” ‘enrol’ should read, ‘enroll.’\n\n“The inclusion criteria of the participants was mothers who have at one child aged 6–12 months..” Should read, “The inclusion criteria of the participants were mothers who have at least one child aged 6–12 months …”\n\nThe authors stated, “A two-stage purposive sampling approach was chosen to enrol mothers that have at least one child aged 6–12 months from Rajshahi district. In the first stage, out of nine upazila of Rajshahi District, one upazila was purposively selected. In the second stage, purposive sampling was used for the selected sample size.” The whole process of the sample size selection is not clear as the authors had stated in the Abstract, “A total of 513 mothers who had at least one child's age (6-12) months from 32 different village hospitals in rural areas of the Rajshahi District, Bangladesh.” Is 32 different village hospitals from one Upazila? Also, the 32 different village hospitals couldn’t have been the sample size and so a step in selecting the village hospitals is missing. This study would have done well with a Probability sampling.\n\nThe authors stated, “The interviews took place at the participants homes.” How is this possible when the authors have already stated that the sample size was from 32 different village hospitals?\nData collection\nThe Sentence, “The survey questionnaires were drafted in Bangla, the national and mother tongue of Bangladesh and was then for research purpose translated…” is not comprehensible. Please rephrase.\n\nIndependent variables\nI would have preferred the raw ages of the women were used so we can have mean age in the study population, before categorization for the inferential statistics purposes.\nStatistical analyses\nIn the statement, “Demographic differences regarding good knowledge and practices of EBF were assessed by χ2 analysis significance..”\n\nIt is proper to put a Chi-Square before the χ2 symbol. Also, the ‘significance’ should be removed.\n\n“95% confidence intervals (Cis)” Cis should read CIs\nResults\nI think the first table for this study should have been a descriptive statistics of all the independent variables. The authors started with an inferential statistics, where they compared the independents and the dependents variables. In their methodology they mentioned, “Descriptive analyses were conducted…” although I didn’t find a separate table for the descriptive analysis, which is the proper thing to do, as it was merged with the inferential statistics table.\n\nThey also mentioned, “The incidence [which I think should be changed to prevalence] of EBF good knowledge and practices was 32.0% and 27.9% among mothers with at least one child age (6-12) months.” No separate descriptive statistics table to verify this information.\nDiscussion\nThe authors stated “Until now, according to the best of our knowledge this type of study has not been performed in Bangladesh, but similar studies have been conducted in different populations.” The phrase ‘similar studies’ [plural] should have more than one reference. And the Reference 22 quoted here has to do with Tuberculosis (TB) and not EBF: [Moya EM, Biswas A, Chávez Baray SM, et al.: Assessment of stigma associated with tuberculosis in Mexico. Public Health Action. 2014; 4(4): 226–32.2].\n\nThe authors incorrectly stated, “The study found that, middle aged mothers (≤30 years) had low knowledge and practices as compared with older respondents (>31 years)… and similar results have been found in other countries”. This is contrary to findings in Tables 1 & 2, and also the findings stated in the Abstract section of the study. Also the phrase, “similar results have been found in other countries” is from a study conducted in one country, India. I do not see the justification for the phrase, ‘other countries.’\n\nThe authors incorrectly stated, “The present study found that hospital delivery respondents had low knowledge and practices..” This is totally in opposite to findings in Tables 1 & 2, and also the findings stated in the Abstract section of the study.\n\n“This study result is consistent with previous other studies.” Was mentioned in the concluding aspect of the third paragraph. Reference 26 is a TB study [Rana M, Sayem A, Karim R, et al.: Assessment of knowledge regarding tuberculosis among non-medical university students in Bangladesh: a cross-sectional study. BMC Public Health. 2015; 15(1): 716.3].\n\nThe authors stated, “This study had a few limitations. Firstly, it was a village based study and people are busy.” I do not see this as a limitation. Appropriate study limitations recommended.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "57778", "date": "16 Dec 2019", "name": "Kishwar Azad", "expertise": [ "Reviewer Expertise Maternal and Neonatal Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article titled, ‘Knowledge and practices about breastfeeding in rural areas of Rajshahi District, Bangladesh’: A cross sectional study, examines the knowledge and practices regarding exclusive breastfeeding among mothers with at least one child aged 6-12 months.\n\nI have the following comments:\nThe authors’ claim that this is the first study of its sort, is unjustified. Several studies have been carried by researchers independently and also based on data obtained during Bangladesh health and demographic survey.\n\nThere are factual errors, e.g. 91% of mothers breastfeeding their babies up to 2 years, is an overestimation.\n\nWhat is a ‘village hospital’?\n\nWhy did the authors use lists used in EPI – why wasn’t household survey carried out?\n\nHow were mothers with ‘psychological disorders’ ruled out?\n\nThe participants were interviewed during ‘routine check-ups’ in their homes. This is not clear. What constituted routine check-up?\n\nDoes under 30 years constitute middle age? Why was this age taken as cut-off point?\n\nWhy was income Tk 6999/ used as a cut-off point?\n\nThe authors do not explain why service holders were less knowledgeable than housewives regarding EBF, and why women who delivered in hospital were less likely to practice EBF than ‘their counterpart’.\n\nDiscussion is very thin.\n\nConclusions: Malnutrition is not solely dependent on poor EBF practices as the authors claim. Poor weaning also contributes to malnutrition.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "56433", "date": "29 Jun 2020", "name": "Zhitao Liu", "expertise": [ "Reviewer Expertise human nutrition", "food safety", "nutrition and health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study surveyed the knowledge and practice of exclusive breastfeeding (EBF) and its relation to various social-economic, demographic factors among mothers with at least one child aged 6-12 months in rural area of Rajshahi district, Bangladesh. Poor knowledge and practice of EBF was reported. Conclusions from this study play an important role in optimizing EBF practices. However, I have some suggestions and major concerns that must be addressed.\nTitle:\nI suggest you change breastfeeding to exclusive breastfeeding. Because your manuscript fully focused on EBF not breastfeeding.\n\nAbstract: In this section, I agree with suggestions from Felix Emeka Anyiam. I think the reviewer had already worked carefully.\n\nIntroduction: In this section, the statement “To the best of our knowledge, in Bangladesh this type of study has not been conducted” is not exactly correct. Because I had found similar studies conducted in Bangladesh when I searched on Pudmed. Such as two articles,\n“Exclusive breastfeeding practice during first six months of an infant’s life in Bangladesh: a country based cross-sectional study” (https://doi.org/10.1186/s12887-018-1076-0)1. It is the Bangladesh Demographic and Health Survey (BDHS-2014) which collected data from 17,863 Bangladeshi married women in reproductive age from the entire country.\n\n“Knowledge and practices of exclusive breastfeeding among mothers in rural areas of Rajshahi district in Bangladesh: A community clinic based study” (https://doi.org/10.1371/journal.pone.0232027)2. It is published on May 2020. The present study was similar with it, including the same study design, sample size and analysis. Is it the same one?\nI suggest the author reported more findings from previous studies and addressed your novelty in this manuscript.\n\nMethods: In this section, I agree with suggestions from Felix Emeka Anyiam. Additionally, there was a minor error. “ten questions about the knowledge of breastfeeding were provided”. But nine questions were mentioned above. Please check it.\n\nResults:\n“a major portion of respondent’s (59.8 %) had a monthly family income <6,999 BDT”. Is it ≤6,999 BDT? Because monthly income was categorized two groups (≤6,999 Bangladeshi Taka (BDT) and ≥7,000 BDT ). Please check it. Also, monthly income was not the same in the four tables (table 1 ≤6,999 >7,000, table 2 <6,999 ≥7,000 table 3 and table 4 ≤6,699 ≥7,000, which one is correct? I thought 6,699 was just a typing error. Is it?).\n\nThe statement “There was a good level of knowledge and practice of EBF\nAmong the mothers that participated in this study”, but I thought it is not good enough (32% vs.27.9%). And you mentioned in the discussion “poor knowledge and practice of EBF was seen”. Is it opposite to results? Please check it.\nFor table 3 and table 4, I have different explanations. For table 3, my interpretations were mothers aged ≥31years, gave birth at home and had ≥7,000 BDT monthly family income were less likely to have good knowledge of EBF compared to their counterparts (p<0.05). Mothers that were literate, service holder and had single families were more likely to have good knowledge of EBF compared to their counterparts (p<0.05). For table 4, my interpretations were mothers aged ≥31 years, gave birth at home, had single families and had ≥7,000 BDT monthly family income were less likely to have good practices of EBF compared to their counterparts (p<0.05). Mothers that were literate, service holder were more likely to have good practices of EBF compared to their counterparts (p<0.05).\n\nBecause in my opinion, If OR>1, 95% CI did not include 1 and p<0.05, the dependent variable was a risky factor. The risk for the dependent variable (which was label as 1) had more times of risks than the dependent variable (which was label as 0). If OR<1, 95% CI did not include 1 and p<0.05, the dependent variable was a protective factor. Please see more information of logistic regression interpretation.\nDiscussion: In this section, I agree with suggestions from Felix Emeka Anyiam. Moreover, I had different explanations for logistic regressions. This discussion may be rewritten if you agreed with me.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1903
https://f1000research.com/articles/8-1900/v1	11 Nov 19	{ "type": "Research Article", "title": "Putative autoantibodies in the cerebrospinal fluid of Alzheimer’s disease patients", "authors": [ "Bryant Lim", "Magda Tsolaki", "Ihor Batruch", "Anna Anastasiou", "Antonis Frontistis", "Ioannis Prassas", "Eleftherios P. Diamandis", "Bryant Lim", "Magda Tsolaki", "Ihor Batruch", "Anna Anastasiou", "Antonis Frontistis", "Ioannis Prassas" ], "abstract": "Background: Recent efforts have described an immunogenic component to the pathobiology of Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, current methods of studying fluid autoantibodies, such as enzyme-linked immunosorbent assays and immunohistochemistry, are hypothesis-driven and not optimal for discovering new autoantibody biomarkers by proteome-wide screening. Recently, we developed a general mass spectrometry-based approach to identify tissue-specific autoantibodies in serum, at a proteome-wide level. In this study, we adapted the method to explore novel autoantibody biomarkers in the cerebrospinal fluid (CSF) of AD and PD patients. Methods: CSF samples were obtained from 10 headache control individuals, 10 AD patients and 10 PD patients. Antibodies present in the CSF were isolated by immobilization to protein-G magnetic beads. These antibodies were incubated with a brain tissue extract, prepared from frontal cortex, pons, cerebellum and brain stem. Protein antigens captured by the protein-G magnetic bead-bound antibodies were digested with trypsin and analyzed using mass spectrometry. Autoantibody candidates were selected by 1) detection in one or less individuals of the control group and 2) identification in at least half of the patient groups. Results: There were 16 putative autoantibody biomarkers selected from the AD group. Glia-derived nexin autoantibody was detected in eight of ten AD patients and was absent in the control group. Other AD pathology-related targets were also identified, such as actin-interaction protein, quinone oxidoreductase, sushi repeat-containing protein, metalloproteinase inhibitor 2, IP3 receptor 1 and sarcoplasmic/endoplasmic reticulum calcium ATPase 2. An additional eleven autoantibody targets were also identified in the present experiment, although their link to AD is not clear. No autoantibodies in the PD group satisfied our selection criteria. Conclusion: Our unbiased mass spectrometry method was able to detect new putative CSF autoantibody biomarkers of AD. Further investigation into the involvement of humoral autoimmunity in AD and PD pathobiology may be warranted.", "keywords": [ "Alzheimer’s disease", "Parkinson’s disease", "cerebrospinal fluid", "autoantibodies", "immuno-mass spectrometry", "biomarkers", "glia-derived nexin", "actin-interacting protein", "quinone oxidoreductase", "sushi repeat-containing protein", "metalloproteinase inhibitor 2", "inositol 1", "4", "5-triphosphate receptor type 1", "sarco/endoplasmic reticulum calcium ATPase 2" ], "content": "Introduction\n\nSignificant efforts have been made on advancing diagnostic protein biomarkers of Alzheimer’s (AD) and Parkinson’s (PD) disease, the most common forms of neurodegenerative diseases. These discoveries inform the underlying pathobiology and innovative therapeutics for AD and PD1,2. Though the causes of neurodegeneration are largely unknown, recent research hints to an autoimmune component to these diseases3.\n\nThe notion of immune privilege of the central nervous system (CNS) has been challenged by studies revealing functional lymphatic systems that drain cerebrospinal fluid (CSF) to peripheral lymph nodes, prompting re-evaluation of the role of adaptive immunity in neurodegenerative diseases4. In studies linking autoimmune mechanisms to AD, D’Andrea observed immunoglobulin G (IgG)-specific neuron degeneration through a classical complement pathway mediated by microglia in AD post-mortem brains5,6. In PD, post-mortem studies of brain tissue showed IgG binding and alterations in CD4+ and CD8+ T cell levels in proximity to dopamine neurons, suggesting a potential autoimmune involvement in PD progression7. Changes in brain-related autoantibody levels in CSF and serum of AD and PD patients have also been identified. The targeted self-antigens include pathology-related protein aggregates, neurotransmitters, surface receptors, glial markers, lipids and cellular enzymes8,9.\n\nCurrently, experimental techniques to identify biofluid autoantibodies are limited. The primary methods to quantify autoantibodies are radiobinding assays, immunohistochemistry, enzyme-linked immunosorbent assays (ELISA), bead-based assays and protein microarrays10. Most of these tools, however, require an a priori hypothesis, and are limited to single-target profiling. High-throughput methods such as human protein microarrays have yielded novel autoantibody markers, but they are costly and require recombinant protein availability and optimization10. Recently, our laboratory developed an unsupervised mass-spectrometry-based protocol using a data-dependent acquisition approach to identify tissue-specific autoantibodies11. Here, we applied this protocol in a preliminary study to identify novel brain-specific autoantibodies in the CSF of AD and PD, using a cohort of 10 headache control individuals, 10 AD and 10 PD patients.\n\n\nMethods\n\nCSF was retrospectively collected from a total of 30 individuals between 2014 and 2019 at the memory and dementia clinic of the 1st and 3rd Department of Neurology, AHEPA and “G. Papanicolaou” Hospitals, School of Medicine, Aristotle University of Thessaloniki, Greece. The study was approved with written informed consent from study individuals and by the Greek Alzheimer Association and Related Disorders (GAARD) scientific and ethics committees, and the Institutional Review Board of the University of Toronto.\n\nThe study participants included 10 control individuals with headache, 10 patients with AD and 10 patients with PD. Clinical diagnosis of probable AD was made based on the NINCDS/ADRDA criteria for probable AD with a threshold cut-off for AD at a Mini-Mental State Examination (MMSE) score of 2612. Clinical diagnosis of PD was made based on the modified Hoehn and Yahr (H-Y) scale13. Functional Rating Scale for Symptoms of Dementia (FRSSD) was also measured to assess the impact of dementia on patients’ daily activities.\n\nFollowing confirmation of diagnosis, CSF samples were collected by lumbar puncture in the morning, centrifuged to remove cellular components and stored at -80°C polypropylene tubes. The samples were then shipped to the Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada and stored at -80°C until further processing.\n\nTotal protein was extracted from four regions of the brain: frontal cortex, pons, cerebellum and brain stem. Each tissue was pulverized in liquid nitrogen using a mortar and pestle. The pulverized tissue was further digested with 0.2% RapiGest SF Surfactant (Waters, Milford, MA, USA) in 50 mM ammonium bicarbonate (ABC) for 30 min on ice, while vortexing every 2–5 min. The homogenate was sonicated on ice for three times, 15 s each, and centrifuged at 15,000 g for 20 min at 4°C. The resulting pellet containing debris and insoluble contaminants was removed. Pierce bicinchoninic acid assay (Thermo Fisher Scientific, San Jose, California) was performed to determine total protein concentration. Fractions from each brain region were pooled in equal parts (in terms of total protein contribution).\n\nThe experimental protocol has been described elsewhere11. Briefly, 50 µL of 10% w/v Protein-G Mag Sepharose Xtra magnetic beads (GE Healthcare) medium slurry was resuspended by vortexing and added to a microcentrifuge tube. The microcentrifuge tube was placed in a magnetic separator, and the storage solution was removed. The magnetic beads were washed with 500 µL PBS. CSF samples were spiked with 100 ng of human kallikrein 6 (HK6) mouse monoclonal antibody, purified in-house with high sensitivity and specificity14, as a positive control and added to the magnetic beads. PBS was added to the mixture to reach a final volume of 300 µL. IgG from the CSF was bound to the beads during a 30 min incubation with gentle rotation. After two washes with 500 µL PBS, 100 µg of the pooled brain lysate was added to the beads, followed by a 2-hour incubation with gentle rotation. Following incubation, the beads were washed three times with 500 µL PBS 0.05% Tween 20, and subsequently washed three more times with 500 µL PBS. The beads were reconstituted in 100 µL PBS.\n\nThe reconstituted beads, along with the captured antibodies and antigens, were reduced by adding 100 mM dithiothreitol (DTT) to a final concentration of 5 mM, and incubated at 56°C for 40 min. For alkylation, 500 mM iodoacetamide (IAA) was added to a final concentration of 15 mM and incubated for 30 minutes in the dark with gentle shaking. For digestion, trypsin was added to each sample in a 1:50 enzyme to substrate ratio and incubated at 37°C overnight with gentle shaking. The supernatant was collected using the magnetic separator, and formic acid was added to a final concertation of 1%, reaching a pH of 2, to stop the reaction.\n\nPeptides were purified by extraction using OMIX C18 tips (Agilent Technologies, Santa Clara, CA), eluted with 3 µL acetonitrile buffer solution (0.1% formic acid in 65% acetonitrile) supplemented with 57 µL of 0.1% formic acid. Using an auto-sampler, 18 µL of sample, run in technical duplicates, was injected from a 96-well plate into a C18 Acclaim PepMap 100 (75 µm x 2 cm, C18 3 µm bead, 100 Å pore size) trap column (Thermo Fisher Scientific, San Jose, California) and peptides were eluted into a 50 cm analytical column (PepMap RSLC C18, 75 μm ID, 2 μm bead, 100 Å pore, ES803, Thermo Fisher Scientific). The liquid chromatography, EASY-nLC 1200 system (Thermo Fisher Scientific), was coupled online to a Q Exactive HF-X (Thermo Fisher Scientific) mass spectrometer with the EASY-Spray ionization source (Thermo Fisher Scientific) with a spray voltage of 2 kV and capillary temperature at 320°C. The 60-minute liquid chromatography (LC) was applied at a flow rate of 250 nl/min with an increasing concentration of buffer D (0.1% formic acid in 95% acetonitrile). In a 60-min data-dependent acquisition (DDA) mode, full MS1 scan was acquired from 400 to 1500 m/z at a resolution of 60,000 in profile mode, followed by MS2 scans of the top 28 parent ions at a resolution of 15,000. Dynamic exclusion was set to 20 s, and 1+ and 6+ or more charge state ions were excluded from MS2 fragmentation. MS method parameters were detailed previously11.\n\nRaw files were uploaded into the Proteome Discoverer v.1.4 (Thermo Fisher Scientific) and searched with Sequest HT search engine against the Human 5640 Swiss-Prot protein database (January 2018) (MaxQuant is an open-source alternative to Proteome Discoverer). The search parameters included: trypsin enzyme with two maximum missed cleavages, cysteine carbamidomethylation as a static modification, precursor mass tolerance of 7 ppm, fragment mass tolerance of 0.02 Da, methionine oxidation as a dynamic modification, 1% false-discovery rate (FDR) at the peptide and protein level using the Percolator node.\n\nAbundant serum proteins that may bind non-specifically to the beads, including hemoglobin, haptoglobin, hemopexin, immunoglobins, keratins, apolipoproteins, serum albumin and complement, were removed from the initial candidate selection. Candidate autoantibody biomarkers were identified by antigens that were 1) absent in the patient control group, defined as identification in a maximum of one out of the 10 control individuals and 2) identified in the patient group, defined as presence in at least half of the patients (5 out of 10).\n\nStatistical analyses for clinical descriptions were performed using GraphPad Prism v. 6.0e. A p-value <0.05 was considered significant. Chi-square test (overall and pairwise) was used to compare categorical demographic characteristics of the three groups. Non-parametric Kruskal-Wallis test by ranks was used to compare characteristics on a continuous scale. Dunn’s multiple comparison test was applied for pairwise comparisons.\n\n\nResults\n\nPatient descriptions are shown in Table 1 and Underlying data15. There were no significant differences in the proportion of males and females. Age was significantly different between groups (p = 0.0002), and pairwise comparisons revealed lower age in the control group than both AD (p = 0.0005) and PD (p = 0.0025) patient groups. MMSE score was significantly different between groups (<0.0001), and multiple comparisons revealed that AD (p < 0.0001) and PD (p = 0.0188) groups had lower MMSE scores than the control group. The median (interquartile range) of the H-Y score in the PD group was 2.0 (1.5-2.8). FRSSD was not significantly different between AD and PD groups.\n\naExpressed as median (25th, 75th percentile)\n\nbMini-mental status examination\n\ncHoehn and Yahr score\n\ndFunctional Rating Scale for Symptoms of Dementia\n\nThe total protein content from human brain regions, frontal cortex, pons, cerebellum and brain stems, ranged from 1.7 mg/mL to 5.3 mg/mL (Extended Data: Supplementary Table 116).\n\nAntibodies from the CSF, bound to protein-G beads, captured putative cognate antigens from the brain tissue-mix. Mass spectrometry identification of these cognate autoantigens infer presence of brain-specific autoantibodies from the CSF. Using a 1% FDR for peptide identification, the number of antigens detected in each individual CSF ranged from 461 to 1192, amounting to 1508, 1754, 1452 total antigens identified in the control, AD and PD groups, respectively. After removal of abundant serum proteins, 1342, 1562 and 1281 antigens were remaining in control, AD and PD respectively. The number of antigens that were uniquely found in control, AD and PD groups was 137, 299 and 129, respectively. A Venn diagram of the putative CSF autoantibody-bound antigens detected in each group, after removal of abundant serum antigens is shown in Figure 1. The positive control, human kallikrein 6, (hK6), was abundantly identified in all samples, with nine to 13 unique peptides (Extended Data: Supplementary Table 216). See Underlying data15 for details of all.\n\nTotal number of identified antigens in all samples was 1854 with 1042 (56%) common antigens in all groups. Number of identified antigens in control, AD and PD groups were 1342, 1562 and 1281 respectively.\n\nIn total, 16 putative autoantibodies fulfilled our aforementioned selection criteria in the AD group. No candidates fulfilled the criteria in the PD group. The candidates, along with the number of unique peptides identified for each antigen, are summarized in Table 2. More details on the identity of each identified peptide per antigen, are shown in Extended Data: Supplementary Table 216.\n\nFor AD, three autoantibodies against brain-specific antigens, glia-derived nexin (SERPINE2), fibromodulin (FMOD) and quinone oxidoreductase (NQO1), were absent in CSF of all patients in the control group and were present in eight, six and five patients with AD, respectively (Table 2). SERPINE2 was identified with an average of 2.3 unique peptides in each individual (Extended Data: Supplementary Table 216), totaling seven unique peptides in the whole patient group. FMOD was identified with an average of 1.5 unique peptides per patient with a total of 4 peptides in the whole patient group. Finally, NQO1 was identified in five patients with one unique (the same) peptide per patient (Extended Data: Supplementary Table 216).\n\nA further 13 putative autoantibodies against brain-specific antigens were found in one out of ten control individuals and at least half of the AD patients. In all cases in the control group, the antigen was identified using only one unique peptide (Table 2 and Extended Data: Supplementary Table 216). Autoantibodies against cathepsin F (CTSF), cadherin-13 (CDH13), and phospholipase D4 (PLD4) were identified in six AD patients, with an average of 2.3, 2 and 1 unique peptide per patient, respectively (Table 2 and Extended Data: Supplementary Table 216). The remaining candidates were identified in five AD patients (Table 2). This included inositol 1,4,5-triphosphate receptor type 1 (ITPR1, or IP3 receptor), sushi repeat-containing protein (SRPX), isoaspartyl peptidase (ASRGL1), heterogeneous nuclear ribonucleoprotein H (hnRNP H), cerebellin-3 (CBLN3), oligodendrocyte-myelin glycoprotein (OMG), metalloproteinase inhibitor 2 (TIMP-2), WD repeat-containing protein 1 (WDR1, or AIP1), 4F2 cell-surface antigen heavy chain (SLC3A2) and sarco/endoplasmic reticulum calcium ATPase 2 (ATP2A2, or SERCA2). The number of unique peptides detected for each antigen ranged from 1 to 1.8 (Table 2 and Extended Data: Supplementary Table 216).\n\n\nDiscussion\n\nThe relevance of autoimmune mechanisms in AD and PD pathobiology is not well understood. In the present study, we adapted an in-house-designed novel mass-spectrometry-based protocol to explore brain-specific autoantibody biomarkers in the CSF of AD and PD patients11. Presence of autoantibodies is inferred by identification of their cognate antigens. To our knowledge, this is one of the first studies using a non-biased mass spectrometry approach for autoantibody discovery in CSF of AD and PD patients.\n\nPutative AD and PD-relevant self-antigens were defined by 1) identification in one or less individuals in the patient control group (n=10) and 2) presence in at least half of the patient group (n=10 each). Using these preset selection criteria, we identified 16 putative brain-specific autoantibodies related to AD. No candidates were identified for PD.\n\nPresence of autoantibodies against SERPINE2 was detected in eight of the ten AD patients with an average of 2.3 unique peptides for the antigen; no peptides were identified in any individuals from the control group. SERPINE2, one of several members in the SERPIN superfamily, is a serine protease inhibitor constitutively secreted by glial cells, and plays a key role in synaptic plasticity for developing and adult CNS17–19. In AD pathology, post-mortem patients show that SERPINE2 levels are related to tau-positive dystrophic neurites and amyloid protein processing in the hippocampus20,21. Furthermore, SERPINE2 is a potent regulator of thrombin, a proximate proinflammatory mediator of blood brain barrier dysfunction implicated in AD22,23. Presence of autoantibodies targeting SERPINE2 may reflect a biological relationship between AD pathogenesis and SERPINE2. Other autoantibodies targeting brain antigens implicated in AD pathology, including WDR1, NQO1, SRPX, TIMP-2, ITPR1 and ATP2A2, were also identified. These proteins are involved in a variety of neurological processes related to AD such as mediating amyloid-beta induced cytotoxicity24,25, antioxidant activity26–29, amyloid plaque co-accumulation30, blocking of Aβ-induced release of lactate dehydrogenase31, maintaining age-related neuronal plasticity32, and mediating presenilin-controlled calcium ion homeostasis33–36. Autoantibodies against FMOD, CDH13, CTSF, PLD4, SRPX, ASRGL1, hnRNP H, CBLN3, OMG and SLC3A2 were also identified in the present study, although their link to AD is not well understood.\n\nInterestingly, no autoantibody biomarker candidates were identified in PD using the same selection criteria. Whether this is due to the insensitivity of the method or to the lesser involvement of autoimmunity in PD cannot be determined.\n\nThere are several limitations with this study. The significantly younger control group could be a confounding factor, leading to lower abundance of autoantibodies in the control group37. The study comprises a small sample size, and therefore candidates would require further verification in a larger cohort using recombinant proteins or orthogonal methods, such as ELISA. Finally, this exploratory method may identify autoantibodies of unknown significance, and is unable to establish a causal link between the identified autoantibodies and disease processes. Functional studies must be conducted to delineate the roles of each autoantibody in disease pathology.\n\nThe role of humoral immunity in the pathogenesis of AD and PD remains a controversial topic. However, given that over 99% of compounds entering phase I trials for AD never reach approval and the mounting evidence of the multi-faceted complexity of AD pathology, innovative research perspectives and technologies are necessary to explore its pathobiology from alternative angles38. Biomarkers identified from these approaches could subsequently inform our understanding of the underlying biology and potential therapeutics. In the present study, we adapted a novel unsupervised proteomic approach to detect potential immunogenic components of AD and PD, and identified promising autoantibody biomarkers of AD. Future studies focusing on an autoimmune pathogenesis of AD, but not PD, are warranted.\n\n\nData availability\n\nHarvard Dataverse: Putative autoantibodies in the cerebrospinal fluid of Alzheimer’s disease patients. https://doi.org/10.7910/DVN/DYMEQR15.\n\nThis project contains the following underlying data:\n\n- Patient descriptives.txt (clinical data, including demographic information and clinical presentation data for each enrolled patient).\n\n- Shotgun mass spectrometry for brain specific autoantibodies.tab (Raw mass spectrometry data showing identified antigens).\n\nHarvard Dataverse: Extended data for \"Putative autoantibodies in the cerebrospinal fluid of Alzheimer’s disease patients\" https://doi.org/10.7910/DVN/JJW0LG16.\n\nThis project contains the following extended data:\n\nSupplementary Table 1 (Total protein concentration in each human brain tissue extract as determined by Pierce BCA Protein Assay).\n\n- Supplementary Table 2 (Brain-specific self-antigens and the number of associated peptides used to identify the protein in each sample. In the expanded spreadsheet, \"I\" indicates that the peptide was identified in the patient CSF sample, while blank cells indicate absence).\n\n- Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBlennow K, Biscetti L, Eusebi P, et al.: Cerebrospinal fluid biomarkers in Alzheimer's and Parkinson's diseases-From pathophysiology to clinical practice. Mov Disord. 2016; 31(6): 836–847. PubMed Abstract \| Publisher Full Text\n\nVemuri P, Fields J, Peter J, et al.: Cognitive interventions in Alzheimer's and Parkinson's diseases: emerging mechanisms and role of imaging. Curr Opin Neurol. 2016; 29(4): 405–411. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindestam Arlehamn CS, Garretti F, Sulzer D, et al.: Roles for the adaptive immune system in Parkinson's and Alzheimer's diseases. Curr Opin Immunol. 2019; 59: 115–120. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin HJ, Shaffer KM, Sun Z, et al.: Glial-derived nexin, a differentially expressed gene during neuronal differentiation, transforms HEK cells into neuron-like cells. Int J Dev Neurosci. 2005; 23(1): 9–14. PubMed Abstract \| Publisher Full Text\n\nKutz SM, Higgins CE, Higgins PJ: Novel Combinatorial Therapeutic Targeting of PAI-1 (SERPINE1) Gene Expression in Alzheimer's Disease. Mol Med Ther. 2012; 1(2): 106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi BH, Kim RC, Vaughan PJ, et al.: Decreases in protease nexins in Alzheimer's disease brain. Neurobiol Aging. 1995; 16(4): 557–562. PubMed Abstract \| Publisher Full Text\n\nNamba Y, Ikeda K, Tomonaga M: Protease Nexin 1 Immunoreactivity in Senile Plaques in Alzheimer Disease and Aged Brain. In:Springer, Boston, MA. 1990; 38A: 55–58. Publisher Full Text\n\nFestoff BW, Sajja RK, van Dreden P, et al.: HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer's disease. J Neuroinflammation. 2016; 13(1): 194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaughan PJ, Su J, Cotman CW, et al.: Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer's disease. Brain Res. 1994; 668(1–2): 160–170. PubMed Abstract \| Publisher Full Text\n\nWang H, Fan L, Wang H, et al.: Amyloid β regulates the expression and function of AIP1. J Mol Neurosci. 2015; 55(1): 227–232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHassan WM, Merin DA, Fonte V, et al.: AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model. Hum Mol Genet. 2009; 18(15): 2739–2747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsvetkov P, Adamovich Y, Elliott E, et al.: E3 ligase STUB1/CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1) accumulation in aged brain, a process impaired in certain Alzheimer disease patients. J Biol Chem. 2011; 286(11): 8839–8845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKapinya KJ, Harms U, Harms C, et al.: Role of NAD(P)H:quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischaemia in vivo. J Neurochem. 2003; 84(5): 1028–1039. PubMed Abstract \| Publisher Full Text\n\nSantaCruz KS, Yazlovitskaya E, Collins J, et al.: Regional NAD(P)H:quinone oxidoreductase activity in Alzheimer's disease. Neurobiol Aging. 2004; 25(1): 63–69. PubMed Abstract \| Publisher Full Text\n\nChhetri J, King AE, Gueven N: Alzheimer's Disease and NQO1: Is there a Link? Curr Alzheimer Res. 2018; 15(1): 56–66. PubMed Abstract \| Publisher Full Text\n\nInoue Y, Ueda M, Tasaki M, et al.: Sushi repeat-containing protein 1: a novel disease-associated molecule in cerebral amyloid angiopathy. Acta Neuropathol. 2017; 134(4): 605–617. PubMed Abstract \| Publisher Full Text\n\nWang XX, Tan MS, Yu JT, et al.: Matrix metalloproteinases and their multiple roles in Alzheimer's disease. Biomed Res Int. 2014; 2014: 908636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcía-González L, Pilat D, Baranger K, et al.: Emerging Alternative Proteinases in APP Metabolism and Alzheimer's Disease Pathogenesis: A Focus on MT1-MMP and MT5-MMP. Front Aging Neurosci. 2019; 11: 244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrajnak K, Dahl R: A new target for Alzheimer's disease: A small molecule SERCA activator is neuroprotective in vitro and improves memory and cognition in APP/PS1 mice. Bioorg Med Chem Lett. 2018; 28(9): 1591–1594. PubMed Abstract \| Publisher Full Text\n\nGreen KN, Demuro A, Akbari Y, et al.: SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production. J Cell Biol. 2008; 181(7): 1107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGazda K, Kuznicki J, Wegierski T: Knockdown of amyloid precursor protein increases calcium levels in the endoplasmic reticulum. Sci Rep. 2017; 7(1): 14512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMak DO, Cheung KH, Toglia P, et al.: Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins. Blackwell KT, ed. PLoS Comput Biol. 2015; 11(10): e1004529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagele EP, Han M, Acharya NK, et al.: Natural IgG autoantibodies are abundant and ubiquitous in human sera, and their number is influenced by age, gender, and disease.Tsokos GC, ed. PLoS One. 2013; 8(4): e60726. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCummings JL, Morstorf T, Zhong K: Alzheimer's disease drug-development pipeline: few candidates, frequent failures. Alzheimers Res Ther. 2014; 6(4): 37. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "58126", "date": "29 Jan 2020", "name": "Kiang-Teck Jerry Yeo", "expertise": [ "Reviewer Expertise Our areas of research encompass blood", "CSF", "urine", "other body fluids biomarkers employed in the diagnosis", "prognosis and treatment of diseases." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlzheimer’s disease (AD) and Parkinson’s disease (PD) are two most common neurodegenerative diseases responsible for dementia. Currently, AD is often diagnosed in the mild dementia stage by a combination of clinical tests and brain imaging studies. Numerous studies have highlighted the use of CSF or blood biomarkers in identifying and diagnosing AD at earlier stages, even in the pre-symptomatic phase. Despite the hallmark findings of accumulation of amyloid-β protein (Aβ) and hyper-phosphorylated tau proteins in AD, the pathogenic mechanism that led to the onset and development of disease are still largely unknown. There is increasing evidence suggesting that autoimmune diseases are linked to increased risk of dementia development. Autoantibodies against a variety of antigens were found to be associated with AD, such as autoantibodies to Aβ, tau proteins, neurotransmitters, glial markers, and lipid molecules. There is still a real unmet need to discover sensitive and specific biomarkers for AD and PD, ideally in less invasive, blood samples. Recently, ELISA and microarray based analysis has been applied in an attempt to develop diagnostic tests for AD.\n\nThe authors described a novel immunoaffinity-based mass spectrometry method to identify potential autoantibody biomarkers in AD and PD. The study limitations, including significant younger age of control group, small sample size (10 in each group), and unknown method sensitivity are discussed. CSF and brain tissue preparation were analyzed using NanoLC-MS, and peptide search are all described in detail. The preset selection criteria for representative autoantibodies in the disease group attempts to identify relatively more disease-specific biomarkers. Overall this study, although small in sample size, is scientifically sound, and presents a good and interesting proteomics approach for biomarker discovery, especially immunoglobulin biomarker study. Several questions are raised by the reviewers regarding this work that may help clarify some aspects of the methods:\n\nAre AD or PD patients truly able to give “informed consent”?\n\nHow do you convince patients with HA to undergo LP for CSF collection, or is this part of the diagnostic workup?\n\nWhy was RapiGest SF surfactant used on brain tissue before incubation with bead-bound IgG? RapiGest is a mild denaturant, which unfolds proteins and makes proteins more susceptible to enzymatic cleavage. Most immunoaffinity-based mass spectrometry methods denature proteins after immunoaffinity precipitation and elution.\n\nThe authors stated measuring protein concentration of extracted brain tissue and applied an equal amount of tissue protein in each sample. However, did the authors also measure CSF protein and CSF IgG concentration? Were equal amounts of CSF IgG applied in each sample? If not, will low CSF IgG concentration in one sample/or one group result in lower sensitivity on autoantibody detection?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "58124", "date": "05 Feb 2020", "name": "Ronald A. Booth", "expertise": [ "Reviewer Expertise My work is in the area of clinical autoimmune diagnostics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a manuscript detailing a novel mass spectrometric method of identifying putative autoantibodies in CSF.\n\nOverall, the manuscript is clear and well written. It is however a very preliminary report for the identification of potential autoantibodies, and the authors have addressed this in their limitations. One potentially significant confounding element is the age difference between controls and patients. The AD and PD groups are significantly older, which has the potential to produce false positive autoantibodies, as it is well known that older healthy individuals have a higher rate of autoantibodies in the blood, and presumably in the CSF. The increased rate of autoantibodies may account for the unique autoantibodies found in the PD and AD groups. Use of a control group of a similar age would be preferred.\n\nI have a few specific comments/questions:\nThe authors included a positive control antibody, human kallikrein 6, at a concentration of 100 ng. The concentration used may be supraphysiological and hence create an inappropriate positive control. Have the authors titrated the control antibody concentration to determine the lower limit of detection?\n\nIn regards to the putative autoantibodies identified in AD patients, have any of the antibodies been confirmed using an alternate technique? ELISA or immunohistochemistry? Without corroboration with an alternate methodology, these may all be false positive results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "58125", "date": "07 Feb 2020", "name": "Stefan Holdenrieder", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a well-written manuscript on a novel approach for exploring novel autoantibody (AAB) biomarkers in the cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD) and Parkinson’s disease (PD). They used an unsupervised mass spectrometry method to identify brain antigens that were bound to AABs in the CSF of these patients but not in headache patients as controls.\n\nThereby they identified several novel candidate markers particularly in AD patients that were present in at least half of AD patients but not or almost not in controls. In PD patients, no relevant marker was identified using these criteria. Generally, this is an interesting approach as it overcomes the targeted hypothesis-based search by immunological methods and leads to the detection of until now unknown markers. For some of these new markers, a link to disease pathophysiology was proposed.\n\nHowever, the authors also emphasize that this is only the first step of novel marker detection strategy and recognize the limitations of their study, e.g. the low patient number, the presence of confounding factors and the need of validation in a larger cohort by orthogonal methods. Finally basic science is required to establish a link of all novel markers with disease pathogenesis and progression.\n\nIn addition, there may be some more general aspects that should be addressed in the discussion section, e.g. the limited sensitivity of the method for less frequent antigens, the importance of appropriate preparation of the brain tissue templates (were dopaminergic structures of the striatum included?) and the variable antibody specificity for interindividually different antigens on brain structures.\n\nFurthermore, the heterogeneity of these diseases and the different stages included in the cohort should be discussed. Most importantly, it may be relevant that also other types of dementia (e.g. cerebrovascular) and neuroinflammatory diseases are included as further control groups.\n\nRegarding the present study it could be discussed whether markers that are present in AD but not in PD (and vice versa) are more relevant as they may be more disease specific. This means that also the other diseased patients could serve as control group. Finally, if then an AAB is present in less than 50% e.g. of PD patients but in none of all other control cohorts, this may be a valuable disease-specific marker although it is not as frequent in the specified disease as other ones.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "56422", "date": "05 Oct 2020", "name": "Mohd M. Khan", "expertise": [ "Reviewer Expertise Systems Proteomics", "Mass Spectrometry-based biomarker discovery and validation", "Toxicoproteomics", "Secretomics", "Host-Pathogen Biology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEach year about 10 million new cases of dementia are reported worldwide. In the US alone, Alzheimer's disease (AD) is the 6th leading cause of death as it affects >5 million Americans. AD is generally diagnosed by conducting tests to assess memory impairment as well as using brain imaging such as Magnetic resonance imaging (MRI), Computerized tomography (CT), and Positron Emission Tomography (PET) based tests. Numerous studies have proposed potential use of biomarkers (e.g., beta-amyloid and tau levels in cerebrospinal fluid (CSF) and brain changes detectable by imaging), however currently there are no validated biomarkers for AD. Recently, several reports suggested that autoantibodies may play role in AD and can be used as diagnostic/prognostic biomarkers for AD as well as understanding AD etiology. In the present work, authors conducted mass spectrometry-based discovery proteomics to identify autoantibodies in CSF of AD subjects that may be used as potential diagnostic markers. The manuscript is well-written and the work is interesting, however following are a few suggestions that will improve the manuscript:\nAuthors should provide additional details on Tissue protein extraction such as which enzyme was used to digest pulverized tissue (as RapiGest SF, a mild denaturant, only solubilizes and unfolds the proteins) \"OR\" authors meant that RapiGest SF was used to solubilize proteins?\n\nImmunoprecipitation: did authors optimize the immunoprecipitation method to minimize binding of non-specific proteins as in the \"Data Analysis\" section they describe several non-specific proteins were detected (Authors did remove these from the final analyses and that may potentially introduce systematic error/bias)?\n\nData Analysis: authors should provide additional details such as how the peptides/proteins were filtered (e.g., use of >2 unique peptides; and also provide details if they have calculated Log2 fold changes in protein expression between groups). It is reported that ~1 unique peptide per protein is detected and that is not as robust data to confidently detect, identify, and quantify unique proteins.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1900
https://f1000research.com/articles/8-1630/v1	12 Sep 19	{ "type": "Case Report", "title": "Case Report: Borderline tumor and primary peritoneal carcinoma - a rare synchronism", "authors": [ "Mariana Rei", "Sofia Raposo", "Paulo Figueiredo", "Rita Sousa", "Luís Sá", "Sofia Raposo", "Paulo Figueiredo", "Rita Sousa", "Luís Sá" ], "abstract": "Ovarian borderline serous tumors present with peritoneal involvement in 20% of cases, either as non-invasive or invasive implants, also known as extraovarian low-grade serous carcinoma. The coexistence of high-grade serous carcinoma is rare, suggesting a synchronous neoplasia with a distinct and independent tumor biology and behavior. We aim to describe a case of a synchronous ovary-peritoneum neoplasia: serous borderline tumor and primary peritoneal high-grade serous carcinoma. A discussion and literature review concerning the optimal diagnostic and therapeutic approach is provided.", "keywords": [ "borderline tumor", "ovarian neoplasm", "peritoneal neoplasm", "high grade serous carcinoma", "low grade serous carcinoma" ], "content": "Introduction\n\nSerous borderline tumors/atypical proliferative serous tumors (SBT/APST) are defined as non-invasive tumors displaying greater epithelial proliferation and cytological atypia than their benign counterparts. However, the SBT/APST show less cellular atypia than low-grade serous carcinoma (LGSC)1. This entity is considered the premalignant lesion of LGSC and usually occurs in women 10 to 15 years younger than those with serous carcinoma. Although there are some similarities in risk-factor associations to high-grade serous carcinoma (HGSC), infertility is more common and there is usually no relation with BRCA1/BRCA2 mutations2,3.\n\nPeritoneal lesions associated with SBT/APST may occur in 20% of cases and were classically defined as non-invasive or invasive implants based on the capacity to infiltrate the underlying tissue. The non-invasive implants are further subdivided into desmoplasic or epithelial types and have almost no negative influence on the 10-year survival rates. Distinctively, the invasive form behaves like LGSC, featuring a poor prognosis with a 50% recurrence rate and a 35% 10-year survival rate2,4,5.\n\nTherefore, the morphology of the peritoneal implants is the main prognostic factor for patients presenting with stage II-III SBT/APST1,5. However, implants are heterogeneous, different histologic patterns may coexist, and unequivocal invasion may be difficult to establish in some cases. A comprehensive histopathological examination of multiple fragments of peritoneal implants is recommended in order to optimize the differential diagnosis between non-invasive and invasive implants.\n\nPrimary peritoneal carcinoma (PPC) resemble low- or high-grade serous ovarian counterpart and occurs in women with a mean age of 62 years. The estimated lifetime risk is 1 per 500 women, and nearly 15% of common epithelial ovarian cancers are in fact PPC1. Histology and immunohistochemistry (IHC) are virtually indistinguishable from epithelial ovarian carcinoma and the most common histological variant is HGSC, although other histologic types have also been reported. In order to meet criteria for PPC, both ovaries and tubes should be macro- and microscopically normal in size or enlarged by a benign process1. From a clinical point of view, the clarification of the tumor origin may not be critical, since the oncologic behavior, treatment and prognosis largely overlap their ovarian counterparts and therefore are addressed similarly. Conversely, the distinction between LGSC and HGSC is of utmost importance concerning diagnostic, therapeutic approaches and prognosis.\n\nThe coexistence of SBT/APST and HGSC is rare, suggesting a synchronous neoplasia with a distinct and independent tumor biology and behavior. We aim to describe a case of a synchronous ovary-peritoneum neoplasia. A discussion and literature review concerning the optimal diagnostic and therapeutic approach is provided.\n\n\nCase report\n\nWe present the case of a 52-year-old postmenopausal woman with no relevant medical history, referred to an oncologic center due to a voluminous adnexal mass. She clinically presented with metrorrhagia, asthenia and anorexia with a significant weight loss. Serum tumor markers Ca125, HE4 and Ca 72.4 were significantly increased (1058 U/mL, 1795 pmol/L and 9.5 U/mL, respectively). Pelvic ultrasonography revealed a large heterogeneous multicystic adnexal mass, with multiple papillae and irregular intern contour. Thoraco-abdominal-pelvic computerized tomography (CT) scan revealed a vascularized heterogeneous adnexal mass measuring 190×100 mm; no ascites or unequivocal signs of peritoneal carcinomatosis or distant dissemination were observed. The validated preoperative diagnosis models ROMA, LR2 and ADNEX were calculated, presenting, respectively, a 34.8%, 72.8% and 85.8% risk of malignancy6–9.\n\nWithin two weeks, the case was discussed in the multidisciplinary gynecologic oncology tumor board, advising for laparotomy with frozen section of the suspicious lesions. The patient was then submitted to exploratory laparotomy, revealing a frozen pelvis and peritoneal carcinomatosis: the right ovary was transformed into a voluminous neoplasia; independently, a large tumor mass involving omentum and the transverse portion of colon, apparently not surgically resectable; the left ovary was macroscopically normal. Intraoperative frozen section of the right ovary revealed a borderline tumor, whereby cytoreductive surgery was performed, including hysterectomy, double adnexectomy, omentectomy and resection of the peritoneal implants. The final cytoreduction was complete for the pelvis but incomplete for the superior abdomen, with over 2 cm of residual disease (R2). The final histology with hematoxylin and eosin staining revealed two synchronous tumors: BST of the right ovary and HGSC of probably primary peritoneal origin, FIGO stage IIIC (Figure 1A–C). The left ovary did not show any signs of either malignancies. The IHC was crucial for the final diagnosis, showing positivity for PAX8, WT-1 and p53, while calretinin and CD10 staining were negative.\n\n(A) Hematoxylin-eosin staining, magnification 10×. High-grade serous carcinoma (HGSC) in a peritoneal implant, of presumably primary peritoneal origin. (B) Hematoxylin-eosin staining, magnification 10×. SBT/APST of the right ovary. Left ovary with no evidence of malignancy. (C) Hematoxylin-eosin staining, magnification 10×. Invasive peritoneal implant of the parietocolic gutter, suggestive of HGSC.\n\nThe post-operative CT scan performed four weeks later revealed rapid peritoneal disease progression, with high-volume ascites and hydronephrosis, resulting in an important decline on clinical status and death before initiating palliative chemotherapy.\n\n\nDiscussion\n\nThe dualistic model of ovarian carcinogenesis recognizes two distinct categories in epithelial ovarian carcinomas: the more common aggressive type II tumors and the less common slow-growing type I tumors10. A revised model takes into account the current histopathologic classification, providing a bridge into the future by integrating emerging molecular genetic findings5. Type I tumors arise mostly from borderline tumors and include endometriosis-related tumors, LGSC, mucinous carcinomas and malignant Brenner tumors. Distinctively, type II tumors seem to develop from tubal intraepithelial carcinomas (STICs) that disseminate as carcinomas involving ovary and extraovarian sites, in most cases corresponding to HGSC. This two-tier system subdivides serous carcinomas into low- and high-grade, implying independent pathogenesis and displaying distinct morphology, IHC and molecular biology2,5,11.\n\nHistopathologically, SBTs are characterized by a hierarchical papillary or micropapillary pattern, low-grade nuclear atypia and low mitotic activity. Most SBTs and LGSCs show PAX2 expression but no aberrant p53 expression and p16 overexpression. The molecular genetic analysis frequently demonstrates KRAS or BRAF mutation but TP53 mutation is rare. In contrast, HGSCs present an invasive growth pattern, high-grade nuclear atypia and intense mitotic activity. Aberrant p53 expression, p16 overexpression and high MIB-1 labeling index are common; molecular studies frequently reveal TP53 mutation, but KRAS/BRAF mutation is infrequently reported12,13.\n\nNevertheless, it is still not clear whether some type II tumors develop from type I tumors. Dehari et al.14 studied the clonality of six cases of HGSC closely related to SBT/APST and invasive micropapillary LGSC from a cohort of 210 ovarian serous tumors. A morphologic continuum between the high-grade and the low-grade tumors was observed in four cases; the same KRAS mutations were found in both the SBT/APST and HGSC component of the tumor, indicating a clonal relationship and suggesting that in rare cases, HGSC may arise from SBT/APST14.\n\nWe report a case of a rare synchronism of neoplasms with distinct carcinogenesis pathways: SBT/APST of the right ovary with preserved contralateral ovary and HGSC identified in peritoneal implants. Peritoneal involvement could be related to multiple origins: non-invasive implants of SBT/APST, invasive implants or extraovarian LGSC, coexistence of other ovarian or secondary malignancy. An accurate histopathological and immunohistochemical analysis is of utmost importance in order to better delineate treatment and prognosis.\n\nIn this case, the ovarian tumor showed positivity for PAX8 and WT1. PAX8 is expressed in ovarian and endometrial neoplasias, whereas WT1 is expressed in ovarian serous tumors and is useful to support ovarian origin. Estrogen receptor expression was also positive. On the other hand, both progesterone receptors and CD10 were not present on IHC analysis.\n\nTP53 mutations integrate the molecular carcinogenesis pathway of STIC and are ubiquitous in HGSC, therefore optimizing the differential diagnosis between HGSC and LGSC. p53 is widely used as a surrogate for TP53 mutation; a recent study showed that it can approach 100% specificity for TP53 mutation, and its high negative predictive value is clinically useful to exclude the possibility of a LGSC2,12. In the current report, peritoneal implants stained positive for this marker, excluding the diagnosis of non-invasive implants of SBT/APST or LGSC and reinforcing the coexistence of HGSC. Identification of HGSC in peritoneal implants with a preserved contralateral ovary and tube strongly suggests a primary peritoneal origin.\n\nThe diagnosis of peritoneal carcinomatosis related to HGSC majorly impacts therapeutic approach. While there is no evidence to recommend neoadjuvant or adjuvant chemotherapy in SBT/APST, the diagnosis of HGSC with a high peritoneal cancer index (PCI) might have altered the course of the surgical approach in favor of chemical cytoreduction with neoadjuvant chemotherapy.\n\nNearly 15 to 20% of HGSC are associated with BRCA1/BRCA2 germline mutations15. Its presence impacts therapy and prognosis, since these tumors are highly sensitive to poly (ADP-ribose) polymerase inhibitors, showing improved survival comparing to non-carriers13. Hence, there is now strong evidence to recommend genetic testing to all women affected with high-grade epithelial non-mucinous ovarian/tubal/peritoneal cancer15.\n\nThe diagnosis of a borderline tumor with peritoneal implants imposes the challenging differential diagnosis between non-invasive implants, LGSC and the rare coexistence of HGSC of ovarian or extraovarian origin. This last entity presents a distinct tumor biology and carcinogenesis profile, with strong implications in therapeutic approach and prognosis. Whenever unresectable disease is strongly suspected, adnexectomy or ovarian biopsy in association with peritoneal implants biopsy would allow an accurate diagnosis of a synchronous neoplasia. A peritoneal disease with high tumor burden due to HGSC could impact the optimal therapeutic approach, namely the possibility of neoadjuvant chemotherapy and interval surgery. The discrepancy between tumor markers, imaging criteria and malignancy scores should raise the clinical suspicion for the coexistence of a synchronous neoplasia with a more aggressive oncologic behavior.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from a relative of the patient.", "appendix": "References\n\nPrat J, Cao D, Carinelli S, et al.: (Chapter 1: Tumours of the ovary). In Kurman RJ, Carcangiu ML, Herrington CS, Young RH (eds), WHO Classification of Tumours of Female Reproductive Organs, 4th edition. IARC: Lyon, 2014; 57–62.\n\nKurman RJ, Shih IM: Pathogenesis of ovarian cancer: lessons from morphology and molecular biology and their clinical implications. Int J Gynecol Pathol. 2008; 27(2): 151–160. PubMed Abstract \| Free Full Text\n\nRisch HA, McLaughlin JR, Cole DE, et al.: Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer. Am J Hum Genet. 2001; 68(3): 700–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaldawy A, Segev Y, Lavie O: Low-grade serous ovarian cancer: A review. Gynecol Oncol. 2016; 143(2): 433–438. PubMed Abstract \| Publisher Full Text\n\nKurman RJ, Shih IeM: The Dualistic Model of Ovarian Carcinogenesis: Revisited, Revised, and Expanded. Am J Pathol. 2016; 186(4): 733–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTimmerman D, Testa AC, Bourne T, et al.: Logistic regression model to distinguish between the benign and malignant adnexal mass before surgery: a multicenter study by the International Ovarian Tumor Analysis Group. J Clin Oncol. 2005; 23(34): 8794–8801. PubMed Abstract \| Publisher Full Text\n\nVan Calster B, Van Hoorde K, Valentin L, et al.: Evaluating the risk of ovarian cancer before surgery using the ADNEX model to differentiate between benign, borderline, early and advanced stage invasive, and secondary metastatic tumours: prospective multicentre diagnostic study. BMJ. 2014; 349: g5920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeys EMJ, Jeelof LS, Achten NMJ, et al.: Estimating risk of malignancy in adnexal masses: external validation of the ADNEX model and comparison with other frequently used ultrasound methods. Ultrasound Obstet Gynecol. 2017; 49(6): 784–792. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraicu EI, Van Gorp T, Nassir M, et al.: Preoperative HE4 and ROMA values do not improve the CA125 diagnostic value for borderline tumors of the ovary (BOT) - a study of the TOC Consortium. J Ovarian Res. 2014; 7: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShih IeM, Kurman RJ: Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol. 2004; 164(5): 1511–1518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalpica A, Deavers MT, Lu K, et al.: Grading ovarian serous carcinoma using a two-tier system. Am J Surg Pathol. 2004; 284): 496–504. PubMed Abstract \| Publisher Full Text\n\nKöbel M, Piskorz AM, Lee S, et al.: Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma. J Pathol Clin Res. 2016; 2(4): 247–258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nImamura H, Ohishi Y, Aman M, et al.: Ovarian high-grade serous carcinoma with a noninvasive growth pattern simulating a serous borderline tumor. Hum Pathol. 2015; 46(10): 1455–1463. PubMed Abstract \| Publisher Full Text\n\nDehari R, Kurman RJ, Logani S, et al.: The development of high-grade serous carcinoma from atypical proliferative (borderline) serous tumors and low-grade micropapillary serous carcinoma: a morphologic and molecular genetic analysis. Am J Surg Pathol. 2007; 31(7): 1007–12. PubMed Abstract \| Publisher Full Text\n\nHampel H, Bennett RL, Buchanan A, et al.: A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med. 2015; 17(1): 70–87. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53847", "date": "14 Oct 2019", "name": "Carla Bartosch", "expertise": [ "Reviewer Expertise Gynecologic pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report, presenting the rare finding of a serous borderline ovarian tumor with a synchronous high grade serous carcinoma in the peritoneum. The introduction and discussion are well written. The case report itself, essentially relies on the pathological diagnosis, which in my opinion is insufficiently demonstrated.\nMy comments are remarks are the following:\nAbstract:\n“either as non-invasive or invasive implants, also known as extraovarian low-grade serous carcinoma.” – change to “the latter also known…”\n\nIntroduction:\n“In order to meet criteria for PPC, both ovaries and tubes should be macro- and microscopically normal in size or enlarged by a benign process” – This is not entirely correct. If the tube has, for example, STIC, the tumor should be considered of tubal origin, yet it may have a perfectly normal size.\n\n“largely overlap their ovarian counterparts” – change to “tubal and ovarian counterparts”\nCase report:\nThe ovarian tumor is large (19cm), was an adequate sampling done to exclude an invasive component?\n\nIn the presence of a HGSC in the peritoneum, total inclusion of the adnexa, particularly the tube following the SEE-FIM protocol, should be done to exclude a tubal primary – was this done? Were there any precursor lesions in the Fallopian tube epithelia? The case reports a “frozen pelvis” – weren’t the tubes involved by the tumor?\n\nHGSC can architecturally simulate LGSC in the peritoneum. The two basic criteria to distinguished HGSC and LGSC are the nuclear atypia and mitotic index (as emphasized in manuscript reference 11), but none of which are mentioned in the case description.\n\nHGSC may also simulate a BST architecture in the ovary, but the cytological features and immunohistochemical profile are very different. Please provide the morphological description (including architectural patterns, cytological features, mitotic index) as well as the immunohistochemical profile of both neoplasias to support the diagnosis.\n\np53 should be report as wild type or aberrant (null or diffusely positive). Variable p53 positivity occurs in wild type patterns. Aberrant p16 overexpression and a very high proliferation index (Ki-67) would also support the differential diagnosis between HGSC and LGSC.\n\nWere there any lesions in the hysterectomy specimen, namely in the endometrium?\n\nHistological pictures are all low-power. A and C show similar aspects. Legend mentions the left ovary, which is not shown – consider deleting this from the legend as it is already in the case report text. I suggest making a panel with low and high power magnifications showing the architectural and cytological features, plus p53 expression, of both neoplasias.\n\nWas any genetic study performed, namely for BRCA1/2 or BRAF mutations?\nDiscussion:\nFirst paragraph – Kurman’s dualistic model and Malpica’s two tier system are different concepts. Thus, the last phrase can not start with “this”.\n\nEstrogen and progesterone expression is mentioned in the discussion, but is not in the case report description. Provide the extension of ER and PR expression in both tumors. PR expression was negative in the BST?\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5012", "date": "08 Nov 2019", "name": "Mariana Rei", "role": "Author Response", "response": "Reply to Reviewers Thank you for your kind reply and consideration of the manuscript “Borderline tumor and primary peritoneal carcinoma: a rare synchronism” for revision. Your comments were highly appreciated and were thoroughly taken into consideration, as following: 1. Additional clinical details were provided in order to demonstrate the pathological diagnosis in which the case relies on.2. Regarding the diagnosis of the SBT in the right ovary,multiple tissue blocks from the ovarian tumor were examined in the microscopy exam and none identified invasive component. Both adnexa weretotally included andanalyzed according to the Sectioning and Extensively Examining the Fimbriated End Protocol (SEE-FIM) and noprecursorlesionsin the fallopian tube epithelia were found.3. More detailed information regarding the morphological and immunohistochemical features of both tumor specimens was provided, allowing to distinguish between SBT and HGSC and to rule out LGSC. Regarding IHC, p16 was not performed in either specimen, therefore we could not provide that data.4. The molecular genetic analysis was not performed, therefore we could not provide that set of data.5. The concepts of ovarian tumor carcinogenesis models described in the first paragraph of the discussion, namely Kurman’s dualistic model and Malpica’s to tier system, were further clarified. 6. Finally, the legend of the histological pictures was modified accordingly and an additional set of images with higher power magnifications plus p53 expression of both tumors was provided. NOTE: The major revision work-up is noticed in the third, fourth and fifth paragraphs of the Case Report section and in the first paragraph of the Discussion section. Thank you in advance for your encouraging and inspiring review. We hope that the manuscript is now appropriate for being considered for publication. Kind regards,Mariana Rei, MD" } ] }, { "id": "54372", "date": "18 Oct 2019", "name": "Maria Dolores Diestro", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFrom the point of view and optics of Oncological Gynaecology, it would highlight the originality of the clinical case presented.\nThe presentation of clinical and surgical data is adequate and meticulous and translates a good level of background knowledge of the author.\nGood writing, clarity in the presentation and good structuring of the discursive dialogue of the discussion.\nPerhaps there would be a short synthesis at the end of the article with the background of the case and a small conclusion.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5013", "date": "08 Nov 2019", "name": "Mariana Rei", "role": "Author Response", "response": "Thank you for your kind reply and consideration of the manuscript “Borderline tumor and primary peritoneal carcinoma: a rare synchronism” for revision. The reviewers’ comments were highly appreciated and were thoroughly taken into consideration.1.Additional clinical details were provided in order to demonstrate the pathological diagnosis in which the case relies on, namely regarding the morphological and immunohistochemical features and a new set of histologic images.2.In the last paragraph of the Discussion section we display a final summary of the main findings and their implications for the clinical practice, while trying to be clear and concise.Thank you in advance for your encouraging and inspiring review. We hope that the manuscript is now appropriate for being considered for publication. Please do not hesitate to contact me if any further information is needed. Kind regards,Mariana Rei, MD" } ] } ]	1	https://f1000research.com/articles/8-1630
https://f1000research.com/articles/7-1827/v1	20 Nov 18	{ "type": "Research Article", "title": "Supramolecular protein-mediated assembly of brain extracellular matrix glycans", "authors": [ "Anthony Tabet", "Kamil Sokolowski", "Jarrod Shilts", "Marlous Kamp", "Nina Warner", "Dominique Hoogland", "Oren A. Scherman", "Anthony Tabet", "Kamil Sokolowski", "Jarrod Shilts", "Marlous Kamp", "Nina Warner", "Dominique Hoogland" ], "abstract": "Background: Hyaluronic acid (HA) is the major component of the extracellular matrix in the central nervous system and the only supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In the majority of cases, HA is covalently functionalized with moieties that can facilitate network formation through physical selfassembly, or photo-catalyzed covalent crosslinking as the polysaccharide does not gel on its own. However, these covalent crosslinks are not the driving force of HA self-assembly in biological tissues. Methods: Oscillatory rheology and dynamic light scattering were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to study mass transfer of a hydrophilic small molecule into the albumin/HA/CS materials. Results: In this work we examine the intermolecular interactions of two major glycans found in the human brain, HA and the lower molecular weight CS , with the protein albumin. We report physiochemical properties of the resulting supramolecular micro- and nanomaterials. Albumin/HA mixtures formed supramolecular gels, and albumin/CS mixtures formed micro- and nanoparticles. We also summarize the concentrations of HA and CS found in various mammalian brains. Conclusions: Simple preparation and combination of commercially available charged biomacromolecules under short time-scales can result in interesting self-assembled materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective and simple models of nervous system electrostatic interactions and as in vitro drug release and mass transfer quantification tools.", "keywords": [ "hyaluronic acid", "chondroitin sulfate", "protein-polymer assembly" ], "content": "Introduction\n\nA major paradigm that has dominated the drug delivery and tissue engineering communities is the development of bio-inspired hydrogels that mimic the intermolecular interactions and mechanical properties of physiological tissue. Glycosaminoglycans (GAGs) are polysaccharides that are critical structural components of the brain extracellular matrix (ECM). One particularly abundant brain GAG, hyaluronic acid (HA) (Figure 1A), has been made into many covalently modified derivatives that have been widely explored in drug delivery and tissue engineering1. However, HA is the only supramolecular brain GAG2, and other ECM components including the more abundant protein-linked GAG chondroitin sulfate (CS) have been comparatively under studied3. HA is often chemically functionalized because the high molecular weight polysaccharide does not gel on its own. Although functional materials based on covalently modified HA exhibit useful features for many applications, these gelation methods do not capture the physiological supramolecular interactions that are endogenously found in the brain extracellular matrix4.\n\n(A) Structures of chondroitin sulfate (CS) and hyaluronic acid (HA). (B) Relative surface charge densities of bovine serum albumin (BSA). Dataset 1: Dynamic light scattering and transmission electron microscopy data7.\n\nIn this work we examine the intermolecular interactions of two major biopolymer components of the brain, HA and CS, with the model protein bovine serum albumin (BSA) (Figure 1B). We exploit known electrostatic interactions between the BSA and GAG polymers5,6 to generate structurally diverse protein-glycan complexes. We examine how these negatively charged biopolymers interact and self-assemble with BSA, and develop supramolecular systems formed from competing HA/BSA, HA/CS, and CS/BSA interactions.\n\n\nMethods\n\nAll starting materials were purchased from Sigma Aldrich and used as received unless stated otherwise.\n\nThe crystal structure of bovine serum albumin (BSA) was downloaded from Protein Data Bank (ID:3V03). The second chain of the homodimer in the crystal structure was removed to display BSA in monomeric form. Using the Adaptive Poisson-Boltzmann Solver (APBS) tool available through PyMOL v2.2.2, the electrostatic surface potential was calculated under default parameters. The section of the protein surface showing the previously-described GAG binding pocket was rendered with PyMOL.\n\nA 10 wt% chondroitin sulfate (C9819 Sigma) solution was prepared by mixing the polymer powder in Milli-Q H2O (18 mΩ) at room temperature overnight. Bovine serum albumin (BSA; 50 mg/mL) was added to the solution and rigorously mixed for 12 hours (1000 RPM) at room temperature. Lower total concentrations of polymer and protein took more than 12 hours to form particles.\n\n4 wt% hyaluronic acid (1.5-1.8 MDa; 53747 Sigma) solutions were prepared by mixing the polymer powder in Milli-Q H2O (18 mΩ) at 40 °C for 40 hours. The solution was sealed and stored at 4 °C until further use. BSA (50 mg/mL) was added to the viscous polysaccharide solution and mixed with a metal spatula for several minutes. The samples were sealed until they appeared transparent, typically for at least 24 hours. It was observed that BSA added without stirring would not result in the same behavior, and appeared to lower the solution viscosity instead. To form HA/CS/BSA systems, dry chondroitin sulfate (10 wt%) was added along with the dry BSA and allowed to homogenize with stirring (200 RPM). Poly(caprolactone) (PCL) blends were formed by mixing poly(caprolactone) diol (Mn = 2 kDa; 5 wt%) with poly(caprolactone) diol melt (Mn = 550 Da) and mixing at 700 RPM at 50 °C overnight. A specified amount of rhodamine B was added to the blend for Ultraviolet–visible spectroscopy (UV-Vis) studies.\n\nAll rheological sweeps were conducted on an AR-G2 Rheometer (TA Instruments, New Castle, DE, USA) with a 40 mm parallel plate geometry at 20.0 °C. Zero gap, rotational mapping (precision bearing mapping; 2 iterations), geometrical inertia, and friction calibrations were done prior to each use of the rheometer. Samples were loaded onto the rheometer with a 600–1000 µm loading gap. A water trap was placed to prevent dehydration. Amplitude sweep were conducted to determine a strain in the linear viscoelastic region.\n\nDynamic light scattering (DLS) measurements were carried out on a Malvern Zetasizer NS90 instrument at room temperature and standard settings. Samples were analysed in a 1.5 mL PS cuvette (Fisher Brand).\n\nTransmission electron microscopy (TEM) was carried out on a FEI Philips Tecnai 20. Samples were prepared on holey carbon grids by pipetting 1 µL of desired aqueous solution and allowing it to evaporate under ambient conditions (drop-casting). Particle size distributions were calculated by counting the diameters of more than 100 particles.\n\nUV-Vis spectroscopy was performed using a Mikropack DH-2000 UV-Vis-NIR Halogen light source and an OceanOptics USB2000 Fiber Optic Spectrometer. Spectra from 375 nm to 750 nm were recorded at 150 ms integration time and time intervals of 60 s.\n\nTo estimate brain chondroitin sulfate (CS) levels, all articles cited as using the Blyscan assay to measure sulfated GAGs were searched against the keywords “brain” and “neural”. A total of 7 articles were found meeting the criteria of measuring sulfated GAGs in mammalian brain tissue. A separate literature search of hyaluronic acid (HA) measurements identified 2 articles. Reported concentration values were converted to molarities, representing the moles of disaccharide repeat units per volume of native brain tissue, assuming a brain density of 1.04 g/ml. In cases where brain weight was reported as dry mass instead of native tissue, masses were converted by assuming that 77% of brain weight is water.\n\n\nResults and discussion\n\nIn this work we explored the electrostatically-driven self-assembly of charged proteins with negatively-charged polysaccharides endogenous to the human brain. To mimic the native interactions these polysaccharides have with surrounding proteins, we introduced BSA, which has electrostatic binding pockets complementary to anionic GAGs, analogous to the binding interfaces of ECM proteins. BSA and CS polymer solutions were mixed overnight and allowed to self-assemble into nanostructures (ESI). Two distinct populations of particles were observed to form (Figure 2, Dataset 17), and were dissimilar to the flocculation of BSA aggregates alone. The smaller particles were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS yielded an average particle diameter (D) of 51 ± 3 nm. These structures were stable for at least 2 days in the parent suspension (Figure S1A, Extended Data8). DLS autocorrelation data suggested that a large diameter species may also be present in the solution (Figure S1B, Extended Data8). TEM was used to characterize these self-assembled microparticles (Figure 2D). The analysis of these micrographs indicated the presence of two distinct populations of assembles, with the mean diameter of D = 60 ± 10 nm, that is consistent with DLS experiments, and an additional one of D = 1.5 ± 0.5 µm. In the brain, CS is covalently scaffolded onto peptide cores (e.g. aggrecan4) and binds with many ECM proteins through non-covalent, supramolecular interactions9. The supramolecular interactions between CS and BSA described here could provide insight into an electrostatic driving force that contributes to GAG aggregation and nanostructure formation in vivo.\n\n(A) Schematic of the formation of dense CS-BSA particles. (B) DLS size plot of dynamic BSA aggregates (top) and CS-BSA particles (bottom). (C–D) TEM image of CS-BSA nanoparticles (C) and microparticles (D).\n\nWe then turned our attention to HA, a linear high molecular weight polysaccharide that is the only supramolecular GAG in human physiology. HA and BSA were mixed overnight and the electrostatic interactions between mixture components led the solution to self-assemble into a supramolecular gel (Figure 3, Dataset 17). Supramolecular gels formed via electrostatic protein-polymer interactions with polysaccharide back-bones have been previously reported10. Solutions of HA alone were highly viscous but did not show gel-like properties. Oscillatory rheological measurements were used to probe the mechanics of this supramolecular gel (ESI). After introduction of BSA, electrostatic-driven network percolation resulted in a major stiffening effect and the formation of a gel with G' > 10 kPa. This material exhibited very clear shear-thinning and recovery behavior (Figure 3C).\n\n(A) Schematic illustration dynamic network formation of HA and BSA. (B) Oscillatory rheological frequency sweep of HA solution alone and HA-BSA gels. (C) Oscillatory time sweep after 100% shear of HA-BSA gels and HA solution alone. (D) DLS size measurements of dynamic BSA particles alone and HA-BSA gel. Dataset 1: Rheology and dynamic light scattering data.\n\nUpon introduction of dry CS into a HA/BSA matrix, a sharp decrease in stiffness was observed (Figure S3, Extended Data8). We hypothesize this effect is due to both interference from CS/BSA interactions and the reduction of entanglement of HA. DLS data showed that when combined with either HA or CS, BSA exhibited hierarchical assemblies that were not observed for BSA alone. Furthermore, when combined with HA, the BSA assemblies were more dynamic and polydispersed than with CS (Figure 3D, S28).\n\nWe then explored whether these HA/CS/CS systems could be used to explore mass transfer of the model hydrophilic drug rhodamine B (rhodB). Many parenteral drug-delivery studies will monitor in vitro release kinetics into saline as a model for in vivo release. Here we explore the feasibility of monitoring release kinetics into a gel instead of saline (Figure 4, Extended Data8). A hydrophobic blend of poly(caprolactone) (PCL) chains at different molecular weights (ESI) loaded with rhodB was carefully added on top of the hydrogel phase (Figure 4, Extended Data8). We found that it was possible to monitor the interfacial concentration of rhodB in the hydrogel phase with UV-Vis spectroscopy. Interestingly, we observed a large bolus release of the hydrophilic drug from the hydrophobic phase to the hydrophilic phase at the interface until the same concentration was reached, subsequently an equilibrium or pseudo-steady-state concentration was reached after 14 hours. Such a system is potentially useful in modeling mass transfer of drugs between hydrophobic drug delivery materials and biological tissue such as the brain11.\n\n(A) Picture of initial two phases measured via UV-Vis spectroscopy. (B) Concentration at interface after large bolus release over time. Hydrophobic phase drug concentration = 10 µg/mL. Average hydrophilic phase equilibrium concentration = 14.5 µg/mL. The periodic fluctuations in concentration were attributed to trapped air interfering with the interface. (Dataset 1: UV-Vis data.\n\nPrior tissue engineering studies have largely neglected the question of how the exact composition of the brain ECM might inform efforts to create biologically-mimetic hydrogels. To estimate the physiological concentrations of CS and HA that occur in vivo (Figure 5, Dataset 17), we summarize the literature for CS and HA measurements of mammalian brain tissue12–19. These estimates clustered in the millimolar range for CS disaccharides, and 100 micromolar range for HA.\n\nThe concentration of HA in healthy tissue, glioblastoma, and astrocytoma is also plotted.\n\n\nConclusions\n\nIn this work we characterize supramolecular interactions between HA, CS, and BSA. We report nano- and microparticle self-assembly, gelation, and network interference be haviors. We also report on the mass transfer of a model drug from a hydrophobic phase into a glycan-based hydrogel. Finally, we summarize reported concentrations of HA and CS in different animal models. This report may inform the development of biomaterials for tissue engineering that capture or exploit supramolecular interactions between brain extracellular matrix glycans.\n\n\nData availability\n\nUnderlying data for this study is available from Open Science Framework\n\nOSF: Dataset 1: Supramolecular Protein-Mediated Assembly of Brain Extracellular Matrix Glycans, https://doi.org/10.17605/OSF.IO/3BXQG7\n\nData is available under a CC0 1.0 License\n\n\nExtended data\n\nElectronic supplementary information (ESI) document is available from Open Science Framework.\n\nOSF: Extended Data. Supramolecular Protein-Mediated Assembly of Brain Extracellular Matrix Glycans ESI https://doi.org/10.17605/OSF.IO/NG6H78\n\nThe ESI contains the following Supplementary Figures -\n\nFigure S1: Autocorrelation function of chondroitin sulfate (CS)/bovine serum albumin (BSA) NPs. (B) Time resolved DLS of CS/BSA NPs in Milli-Q water.\n\nFigure S2: Time resolved dynamic light scattering of hyaluronic acid (HA)/bovine serum albumin (BSA) gels.\n\nFigure S3: (A) Oscillatory frequency sweeps of hyaluronic acid (HA) systems loaded with bovine serum albumin (BSA) and with or without chondroitin sulfate (CS). (B) Time-resolved dynamic light scattering experiment of CS/BSA particles loaded with HA and sheared.\n\nExtended data is available under a CC0 1.0 License", "appendix": "Grant information\n\nAT and JS thank The Winston Churchill Scholarship Foundation of the United States for funding support.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBurdick JA, Prestwich GD: Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011; 23(12): H41–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSasarman F, Maftei C, Campeau PM, et al.: Biosynthesis of glycosaminoglycans: associated disorders and biochemical tests. J Inherit Metab Dis. 2016; 39(2): 173–88. PubMed Abstract \| Publisher Full Text\n\nDjerbal L, Lortat-Jacob H, Kwok J: Chondroitin sulfates and their binding molecules in the central nervous system. Glycoconj J. 2017; 34(3): 363–376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNovak U, Kaye AH: Extracellular matrix and the brain: components and function. J Clin Neurosci. 2000; 7(4): 280–290. PubMed Abstract \| Publisher Full Text\n\nCooper CL, Goulding A, Kayitmazer AB, et al.: Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles. Biomacromolecules. 2006; 7(4): 1025–1035. PubMed Abstract \| Publisher Full Text\n\nLenormand H, Deschrevel B, Tranchepain F, et al.: Electrostatic interactions between hyaluronan and proteins at pH 4: how do they modulate hyaluronidase activity. Biopolymers. 2008; 89(12): 1088–1103. PubMed Abstract \| Publisher Full Text\n\nTabet A: Supramolecular protein-mediated assembly of brain extracellular matrix glycans. 2018. Publisher Full Text\n\nTabet A: Supramolecular protein-mediated assembly of brain extracellular matrix glycans ESI. 2018. Publisher Full Text\n\nMizumoto S, Yamada S, Sugahara K: Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr Opin Struct Biol. 2015; 34: 35–42. PubMed Abstract \| Publisher Full Text\n\nDubbin K, Tabet A, Heilshorn SC: Quantitative criteria to benchmark new and existing bio-inks for cell compatibility. Biofabrication. 2017; 9(4): 44102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTabet A, Wang C: Gels Without Vapor Pressure: Soft, Nonaqueous and Solvent-Free Supramolecular Biomaterials for Prospective Parenteral Drug Delivery Applications. Adv Healthcare Mater. 2018; 1800908. Publisher Full Text\n\nSood D, Chwalek K, Stuntz E, et al.: Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue. ACS Biomater Sci Eng. 2016; 2(1): 131–140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeQuach JA, Yuan SH, Goldstein LS, et al.: Decellularized porcine brain matrix for cell culture and tissue engineering scaffolds. Tissue Eng Part A. 2011; 17(21–22): 2583–2592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedberry CJ, Crapo PM, Siu BF, et al.: Hydrogels derived from central nervous system extracellular matrix. Biomaterials. 2013; 34(4): 1033–1040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKočí Z, Výborný K, Dubišová J, et al.: Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues. Tissue Eng Part C Methods. 2017; 23(6): 333–345. PubMed Abstract \| Publisher Full Text\n\nBaiguera S, Del Gaudio C, Lucatelli E, et al.: Electrospun gelatin scaffolds incorporating rat decellularized brain extracellular matrix for neural tissue engineering. Biomaterials. 2014; 35(4): 1205–1214. PubMed Abstract \| Publisher Full Text\n\nTubert AR, López FB, Matías ÉA, et al.: Vectors and sequences for the treatment of diseases. 2016.\n\nLaurent UB, Tengblad A: Determination of hyaluronate in biological samples by a specific radioassay technique. Anal Biochem. 1980; 109(2): 386–394. PubMed Abstract \| Publisher Full Text\n\nDelpech B, Maingonnat C, Girard N, et al.: Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma. Eur J Cancer. 1993; 29A(7): 1012–1017. PubMed Abstract \| Publisher Full Text" }	[ { "id": "40915", "date": "03 Dec 2018", "name": "Laura De Laporte", "expertise": [ "Reviewer Expertise hydrogels", "tissue engineering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper looks at the interaction of natural GAG, such as HA and CS, with BSA as model protein. Overall the data is clear but some improvements can be made.\n\nAre the particles formed with CS/BSA hard particles or nano, microgels? You can do AFM to check this, by measuring the swelling behavior in media and determining the Young's modulus.\nFor the HA/BSA interaction, gels are formed. Here more analysis can be done. The rheometer data only shows one data point. The G' for multiple gels can be measured to do statistics. Ideally these measurements should also be compared with media as the salts will affect the mechanical properties of the gel. Also, the gels seem to be very stiff for these natural materials. You decrease the concentrations to check whether you can vary the stiffness. Nervous tissue is usually in the range below 1 kPa. The authors mention the gel is shear thinning but this is not proven with rheology. Even a video of the gel would be helpful to have an idea about its properties, like transparency, shear thinning properties via pipetting, and gelation. In addition, even though SEM may cause some artifacts, it will still give you an idea about the internal structure of these gels compared to other gels reported in the literature. In Figure 3C, there is not legend and unclear what the different conditions are. For Figure 3D, as you have a gel, how can you also have individual particles for DLS measurements? Please make a note about this in the text.\nFigure 4: As Rhodamine is fluorescent, you can show fluorescent images to demonstrate the diffusion of the molecule into the gel.\nSome minor comments to improve the text: Abstract:\nSelf-assembly HA has been crosslinked covalently via other mechanisms than photo-catalyzed 1,2,3.\nIntroduction:\nThe authors mention that HA has been made into many covalently modified derivatives. Do they mean that functional groups were covalently bound to the HA backbone to make derivatives, or that functionalized HA can covalently bind to other molecules to form drug delivery and TE materials. This is repetitive with a sentence later in the intro: 'HA is often chemically modified…….'\nMethods:\nThe GAG binding pocket of BSA is not shown in Figure 1B. BSA should only be written full once, after that just use the abbreviation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "42740", "date": "12 Feb 2019", "name": "Jessica CF. Kwok", "expertise": [ "Reviewer Expertise Extracellular matrix", "glycosaminoglycan", "central nervous system", "neuroplasticity", "neuroregeneration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article ‘Supramolecular protein-mediated assembly of brain extracellular matrix glycans’ by Tabet et al., describes the self-assembly of glycosaminoglycans hyaluronan (HA) and chondroitin sulfate (CS) in the presence of bovine serum albumin (BSA). The authors report that HA/BSA forms hydrogel upon mixing while CS/BSA forms nano- and micro-particles.\n\nThe recognition of supramolecular assembly with biological materials has increased our understanding of how biological systems function in the last years. Here, the authors aim to address how HA and CS self-assemble for their function, emphasising on their role in the central nervous system. However, the study appears partial and the hypothesis is not clear.\n\nHypothesis is not clear: Why do you use serum albumin? Serum albumin is not normally present in the brain tissue but confines inside the blood vessels in the brain. The penetration of serum albumin to the brain is protected by the blood-brain barrier. Extravasation of albumin to the brain will only happen in pathological conditions. This means that serum albumin is not the normal contributor to brain extracellular matrix (ECM) glycan assembly. This needs to be stated clearly in the article. Resulting from this, the title is also misleading, because the model described is thus not related to brain ECM.\n\nPlease define clearly the meaning of ‘supramolecular’ in the article. In both the Abstract and the Introduction, the authors claimed that HA is the ‘only supramolecular glycosaminoglycan’. What do you mean by this? However, in the main text, the authors described CS/albumin interaction as ‘supramolecular interactions’. Please define clearly the meaning of the term.\n\nRegarding the CS/albumin interaction, the authors attribute particle formation from mixing CS with serum albumin is due to supramolecular interactions between CS and BSA. How can you rule out that this is not due to phase separation of your materials instead? Are the nano- and micro-particles stable over time?\n\nRegarding the model of drug release, the authors used rhodamine by laying it on top of the hydrogel. This is showing diffusion of molecules through the gel, but not the release of rhodamine from the gel. Please correct the terminology accordingly.\nRegarding the title, please change glycans to glycosaminoglycans.\n\nMinor changes:\n\nIn the abstract, the first sentence: HA is ‘a’ major component of ECM in the CNS, but not “the” major component. CS is present in higher concentration than HA in the CNS, which is also indicated by the analysis reported in Figure 5 in the article. Please correct the sentence.\n\nFigure 1: The CS shown in Figure 1A is chondroitin 6-sulfate, while the CS used in the experiment (Sigma C9819) is a chondroitin 4-sulfate. Could you please amend the figure to represent the structure of the CS used?\n\nFigure 1: The HA structure is wrong. HA is formed by disaccharide repeats of [GlcNAc-β1,4-GlcA-β1,3]. The figure shows a [GlcNAc-β1,4-GlcA-β1,4] linkage. Please correct this accordingly.\n\nPage 4, first paragraph in the result section: ‘CS is covalently scaffolded onto peptide cores (e.g. aggrecan)’. Please change peptide into protein. Aggrecan is a big protein with molecular weight larger than 250 kDa even without the attachment of glycosaminoglycan chains.\n\nFigure 3C: what are the three lines in Figure C? Please state clearly, either in the figure or in the legend.\n\nPage 5, I assume ‘HA/CS/CS’ systems actually mean ‘HA/CS/BSA’ systems?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-1827
https://f1000research.com/articles/8-240/v1	01 Mar 19	{ "type": "Research Article", "title": "Preliminary assessment of the utilization of durian peel liquid smoke as a natural preservative for mackerel", "authors": [ "Muhammad Faisal", "Asri Gani", "Farid Mulana", "Asri Gani", "Farid Mulana" ], "abstract": "Background: Durian peel is a type of biomass waste that contains cellulose, hemicelluloses, and lignin. The pyrolysis of these compounds results in production of liquid smoke which can be used as a natural preservative to replace current synthetic preservatives. This research assessed the ability of liquid smoke produced during pyrolysis of durian peel to preserve fish. Methods: Dry durian peel waste underwent batch reactor pyrolysis at 340°C and 380°C, resulting in production of liquid smoke (grade 3), charcoal, and tar. This liquid smoke was then distilled at 190°C to produce grade 1 liquid smoke, which was used to preserve mackerel. The preservation process was conducted by soaking the mackerel samples in liquid smoke at 0.5, 1, 2, and 3% concentration levels followed by observations every 6 hours. Tests to determine the total volatile base (TVB) content, antibacterial quality of the liquid smoke and organoleptic quality of the fish were conducted in order to assess the preservation properties of the liquid smoke. Results: Tests on the antibacterial effects showed that the liquid smoke inhibited the growth of Escherichia coli and Staphylococcus aureus on fish even at low concentrations. At 54 hours, the TVB values remained below 30 mg nitrogen/g, indicating that the fish was still safe for human consumption. Results from the organoleptic tests showed that the concentration of liquid smoke influenced the preservation effects. Conclusions: At a concentration of 2–3%, the fish samples possessed acceptable flavor, taste, color and texture for up to 48 hours of soaking. However, the best conditions were obtained with a 3% concentration of liquid smoke (produced with 340°C pyrolysis), as the fish was still considered acceptable for up to 42 hours.", "keywords": [ "durian peel", "pyrolysis", "liquid smoke", "natural preservatives", "TVB", "organoleptic quality" ], "content": "Introduction\n\nIndonesia is located at the equator and is rich in abundant plantation produce as well as other natural resources, such as durian. Although seasonal, durian production in Indonesia continues throughout the year. High consumption of durian can lead to environmental issues because there is no proper management of durian peel waste. In general, durian peel contains a high level of cellulose (50–60%), starch (20%) and lignin1, and has the potential to be used as a raw material for production of liquid smoke.\n\nA common method to produce liquid smoke is pyrolysis, in which cellulose, lignin, and starch are processed into various chemical compounds2. This process occurs in various stages: (i) hemicelluloses are disintegrated at 200–315°C, resulting in formation of furan, acetic acid and its derivatives; (ii) cellulose is disintegrated at 240–350°C, resulting in carbonic acid formation; (iii) lignin is disintegrated at 280–500°C, resulting in production of phenol, phenolic ether and its derivatives2. In principle, any wood material can be used in pyrolysis to produce liquid smoke, including palm kernel shells, sugar cane fibers, empty fruit bunches, rice husks, and coconut peel3–10. Previous research11–13 has shown that the liquid smoke produced from pyrolysis of palm kernel shells contained phenol, carbonyl and other acids. These compounds have antimicrobial properties that can help preserve food14–17; they inhibit damaging and spoilage microbes and therefore increase the shelf-life of food products. In addition, liquid smoke can contribute a unique flavor, taste and color to foods. Several researchers have studied the effects of liquid smoke produced from palm kernel shells to preserve mackerel16, fish ball18 and tofu19. However, to our knowledge, there is no research reported on the utilization of durian peel biomass as a natural preservative, despite the wide availability and potential usefulness of durian peel waste.\n\nFish is a staple food for people living in Indonesia’s coastal and maritime areas. The fish is usually consumed fresh or as processed products. Fish has highly nutritious and beneficial for health, however, it decomposes easily (in approximately 8 hours) due to the activity of spoilage microorganisms. Traditional fishermen use formaldehyde as a preservative to lengthen the storage life, but this compound is dangerous to human health. In recent years because of concern regarding the use of chemical preservatives, there has been much research conducted on natural antimicrobials to prevent microbial growth and food spoilage. One natural alternative is liquid smoke, which contains antimicrobial properties. The application of liquid smoke to preserve fish is simple, can be used repeatedly and has the potential to replace commonly used harmful man-made preservatives. This research aims to study the potential usefulness of liquid smoke resulting from durian peel pyrolysis as a natural mackerel preservative.\n\n\nMethods\n\nLiquid smoke was produced in a pyrolysis reactor as explained in previous research1,16. As much as 3 kg of dried durian peel was placed in the reactor set at 340°C and 380°C. The resulting smoke was condensed to produce tar, charcoal and grade 3 liquid smoke. The next step was distillation at 190°C to separate the liquid smoke from tar, resulting in production of grade 1 liquid smoke. This was then used as the preservative by soaking mackerel in different concentrations: 0.5%, 1%, 2%, and 3%. The following analysis tests were conducted during storage: measurement of TVB, organoleptic quality, antibacterial activities and total aerobic mesophilic counts.\n\nTVB is a method to determine the freshness of fish based on its spoilage due to microbial growth and loss of fats or proteins20. TVB measurement was carried out by placing a Conway petri dish sideways with its lid half open inside an incubator at 35°C for 35 minutes. The petri dish contained the liquid smoke used to soak the fish, and K2BO3, and H3BO3 in each partition. After incubation, the dish was covered and shaken, before further incubation at 35°C for 8 hours. Afterwards, 0.1 ml boric acid was exposed to every indicator and left for 2 hours. Titrations with HCl (0.01 N) were performed until the color turned pink.\n\nThe organoleptic tests involved examining the samples using the senses of volunteer panelists, including examining the color, smell, taste and texture of the fish meat. These tests were carried out in order to identify how much people liked the liquid smoke preserved mackerel and to determine how long the fish would last. Testing was carried out in compliance with organoleptic testing standard manuals SNI 01-2346-200621. The number of panelists used in this research was 30 people, consisting of 23 non-standard subjects (people who were not trained in performing organoleptic assessment/testing, recruited from the pool of chemical engineering students at Syiah Kuala University) and seven standard subjects (people with high sensitivity towards testing product quality, and possessed knowledge and experience in assessing product quality, recruited from the Health Laboratory, Banda Aceh ). The panelists were given briefing and training prior to performing the tests. The resulting values were then processed using hedonic tests.\n\nAverage quality value: x=∑xin\n\nWhere x = average quality scores, xi = value of organoleptic quality testing; panels i, and n = number of panelists\n\nFlavor testing on fish was carried out after the samples had been steamed (at 90–100°C for 15 min) without changing the flavor. Table 1 describes the scale used to determine the flavor of fish samples.\n\nThe antibacterial activity testing was carried out to identify the activity of durian peel liquid smoke against Escherichia coli and Staphylococcus aureus bacteria. The method used was the disk diffusion (Kirby–Bauer) assay, as described by Tendencia22, involving the use of Mueller Hinton (MH) media (Merck, KgaA, Germany), performed by the Health Laboratory (Banda Aceh, Indonesia). This method was chosen because of its simplicity and the ability to see the formation of inhibition zones as clear areas around the antimicrobial disks.\n\n\nResults and discussion\n\nThe TVB values in fish after being treated with liquid smoke are shown in Figure 1 and Figure 2. The higher the concentration of liquid smoke, the lower the TVB value and the greater the antibacterial inhibition produced. The lowest TVB value was observed with 3% liquid smoke. Figure 2 shows that the TVB values resulting from the use of liquid smoke produced at 340°C at 0.5%, 1%, 2%, and 3% concentrations were low at 2.814, 1.407, 1.407 and 1.407 mg nitrogen/g (mgN/g), respectively. In the meantime, liquid smoke produced at 380°C (Figure 3) at the same concentrations resulted in the following TVB values: 2.814; 1.407; 1.407 and 0.7035 mgN/g within 6 hours. Within 54 hours, the TVB values for each soaking time were still within the safe limits allowed. After 60 hours, the TVB values increased to levels greater than 33 mgN/g, which is above the acceptable consumer standard of 30 mgN/g. These results are comparable to those obtained in previous research16 using liquid smoke from oil palm kernel shells. A TVB value of 0–30 mgN/g in fresh produce signifies good quality in compliance with National Indonesian Standards for food (SNI 01-2729-1992). TVB values increase due to a bacterial enzyme which degrades proteins into amino acids, and short peptide bonds resulting in production of a number of bases including, amine, ammonia, and trimethylamine which produce foul odors in foods23. Longer soaking periods will result in greater bacterial activities, which in turn produce more bases and increase TVB values16.\n\nColor. Results from organoleptic testing on color of samples showed that mackerel soaked in various concentrations of liquid smoke changed color depending on the soaking duration (Table 2). With 0.5–3% liquid smoke concentrations produced at 340°C pyrolysis, the color changed from pale white to yellowish–cream in 36 hours. The same concentrations for 380°C pyrolysis caused the color to change from pale white to yellowish cream in 42 hours. A food product that has high nutritional value, good flavor and good taste will have little interest from customers if the product does not also have an attractive color. Fish soaked in 0.5–3% liquid smoke produced at 380°C had the most optimum results, probabaly due to a high content of phenol and acetic acid, which maintained the freshness of the fish16,19. For comparison, fish samples that did not receive liquid smoke treatment turned a cream color after only 8 hours. Raw data are available on Zenodo24.\n\nFlavor and aroma. The hedonic test results for flavor and aroma are shown in Table 3 and Table 4. High concentrations of liquid smoke slowed down the occurrence of foul smells and bad flavors in mackerel for up to 30 hours (Table 3 and Table 4). With regards to aroma, the use of 2–3% liquid smoke produced at 380°C maintained a desirable aroma for up to 48 hours of soaking, although the aroma grew thick (similar to the smell of liquid smoke). The smoked scent seeped into the mackerel and grew stronger due to a reduced content of acetic acid in the fish. Production of foul odors can also be used to indicate food spoilage caused by oxidation and fat oxidation lead to production of foul odors in fish25. With regards to taste, liquid smoke produced a smoky smell in the fish but without smoke treatment, the deterioration in changes in taste and smell would have occurred in less than 12 hours. Saloko et al.17 stated that the use of 5% liquid smoke in a nanocapsule from chitosan and maltodextrin had the potential to maintain the freshness of mackerel for up to 24 hours.\n\nTexture. Texture tests can be carried out orally as well as by touching with hands aimed at feeling the texture of a food product. Table 5 shows that the best texture of mackerel occurred when 3% liquid smoke produced at 380°C was used for up to 48 hours. The fish texture was still quite chewy up to 42 hours in fish treated with 2–3% liquid smoke produced at 340°C. At both pyrolysis temperatures, the fish texture became rigid after 48 hours. At a low concentration (0.5%), the texture of the fish started to change within 36 hours. The change in texture was influenced by the speed of bacterial growth. Fish quality decreased when the texture became tender due to the effects of cathepsin and collagenase enzymes on muscle tissues. Cathepsin in fish degrades protein and causes the meat to become tender, while collagenase degrades polypeptide bonds when protein is not denatured25.\n\nTable 6 shows the effects of liquid smoke on bacterial growth (E. coli and S. aureus). A concentration of 0.5% liquid smoke produced at 340°C did not inhibit bacterial growth; however, bacterial inhibiting properties were shown at a concentration of 1%. The same antibacterial properties were seen in liquid smoke produced at 380°C even at lower concentrations. Kim et al.26 stated that the use of 0.1–1% liquid smoke produced from rice husks inhibited Salmonella growth while Milly et al.27 demonstrated that 1.5-9% liquid smoke produced from cinnamon can also inhibit bacteria. In addition, Saloko et al.17 showed that 5% liquid smoke from chitosan and maltodextrin in a nanocapsule inhibited E. coli and P. fluorescens growth. The type of wood biomass used to produce liquid smoke will result in production of different phenol, carbonyl and acid compounds, which in turn will determine the antibacterial properties, as well as the sensitivity of the pathogenic bacteria to the liquid smoke raw material14.\n\nThe total number of bacterial counts in the mackerel will determine whether or not the product is acceptable for human consumption. Plate count agar (PCA) was used to determine the total bacterial counts on mackerel samples. The number of microbes present after soaking must fall within the safe limits for consumption, namely 5×105 colonies/g, in accordance with SNI 02-2725-199228. Table 7 shows that 12 hours of soaking did not result in any significant changes in the number of counts; levels ranged from 3.2×105 to 3.5×105 colonies/g, indicating that the products were considered to be safe. However, after 42 hours the number of colonies increased to levels of 5.02 × 105 colonies/g, making the fish unsafe for human consumption. Liquid smoke can inhibit the growth of bacteria due to the presence of phenols, acids and carbonyl compounds working together to inhibit degradation and spoilage. In particular, acetic acid can penetrate the cell membrane and neutralize the pH gradient29.\n\n\nConclusion\n\nSmoke produced from durian peel pyrolysis had inhibitory effects against bacteria even when applied at low concentrations. This showed that the liquid smoke treatment had the potential to be used as a food preservative, especially for fish. The organoleptic tests carried out showed that the preservation of mackerel depended on the concentration and pyrolysis temperature used during the liquid smoke production. At a concentration of 3% liquid smoke produced at 340°C, the fish stayed acceptable for consumption for 42 hours, based on its color (cream), flavor, aroma and texture, and the total bacterial counts were also acceptable and within the safe limits. Meanwhile, TVB tests showed that the fish remained of acceptable quality for 54 hours, with a TVB value of less than 30 mgN/g.\n\n\nData availability\n\nData associated with this study, stratified by table, are available on Zenodo. Data include raw organoleptic test results for the quality of fish and effects of liquid smoke on bacterial counts. Bacterial count data are provided as mean values, since this is the output generated by the external Health Laboratory. DOI: https://doi.org/10.5281/zenodo.255648224.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis research received funding from The Ministry of Research, Technology and Higher Education of the Republic of Indonesia.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe author is grateful to the Syiah Kuala University and the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for the research funding. Thanks very much to Hera Desvita and A.R Yelvia Sunarti for performing the experiments and analyses.\n\n\nReferences\n\nFaisal M, Yelvia Sunarti AR, Desvita H: Characteristics of liquid smoke from the pyrolysis of durian peel waste at moderate temperatures. Rasayan J Chem. 2018; 11(2): 871–876. Publisher Full Text\n\nShen DK, Gu S: The mechanism for thermal decomposition of cellulose and its main products. Bioresour Technol. 2009; 100(24): 6496–6504. PubMed Abstract \| Publisher Full Text\n\nYang YB, Phan AN, Ryu C, et al.: Mathematical modelling of slow pyrolysis of segregated solid wastes in a packed-bed pyrolyser. Fuel. 2007; 86(1–2): 169–180. Publisher Full Text\n\nFaisal M, Gani A, Husni, et al.: Pyrolysis of oil palm kernel shell into liquid smoke and its application to control anthracnose disease on chili (Capsicum annum L.). J Eng Appl Sci. 2016; 11(12): 2583–2587. Reference Source\n\nFaisal M, Gani A, Husni, et al.: A preliminary study of the utilization of liquid smoke from palm kernel shells for organic mouthwash. Int J GEOMATE. 2017; 13(37): 116–120. Publisher Full Text\n\nFaisal M, Chamzurni T, Daimon H: A study on the effectiveness of liquid smoke produced from palm kernel shells in inhibiting black pod disease in cacao fruit in vitro. Int J GEOMATE. 2018; 14(43): 36–41. Publisher Full Text\n\nBudaraga K, Marlida Y, Bulanin U: Liquid Smoke Production Quality from Raw Materials Variation and Different Pyrolysis Temperature. Int J Adv Sci Eng Inf Technol. 2016; 6(3): 306–15. Publisher Full Text\n\nAbnisa F, Daud WW, Sahu JN: Optimization and characterization studies on bio-oil production from palm shell by pyrolysis using response surface methodology. Biomass Bioenergy. 2011; 35(8): 3604–3616. Publisher Full Text\n\nAbdullah N, Gerhauser H: Bio-oil derived from empty fruit bunches. Fuel. 2008; 87(12): 2606–2613. Publisher Full Text\n\nAkhtar J, Amin NAS: A review on process conditions for optimum bio-oil yield in hydrothermal liquefaction of biomass. Renew Sust Energ Rev. 2011; 15(3): 1615–1624. Publisher Full Text\n\nZuraida I, Sukarno I, Budijanto S: Antibacterial activity of coconut shell liquid smoke (CS-LS) and its application to fish ball preservation. Int Food Res J. 2011; 18: 405–410. Reference Source\n\nKim SJ, Jung SH, Kim JS: Fast pyrolysis of palm kernel shells: influence of operation parameters on the bio-oil yield and the yield of phenol and phenolic compounds. Bioresour Technol. 2010; 101(23): 9294–9300. PubMed Abstract \| Publisher Full Text\n\nAbnisa F, Arami-Niya A, Daud WW, et al.: Characterization of Bio-oil and Bio-char from Pyrolysis of Palm Oil Wastes. Bioenerg Res. 2013; 6(2): 830–840. Publisher Full Text\n\nLingbeck JM, Cordero P, O'Bryan CA, et al.: Functionality of liquid smoke as an all-natural antimicrobial in food preservation. Meat Sci. 2014; 97(2): 197–206. PubMed Abstract \| Publisher Full Text\n\nGuillen MD, Ibargoitia ML: New components with potential antioxidant and organoleptic properties, detected for the first time in liquid smoke flavoring preparations. J Agri Food Chem. 1998; 46(4): 1276–1285. Publisher Full Text\n\nFaisal M, Gani A, Husni: Utilization of Liquid Smoke from Oil Palm Kernel Shell to Preserve Mackerel. Rasayan J Chem. 2018; 11(3): 1120–1125. Publisher Full Text\n\nSaloko S, Darmadji P, Setiaji B, et al.: Antioxidative and antimicrobial activities of liquid smoke nanocapsules using chitosan and maltodextrin and its application on tuna fish preservation. Food Biosci. 2014; 7: 71–79. Publisher Full Text\n\nGinayanti L, Faisal M, Suhendrayatna: Pemanfaatan Asap Cair Dari Pirolisis Cangkang Kelapa Sawit Sebagai Pengawet Alami Tahu. Jurnal Teknik Kimia USU. 2015; 4(3): 7–11. Reference Source\n\nFaisal M, Gani A: The Effectiveness of Liquid Smoke Produced from Palm Kernel Shells Pyrolysis as a Natural Preservative in Fish Ball. Int J GEOMATE. 2018; 15(47): 145–150. Publisher Full Text\n\nFraser OP, Sumar S: Compositional changes and spoilage in fish (part II)-microbiological induced deterioration. Nutr Food Sci. 1998; 98: 325–329. Reference Source\n\nStandar Nasional Indonesia 01-2346-2006: Petunjuk Pengujian Organoleptik/Sensori. Jakarta: BSN. Reference Source\n\nTendencia EA: Disk diffusion method. In Laboratory manual of standardized methods for antimicrobial sensitivity tests for bacteria isolated from aquatic animals and environment. Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian Fisheries Development Center. 2004; 13–29. Reference Source\n\nFarahita Y, Junianto, Nia K: Karakteristik Kimia Caviar Nilem dalam Perendaman Campuran Larutan Asam Asetat dengan Larutan Garam selama Penyimpanan Suhu Dingin (5-10°C). Jurnal Perikanan dan Kelautan. 2012; 3(4): 165–170. Reference Source\n\nFaisal M: Raw data Faisal for f1000. 2019. http://www.doi.org/10.5281/zenodo.2556482\n\nTaher N: Organoleptic quality assesment of fresh tilapia fFish (Tilapia mossambica) with different size during the cold storage. Jurnal Perikanan dan Kelautan. 2010; VI(1): 8–12. Reference Source\n\nKim SP, Yang JY, Kang MY, et al.: Composition of liquid rice hull smoke and anti-inflammatory effects in mice. J Agric Food Chem. 2011; 59(9): 4570–4581. PubMed Abstract \| Publisher Full Text\n\nMilly PJ, Toledo RT, Ramakrishnan S: Determination of minimum inhibitory concentrations of liquid smoke fractions. J Food Sci. 2005; 70(1): M12–M17. Publisher Full Text\n\nStandar Nasional Indonesia 02-2729-1992: Persyaratan Mutu Produk Pangan Asapan. Jakarta: BSN.\n\nLeha MA: Aplikasi asap cair sebagai biopreservative dalam bahan pangan (Ikan Cakalang Asap). Prosiding Basic Science. 2010; ISBN: 978-602-97522-0-5." }	[ { "id": "50707", "date": "15 Jul 2019", "name": "Maryam Mahmoudzadeh", "expertise": [ "Reviewer Expertise Quality control tests in food science" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhat was your grade 1 liquid smoke composition? I think the biggest gap of your work is the lack of any characterization method for liquid smoke.\n\nAccording to which reference you have chosen the TVBN method?\n\nAs far as I know, \"TVBN\" is correct, not \"TVB\".\n\nWhat is the compositional difference between 340°C and 380°C smoke?\n\nFigure numbers are inaccurately typed in the text - where is Figure 3?\n\nTVBN data reported in the text are equal for 2 temperatures.\n\nHow long did you soak treatments in liquid smoke?\n\nWhere is the total count assay in the method and materials section?\n\nYou should have control samples in all exams, I couldn't see anything like this.\n\nIf you have done the antibacterial test by discs, why didn't you explain it in method and materials? Also, the reported results in Table 6 are not acceptable (you should report inhibition percent data).\n\nThere isn’t any statistical comparison in your work, and I think a big mistake like this is not acceptable.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4757", "date": "22 Jul 2019", "name": "Muhammad Faisal", "role": "Author Response", "response": "The composition of liquid smoke grade 1 produced at various temperatures has been published in our previous work. Please see reference 1. The composition includes; 2-Propanone, Acetic acid, 2,4-Pentadienenitrile, 2-Oxepanone, Phenolic compound etc. We have added the reference information in the manuscripts. The TVB-N was determined according to the procedure established by Susanto et al. with a slight modification. We have added the reference in the manuscript. Thank you very much for your correction, the TVB has been changed to TVB-N. The composition of liquid smoke at various temperature pyrolysis has been discussed in a previous article (see ref. 1). Analyses results showed that the temperature pyrolysis affected the chemical composition of liquid smoke. For example; at 340°C, liquid smoke contains Phenol=0.79 % and Acetic Acid=3.4%, at 380°C, liquid smoke contains Phenol=1.73 % and Acetic Acid=8.51%. Thank you very much for your correction. We have revised the Figure 3 to become Figure 2. The data is correct, because these data are at 6 hours. The TVB-N values are still low for all liquid smoke. The TVB-N then increased with increasing preservation time. We used soaking method for preservation, so the fish were soaked until end of preservation time. We have added the information of total bacterial counts in the method. Without preservation with liquid smoke, the fish can stand only to about 8 hours. This information can be found in the manuscript. I hope this information can be used as a control comparison. We only focused on after treatment. We are very sorry about this matter. We only conducted simple analysis for the antibacterial test. Detailed analysis will be done in the future. We are very sorry for comparison with other researches, because this investigation is still in a preliminary step. And to our knowledge the application of durian peel liquid smoke for fish preservation is not available in the literature. Therefore we only compare our results from a few related references." } ] } ]	1	https://f1000research.com/articles/8-240
https://f1000research.com/articles/8-1878/v1	07 Nov 19	{ "type": "Research Article", "title": "Urine cytological examination: an appropriate method that can be used to detect a wide range of urinary abnormalities", "authors": [ "Emmanuel Edwar Siddig", "Nouh S. Mohamed", "Eman Taha Ali", "Mona A. Mohamed", "Mohamed S. Muneer", "Abdulla Munir", "Ali Mahmoud Mohammed Edris", "Eiman S. Ahmed", "Lubna S. Elnour", "Rowa Hassan", "Nouh S. Mohamed", "Eman Taha Ali", "Mona A. Mohamed", "Mohamed S. Muneer", "Abdulla Munir", "Ali Mahmoud Mohammed Edris", "Eiman S. Ahmed", "Lubna S. Elnour", "Rowa Hassan" ], "abstract": "Background: Urine cytology is a method that can be used for the primary detection of urothelial carcinoma, as well as other diseases related to the urinary system, including hematuria and infectious agents. In this study we aimed at investigating urine abnormalities among Sudanese patients attending Omdurman teaching hospital. Methods: A descriptive cross-sectional study was conducted from November 2016 to October 2017. A voided urine samples were collected and stained using Papanicolaou stain. Results: A total of 1238 urine samples were meticulously examined, 832 (67.2%) from males (mean age 41.7±12.67), and 406 (32.8%) from females (mean age 43.8±10.94). 147 (11.9%) patients had an underlying medical condition, either AIDs, diabetes mellitus type 2 or historical renal transplantation. Hematuria was more frequent amongst males than females, 100 (68.9%) and 45 (31.1%), respectively. Urine cytology diagnosis was 43 cases (3.5%) of candidiasis, 36 cases (2.9%) of reactive urothelial cells, 11 cases (0.9%) of cryptococcosis, 9 cases (0.7%) of urothelial carcinoma, 9 (0.7%) human papilloma virus (HPV) diagnoses, 8 (0.6%) polyomavirus (BKV) diagnoses, 6 (0.5%) schistosomiasis diagnoses and 3 cases (0.2%) of low grade urothelial cells. Conclusion: Urine cytology seems to be a non-invasive technique that is suitable for all patients with urinary tract infections; those with diabetes, renal transplants, and HIV; and patients with macroscopic or microscopic hematuria for the detection of infectious agents and malignancy.", "keywords": [ "Urine cytology", "Urinary abnormalities", "Urothelial carcinoma", "Khartoum", "Sudan" ], "content": "Introduction\n\nUrine cytology is a safe and inexpensive method to diagnose urinary tract abnormalities. It is similarly used for the detection of primary and recurrent urothelial carcinoma1,2; as well as in many other diseases related to the urinary system such as urinary schistosomiasis3 and bacterial infections4. Urine cytology has variable sensitivity and specificity depending on the sample collection method and tumor grade in the case of malignancy5–7. Although the determination of cell morphology through microscopy remains the gold standard8, false positive and negative results have been reported. The false-positive results from urine cytology may be attributed to the presence of viral infections, such as polyomavirus. False negative results have been linked to the sampling method used, the number of the samples obtained from the patient and the volume of the urine being processed1,6–10. Urothelial carcinoma is one of the most commonly reported cancers11. The major clinical concern for the management of bladder cancer patients is that most of the patients relapse12,13. Therefore, regular follow-up is required for patients who are at risk of urothelial carcinoma14. Despite many attempts to develop tests with higher sensitivity and specificity, cytology remains one of the best ways to diagnose a variety of bladder lesions15.\n\nHematuria is one of the common indications for urine cytology examination. It is also known to be caused by an underlying malignancy in 5% to 10% of patients16. Urine cytology is also used for the detection of abnormalities related to diseases like schistosomiasis3, candidiasis17, polyomavirus (BK virus (BKV)) infection18,19, Human Papillomavirus (HPV)20,21 and cryptococcosis22. In this study, we aimed at investigating the frequency of urine abnormalities among Sudanese patients attending the outpatient clinic at Omdurman Teaching Hospital, Khartoum, Sudan using urine cytology.\n\n\nMethods\n\nA descriptive cross-sectional study conducted from November 2016 to April 2017, an organized mass screening programme for urine cytology was introduced during the study period. The target screening population consists of the entire patients attending the outpatient’s clinic and agreed to participate in the study. A total of 1238 Patients were recruited from the outpatient clinic at Omdurman Teaching Hospital in Khartoum state, Sudan. All the patients attending the outpatients clinic were assessed by our research team and we discussed with all the patients the objective of our research. After obtaining written informed consent from the patients whom were willing to participate our research team gathered the patients information and demographic data including name, age, and any clinical signs or symptoms using a questionnaire, the process of filling and collecting the patients clinical data were conducted by our research team and it took place in the patients waiting area in the clinic, focusing on patients with HIV, diabetes mellitus and transplant recipients, and any patients with other conditions mentioned in the questionnaire.\n\nEarly in the morning, first pass urine sample was rejected as the cells may be abnormal, with features such as enlarged nuclei and washing out of the chromatin, features that mimic malignant cells; this is due to the pH of the urine23–25. Accordingly, the patients were asked if they passed urine before and if they drank water? Then each patient was provided with a sterile screw cap urine container and asked to pass some urine into the toilet at first, then without stopping the flow of the urine catch some in the provided container.\n\nThe entire volume of the urine samples were centrifuged (using Hettich® EBA 20 centrifuge, Germany) for five minutes at 3000rpm then the supernatant was discarded leaving the sediment, this was followed the addition of suspending media (74 ml saline, 6 ml Glacial acetic acid and 20 ml of 100% ethanol) to the sediment. The quantity of the suspending media was proportional to the quantity of the obtained sediment this is done by measuring the sediment quantity using automated pipette and the same quantity of the sediment was added the suspending media (after centrifugation we measured the quantity with a pipette). It was left for 30 minutes, followed by centrifugation for 5 mins at 3000 rpm. The supernatant was discarded and 1% acid alcohol (1 ml of Hydrochloric acid added to 99 ml of 70% alcohol) was added to the sediment and left for 30 minutes, followed by centrifugation at 3000 rpm for 10 mins, and discarding of the supernatant again. The resulting sediment was then used for smears by spreading the entire obtained volume on a microscopical slide, and then the slide were left to concentrate. Then the smears were fixed in 95% ethyl alcohol (V/V) for 15 mins.\n\nThe smears were stained using Papanicolaou staining procedures as follow: the smears were immersed in a descending gradient of alcohol from 80%, 70% to 50% (V/V) and then immersed the smear in tap water for 2 mins. Then the smears were stained using Harris Hematoxylin for 2 mins, after that the smears were washed in tap water and differentiated in 1% acid alcohol for seconds. This was followed by bluing by leaving it in a bath of running water for 10 mins, then immersed in 80% and 95% alcohol and stained with Orange G solution (O.G.6) for 2 mins. This is followed by washing in 95% alcohol, and then staining with Eosin Azure (EA.50) for 4 mins and washed again in 95% alcohol. Then rinsed quickly in 100% alcohol and blot using a sterile filter paper to dry. Clear in Xylene and mounting in distyrene (a polystyrene), a plasticiser (tricresyl phosphate), and xylene (D.P.X) (Catalog No. 100579; sigma – Aldrich, USA)14.\n\nFor reporting the cytological smears, two cytopathologist (EES, MAM) examined the entire slides blindly using Olympus light microscope (Model No. CX21FS1, Olympus, Tokyo, Japan). The cytological diagnosis was grouped into 5 categories according to The Paris System for Reporting Urinary Cytology (PSRUC)23, categories included nondiagnostic/unsatisfactory, negative for high-grade urothelial carcinoma (NHGUC), atypical urothelial cells, suspicious for high-grade urothelial carcinoma (SHGUC), high-grade urothelial carcinoma (HGUC) and Low-grade urothelial neoplasm (LGUN). The presence of koilocytosis, decoy cells, and pseudo-hyphae, as well as detecting the variable sizes of cells with clear hollow and narrow base with single budge were used as a diagnostic remark for the presence of other urinary abnormalities such as HPV and BKV. Urine cytology results obtained by the cytologists were sent to the physician who made the final decision on the patients’ diagnosis for proper patients management according to their findings of the cytological smears.\n\nStatistical analysis was performed using the Statistical Package for Social Sciences (SPSS. Version 16). Chi-square test was used to compare urine cytology results with age, gender and to test the odds ratios of patients showing abnormalities in relation to their medical condition (A p value of <0.05 was considered to be statistically significant).\n\n\nResults\n\nA total of 1238 urine samples were examined, of them, 832 (67.2%) were males (mean age 41.7±12.67), and 406 (32.8%) were females (mean age 43.8±10.94). The age range of the patients was 10 to 77 years, with a median age of 43 years. Patients were grouped according to their age into 4 groups; 46 (3.7%) were ≤ 20 years, 539 (43.5%) were 21 to 40 years, 522 (42.2%) were 41 to 60 years and the last age group were those aged more than 60 years; 131 (10.6%). Of the 1238 patients, 1091 (88.1%) reported no significant medical history, the remaining 147 (11.9%) patients reported an underlying medical condition which included HIV/AIDS, diabetes mellitus type 2 (DM2), and renal transplantation history. A total of 13 (1.1%) had HIV, of which 12 were male (92.3%) and one was a female (7.7%). Regarding the gender distribution among the DM2 and renal transplantation, 64 (92.8%) and 57 (87.7%) were males, respectively; and 5 (7.2%) and 8 (12.3%) were females, respectively.\n\nUrine cytology showed a total of 1113 (89.9%) with normal urine findings. The remaining 125 (10.1%) patients showed a variety of findings; 43 (3.5%) cases of candidiasis, 36 (2.9%) with reactive urothelial cells, 11 (0.9%) with cryptococcosis, 9 (0.7%) with urothelial carcinoma, 9 (0.7%) with HPV, 8 (0.6%) with BKV, 6 (0.5%) with schistosomiasis and 3 (0.2%) with low grade urothelial cells. Frequency of cytological findings from urine analysis was higher in males than females and the results were found to be statistically significant (p value= 0.0001). (Table 1).\n\nRegarding the relationship between age and cytological findings, our study showed that candida infection, Schistosomiasis, Polyoma virus and HPV were highest among the age group 41 – 60 years; while Cryptococcus infection was highest among 21 – 40 years old patients.\n\nA representative microscopic result of urine cytology diagnosis is shown in Figure 1, and the distribution of gender, presence of hematuria, medical condition and urine cytology diagnosis based on age groups is described in Table 2.\n\nA: Cytopathic effect of human papilloma virus (HPV) with the eosinophilic intranuclear inclusion and a perinuclear halo and koilocytosis. B: Cryptococcus infection showed the organism with double contours. C: Polyomavirus cytopathic effect, the presence of decoy cells and pseudo hyphae and cells exhibit threads and small, round clumps of degenerating chromatin. D: Papillary cluster and single urothelial cells from low-grade urothelial cells. E: Cytological smears showed a monolayer sheet of cells with hyperchromasia and irregular distribution of chromatin and prominent nucleoli features of high-grade urothelial carcinoma. F: Schistosoma haematobium egg. G: Candida infection with bacterial. H and I: The counterpart diagnosis of D and E for the detected low-grade urothelial neoplasm and high-grade urothelial carcinoma using Hematoxylin and Eosin (H&E) histopathology staining (x40). H: Atypical urothelial cells. I: High-grade urothelial carcinoma showing very large, irregular and hyperchromatic nuclei. A–G: Staining using Papanicolaou stain (x100).\n\nOf the 1238 patients, a relative risk estimation was conducted for both male and female with any of abnormal cytological finding and the results showed a significant proportion of females to be infected with cryptococcosis and HPV; p value = 0.001, odds ratio was 0.106 [95% CI, 0.023-0.494] for cryptococcosis, and p value = 0.001, odds ratio was 0.060 [95% CI, 0.007-0.480] for HPV. Risk estimates of HGUC, atypical urothelial cells, presence of hematuria, polyomavirus, and schistosomiasis are presented in Table 3.\n\n* HGUC; High-grade urothelial carcinoma, AUC; Atypical urothelial cells.\n\nThe results of risk estimation for patients having a current or history of a medical condition showed that patients with AIDs are particularly vulnerable to candidiasis and HPV; p value= 0.009 and 0.005, respectively. While those with DM2 are more at risk of candidiasis and atypical urothelial cells, p values = 0.000 and 0.003, respectively. On the other hand, patients with a medical history of renal transplant showed increased risk for having BKV; p value= 0.000, and candidiasis, p value= 0.022. Results of risk estimation based on current medical condition are shown in Table 4.\n\n* Calculated for patients having medical condition or medical history.\n\n- HGUC; High grade Urothelial Carcinoma. AUC; Atypical Urothelial Carcinoma. HPV; Human Papilloma Virus. BKV; Polyomavirus BK virus.\n\n\nDiscussion\n\nUrine cytology is considered a noninvasive, cost effective technique that is able to diagnose a wide range of abnormalities including infections, atypical cellular changes and malignancy26,27. However, in this study, we investigated a varied array of urine abnormalities among Sudanese patients attending the outpatient clinic at Omdurman Teaching Hospital, Khartoum-Sudan using urine cytology.\n\nIn this study, we observed that urine cytology could easily differentiate benign from malignant cells showing high specificity for detecting HGUC; this result agrees with previous reports8,28–30. HGUC are aneuploid and are detected with high sensitivity, as compared to atypical urothelial cells29–31. The cytological features of HGUC identified in this study showed that the smears exhibited high cellularity and dehiscence cellular pattern. Malignant cells were larger than normal cells and showed pleomorphic nuclei with prominent nucleoli. Also, the cytoplasm ranged from homogenous to scant vacuolated one. Depending on these features, previous studies have indicated that detection of malignant cells using urine cytology is the gold-standard method for diagnosing urothelial cancer.2,29,32.\n\nIn this report we are also able to identify Candida spp with great confidence by the presence of the budding yeast and pseudohyphae which is considered as the cytomorphological characteristic of this fungus, however if the organism was not identified, cytological changes such as stain anomaly, nuclear degeneration and bacterial background may pinpoint the presence of candida.\n\nCryptococcus infection was detected in 2 out of 13 HIV patients; 0 out of 69 DM2 patients and 2 out of 65 renal transplant recipients. The organism was correctly identified by the presence of a semitransparent fungal organism with double contours in an inflammatory background22,31,33.\n\nPolyoma virus (BKV) was detected in 0 out of 13 (0%) HIV positive patients; 2 out of 69 (2.89%) DM2 patients and 6 out of 65 (9.2%) renal transplant recipients. The key feature for the existence of polyomavirus is depending on the presence of the unique cytomorphological features, i.e., presence of the typical decoy cells, the nuclei of this cell appeared as uniform with washed chromatin34–42. The results obtained by this study demonstrated that urine cytology can be used as a simple monitoring test for the renal transplant recipient for the presence of polyoma virus, since polyoma virus infection in renal transplant recipient is considered as one of the factors that can lead to graft rejection42–48. Our results are in concordance to that conducted by Kapila and his associates; they investigated the diagnostic efficacy of urine cytology in detecting Polyoma virus among renal transplant recipients comparing urine cytology with molecular diagnosis and their results showed that urine cytology can be used as an easy tool for monitoring the patients49. Furthermore, our results showed that no polyoma virus was been detected among HIV patients, which is in contrast to the findings of Boldorini and his associates as they screened 78 patients with HIV for the presence of polyoma virus using urine cytological examination and molecular and immunohistochemistry, there results demonstrated that out of 78 patients, only 17 patients were positive for Polyoma virus using cytology50.\n\nFurthermore, Human papilloma virus was been detected among 9 out of 1238 (0.7%) of whom 8 were females and one was a male; the results obtained here should be evaluated with care especially in the settings of female individuals as it is very difficult if not impossible to differentiate whether these koilocytic cells originated from the urinary tract or they belong to the uterine cervix and therefore in such circumstances we highly recommend that these females should go for pap smear to locate the exact site of HPV infections.\n\n\nConclusion\n\nUrine cytology is a safe, noninvasive, and reliable diagnostic tool for identifying viral cytopathic effects in urothelial cells, and deserves more widespread use in the monitoring of patients, especially those with renal transplants. It now seems that urine cytology needs to be implemented in clinical settings for the benefit of patients and urologists. Polyoma virus infection detection among transplant patients helps urologists to identify and manage renal graft rejection early which is of invaluable benefit to patients, physicians and economically. Due to the risk of the presence of polyoma virus among renal transplant recipient and based on the fact that 50% of the cases with Polyoma virus was associated with the graft rejection we highly recommended to use urine cytology as an early screening tool for routine checks among renal transplant patients. Furthermore, Pap staining was considered as an excellent cytological stain that provided excellent morphological quality for the visualization of the nuclear changes.\n\n\nEthics approval and consent to participate\n\nThe study was approved by the Faculty of Medical Laboratory Sciences Research Ethics Committee – University of Khartoum, Sudan (Nov/2016/102). Written informed consent was obtained from each participant prior to participation.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Urine cytology in our daily practice\", https://doi.org/10.7910/DVN/JHZCCM51.\n\nHarvard Dataverse: Replication Data for: Urine cytology in our daily practice\", https://doi.org/10.7910/DVN/JHZCCM51.\n\nThis project contains the following extended data:\n\n- Supplementary Table 1: Paris Reporting System for Urinary Cytology based on age groups and gender of patients.\n\n- Supplementary Table 2: Gender risk estimation for having urine abnormalities\n\n- Supplementary Table 3: Patients medical condition or medical history and risk estimation for having urine abnormalities\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank the staff of Omdurman Teaching Hospital in Khartoum state, Sudan for their kind collaboration and assistance during patients’ recruitment and sampling. We also thank all participants that contributed to this work.\n\n\nReferences\n\nRaab SS, Grzybicki DM, Vrbin CM, et al.: Urine cytology discrepancies: frequency, causes, and outcomes. Am J Clin Pathol. 2007; 127(6): 946–53. PubMed Abstract \| Publisher Full Text\n\nBarlandas-Rendón E, Müller MM, García-Latorre E, et al.: Comparison of urine cell characteristics by flow cytometry and cytology in patients suspected of having bladder cancer. Clin Chem Lab Med. 2002; 40(8): 817–23. PubMed Abstract \| Publisher Full Text\n\nBellafiore S, Zanichelli M, Piana S: Schistosoma haematobium in Urine Cytology: Diagnosis Is Possible. Am J Med. 2018; 131(3): e87–e88. PubMed Abstract \| Publisher Full Text\n\nCheung F, Loeb CA, Croglio MP, et al.: Bacteria on Urine Microscopy Is Not Associated with Systemic Infection in Patients with Obstructing Urolithiasis. J Endourol. 2017; 31(9): 942–945. 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PubMed Abstract \| Publisher Full Text\n\nGiuliano AR, Nielson CM, Flores R, et al.: The optimal anatomic sites for sampling heterosexual men for human papillomavirus (HPV) detection: the HPV detection in men study. J Infect Dis. 2007; 196(8): 1146–1152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKawaguchi S, Shigehara K, Sasagawa T, et al.: Liquid-based urine cytology as a tool for detection of human papillomavirus, Mycoplasma spp., and Ureaplasma spp. in men. J Clin Microbiol. 2012; 50(2): 401–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaziarz EK, Perfect JR: Cryptococcosis. Infect Dis Clin North Am. 2016; 30(1): 179–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiddappa S, Mythri K, Kowsalya R: Cytological findings in routine voided urine samples with hematuria from a tertiary care center in south India. J Cytol. 2012; 29(1): 16–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrees S, Bidnur S, Metcalfe M, et al.: Effect of contrast media on urinary cytopathology specimens. Can Urol Assoc J. 2016; 10(7–8): 228–233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan PS, Chan JB, Levin MR, et al.: Urine cytology and adjunct markers for detection and surveillance of bladder cancer. Am J Transl Res. 2010; 2(4): 412–440. PubMed Abstract \| Free Full Text\n\nBarkan GA, Wojcik EM, Nayar R, et al.: The Paris System for Reporting Urinary Cytology: The quest to develop a standardized terminology. Acta Cytol. 2016; 60(3): 185–197. PubMed Abstract \| Publisher Full Text\n\nXie Q, Huang Z, Zhu Z, et al.: Diagnostic Value of Urine Cytology in Bladder Cancer. A Meta-Analysis. Anal Quant Cytopathol Histpathol. 2016; 38(1): 38–44. PubMed Abstract\n\nBrown FM: Urine cytology: is it still the gold standard for screening? Urol Clin North Am. 2000; 27(1): 25–37. PubMed Abstract \| Publisher Full Text\n\nOeyen E, Hoekx L, De Wachter S, et al.: Bladder Cancer Diagnosis and Follow-Up: The Current Status and Possible Role of Extracellular Vesicles. Int J Mol Sci. 2019; 20(4): pii: E821. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXing J, Reynolds JP: Diagnostic Advances in Urine Cytology. Surg Pathol Clin. 2018; 11(3): 601–610. PubMed Abstract \| Publisher Full Text\n\nPalaoro LA, Angerosa M: Correlation between the cytology of urine sediment in fresh sample and smears stained by Papanicolaou and Giemsa methods. J Cytol. 2014; 31(1): 25–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSekita N, Shimosakai H, Nishikawa R, et al.: [Comparison of the Conventional Centrifuged and Filtrated Preparations in Urine Cytology]. Hinyokika Kiyo. 2016; 62(3): 111–6. PubMed Abstract\n\nFisher JF, Kavanagh K, Sobel JD, et al.: Candida urinary tract infection: pathogenesis. Clin Infect Dis. 2011; 52(Suppl 6): S437–51. PubMed Abstract \| Publisher Full Text\n\nSahay K, Mehendiratta M, Rehani S, et al.: Cytological artifacts masquerading interpretation. J Cytol. 2013; 30(4): 241–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNickeleit V, Hirsch HH, Binet IF, et al.: Polyomavirus infection of renal allograft recipients from latent infection to manifest disease. J Am Soc Nephrol. 1999; 10(5): 1080–9. PubMed Abstract\n\nNickeleit V, Singh HK, Mihatsch MJ: Polyomavirus nephropathy: morphology, pathophysiology, and clinical management. Curr Opin Nephrol Hypertens. 2003; 12(6): 599–605. PubMed Abstract \| Publisher Full Text\n\nRamos E, Drachenberg C, Portocarrero M, et al.: BK virus nephropathy diagnosis and treatment: experience at the University of Maryland Renal Transplant Program. Clin Transpl. 2002; 143–53. PubMed Abstract\n\nBuehrig CK, Lager DJ, Stegall MD, et al.: Influence of surveillance renal allograft biopsy on diagnosis and prognosis of polyomavirus-associated nephropathy. Kidney Int. 2003; 64(2): 665–73. PubMed Abstract \| Publisher Full Text\n\nHirsch HH, Knowles W, Dickenmann M, et al.: Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med. 2002; 347(7): 488–96. PubMed Abstract \| Publisher Full Text\n\nSalih M, Hassan O, Gasim G, et al.: Polyomavirus among Transplanted Sudanese Patients. Virology and Emerging Diseases. 2015; 1(1). Reference Source\n\nAlmobarak AO, Mohammed MH, Ahmed AO, et al.: Urinary cytopathologic findings in renal allograft recipients in Sudan: trends, outcome and challenges. Saudi J Kidney Dis Transpl. 2014; 25(2): 410–1. PubMed Abstract \| Publisher Full Text\n\nHirsch HH, Drachenberg CB, Steiger J, et al.: Polyomavirus-associated nephropathy in renal transplantation: critical issues of screening and management. Adv Exp Med Biol. 2006; 577: 160–173. PubMed Abstract \| Publisher Full Text\n\nTrofe J, Gordon J, Roy-Chaudhury P, et al.: Polyomavirus nephropathy in kidney transplantation. Prog Transplant. 2004; 14(2): 130–142. Publisher Full Text\n\nBonvoisin C, Weekers L, Xhignesse P, et al.: Polyomavirus in renal transplantation: a hot problem. Transplantation. 2008; 85(7 Suppl): S42–8. PubMed Abstract \| Publisher Full Text\n\nShigehara K, Sasagawa T, Kawaguchi S, et al.: Prevalence of human papillomavirus infection in the urinary tract of men with urethritis. Int J Urol. 2010; 17(6): 563–8. PubMed Abstract \| Publisher Full Text\n\nNakamura K, Kasraeian A, Iczkowski KA, et al.: Utility of serial urinary cytology in the initial evaluation of the patient with microscopic hematuria. BMC Urol. 2009; 9(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrossfeld GD, Litwin MS, Wolf JS Jr, et al.: Evaluation of asymptomatic microscopic hematuria in adults: the American Urological Association best practice policy--part II: patient evaluation, cytology, voided markers, imaging, cystoscopy, nephrology evaluation, and follow-up1. Urology. 2001; 57(4): 604–10. PubMed Abstract \| Publisher Full Text\n\nTan WS, Sarpong R, Khetrapal P, et al.: Does urinary cytology have a role in haematuria investigations? BJU Int. 2019; 123(1): 74–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKapila K, Nampoory MR, Johny KV, et al.: Role of urinary cytology in detecting human polyoma bk virus in kidney transplant recipients. A preliminary report. Med Princ Pract. 2007; 16(3): 237–9. PubMed Abstract \| Publisher Full Text\n\nBoldorini R, Zorini EO, Viganò P, et al.: Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients. Acta Cytol. 2000; 44(2): 205–10. PubMed Abstract \| Publisher Full Text\n\nSiddig E: \"Replication Data for: Urine cytology in our daily practice\". Harvard Dataverse, V1, UNF:6:QI5RxH19bMjvAVYeI5Pg2g== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/JHZCCM" }	[ { "id": "66661", "date": "27 Jul 2020", "name": "Mohammad Hossein Anbardar", "expertise": [ "Reviewer Expertise Pathology", "cytopathology", "GI", "liver", "transplant." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a descriptive cross-sectional study evaluating urine cytology in patients referee to the outpatient clinic. The authors analyzed the data according to age, gender, and underlying medical disease. Below comments should be considered and answered:\nIn the method it is mentioned that 2 cytopathologists examined the smears, is there any disagreement between them especially in atypical, suspicious and positive cases? If present, what is your approach?\n\nFigure 1A is not representative of HPV. It must be changed with the proper one.\n\nIn 2nd paragraph of \"Discussion\", the authors said: 'In this study, we observed that urine cytology could easily differentiate benign from malignant cells showing high specificity for detecting HGUC'. To determine specificity we have to compare the results with pathologic examination. If there is a cytology-pathology correlation in this study, it is better to mention in \"method and result\" sections with proper tables.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "70229", "date": "04 Sep 2020", "name": "Torill Sauer", "expertise": [ "Reviewer Expertise Cytopathology in general with fine needle aspirations as speciality. This includes pathologist handled FNAC", "both palpable lesions and by doing my own ultrasound-guided FNC. In histology my speciality is breast pathology. I have been involved in the Norwegian screening programmes for cervical cancer and mammography screening for breast cancer. Main research in breast cytopathology and histopathology. In addition head and neck as well as thyroid cytopathology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well conducted study and a well written manuscript; easy to read, but with scattered linguistic errors.\nI have a few comments: Your study population are patients referred to the out patient clinic, I presume. I see that only a minority have a condition, but they must have had some symptom(s) or cause for their referral. Even if it turned out that there was no specific condition, you should state the cause for referral.\nYou have 3 cases with a cytological diagnosis of low grade (atypical) urothelial cells. The Paris system is only concerned about high grade atypia. In which category did you put these three cases?\nUnder discussion: second last paragraph: ...conducted by Kapila and her associates. She is a woman (and a friend of mine).\nUnder conclusions, you could add a sentence or two about the value of urinary cytology in the diagnosis of infectious conditions as you so nicely demonstrated with cryptococcus and Schistosomiasis. This would be most valuable in areas where tropic/subtropic infections of the urinary tract are widespread.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1878
https://f1000research.com/articles/8-1204/v1	26 Jul 19	{ "type": "Research Article", "title": "An audit of electron microscopy in the diagnosis of focal segmental glomerulosclerosis: are current pathological techniques missing important abnormalities in the glomerular basement membrane?", "authors": [ "Justin Davis", "Alwie Tjipto", "Katharine Hegerty", "Andrew Mallett", "Alwie Tjipto", "Katharine Hegerty", "Andrew Mallett" ], "abstract": "Background: There is an increasing appreciation that variants of the collagen IV genes may be associated with the development of focal segmental glomerulosclerosis (FSGS). On electron microscopy, such variants may produce characteristic changes within the glomerular basement membrane (GBM). These changes may be missed if glomerular lesions histologically diagnosed as FSGS on light microscopy are not subjected to electron microscopy. Methods: We conducted a retrospective cohort analysis of all patients presenting to two hospitals who received a primary histological diagnosis of FSGS to see if these samples underwent subsequent electron microscopy. Each such sample was also scrutinised for the presence of characteristic changes of an underlying collagen IV disorder Results: A total of 43 patients were identified. Of these, only 30 underwent electron microscopy. In two samples there were histological changes detected that might have suggested the underlying presence of a collagen IV disorder. Around one in three biopsy samples that had a histological diagnosis of FSGS were not subjected to electron microscopy. Conclusion: Renal biopsy samples that have a histological diagnosis of primary FSGS not subjected to subsequent electron microscopy may potentially miss ultrastructural changes in the GBM that could signify an underlying collagen IV disorder as the patient’s underlying disease process. This could potentially affect both them and their families’ investigative and management decisions given potential for implications for transplant, heritability and different disease pathogenesis. This represents a gap in care which should be reflected upon and rectified via iterative standard care and unit-level quality assurance initiatives.", "keywords": [ "Collagen IV", "Electron Microscopy", "Glomerular Basement Membrane", "Focal Segmental Glomerulosclerosis", "Renal Biopsy." ], "content": "Abbreviations\n\nChronic kidney disease (CKD); end stage kidney disease (ESKD); focal segmental glomerulosclerosis (FSGS); glomerular basement membrane (GBM); not otherwise specified (NOS); thin basement membrane nephropathy (TBMN).\n\n\nIntroduction\n\nFocal segmental glomerulosclerosis (FSGS) is a pathological diagnosis which underpins a variety of progressive renal diseases that commonly result in proteinuria, chronic kidney disease (CKD) and potential end stage kidney disease (ESKD) requiring renal replacement therapy1. FSGS occurs in either a primary. secondary, or genetic form. Primary FSGS is thought to be largely immunological in nature perhaps driven by an elusive permeability factor and secondary FSGS caused by compensatory hyperfiltration due to a previous glomerular injury2. In recent years there has been a greater appreciation of the contribution of underlying genetic causes of this histological pattern. Many of these genetic aetiologies for FSGS have potential clinical significance for at-risk family members, subsequent genetic counselling, future living related kidney transplantation and therapeutics including potential avoidance of immunosuppressive therapies otherwise used for primary immunologically-mediated FSGS. Although initial focus has been on genes that are involved in the maintenance of podocyte structure and function it has become apparent that abnormalities in genes responsible for the structural integrity of the glomerular basement membrane (GBM) may underpin a significant minority of cases of adult-onset FSGS3. Variants in COL4A3, COL4A4 and COL4A5, which encode the α3, α4 and α5 chains of collagen IV, respectively, the major constituent of the GBM, previously linked to both Alport syndrome and thin basement membrane nephropathy (TBMN), may also underlie cases of FSGS4. This suggestion has biological plausibility. Podocytes, the glomerular epithelial cells responsible for maintenance of the filtration barrier of the glomerulus through which plasma is ultrafiltrated, are not only a source for the α-chains secreted to form the GBM but also are required to be anchored to this structure in order to maintain the aforementioned filtration barrier2,5. It is not inconceivable then that inherited structural abnormalities of the GBM may lead to subsequent podocyte dysfunction and ultimately the lesion histologically characterised as FSGS.\n\nThe relationship between the three renal conditions intertwined around variants in the collagen IV genes, Alport syndrome, TBMN, and FSGS is complex and incompletely understood. Whilst previously it was believed that patients heterozygous for variants in COL4A3 and COL4A4 would develop TBMN with persistent microscopic haematuria and an otherwise benign prognosis, this traditional thinking has been overturned by the discovery that some pedigrees with these variants will go on to develop significant proteinuria, FSGS lesions and the potential for progressive CKD or ESRD6,7. More recently, targeted gene sequencing of adults thought to have primary FSGS or steroid resistant nephrotic syndrome (the paediatric equivalent) found that pathogenic or likely pathogenic variants in collagen IV genes were present in up to 38% of families with familial FSGS, and 3% of those with sporadic FSGS3,8. These findings highlight the importance of considering variants in these genes in the diagnosis and subsequent management of patients found to have FSGS histologically.\n\nFSGS is a clinicopathological diagnosis that is made on the review of renal tissue obtained by percutaneous biopsy. This tissue is processed and subjected to several different staining techniques and methods of microscopy, including light, immunofluorescence and electron microscopy9. FSGS, suggested on light microscopy by the finding of focal, segmental lesions with sclerosis, and an increase in the glomerular matrix with obliteration of the capillary lumens only requires this finding in one glomerulus and light microscopy to diagnose2. As such, some specimens may previously have not been sent for further processing and review with other techniques such as electron microscopy even though this histopathology has heterogenous causative aetiologies. The other potential renal lesions that may be caused by collagen IV gene variants, Alport syndrome and TBMN, cause thinning, lamellation and fraying of the GBM and may also be associated with podocyte foot process effacement, all of which require electron microscopy to visualise4. It should be noted that for Alport syndrome there are other techniques aside from electron microscopy available for identifying the diagnosis. Immunostaining of a renal biopsy specimen for type IV collagen may demonstrate the absence of alpha 3,4 or 5 chains in up to two thirds of patients, or a skin biopsy may show an absence of an alpha 5 chain10,11, in addition to the aforementioned genetic testing. Such absence by immunostaining does not appear be present in FSGS12. Given that the pathological diagnosis of FSGS does not routinely require electron microscopy it is therefore conceivable that GBM lesions potentially associated with an underlying collagen 4 variant may have been missed. This represents an opportunity to audit our prior clinical behaviour to see if our multidisciplinary diagnostic practice may require improvement. We conducted a retrospective audit of prior renal biopsy results in two tertiary centres to see how many of those that had been given a histological diagnosis of FSGS were sent for subsequent electron microscopy.\n\n\nMethods\n\nOur study was a retrospective cohort analysis across two tertiary hospital sites involving the review of prior renal biopsy results that had been given the histological diagnosis of FSGS on light microscopy and how many of these samples subsequently went on to be processed for electron microscopy. In addition, of those samples that were subjected to electron microscopy, we reviewed how many displayed evidence of a potential collagen IV disorder. This project had ethics approval through Barwon Health’s Research Ethics Governance and Integrity unit and the Royal Brisbane and Women’s Hospital Human Research and Ethics Committee (HREC 18/131 and HREC/18/QRBW/258, respectively). Participants consent was waived by both ethics committees due to the negligible risk nature of data acquisition via retrospective datasets already maintained on electronic health records at both institutions.\n\nPatients aged over the age of 18 who received a histological diagnosis of FSGS on native kidney biopsy during the time period of January 1st 2008 until July 31st 2018 at the first participating hospital and from the 1st of October 2013 until the 29th of December 2018 at the second were eligible to be included in the retrospective audit. Participants were excluded from the audit if they were less than 18 years of age, underwent kidney transplant biopsy, were defined clinically to have secondary FSGS as opposed to primary disease, received their diagnosis of FSGS or underwent a renal biopsy and pathological review outside of the prespecified time period for each institution.\n\nSamples for biopsy were placed in the following fixatives for processing; formalin for light microscopy, saline soaked gauze with freezing for immunofluorescence, and glutaraldehyde for electron microscopy. Samples for light microscopy are embedded and sectioned at 2- to 3-µm thickness with hematoxylin-eosin, Jones silver, periodic acid-schiff and trichrome staining performed. Immunofluorescence samples are sectioned within a cryostat and placed on prelabelled air-dried slides of the antigen in question. Electron microscopy tissue is processed into plastic, trimmed, cut into a 1-µm section and stained with toludine blue. The images are reviewed in a digital medium13.\n\nA search strategy was developed for this retrospective cohort study to identify appropriate patients through local electronic medical record systems using the search term ‘focal segmental glomerulosclerosis’ with the AND operator to combine with ‘glomerulonephritis’ or ‘hereditary nephritis’. These patient results were subsequently reviewed for evidence of a previous percutaneous renal biopsy and report. The data were independently extracted from the included patients by the primary reviewer and collated in a Microsoft Excel document that included information on age, primary diagnosis, included use of electron microscopy and any changes that may be consistent with an underlying collagen disorder including thinning, lamellation and fraying of the GBM. The data extracted was verified by the other three co-authors with all discrepancies resolved through discussion and consensus.\n\n\nResults\n\nFrom January 2008 through to July 2018 at the first centre and October 2013 until December 2018 at the second, a total of 43 patients were identified as having primary FSGS. The baseline characteristics of the study cohort are provided in Table 1. The median age was 49 years and the patients were predominantly male (55%). The most common underlying histological diagnosis reported in the renal biopsy clinical pathology reports was FSGS not otherwise specified (NOS), followed by familial FSGS with no underlying genetic disorder ascertained. There were small numbers of the cellular, collapsing, perihilar and tip FSGS variants noted. The two most common stages of CKD at the time of presentation were I and III. The results extracted and analyses in this study are available as Underlying data14.\n\n*Values given as median (range).\n\nOf the 43 patients identified, samples from 30 underwent electron microscopy after initial light microscopy and immunofluorescence. Two of these samples showed signs on electron microscopy that might be consistent with an underlying collagen IV glomerular basement membrane disorder. Microscopic haematuria was not noted in either of these two patients. Of the 13 samples that did not undergo electron microscopy, four had no glomeruli present within the processed core. The remaining nine samples had no documented reason.\n\nThe overall percentage of biopsy samples for which data were available that were not subjected to electron microscopy was 30%, of which close to 21% had no documented reason for not undergoing electron microscopy. The annualised rate of biopsy samples that were not subjected to electron microscopy varied with time (Figure 1). The number of biopsy reports available for analysis prior to 2013 was only one per year at most; however, from 2013 onwards the rate progressively increased to between 3 and 11 cases of FSGS diagnosed at histopathology between the two institutions per year. The annualised rate of electron microscopy from 2013 onwards varied each year, ranging from 50 to 87%. Overall 30% of biopsy samples did not receive electron microscopy. The overall rate of electron microscopy did not significantly change across the study period between the two centres.\n\n\nDiscussion\n\nThis retrospective cohort analysis demonstrated that about two-thirds of native kidney biopsy samples across two institutions that were deemed to have primary FSGS underwent subsequent electron microscopy. Of those that did, two were reported to have characteristics that might be consistent with an underlying collagen IV disorder. In both samples, electron microscopy revealed a diffusely thin GBM, with the second additionally identifying early focal splitting of the GBM. The first sample was suggested to be consistent with TBMN whereas there was no pathological comment made about the second. FSGS (NOS) was the most common lesion described in this study, which is consistent with prior reports2. Notably, close to one in three cases of primary FSGS were not proceeding to electron microscopy despite an indication to do so and 1 in 20 cases within our cohort had structural changes that were consistent with an underlying collagen IV variant. Whilst some samples were unable to undergo electron microscopy due to a lack of glomeruli in the biopsy core, in the majority of the others it is unclear why subsequent electron microscopy did not occur. The annualised rate of biopsy samples not subjected to electron microscopy varied by year, but on average around one in three samples were not subjected to electron microscopy despite receiving a histological diagnosis of FSGS.\n\nCuriously, neither of the two patients in whom electron microscopy was suggestive of an underlying collagen IV disorder was noted to have haematuria on their urinalysis at the time of presentation. Both of these samples were noted to have a thin basement membrane on electron microscopy, with focal splitting noted in one with an average thickness of 230.72 nm given. Unfortunately, due to the retrospective nature of this study we were unable to send any samples for immunostaining of collagen IV.\n\nThere is an increasing body of evidence indicating that inheritable variants in collagen IV genes may underlie a proportion of cases of FSGS, with up to 12.5% cases of autosomal dominant FSGS attributable to COL4A3 in some cohorts15. Not subjecting these renal biopsy samples to electron microscopy represents a potential gap in the investigation and subsequent management of such patients given they are much less likely to respond to immunosuppressive therapy16 which has otherwise been classically indicated.\n\nThis study was designed as a retrospective cohort study looking at the number of samples sent for electron microscopy, as well as any potential changes which might be consistent with a collagen IV glomerular basement membrane disorder. It is important to recognise that not all groups have found the characteristic changes associated with the collagen IV disorders such as Alport’s Syndrome or TBMN on electron microscopy. One study described the typical pathological changes of FSGS but not the glomerular basement membrane abnormalities characterising Alport syndrome or TBMN in patients known to have variants in either COL4A3 or COL4A47. It is thus possible that a lack of classical findings for a collagen IV glomerular basement membrane disorder may have accounted for the low number of those with GBM features on electron microscopy consistent with a collagen 4 disorder noted within our study. Indicating against this, however, another study suggested that biopsy samples from patients with the classical features of Alport Syndrome or TBMN showed podocyte detachment which might be expected and subsequently cause FSGS-type lesions4. Other studies which have looked at electron microscopy in FSGS cases have similarly found low numbers of abnormalities that may be consistent with an underlying collagen 4 disorder3,8, which suggests the overall number of abnormalities to be found via electron microscopy may be low.\n\nThe process by which variants within the collagen IV genes might cause FSGS remains unclear, particularly given their clear association with Alport Syndrome and TBMN. One proposal is that the ultrastructural changes induced by the collagen IV variants, perhaps under the influence of modifier genes such as laminin, result in impaired podocyte attachment to the glomerular basement membrane which leads to accelerated podocyte detachment, subsequent foot process effacement as a response to the increased shear stress induced by the denuded basement membrane and at a critical level of podocyte loss collapse of the capillary network with the appearance of the classical segmental sclerotic lesion2,4,17. It also remains unclear as to whether the changes of FSGS are a secondary process occurring in those with TBMN or whether the collagen 4 variants are capable of causing primary FSGS7,18. FSGS occurring as a secondary process to other basement membrane abnormalities may explain why immunosuppressive therapy has traditionally been less effective in inherited forms of FSGS, although there are case reports of the successful use of the calcineurin inhibitor cyclosporine for some patients harbouring inheritable collagen 4 disorders6.\n\nIn summary, this study has found that not all biopsy samples that had primary FSGS as a histological diagnosis were subjected to subsequent electron microscopy. This may have potentially led to inadvertently overlooking characteristic basement membrane abnormalities, which may suggest an underlying and heritable collagen IV disorder. These findings reflect an opportunity to change practice in order to better investigate, counsel and provide clinical management to these and future patients.\n\n\nData availability\n\nFigshare: Davis et al. FSGS biopsy audit.xlsx. https://doi.org/10.6084/m9.figshare.8949032.v114.\n\nThis project contains the variables extracted for each individual retrospectively assessed in this study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nAll authors made substantial intellectual contributions to the manuscript. The audit was overseen by the lead authors. All authors participated in the interpretation of data and vouch for the completeness and accuracy of the data. The first author (JD) wrote the first draft with all authors involved in the conception and design of the manuscript. All authors participated in the development of this manuscript and made the decision to publish the results.\n\nThe acquisition, analysis and interpretation of data was led by the corresponding author JD with equally important contributions and oversight including the resourcing of key references from AT, KH and AM. All authors were both involved in the drafting of the manuscript and critical revision for important intellectual content. All authors have given final approval for the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content and both authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in funding this work.\n\n\nReferences\n\nHines SL, Agarwal A, Ghandour M, et al.: Novel variants in COL4A4 and COL4A5 are rare causes of FSGS in two unrelated families. Hum Genome Var. 2018; 5: 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Vriese AS, Sethi S, Nath KA, et al.: Differentiating Primary, Genetic, and Secondary FSGS in Adults: A Clinicopathologic Approach. J Am Soc Nephrol. 2018; 29(3): 759–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGast C, Pengelly RJ, Lyon M, et al.: Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis. Nephrol Dial Transplant. 2016; 31(6): 961–70. PubMed Abstract \| Publisher Full Text\n\nWickman L, Hodgin JB, Wang SQ, et al.: Podocyte Depletion in Thin GBM and Alport Syndrome. PLoS One. 2016; 11(5): e0155255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorstanje R, Caputo CR, Doty RA, et al.: A mouse Col4a4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers. Kidney Int. 2014; 85(6): 1461–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPierides A, Voskarides K, Athanasiou Y, et al.: Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis. Nephrol Dial Transplant. 2009; 24(9): 2721–9. PubMed Abstract \| Publisher Full Text\n\nMalone AF, Phelan PJ, Hall G, et al.: Rare hereditary COL4A3/COL4A4 variants may be mistaken for familial focal segmental glomerulosclerosis. Kidney Int. 2014; 86(6): 1253–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYao T, Udwan K, John R, et al.: Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS. Clin J Am Soc Nephrol. 2019; 14(2): 213–223, pii: CJN.08750718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHogan JJ, Mocanu M, Berns JS: The Native Kidney Biopsy: Update and Evidence for Best Practice. Clin J Am Soc Nephrol. 2016; 11(2): 354–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKashtan CE: Alport syndrome. An inherited disorder of renal, ocular, and cochlear basement membranes. Medicine (Baltimore). 1999; 78(5): 338–60. PubMed Abstract \| Publisher Full Text\n\nMassella L, Onetti Muda A, Faraggiana T, et al.: Epidermal basement membrane alpha 5(IV) expression in females with Alport syndrome and severity of renal disease. Kidney Int. 2003; 64(5): 1787–91. PubMed Abstract \| Publisher Full Text\n\nCai YI, Sich M, Beziau A, et al.: Collagen distribution in focal and segmental glomerulosclerosis: an immunofluorescence and ultrastructural immunogold study. J Pathol. 1996; 179(2): 188–96. PubMed Abstract \| Publisher Full Text\n\nWalker PD: The renal biopsy. Arch Pathol Lab Med. 2009; 133(2): 181–8. PubMed Abstract\n\nDavis J: Davis et al FSGS biopsy audit.xlsx. figshare. Dataset. 2019.\n\nXie J, Wu X, Ren H, et al.: COL4A3 mutations cause focal segmental glomerulosclerosis. J Mol Cell Biol. 2014; 6(6): 498–505. PubMed Abstract \| Publisher Full Text\n\nPapazachariou L, Papagregoriou G, Hadjipanagi D, et al.: Frequent COL4 mutations in familial microhematuria accompanied by later-onset Alport nephropathy due to focal segmental glomerulosclerosis. Clin Genet. 2017; 92(5): 517–27. PubMed Abstract \| Publisher Full Text\n\nVoskarides K, Pierides A, Deltas C: COL4A3/COL4A4 mutations link familial hematuria and focal segmental glomerulosclerosis. glomerular epithelium destruction via basement membrane thinning? Connect Tissue Res. 2008; 49(3): 283–8. PubMed Abstract \| Publisher Full Text\n\nBraunisch MC, Buttner-Herold M, Gunthner R, et al.: Heterozygous COL4A3 Variants in Histologically Diagnosed Focal Segmental Glomerulosclerosis. Front Pediatr. 2018; 6: 171. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "53353", "date": "18 Sep 2019", "name": "James McCaffrey", "expertise": [ "Reviewer Expertise Paediatric nephrology", "matrix biology", "glomerular cell biology", "histopathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a retrospective study of kidney biopsies from 43 patients over a 10-year interval. The authors retrieved native biopsies from patients with a primary histological diagnosis of FSGS in two tertiary hospitals. They determined the number of biopsies that had both histological evidence of FSGS and accompanying ultrastructural analysis with electron microscopy (EM). 13/43 biopsies did not have evaluation by EM. In 2 biopsies that had both histology and EM, abnormalities of the glomerular basement membrane (GBM) were identified. The authors advocate an increase in the use of EM with a diagnosis of FSGS to identify a subset of patients with an underlying genetic disorder affecting type IV collagen.\n\nOverall the study is a simple, retrospective review design and it is well presented. It highlights the important observation and increasing recognition that pathogenic variants in COL4A genes are present in a high proportion of families with familial FSGS and also in individuals with sporadic FSGS. There is a pressing clinical need to identify these patients and, as such, this study comes at an opportune time. We have the following questions and suggestions for improvement:\n\nThe authors could present a stronger argument for EM being the investigation of choice for identifying patients with COL4A mutations. Although the authors discuss the variable EM findings in patients with Alport syndrome or TBMN, this could be expanded. How sensitive are the GBM findings for COL4 mutations? Not all patients with COL4A variants have typical EM findings, and furthermore suggestive GBM abnormalities may be found in other genetic disorders including mutations in MYH9 and LMX1B.\n\nThere would be additional value if the authors could propose a pros/cons comparison of EM versus genetic screening with for example an NGS gene panel to identify these patients. This could be presented as a proposed workflow for investigation.\n\nThe authors identify 2 patients with EM findings suggestive of a COL4A disorder. However, there are no data detailing whether these patients did in fact harbour a COL4A variant (notably neither of them had haematuria at presentation). Are any data available, for example, on clinical response to immunosuppression, or any sequencing data?\n\nMinor comments\n\nThe title described the study as an 'audit' but the text repeatedly refers to a 'retrospective cohort analysis.' As there are no defined standards against which clinical practice is being measured here, perhaps remove 'audit' from the title?\n\nThe Y-axis in Figure 1 should be labelled.\n\nWhich FSGS variant did the 2 patients with EM abnormalities fall into?\n\nImprove consistency with nomenclature—use of conventional gene (COL4A) or protein names (type IV collagen).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5014", "date": "07 Nov 2019", "name": "Justin Davis", "role": "Author Response", "response": "To the first reviewer who kindly reviewed our article; On behalf of my co-authors, I wish to thank you for taking the time out of your busy work schedule to read through our manuscript and make suggestions which only serve to strengthen the article overall and make it a better piece for submission. We would like to respond to each of the suggestions listed below. Major changes include: 1. The authors agree that the extrapolation of EM changes in COL4A variants could be further elaborated on and have introduced a new section in the introduction looking at a Chinese cohort with both confirmed EM and COL4A mutations. EM is unlikely to be the preferred investigation for such cases, but if suggestive abnormalities are found by happenstance then investigation for a potential COL4A variant seems appropriate based on our current understanding. Unfortunately, due to small sample sizes, the sensitivity of such findings cannot be commented on at this time and forms a body of ongoing research into these potential links. 2. The authors have included a new figure (Figure 2) with a proposed worksheet for investigation into patients whom may present in this manner. 3. Unfortunately, the patients whom had EM abnormalities suggestive of a type IV collagen disorder have been lost to follow up, and the data on them is simply not able to extrapolated for this particular article (a shame as the authors were curious about such things too). Minor changes include: 1. The title has been reworded to avoid confusion, and text within the paragraphs altered to reflect this change away from audit. 2. The Y axis in figure 1 is now labelled appropriately. 3. Tip and NOS, now specified within the manuscripts main paragraphs. 4. The authors thank the reviewers for pointing out small errors of inconsistency which can creep into a document like this, and have amended them appropriately Again, many thanks for taking the time out to read through this manuscript." } ] }, { "id": "54539", "date": "28 Oct 2019", "name": "Constantinos Deltas", "expertise": [ "Reviewer Expertise Human molecular and medical genetics", "nephrogenetics", "cell biology and biochemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis retrospective study focuses on the fact that renal biopsies are not always evaluated by electron microscopy (EM). The authors found 43 renal biopsies in the records of two hospitals, where the diagnosis of FSGS was made, for which EM had been performed only in 30. Most importantly, in two of the biopsies studied by EM, there was evidence for a collagen IV disorder but had not been followed up. This has important implications in view of numerous reports in the past decade and more, according to which the histological diagnosis of FSGS could be on the background of collagen IV mutations, which cause Alport syndrome or thin basement membrane nephropathy.\nI agree with all points raised by the previous reviewer and which I do not intend to repeat here. I would only emphasize two things:\nI believe there should be no renal biopsy attempted if it is not going to be studied fully by light microscopy, immunofluorescence studies and EM. It is an invasive procedure accompanied by some risk and when performed it must be fully exploited as very significant results may come up, which bear implications on treatment, inheritance patterns and family planning.\n\nThe authors found two patients who had undergone EM and there were findings indicative of a collagen IV disorder. They should have proceeded to genetic testing to verify this and strengthen their case. Even belatedly, this could be of benefit not only to the patients but also to family members.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5015", "date": "07 Nov 2019", "name": "Justin Davis", "role": "Author Response", "response": "To the second reviewer who kindly reviewed our article;On behalf of my co-authors, I wish to thank you for taking the time out of your busy work schedule to read through our manuscript and make suggestions which only serve to strengthen the article overall and make it a better piece for submission. We would like to ensure we have addressed both concerns pointed out below.1. The authors agree with this point and feel most (if not all, at least in the case of native kidney) biopsies should be subjected to appropriate microscopic techniques which includes electron microscopy. It is unclear why some of our samples unfortunately were not, and remains a clear source of potential quality improvement that can be fed back, particularly when suggestive abnormalities may be found that influence further management. 2. The authors too are curious as to the genetic status of the two individuals who had suggestive EM changes noted within the body of the manuscript. Unfortunately on perusal of the data available they have been lost to follow up without a clear genetic diagnosis at this time.Once again, many thanks for taking the time to review our work" } ] } ]	1	https://f1000research.com/articles/8-1204
https://f1000research.com/articles/8-953/v1	24 Jun 19	{ "type": "Research Article", "title": "The performance of bovine serum albumin filtration by using polyethersulfone-Tetronic 304 blend Ultrafiltration Membrane", "authors": [ "Fachrul Razi", "Sri Mulyati", "Nasrul Arahman", "Fachrul Razi", "Sri Mulyati" ], "abstract": "Background: Membrane technology has been widely applied for protein purification. In applications for protein separation, a membrane with stable filtration performance is necessary. In this work, two types of hollow fiber membranes with different characteristic were used to study the filtration profile of bovine serum albumin. Methods: A single piece of hollow fiber module was used for ultrafiltration testing using UF0 and UFT304 membranes. Flux and rejection of BSA solution were collected based on a pressure-driven inside filtration model. Results: Ultrafiltration experiments showed that the flux of UFT304 membrane was higher than that of UF0 membrane in all applied pressure condition. Solute rejection was achieved up to 90% for ultrafiltration of BSA solution by using UF0 membrane obtained on the operating pressure of 0.5 atm. Conclusion: In general, UFT304 membranes has better ultrafiltration performance for BSA separation than UF0 membranes. The UFT304 membrane has a more stable flux for up to two hours of filtration.", "keywords": [ "albumin", "solute rejection", "polyethersulfone", "ultrafiltration" ], "content": "Introduction\n\nAlbumin proteins are one of the most important substances in the human body. Albumin maintains the balance of vascular fluid, forms new cell tissue, and functions acts as an antibody1. Albumin can help to reduce the risk of stroke and coronary heart disease2. Continuous experimentation is performed to establish the best method for isolation of protein from egg white to get the best quality albumin. The most popular technique is precipitation with Na2SO4 or (NH4)2SO4 at 25°C and pH 4-7. The protein-purifying method used in the precipitation technique can result in a large albumin yield, but the protein product contains the residue of precipitate materials, namely ammonium sulfate or sodium sulfate, which can potentially harmful3. The low purity and loss of albumin during the purification process has led researchers to search for alternative methods to address these short-comings.\n\nRapid development in the field of biotechnology has allowed for the separation of proteins with safer and more efficient methods, namely purification using membrane technology (ultrafiltration, UF). This method has been reported to successfully achieve protein fractionation through UF with several advantages compared with the conventional separation using biology/chemistry methods4–6. The main advantage being generating a product with high purity level and low toxicity7.\n\nDespite these advantages, ultrafiltration technology also has some drawbacks. In many cases, protein molecules are easily absorbed on to the surface of hydrophobic membranes, such as polyethersulfone (PES), resulting in fouling. As a consequence, membrane flux decreases drastically along with the operation time. Modification of membrane surface traits is one solution to minimize the fouling of the membrane surface8,9.\n\nIn this research, the performance of two type hollow fiber membranes for BSA separation was investigated, one made of PES/ N-Methyl-2-pyrrolidone (NMP) and PES/MNP/Tetronic 304 system. Tetronic 304 is a non-ionic surfactant with two bonds of polyethylene oxide (PEO) and two bonds of polypropylene oxide (PPO) to ethylene diamine group. This surfactant potentially improves hydrophilic traits of the PES membrane due to its PEO group. This study aimed to investigate and establish the profile of BSA solution filtration using a standard PES membrane and a membrane modified with Tetronic 304 surfactant for permeability and rejection parameters.\n\n\nMethods\n\nWe used two types of hollow fibre membrane made of polyethersulfone (PES, E6020P: BASF Co. Ludwigshafen, Germany; FN. 218189-009)/N-metilpirrolidon (Merck Germany, 606-021-00-7) (labeled as UF0), and PES/NMP/Tetronic 304 (BASF, Germany) system (labeled as UFT304). UF0 membrane has inner and outer diameters of 699 and 776 µm, respectively. UFT304 membrane has an inner diameter of 705 µm and the outer diameter of 796 µm. The hydrophilicity characteristics based on water contact angle data are 74.0O and 62.0O for UF0 and UFT304 membrane, respectively. Protein model was bovine serum albumin (BSA) Cohn fraction V, pH 7 (WAKO Pure Chemical Industries, Japan; 017-23294).\n\nMembrane characterization was performed to establish the morphology of all membrane types using scanning electron microscopy (SEM, Hitachi Co, S-800, Japan) with voltage acceleration at 20 kV, with a convergence angle of -30O, beam current x. 6.09 mm; y.5.79 mm, and diameter of the beam of 0.006 nm. Before the scanning electron microscopy (SEM) test, the membrane was soaked in liquid nitrogen for around five minutes, after the membrane turned rigid. This treatment was performed to prevent any change in the membrane structure when placing the sample on the SEM plate. The membrane was dried in freeze-drier overnight. One piece of the membrane was taken randomly and mounted on the sampling tubs (Hitachi, 10-004139-1, stubs Ø25 x 16mm x M4, 2 x 90°) wrapped with carbon tape, and then coated with osmium coater (Neoc-STB, Meiwafosis Co., Ltd., Japan) sputtering. The sample was placed on the SEM panel for the picture-capturing process at 100 and 3000 magnification for whole and enlarged cross-section, respectively. Captured images were processed using Irfan View v.3.45\n\nThe ultrafiltration module was designed using a single membrane module as previously described10. Ultrafiltration module consisted of a peristaltic pump (Watson Marlow, SciQ 323), a circuit of hose for feed flow, two pressure gauges (WIKA 213.53), valve, and hollow fibre membrane installed in the circuit. It was also equipped with feed, permeate, and retentate container.\n\nBSA solution was made at a concentration of 500 ppm at constant pH of 4.5. The filtration of BSA solution was conducted by flowing the feed solution to the membrane using a peristaltic pump (Watson Marlow, SciQ 323). The permeate has recorded a result of filtration, and the retentate was returned to the feed tank. The permeate was collected every 10-minute filtration, and its weight was measured by an auto digital balance (Shimadzu, Japan; ATY224). The received data of permeate weight were then converted in a volume unit. Flux and permeability of albumin solution were measured with Equation 1) and Equation 2)11. The filtration process is carried out at several operating pressures, which are 0, 5; 1.00; and 1.5 Atm.\n\nFlux=VA.t(1)\n\nPermeability=VA.t.P(2)\n\nV is a permeate volume, A is the membrane surface area, t is filtration time, and P is operational pressure. BSA concentration in permeate and retentate was then analyzed using a spectrophotometer. Rejection capability of the membrane can be determined using the equations as follows11:\n\nRejection=CF-CpCF×100%(3)\n\nCF and Cp are protein concentration in sample and permeate analyzed by a spectrophotometer UV-Vis (Shimadzu UV-1800) at a wavelength of 278.0.\n\n\nResult\n\nSEM images of the two membrane are shown in Figure 1 (see underlying data12). The morphological structure of membrane of the inner and outer surface were recorded with SEM at 3000x magnification as shown in Figure 1b and Figure 1c. Differences in pore structure and macrovoid pattern are clearly visible. The UFT304 membrane had larger pores with greater quantity of longer macrovoids. This likely effected ultrafiltration process of BSA protein as discussed below. Figure 1b and Figure 1c also shows a large sponge structure in the middle of UF0 membrane which is absent in the UFT304 membrane. The increase in number and size of macrovoid was caused by the addition of polymeric surfactant. During the solidification process of the membrane in the coagulation batch, part of the additive leached out of the polymer system. Therefore, a large macrovoid structure was formed.\n\nOne important parameter for the membrane performance is its capability to filter solutions, referred to as flux or permeability. Another important parameter is the capability of membrane to remove components or particles from the solution, commonly known as solute rejection. In this study the ultrafiltration capability of the membrane was tested using a single membrane of hollow fibre (single module) designed with pressure driven inside (PDI) flow. The filtration profile of the albumin protein solution using UF0 and UFT304 membranes at various operational pressures is shown in Figure 2a (underlying data13). The flux of UFT304 membrane at the beginning of this experiment with an operating pressure of 0.3 atm was 63.60 L/m2.h. Flux increased significantly with the increase of the operating pressure which reached 161.00 L/m2.hour at a pressure of 1.5 atm. The flux of UF0 membrane at the starting point (operating pressure 0.3 atm) was much lower, 32.0 L/m2.h. At the highest operating pressure (1.5 atm), the flux of UF0 membrane was about 53.0 L/m2.h.\n\nThe ultrafiltration performance of membrane in term of flux (a), and solute rejection (b).\n\nThe difference of flux between the two membranes is likely due to the differences in pore size. Tetronic 304 is a polymeric additive which acts as a pore-forming media. This additive is usually called the membrane-modifying agent (MMA). The increase of membrane pore size with blending MMA into the polymer solution is one of the easiest methods to improve membrane performance fabricated by the phase inversion method14.\n\nThe rejection capability of albumin solution of the two membranes is shown in Figure 2b (underlying data15). Albumin rejection by UF0 membrane >90% was achieved at 0.5 atm. Increasing the operating pressure resulted more albumin particles slipping into the permeate section. For all of the tested operating pressures the rejection of albumin solution by the two membranes remained above 80%.\n\nFiltration stability is the capability of a membrane to filter solute per time unit and operating pressure. This test corresponds to the durability of the membrane (lifetime). The result of stability test of the membrane on BSA filtration using UF0 and UFT304 membranes at 0.5 atm are shown in Figure 3 (underlying data16).\n\nThe permeability of the UFT304 membrane at the initial operating pressure of 0.3 atm was 90.75 L/m2.h.atm. Meanwhile, the permeability of UF0 membrane at 0.3 atm much lower, 14.38 L/m2.h.atm. The performance of filtration in these two membrane types was closely related to the morphological structure of the membrane as shown in Figure 1. Compared with the UF0 membrane, the UFT304 membrane had a larger pore structure with a higher pore density. The structure of finger-like macrovoid was also longer, and there was no sponge structure in the middle of membrane section. This difference of pore structure likely resulted in the UFT304 membrane’s higher filtration capability.\n\nAlong with longer filtration time, the permeability of these two membrane types decreased due to the fouling in membrane pore surface. However, the permeability of UFT304 membrane was still at 5.60 L/m2.h.atm after 120 minutes of filtration. In contrast the permeability of the UF0 membrane which was almost 0. The filtration profile of UFT304 membrane is more stable than UF0 membrane. The stability of filtration for UFT304 membrane was closely related to the hydrophilicity property of this membrane. UFT304 membrane was more hydrophilic with water contact angle of 62.0°, compared with UF0 membrane with water contact angle of 74.0°. The hydrophilic membrane has the capability to hold the fouling better than hydrophobic membrane17.\n\nThe capability to reduce fouling in this hydrophilic membrane is related to the interaction force between membrane surface and solution particle (protein). The content of polyethylene oxide (PEO) chain in the Tetronic additive becomes a block (steric repulsion) for protein molecules, preventing them reaching the membrane surface18. Thus, the protein molecule unlikely to attach the membrane surface. Therefore, the fouling process can be minimized.\n\n\nConclusion\n\nThe application of ultrafiltration membrane technology to separate albumin protein was conducted with two types of hollow fibre membranes, namely UF0 and UFT304. Based on the results of SEM analysis, UFT304 membrane has more finger shape structures than UF0 membrane. The results of the ultrafiltration test showed that the UFT304 membrane has better performance in terms of the stability of the flux. The rejection of the BSA solution was higher than 80% for both membranes.\n\n\nData availability\n\nFigshare: Supplementary data 1. https://doi.org/10.6084/m9.figshare.8109335.v113\n\nThis project contains the following underlying data:\n\n1. Membrane flux.csv (Raw data for membrane flux underlying Figure 2a)\n\nFigshare: Supplementary data 2. https://doi.org/10.6084/m9.figshare.8109332.v315\n\nThis project contains the following underlying data:\n\n2. Membrane Rejection.csv (Raw data for solute rejection underlying Figure 2b)\n\nFigshare: Supplementary data 3. https://doi.org/10.6084/m9.figshare.8109338.v216\n\nThis project contains the following underlying data:\n\n3. Water permeability of BSA solution.csv (Raw data for BSA filtration profile underlying Figure 3)\n\nFigshare: SEM images. https://doi.org/10.6084/m9.figshare.8215862.v212\n\nThis project contains the following underlying data:\n\nSEM images.pdf (Raw SEM images)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgement\n\nWe express our gratitude to Syiah Kuala University for the funding to conduct this research.\n\n\nReferences\n\nTini M, Jewell UR, Camenisch G, et al.: Generation and application of chicken egg-yolk antibodies. Comp Biochem Physiol A Mol Integr Physiol. 2002; 131(3): 569–74. PubMed Abstract \| Publisher Full Text\n\nShaper AG, Wannamethee SG, Whincup PH: Serum albumin and risk of stroke, coronary heart disease, and mortality: the role of cigarette smoking. J Clin Epidemiol. 2004; 57(2): 195–202. PubMed Abstract \| Publisher Full Text\n\nDatta D, Bhattacharjee S, Nath A, et al.: Separation of ovalbumin from chicken egg white using two-stage ultrafiltration technique. Sep Purif Technol. 2009; 66(2): 353–61. Publisher Full Text\n\nJana S, Purkait MK, Mohanty K: Clay supported polyvinyl acetate coated composite membrane by modified dip coating method: Application for the purification of lysozyme from chicken egg white. J Memb Sci. 2011; 382(1–2): 243–51. Publisher Full Text\n\nEmin C, Kurnia E, Katalia I, et al.: Polyarylsulfone-based blend ultrafiltration membranes with combined size and charge selectivity for protein separation. Sep Puriﬁcation Technol. 2018; 193: 127–38. Publisher Full Text\n\nIshak NF, Hashim NA, Monash P, et al.: Recent progress in the hydrophilic modiﬁcation of alumina membranes for protein separation and puriﬁcation. Ceram Int. 2017; 43(1 Part B): 915–25. Publisher Full Text\n\nMayani M, Filipe CDM, Ghosh R: Cascade ultrafiltration systems — Integrated processes for purification and concentration of lysozyme. J Memb Sci. 2010; 347(1–2): 150–8. Publisher Full Text\n\nArahman N, Mulyati S, Lubis MR, et al.: Modification of polyethersulfone hollow fiber membrane with different polymeric additives. Membr Water Treat. 2016; 7(4): 355–65. Publisher Full Text\n\nArahman N, Maimun T, Bilad MR: Fabrication of Polyethersulfone Membranes Using Nanocarbon as Additive. Int J Geomate. 2018; 15(50): 51–57. Publisher Full Text\n\nArahman N, Satria S, Razi F: The Effect of Ca and Mg Ions on the Filtration Profile of Sodium Alginate Solution in a Polyethersulfone-2-(methacryloyloxy) Ethyl Phosphorylchloline Membrane. Water. 2018; 10(9): 1207. Publisher Full Text\n\nLv C, Su Y, Wang Y, et al.: Enhanced permeation performance of cellulose acetate ultrafiltration membrane by incorporation of Pluronic F127. J Memb Sci. 2007; 294(1–2): 68–74. Publisher Full Text\n\nArahman N, Razi F, mulyati S: SEM images. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.8215862.v2\n\nArahman N, Razi F, mulyati S: Supplementary data 1. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8109335.v1\n\nLoh CH, Wang R, Shi L, et al.: Fabrication of high performance polyethersulfone UF hollow fiber membranes using amphiphilic Pluronic block copolymers as pore-forming additives. J Memb Sci. 2011; 380(1–2): 114–23. Publisher Full Text\n\nArahman N, Razi F, mulyati S: Supplementary data 2. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8109332.v3\n\nArahman N, Razi F, mulyati S: Supplementary data 3. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8109338.v2\n\nWang YQ, Su YL, Ma XL, et al.: Pluronic polymers and polyethersulfone blend membranes with improved fouling-resistant ability and ultrafiltration performance. J Memb Sci. 2006; 283(1–2): 440–7. Publisher Full Text\n\nJeon SI, Andrade JD: Protein—surface interactions in the presence of polyethylene oxide: II. Effect of protein size. J Colloid Interface Sci. 1991; 142(1): 159–66. Publisher Full Text" }	[ { "id": "51291", "date": "06 Aug 2019", "name": "Lisendra Marbelia", "expertise": [ "Reviewer Expertise Membrane Technology", "Wastewater Treatment", "Bioprocess Engineering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article tries to compare the performance of two membranes type for BSA filtration. The two membranes are PES based membranes, UF0 and UFT304. Both membranes are PES/NMP based membranes, in which UFT304 is modified with Tetronic 304.\n\nFrom the characterization in this study, it was seen that UFT304 is more hydrophilic (lower contact angle) and also more porous (fingerlike structure in SEM). Because of these properties, filtration results showed that UFT 304 has higher flux (fig 2.a) and also more sustained (fig 3) for a longer BSA filtration test, but with a slightly lower solute rejection (fig 2.b).\n\nThese above-mentioned finding and conclusions are obtained, shown and well explained in this article.\nSome suggestions and revision which should be applied to improve the quality of the paper are as follows:\n\nIn the abstract, results part...It is written that UFT304 is better since it has higher flux. But for the rejection it is only mentioned for the UF0 membranes. Better mention for the UFT304. In the introduction, last paragraph about the Tetronic modified PES membranes... please add reference here. In the method, more detailed information should be added on how the data were recorded. First, for the filtration with several pressure, how long was the filtration? Is it 10 minutes? Please write it accordingly. Also, related to the filtration experiments, it is not mentioned about the replications (in supplementary, it is shown that there are data replications). The authors should add this information in the methodology. How many replications and how the average data was calculated. Furthermore, the authors should add error bar to the graphs (2a, 2b and 3). In the result, when discussing flux and rejection of BSA, it is written that the differences between the two membranes is due to differences in pore size. The authors should differentiate between pore size and porosity. Also in the discussion, it is mentioned pore density...It would be more appropriate to refer to this as porosity (finger like structure seen on SEM), otherwise it might be confused with pore size and density on the surface which are not observed in this study.\n\nTypos:\nIntroduction, last paragraph - NMP instead of MNP Method, first paragraph - N-Methyl-2-pyrrolidone instead of N-metilpirrolidon\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5001", "date": "07 Nov 2019", "name": "Nasrul Arahman", "role": "Author Response", "response": "Comment 1In the abstract, results part...It is written that UFT304 is better since it has higher flux. But for the rejection it is only mentioned for the UF0 membranes. Better mention for the UFT304. ResponseThank You for comments. We revise the sentence as follow: Solute rejection reaches 90 and 88% for ultrafiltration of BSA solution on the operating pressure of 0.5 atm using UF0 and UFT304 membranes, respectivelyComment 2. In the introduction, last paragraph about the Tetronic modified PES membranes... please addreference here. ResponseThank You for your suggestion. We putted a reference in last paragraph of introduction about the Tetronic modified PES membranesComment 3.In the method, more detailed information should be added on how the data were recorded.first for the filtration with several pressure, how long was the filtration? Is it 10 minutes? Please write it accordingly. Also, related to the filtration experiments, it is not mentioned about the replications (in supplementary, it is shown that there are data replications). The authors should add this information in the methodology. How many replications and how the average data was calculated. Furthermore, the authors should add error bar to the graphs (2a, 2b and 3). Response: Thank You for reviewer suggestion. We have added the information in methods part. We have revised the graphs in Fig. (2a, 2b, 3) by including error bar. Comment 4.In the result, when discussing flux and rejection of BSA, it is written that the differences between the two membranes is due to differences in pore size. The authors should differentiate between pore size and porosity. Also in the discussion, it is mentioned pore density.It would be more appropriate to refer to this as porosity (finger like structure seen on SEM), otherwise it might be confused with pore size and density on the surface which are notobserved in this study. Response: The point raised by the reviewer is correct. We have revised our explanation as porosity.Comment 5.Typos:Introduction, last paragraph - NMP instead of MNPMethod, first paragraph - N-Methyl-2-pyrrolidone instead of N-metilpirrolidon Response: Thank You for the correction. We have revised the typo" } ] }, { "id": "54407", "date": "01 Nov 2019", "name": "Alagumalai Nagendran", "expertise": [ "Reviewer Expertise Ultrafiltration Membranes", "Proton Exchange Membranes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nF Razi et al. reported the performance of BSA filtration by PES-Tetronic 304 UF membranes. Authors have reported enough characterization and comprehensive discussion. It may be indexed and authors should consider the following issues:\nCommercial membranes used made it less attractive. If we synthesize, it can optimize the performance characteristics much better. For example: BSA Rejection reported in this study is 80%. If we fabricate, still higher rejection can be achieved\n\nThe role of Tetronic 304 in the modified membrane may be clearly explained in discussion section. Is it ok for long term operation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5002", "date": "07 Nov 2019", "name": "Nasrul Arahman", "role": "Author Response", "response": "Commercial membranes used made it less attractive. If we synthesize, it can optimize the performance characteristics much better. For example: BSA Rejection reported in this study is 80%. If we fabricate, still higher rejection can be achieved Response: Thank You. The statement raised by the reviewer is correct that the synthesized membrane can optimize the characteristic of membrane and its performance on filtration The role of Tetronic 304 in the modified membrane may be clearly explained in discussion section. Is it ok for long term operation? Response: thank you for the valuable comments. In this study we examined for 2 hours filtration. More examination for long term operation is planned in our future study." } ] } ]	1	https://f1000research.com/articles/8-953
https://f1000research.com/articles/8-1856/v1	05 Nov 19	{ "type": "Correspondence", "title": "iCOMPARE, what value does it add to resident duty-hour discussions?", "authors": [ "Zachary H. Hopkins", "Aaron M. Secrest", "Aaron M. Secrest" ], "abstract": "Discussions regarding resident duty-hour restrictions have been ongoing and heated. One influential argument for restrictions has been patient safety. Two trials, FIRST and iCOMPARE, were performed to investigate this relationship with surgical and medicine training, respectively. As the authors are approaching this discussion from a medicine-based perspective, iCOMPARE will serve as the primary basis of our discussion. Results from the iCOMPARE trial comparing flexible (28-hour shifts allowed) to the original 2011 ACGME shift requirements (maximum 16 hours) were recently published in the New England Journal of Medicine. This non-inferiority trial used 30-day post-hospitalization mortality as its primary endpoint. Results met qualifications for non-inferiority, and ACGME policy was changed to allow for 28-hour shifts for medicine residents. iCOMPARE results were highly lauded and used as primary justification for extending resident duty hours. Despite this sweeping impact, few have critically evaluated what this study actually adds to the literature. Herein, we argue that serious questions regarding trial design are apparent. Most importantly, the non-inferiority margins chosen were large, and represent an ambiguous marker of resident performance. Additionally, we question the lack of both patient consenting and direct patient-reported or patient-centered outcomes within the hospital stay. As more discussion arises in the medical literature surrounding patient-reported outcomes and shared decision making, we argue that the results of iCOMPARE disregarded the patient perspective or meaningful patient outcomes in an attempt to maintain status quo. Lastly, we discuss how iCOMPARE missed the broader question of actual duty-hour restrictions, and some practical methods already in practice at some programs, which may more directly balance resident work hours with patient care and resident learning.", "keywords": [ "General Medial Education", "Resident Work Hours", "Resident Duty Hours", "Resident Wellness", "Medical Education", "iCOMPARE" ], "content": "\n\nMr. C was friendly and rapport came quickly. He ultimately needed thoracentesis and during the ensuing discussion he perfunctorily asked, “How long have you been on your shift?” After replying, “12 hours,” he responded, “I understand the necessity, but I want someone performing their best for this procedure. Can you wait until morning?” Fortunately, his case could be safely deferred until morning. This was neither the first, nor the last, time during my training a patient asked this question.\n\nRecently, the New England Journal of Medicine published the final primary endpoint analysis for iCOMPARE with an accompanying editorial1,2. This trial sought to evaluate the impact of flexible (program directed shift lengths up to 28 hours) versus 16-hour capped shifts on 30-day post-hospitalization patient mortality. As the only trial investigating duty-hour effects on internal medicine patients, its impact is large. Indeed, in the ACGME’s new duty-hour guidelines, results from the iCOMPARE and FIRST (similar study from surgical programs), are cited as a major justification for this change3. However, as with any practice changing study, critical appraisal of its results and application is required.\n\nFirst, iCOMPARE lacked informed consent from either patients or residents. This is concerning with a primary endpoint of patient mortality4. One participant institution’s IRB application stated that they were not involved in human research because the residency program itself was the research subject—this claim seems implausible4. Patients are both interested in, and concerned by, the length of their doctors’ shifts5. Indeed, in one study, a significant number of patients stated they would like to be informed of any clinician who had worked over 12 hours6. Patients and residents deserve to be informed of factors affecting their care, particularly in a trial with the primary endpoint involving their 30-day mortality. For residents, this is especially concerning given their vulnerable workforce position. A 2004 lawsuit against the National Resident Matching Program asserted that residents were “forced to participate in a system that ensures they work long hours for low wages.”7 However, after a strong lobbying effort, national legislation exempted the residency match process from anti-trust laws. In this setting the potential for exploitation is high, and policies should err on the side of fair work conditions.\n\nThird, the 1% non-inferiority margin utilized suggests that 2,646 additional deaths is an acceptable justification for longer shifts. Extrapolating this margin to the U.S. population, nearly 80,000 deaths annually would be considered “non-inferior.” Understandably, limitations in recruiting and study design apply, especially for non-inferiority. However, we also question the appropriateness of a non-inferiority design for this question. A proposed ethical prerequisite for using non-inferiority analysis is that an experimental therapy “must have known advantages such as reduced cost, greater convenience, or fewer side effects to justify the randomization of patients to a therapy with unknown efficacy.”8–11 Long duty-hours have been linked to several resident, patient, and community safety concerns12–15. Likewise, patients do not appear to feel comfortable with physicians who worked long hours5,6. With these concerns, we argue that iCOMPARE should have demonstrated superiority of unrestricted shift lengths. Indeed, one oft-used argument for extended shift hours is fewer handoffs and improved continuity. Inherent in the design appears to be a belief of superiority otherwise why put people through it? Furthermore, the hours worked between the two groups were the same, thus it is wholly expected that no difference in endpoint be observed16. Why not choose a restricted hour work week, as is done in Europe, for the comparator?17 Or, if extended lengths of shift are believed to be better for patients, why not use this shift structure as the comparator? Lastly, we suspect superiority in patient clinical endpoints would be the only results acceptable to patients for continuing a training system of which they do not approve.\n\nFinally, the primary endpoint of iCOMPARE is inadequately justified. Residency training should have sufficiently robust supervision that shift length has minimal effect on 30-day mortality. Orders made by an intern pass through supervising residents, attendings, pharmacists, and nurses. While errors can and do get through, post-hospitalization mortality is buffered from the source of errors in question. This is supported by one study showing an increased length of hospital stay and increased number of ICU admissions with long resident work hours, but a non-significant change in hospital readmissions and within-hospital mortality18. Likewise, we suspect 30-day mortality is not the only issue for which patients are concerned. Quantifying errors and near-misses made within the hospital as well as the patient experience could more directly evaluate causal links with resident performance. Additionally, while markers of resident performance (testing performance, wakefulness, etc.) were evaluated, we argue that with work hours being equal and minimal implementation of 28 hour shifts occurring between the two groups, no meaningful comparison between these groups can be derived19,20. With no difference in hours worked and a large non-inferiority margin of an ill-defined primary endpoint, we argue that little can be gained in regards to the preceeding data.\n\nWhile the idea of flexible shift structure is sensical as policy, it severely diluted the applicability and generalizability of the results. Currently, policy extends further than data. A program could, in theory, make a majority of its rotations utilize a 28-hour shift structure and this would be within policy. However, the safety of this structure is untested, as this was not directly evaluated in iCOMPARE. Further, even if the results were to be taken at face value, the reciprocity of the results is seemingly ignored. This trial, as a non-inferiority trial, demonstrated that capped shifts, with the changes in care they bring, do not worsen patient mortality. However, what if even these are too long? Perhaps both capped and flexible structures as they currently stand with 80-hour work week restrictions are inferior to a more restricted system? We posit that this is a more pressing and sensible question.\n\nWe believe that the results of iCOMPARE are critically lacking—we can and must do better (Table 1). We do not argue in favor of 2011 ACGME requirements nor the new adjustments. Rather, we suggest that iCOMPARE itself was constructed to test an artificial argument, and did so poorly. Thoughtfully tested data is necessary to further duty hour discussions and guide further policy refinement as well as an openness to consider new paradigms. Let us work to cultivate a group of compassionate, empathetic, and well-trained physicians; armed with not only with technical prowess, but the emotional reserve to respond to and alleviate patient suffering. In contrast to this goal, the iCOMPARE study is sadly a self-fulfilling prophecy—a superficial justification to continue the status quo.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe would like to thank Dr. Vinay Prasad for his helpful editorial comments on this manuscript.\n\n\nReferences\n\nRosenbaum L, Lamas D: Eyes Wide Open — Examining the Data on Duty-Hour Reform. N Engl J Med. 2019; 380(10): 969–970. PubMed Abstract \| Publisher Full Text\n\nSilber JH, Bellini LM, Shea JA, et al.: Patient Safety Outcomes under Flexible and Standard Resident Duty-Hour Rules. N Engl J Med. 2019; 380(10): 905–914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMallory K, Brigham T, Nasca T: ACGME’s 2017 Revision of Common Program Requirements. Patient Safety Network. 2017; Published August 2017. Accessed August 12: 2019. Reference Source\n\nShepherd L, Macklin R: Erosion of informed consent in U.S. research. Bioethics. 2019; 33(1): 4–12. PubMed Abstract \| Publisher Full Text\n\nBlum AB, Raiszadeh F, Shea S, et al.: US public opinion regarding proposed limits on resident physician work hours. BMC Med. 2010; 8: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrolet BC, Hyman CH, Ghaderi KF, et al.: Hospitalized Patients’ Perceptions of Resident Fatigue, Duty Hours, and Continuity of Care. J Grad Med Educ. 2014; 6(4): 658–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson S: Antitrust Lawsuit Over Medical Residency System is Dismissed. The New York Times. 2004; A00016. Reference Source\n\nAberegg SK, Hersh AM, Samore MH: Do non-inferiority trials of reduced intensity therapies show reduced effects? A descriptive analysis. BMJ Open. 2018; 8(3): e019494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAberegg SK, Hersh AM, Samore MH: Empirical Consequences of Current Recommendations for the Design and Interpretation of Noninferiority Trials. J Gen Intern Med. 2018; 33(1): 88–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarattini S, Bertele’ V: Non-inferiority trials are unethical because they disregard patients’ interests. The Lancet. 2007; 370(9602): 1875–7. PubMed Abstract \| Publisher Full Text\n\nPrasad V: Non-Inferiority Trials in Medicine: Practice Changing or a Self-Fulfilling Prophecy? J Gen Intern Med. 2018; 33(1): 3–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSen S, Kranzler HR, Krystal JH, et al.: A prospective cohort study investigating factors associated with depression during medical internship. Arch Gen Psychiatry. 2010; 67(6): 557–565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyas NT, Barger LK, Cade BE, et al.: Extended work duration and the risk of self-reported percutaneous injuries in interns. JAMA. 2006; 296(9): 1055–1062. PubMed Abstract \| Publisher Full Text\n\nBarger LK, Cade BE, Ayas NT, et al.: Extended work shifts and the risk of motor vehicle crashes among interns. N Engl J Med. 2005; 352(2): 125–134. PubMed Abstract \| Publisher Full Text\n\nWare JC, Risser MR, Manser T, et al.: Medical resident driving simulator performance following a night on call. Behav Sleep Med. 2006; 4(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nSchumi J, Wittes JT: Through the looking glass: understanding non-inferiority. Trials. 2011; 12(1): 106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodriguezJareno MC, Demou E, Vargas-Prada S, et al.: European Working Time Directive and doctors’ health: a systematic review of the available epidemiological evidence. BMJ Open. 2014; 4(7): e004916. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOuyang D, Chen JH, Krishnan G, et al.: Patient Outcomes when Housestaff Exceed 80 Hours per Week. Am J Medicine. 2016; 129(9): 993–999.e1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesai SV, Asch DA, Bellini LM, et al.: Education Outcomes in a Duty-Hour Flexibility Trial in Internal Medicine. N Eng J Med. 2018; 378(16): 1494–1508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasner M, Asch DA, Shea JA, et al.: Sleep and Alertness in a Duty-Hour Flexibility Trial in Internal Medicine. N Engl J Med. 2019; 380(10): 915–923. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88690", "date": "05 Jul 2021", "name": "Annina Ropponen", "expertise": [ "Reviewer Expertise Occupational epidemiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this commentary have chosen very dramatic words, which I would prefer being more moderate/humble. Studying working hours at hospitals is always tricky and especially if the data is gathered via self-reports. Only until recently, few studies exist utilizing objective, pay-roll based working hours. Furthermore, besides very long working hours among physicians or residents, also combinations of work shifts may cause difficulties for recovery and sleep. Hence the studies should not only focus on the length of working hours, but also on shift intervals between work shifts. What comes to the informal consents, the procedures may vary. If only register data is utilized, that could be done even without a consent. Hence I would like to suggest to put more emphasis on discussing the design and analyses, than on the permissions for the study.\n\nIs the rationale for commenting on the previous publication clearly described? Partly\n\nAre any opinions stated well-argued, clear and cogent? Partly\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1856
https://f1000research.com/articles/8-564/v1	26 Apr 19	{ "type": "Research Article", "title": "Human aldose reductase unfolds through an intermediate.", "authors": [ "Gurprit Sekhon", "Ranvir Singh", "Gurprit Sekhon" ], "abstract": "Background: Human aldose reductase (hAR) converts glucose to sorbitol under hyperglycemic conditions. Aldose reductase is first and rate limiting enzyme of polyol pathway. Under hyperglycemia, increased flux of glucose through this pathway has been implicated in development of secondary complication in diabetes. Due to this clinical implication, aldose reductase attracted considerable attention from drug discovery perspective. In spite of extensive characterization of the biochemical and structural context, little is known about the unfolding behavior of aldose reductase. This study reports equilibrium unfolding studies of human aldose reductase. Methods: We carried out thermal and chemical induced equilibrium unfolding studies of human aldose reductase monitored by circular dichroism and tryptophan and ANS fluorescence spectroscopy. Results: Thermal unfolding studies present a classical picture of two state unfolding from native to unfolded state. The data was used to derive thermodynamic parameters and study thermostability of aldose reductase. Urea and GuHCl induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2 M of urea and GuHCl, respectively. Thermodynamic parameters from chemical induced unfolding are in agreement with those obtained from thermal unfolding. Conclusion: This study revealed that aldose reductase unfolds from native to unfolded state via an intermediate. Assessment of thermodynamic stability of native, intermediate and unfolded state shows that three states are separated by significant energy barriers that ensure cooperativity of unfolding. As hAR functions in cells which are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under physiological state.", "keywords": [ "Aldose reductase", "Protein unfolding", "Folding intermediate", "Cooperativity", "Tryptophan fluorescence", "ANS fluorescence", "Thermal unfolding" ], "content": "Abbreviations\n\nGuHCl, guanidine hydrochloride; TCEP, (tris(2-carboxyethyl)phosphine); ANS, 8-anilino-1-naphthalenesulfonic acid ammonium salt; IPTG, isopropyl β-D-1-thiogalactopyranoside; Trp, tryptophan.\n\n\nIntroduction\n\nHuman aldose reductase (hAR) (EC 1.1.1.21) is an NADPH-dependent oxidoreductase that belongs to super family of aldo-keto reductases1. Being the first and rate limiting enzyme of polyol pathway, hAR converts glucose to sorbitol2. Under hyperglycemic conditions, the polyol pathway is up-regulated and a significant proportion of glucose gets fluxed through this pathway, which leads to accumulation of sorbitol, consumption of NADPH and redox imbalance of NADPH/NADP+ ratio. All these factors have been linked with various tissue based pathologies associated with secondary complications of diabetes mellitus3. Due to its clinical importance, hAR has been widely studied from the perspective of development of potent inhibitors so as to prevent or delay the onset of secondary diabetic complications4.\n\nExtensive information is available in the literature about the structure and function of hAR, particularly related to active site of hAR from ultra-high-resolution crystal structures with a number of potential inhibitors5, flexibility in the hAR binding site pocket6 and the thermodynamics of closing/opening of the specificity pocket within binding site pocket of hAR7. Nevertheless, there is little investigation related to the folding/unfolding mechanism of hAR. Understanding the capability of a polypeptide chain to spontaneously fold into a compact tertiary structure on biological relevant time scale is a long-standing challenge in protein science8.\n\nUnder physiological conditions, protein structure fluctuates among different native conformations separated by close free energy barriers9. Since hAR activity leads to sorbitol accumulation, leading to osmotic stress; it seems to function under stress conditions which might perturb its native conformation ensemble. Here we report on thermally and chemically induced unfolding studies of hAR. Thermal unfolding revealed simple two-state transition whereas chemical induced unfolding led us to discover an intermediate state during hAR unfolding.\n\n\nMethods\n\nAll chemicals were reagent grade and purchased from Sigma-Aldrich.\n\nThe hAR cDNA cloned into expression vector pET-15b (Novagen) was a kind gift from Dr. Alberto Podjarny (Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, France). The plasmid, coding for a hexahistidine-tagged protein, was expressed into E. coli strain BL21 (DE3) (Novagen). The cells with recombinant plasmid were grown with 100 μM ampicillin at 37°C to an OD600 nm value of 0.7 and protein expression was induced by adding 1 mM IPTG. Cells were grown for further 3 hours at 37°C. All further operations were carried out at 4°C unless otherwise stated. Cells were centrifuged, re-suspended and lysed by sonication. A Ni-NTA affinity column (GE Healthcare) was used for protein purification. The material used for stationary phase for the column was Ni-Sepharose and the flow rate of column was adjusted to 0.5 ml min-1. Imidazole and other salts were removed by repeated dialysis in 50 mM potassium phosphate, pH-7 buffer containing 50 mM NaCl. Protein concentration was estimated using the molar extinction coefficient and absorbance reading at 280 nm. The histidine tag from recombinant protein was removed by thrombin (4 units of thrombin per mg of recombinant protein at room temperature for 3 hours). Cleaved protein was passed through the Ni-NTA column, and purified protein without tag was collected as flow-through. Enzyme activity was checked as per standard assay10. Homogeneity and molecular weight of hAR with and without histidine tags was analyzed under denaturing conditions on 15% SDS-PAGE. Purified hAR was stored at -20°C for further studies.\n\nThermal unfolding was carried out at a final concentration of 2.8 µM protein in 50 mM potassium phosphate buffer, pH 7.0 containing 50 mM NaCl and 0.1mM TCEP. Transition between 20–70 °C was followed using a far-UV circular dichroism (CD) signal at 222 nm by using 0.1 cm path length cuvette at sampling rate of 1.0 °C min-1 in a Jasco J-810 spectropolarimeter. Buffer blank was duly subtracted before reporting the change in ellipticity (millidegree) at 222 nm.\n\nSamples with 1.4 μM protein concentration were prepared in phosphate buffer (described earlier) containing different concentrations of GuHCl/urea. Samples were incubated for 12 hours to reach equilibrium at 25°C after which no change in signal occurred either in fluorescence or CD spectra. Trp fluorescence (excitation at 295 nm and emission recorded between 300 nm to 400 nm) and ANS fluorescence (excitation at 370 nm and emission recorded between 400 nm to 600 nm) measurements were performed using a Hitachi F-7000 fluorescence spectrophotometer for GuHCl samples and Jasco J-815 spectropolarimeter for urea samples. Far-UV CD measurements were performed using Jasco J-810 spectropolarimeter for GuHCl samples and Jasco J-815 spectropolarimeter for urea samples. Quartz cuvette of 1 cm and 0.5 cm path length were used for fluorescence and CD measurements respectively. All measurements were done at 25 °C. Spectra were reported as ellipticity (millidegree) after baseline correction.\n\nΔCp value for unfolding of hAR was calculated from change in accessible surface area (ΔASA) according to Equation 111\n\nΔCp=−251+0.19×[ΔASA](Equation 1)\n\nProtSA web server was used to calculate change in accessible surface area from native to unfolded conformation of hAR12.\n\nGraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla, California) was used for analysis of thermal and chemical induced unfolding data on the basis of two and three state model respectively as described in following sections.\n\nData was fitted by least square analysis to Equation 213.\n\nY=(An+bN×T)+(Au+bU×T)×exp((ΔHgR)×(1Tg−1T))1+exp⁡((ΔHgR)×(1Tg−1T))(Equation 2)\n\nWhere An and Au are native and unfolded state baseline intercepts respectively and bN and bU are native and unfolded baseline slopes respectively. ΔHm is enthalpy change at melting temperature (Tg). T is absolute temperature and R is the gas constant.\n\nSignal for native (YN = An + bN × T) and unfolded baseline (YU = Au + bU × T) for every point in transition region was calculated from Equation 2. If Y is signal for a particular point in transition region, then fraction of unfolded protein (Fu) at this point is given by Equation 3.\n\nFu=Yn−YYn−Yu(Equation 3)\n\nThe equilibrium constant (k) can be calculated from relative population of species using Equation 4.\n\nk=Fu1−Fu(Equation 4)\n\nΔG can be calculated as a function of temperature using Equation 5.\n\nΔG=−RT ln k(Equation 5)\n\nThe thermal stability curve of hAR was constructed on the basis of Equation 6–814.\n\nΔHT=ΔHg+ΔCp(T−Tg)(Equation 6)\n\nΔST=ΔHgTg+ΔCp×LN(TTg)(Equation 7)\n\nΔGs=ΔHg×(1−TTg)+ΔCp×(T−Tg−(T×LN(TTg)))(Equation 8)\n\nWhere ΔHT and ΔST are enthalpy and entropy change respectively at temperature T with reference to Tg. Th, Ts and Tg are the temperatures at which ∆H, ∆S, and ∆G are zero respectively. ∆Gs is the stabilization free energy of the native state relative to the unfolded state.\n\nData was fitted by least square analysis to Equation 913.\n\nY=(An+bN×[D])+(Ai+bI×[D])×exp⁡(−(ΔG(N−I)RT−m(N−I)×[D]RT))+(Au+bU×[D])×exp⁡(−(ΔG(N−I)R×T−m(N−I)∗ [D]R×T))×exp⁡(−(ΔG(I−U)R×T−m(I−U)×[D]R×T))1+exp⁡(−ΔG(N−I)R×T−m(N−I)×[D]R×T)−exp⁡(−(ΔG(N−I)R×T−m(N−I)×[D]R×T))×exp⁡(−(ΔG(I−U)R×T−m(I−U)×[D]R×T))(Equation 9)\n\nWhere An, Au, and Ai are the native, unfolded and intermediate baseline intercepts, respectively, and bN, bU and bI are the native, unfolded and intermediate baseline slopes, respectively. [D] is denaturant concentration in molar. m(N-I) and m(I-U) are denaturant gradient for native to intermediate and intermediate to unfolded state respectively. ∆G(N-I) and ∆G(I-U) are stabilization free energy of intermediate state relative to native and unfolded state respectively.\n\n\nResults\n\nChange in ellipticity at 222 nm fitted well on the basis of a two-state model (Figure 1A). This analysis gave values for ΔHg and Tg which along with ΔCp value calculated from Equation 1 were used for non-linear regression of transition region (±5 kJ mol-1) to Equation 8 (Figure 1C). Values of ΔH and ΔS were calculated over extended range of temperature by using Equation 7 and Equation 8, respectively (Figure 1D). Thermal stability curve is extrapolation of transition region assuming constant ΔCp during unfolding transition (Figure 1E). The relationship between Ts, Th and ∆G (Ts – Th = ∆Gs/∆Cp) is presented in Figure 1F. Thermodynamic parameters obtained from analysis of thermal unfolding data are listed in Table 1. All raw data are available as Underlying data15.\n\n(A) Change in ellipticity at 222 nm plotted as a function of temperature. (B) Fraction of protein folded (green dots) and unfolded (red dots) plotted against temperature. (C) Portion of transition curve used in van't Hoff analysis. (D) Plots of ΔH and ΔS as function of temperature. (E) thermal stability curve of hAR. (F) Triangular relationship among Th, Ts and ∆Gs. Explanation for Tg, Tg’, Th and Ts is given in text. Dashed lines are extrapolations. Solid line represents fit to unfolding transition, filled symbols represent data points from unfolding experiments and red symbols represent outliers.\n\nThere are six Trp residues in hAR, out of which four are part of the hydrophobic active site pocket in the core of the β-barrel and two are buried in alpha helices surrounding the barrel. Their fluorescence provided global signal of change in tertiary structure. ANS has been extensively used as a probe for non-native, partially unfolded conformations of protein. The binding of ANS to hydrophobic regions results in a significant enhancement of ANS fluorescence and a pronounced blue-shift of the λmax16.\n\nFluorescence emission profiles of hAR equilibrated with different concentrations of denaturants are presented in Figure 2 (Figure 2A and Figure 3A for urea and GuHCl, respectively). A plot of λmax against denaturant concentration indicated cooperative transition from native to unfolded state (Figure 2B and Figure 3B for urea and GuHCl, respectively). In case of ANS fluorescence, significant blue-shift of around 20 nm and 10 nm from the native to intermediate state was observed for urea and GuHCl, respectively (Figure 2B2 and Figure 3B2 for urea and GuHCl, respectively).\n\n(A1) Trp fluorescence scans and (A2) ANS fluorescence scans for all the samples. (B1) λmax (Trp fluorescene) and (B2) λmax (ANS fluorescence) against [urea]. (C1) Imax (Trp fluorescenc) and (C2) Imax (ANS fluorescence) against [urea]. (D1) I295/314 (Trp fluorescence) and (D2) I370/480 (ANS fluorescence) against [urea]. (E1) Trp fluorescence and (E2) ANS fluorescence of samples in native (green), intermediate (cyan) and unfolded state (red). Solid lines represent fit to the unfolding transitions, filled symbols represent data points from unfolding experiments, red symbols represent outliers.\n\nPlot of Imax against denaturant concentration indicated presence of an intermediate during unfolding transition (Figure 2C and Figure 3C for urea and GuHCl, respectively). Imax in case of ANS fluorescence fits satisfactorily on the basis of three-state model (Figure 2C2 and Figure 3C2 for urea and GuHCl, respectively). For both urea and GuHCl induced unfolding, Trp fluorescence emission intensity at 314 nm fits satisfactorily to three-state model. In case of ANS fluorescence, both Imax and fluorescence emission intensity at 480 nm fit equally well on the basis of three-state model. Thus, Trp fluorescence emission intensity at 314 nm and ANS fluorescence emission intensity at 480 nm were analyzed on the basis of three-state model to evaluate thermodynamic stability of hAR (Figure 2D and Figure 3D for urea and GuHCl, respectively). The thermodynamic parameters obtained from fitting are listed in Table 1. Trp and ANS fluorescence clearly demonstrate presence of an intermediate state populated at 3.5-4.0 M and 0.7-2 M urea and GuHCl concentration respectively, apart from the native and unfolded states (Figure 2E and Figure 3E for urea and GuHCl, respectively).\n\n(A1) Trp fluorescence scans and (A2) ANS fluorescence scans. (B1) λmax (Trp fluorescence) and (B2) λmax (ANS fluorescence) against [GuHCl]. (C1) Imax (Trp fluorescence) and (C2) Imax (ANS fluorescence) against [GuHCl]. (D1) I295/314 (Trp fluorescence) and (D2) I370/480 (ANS fluorescence) against [GuHCl]. (E1) Trp fluorescence and (E2) ANS fluorescence of samples in native (green), intermediate (cyan) and unfolded state (red). Solid lines represent fit to the unfolding transitions, filled symbols represent data points from unfolding experiments, red symbols represent outliers.\n\nUnfolding profiles of hAR equilibrated at different denaturants concentrations in far-UV CD are presented in Figure 4A1 and 4A2 for GuHCl and urea, respectively. Thermodynamic stability of hAR was determined on the basis of three state model by plotting change in ellipticity at 219/222 nm as a function of denaturant concentration (Figure 4B1 and 4B2 for GuHCl and urea, respectively). The transition determined by far-UV CD detected intermediate state at similar concentrations of denaturant as interrogated by fluorescence spectroscopy. All three states can be clearly distinguished from Far-UV CD profiles (Figure 4C1 and 4C2 for GuHCl and urea, respectively). Thermodynamic parameters derived from far-UV CD data are listed in Table 1.\n\n(A1) far-UV CD scans recorded for GuHCl and (A2) far-UV CD scans recorded for urea for all the samples. (B1) change in ellipticity at 219 nm against [GuHCl] and (B2) change in ellipticity at 221.8 nm against [urea]. (C) CD spectra of samples in native (green), intermediate (cyan) and unfolded state (red) for (C1) GuHCl and (C2) urea, respectively. Solid line represents fit to unfolding transitions, filled symbols represent data points from unfolding experiments and red symbols represent outliers in data fitting.\n\n\nDiscussion\n\nThe intermediate state with enhanced ANS fluorescence and significant blue shift of λmax pointed to an intermediate state with some sort of ‘molten’ nature during hAR unfolding. Far-UV CD studies strongly suggest that the intermediate state retains significant secondary structure during urea- and GuHCl-induced unfolding.\n\nDuring chemical induced unfolding hAR unfolds through an intermediate state which is absent during thermal unfolding. Moderate concentration of denaturant is known to stabilize native or intermediate state17. Absence of such stabilizing agent may be the reason that the intermediate state was not detected during thermal unfolding.\n\nIn all three probes used in studying unfolding, value of ΔG(N-I) obtained is ~30 kJ mol-1 and ~15 kJ mol-1for urea- and GuHCl-induced unfolding respectively while a ∆Gs of ~70 kJ mol-1 is almost same for both denaturants (Table 1). Thus, while urea seems to stabilize the native state with respect to the intermediate state, GuHCl seems to stabilize the intermediate state with respect to the native state.\n\nIt is known that small molecules change the free energy landscape of protein upon binding by selectively stabilizing native or intermediate/unfolded state18. Difference in value of ∆Gs obtained from thermal and chemical induced unfolding is ~20 kJ mol-1 (Table 1), which is most likely due to free energy of stabilization and destabilization by urea and GuHCl, respectively.\n\nValues of ΔG(N-I) obtained from analysis of ANS fluorescence data are 16.48 and 28.61 kJ mol-1 for GuHCl and urea, respectively (Table 1), which indicate that intermediate state is not separated by a steep energy barrier from native state. Values for ΔG(I-U) obtained from ANS fluorescence are 57.26 and 38.6 kJ mol-1 for GuHCl and urea respectively (Table 1), which indicate that the intermediate state is separated from unfolded state by a high energy barrier. Thus, the intermediate state of hAR is close to its native state which makes it functionally more relevant.\n\nIn summary, equilibrium unfolding studies of hAR have led us to discover that hAR unfolds through an intermediate state, which is close to native state, and might have physiological relevance under hyperglycemic conditions in diabetes.\n\n\nData availability\n\nFigshare: data_f1000_hAR_unfolding.zip. https://doi.org/10.6084/m9.figshare.8001998.v115.\n\nThis project contains raw data for chemically and thermally induced unfolding studies on human aldose reductase.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe acknowledge Dr. Alberto Podjarny (Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, France.) for hAR plasmid as a kind gift, Dr. Yogendra Sharma, at CSIR-CCMB, Hyderabad and Chairperson, Department of Biotechnology, Panjab University, for helping us in spectroscopic data collection. Gurprit acknowledges research fellowship from UGC, Govt. Of India (UGC science JRF).\n\n\nReferences\n\nPenning TM: The aldo-keto reductases (AKRs): Overview. Chem Biol Interact. 2015; 234: 236–246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrownlee M: The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005; 54(6): 1615–1625. PubMed Abstract \| Publisher Full Text\n\nYan LJ: Redox imbalance stress in diabetes mellitus: Role of the polyol pathway. Animal Model Exp Med. 2018; 1(1): 7–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaccari R, Ottanà R: Targeting aldose reductase for the treatment of diabetes complications and inflammatory diseases: new insights and future directions. J Med Chem. 2015; 58(5): 2047–2067. PubMed Abstract \| Publisher Full Text\n\nHoward EI, Sanishvili R, Cachau RE, et al.: Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A. Proteins. 2004; 55(4): 792–804. PubMed Abstract \| Publisher Full Text\n\nSotriffer CA, Krämer O, Klebe G: Probing flexibility and “induced-fit” phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations. Proteins. 2004; 56(1): 52–66. PubMed Abstract \| Publisher Full Text\n\nRechlin C, Scheer F, Terwesten F, et al.: Price for Opening the Transient Specificity Pocket in Human Aldose Reductase upon Ligand Binding: Structural, Thermodynamic, Kinetic, and Computational Analysis. ACS Chem Biol. 2017; 12(5): 1397–1415. PubMed Abstract \| Publisher Full Text\n\nDill KA, MacCallum JL: The protein-folding problem, 50 years on. Science. 2012; 338(6110): 1042–6. PubMed Abstract \| Publisher Full Text\n\nCremades N, Sancho J, Freire E: The native-state ensemble of proteins provides clues for folding, misfolding and function. Trends Biochem Sci. 2006; 31(9): 494–496. PubMed Abstract \| Publisher Full Text\n\nBalendiran GK, Sawaya MR, Schwarz FP, et al.: The role of Cys-298 in aldose reductase function. J Biol Chem. 2011; 286(8): 6336–6344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMyers JK, Pac CN, Scholtz JM: Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995; 4(10): 2138–2148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEstrada J, Bernadó P, Blackledge M, et al.: ProtSA: a web application for calculating sequence specific protein solvent accessibilities in the unfolded ensemble. BMC Bioinformatics. 2009; 10: 104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrimsley GR, Trevino SR, Thurlkill RL, et al.: Determining the conformational stability of a protein from urea and thermal unfolding curves. Curr Protoc Protein Sci. 2013; Chapter 28: Unit28.4. PubMed Abstract \| Publisher Full Text\n\nBecktel WJ, Schellman JA: Protein stability curves. Biopolymers. 1987; 26(11): 1859–1877. PubMed Abstract \| Publisher Full Text\n\nSekhon G, Singh R: data_f1000_hAR_unfolding.zip. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8001998.v1\n\nCattoni DI, Kaufman SB, González Flecha FL: Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins. Biochim Biophys Acta. 2009; 1794(11): 1700–1708. PubMed Abstract \| Publisher Full Text\n\nBhuyan AK: Protein stabilization by urea and guanidine hydrochloride. Biochemistry. 2002; 41(45): 13386–13394. PubMed Abstract \| Publisher Full Text\n\nStreet TO, Bolen DW, Rose GD: A molecular mechanism for osmolyte-induced protein stability. Proc Natl Acad Sci U S A. 2006; 103(38): 13997–4002. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "50315", "date": "24 Jun 2019", "name": "Jonathan W. Mueller", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrs Sekhon and Singh have presented an in-vitro study \"Human aldose reductase unfolds through an intermediate\" to F1000Research to be considered for indexing. Foremost, the authors should describe the newly described intermediate more with respect to its potential physiological relevance to stress the relevance of this study.\nPlease remove \"ultra\" from any description for protein crystal structures, but add the actual resolution of that structure, 0.66 A from Ref. 5, and explain what additional features were derived from these, compared to average crystal structures.\n\"Understanding the capability of a polypeptide...\" - this sentence only has limited linkage to the sentence beforehand and should be deleted. Instead, novel concepts of protein folding and stability should be briefly covered.1, 2\nFigure 1:\nA) it is not clear what the blue and red dots mean. B) Please do not use green and red as colors.\n\nFigure 2:\nA+B) Please label the different graphs within the diagrams.\n\nThe manuscript needs careful copy-editing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "50821", "date": "08 Jul 2019", "name": "Vladimir Uversky", "expertise": [ "Reviewer Expertise Protein physics", "protein folding", "partially folded proteins", "folding intermediates", "protein misfolding' protein aggregation", "conformational diseases", "intrinsically disordered proteins" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Gurprit Sekhon and Ranvir Singh describe the results of the equilibrium unfolding of human aldose reductase. The authors conclude that urea- and GuHCL-induced unfolding of this protein is characterized by the presence of partially folded intermediate. This is an interesting study with some potential. However, additional experiments are needed to provide more evidence for the existence of an intermediate state and also to provide better description of the structural properties of this protein.\n\nThe authors are encouraged to add near-UV CD spectroscopy to the arsenal of techniques utilized in their study. This will give very important information on the effect of denaturants on the tertiary structure of a protein. In fact, combined use of near- and far-UV spectroscopy is the accepted practice in studies on the conformational stability of proteins.\n\nQuality of the reported far-UV CD spectra is very low. Spectra are very noisy and their utilization for the analysis of the effects of urea and GuHCl on secondary structure of human aldose reductase is questionable. The authors have to change settings to generate more reliable and less noisy spectra. Probably, protein concentrations should be increased too.\n\nFar-UV CD spectra of completely unfolded forms induced by high concentrations of urea and GuHCl are very different, suggesting that these unfolded forms are not similar. Why?\n\nCompactness of an intermediate state should be evaluated. This can be done by a whole host of hydrodynamic techniques.\n\nThe authors should make sure that the protein does not aggregate at the conditions promoting formation of a partially unfolded intermediate. This can be done by simple light scattering experiments.\n\nData shown in Figure 3 suggest that very low GuHCl concentrations cause very noticeable changes in some of the analyzed parameters. Similar behavior was described previously1 and was attributed to the aggregation of partially unfolded species. This phenomenon should be discussed.\n\nThe presence of partially folded intermediates can be visualized using \"phase diagram\" method described in previous work by Kuznetsova et al.2 The authors are encouraged to use this approach for the analysis of their data.\n\nThe authors are encouraged to reconsider the use of terms \"denatured/denaturation\" and \"unfolded/unfolding\". Typically, unfolding is attributed to the process resulting in the formation of coil-like conformation, whereas denaturation is referred to the elimination of functional tertiary structure. Obviously, these terms are not equivalent - molten globule is denatured, but is not unfolded. Furthermore, although temperature increase typically causes melting of a protein tertiary structure, temperature-denatured species are often rather compact and preserve high levels of secondary structure.\n\nThe manuscript contains some linguistic issues and errors and definitely needs editing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "51586", "date": "01 Aug 2019", "name": "Yong-Bin Yan", "expertise": [ "Reviewer Expertise Biophysics", "biochemistry", "cell biology", "enzymology", "spectroscopy", "protein folding", "misfolding and aggregation", "aggregation diseases", "proteostasis", "cataract", "mRNA decay", "deadenylation", "translation regulation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this research, the authors studied the equilibrium unfolding pathway of human aldose reductase (hAR) by spectroscopic methods. Although this pure unfolding study may provide information of how hAR unfolds, the underlying physiological relevance is unclear. Furthermore, the present study needs to be improved in the quality of the data and interpretation.\n\nDuring my preparation of the comments, Professor Uversky and Professor Mueller have published their excellent comments and suggestions. I agree with those points raised in their reports. Below are some additional comments that could be considered by the authors to improve their work.\nAlthough the authors have mentioned the importance of hAR in the Abstract and Introduction, especially for that it is an enzyme functions under osmotic stresses, it is unclear for the physiological relevance of the information obtained here using heat and chemical denaturants. I suggest the authors considered the physiologically relevant factors such as osmotic stress in their revised or future work.\n\nhAR is an enzyme. I suggest the authors monitor the activity of the enzyme during unfolding to provide information regarding the local structural changes around the active site.\n\nI am not convinced by the calculations of the thermodynamic parameters. The equations listed in the manuscript are derived from reverse folding processes. However, the authors did not show that the folding of hAR was reversible or not.\n\nThe authors concluded that the thermal unfolding of hAR was a two-state process by far-UV CD spectra analysis. However, only one method/probe is difficult for the definition of a two-state transition. It’s better to study the transition with two or more probes and pure two-state transition will give similar transitions as well as thermodynamic parameters for all probes.\n\nThe authors concluded that hAR undergo a three-state transition during unfolding by chemical denaturants. The present data could not support this statement. The emission maximum wavelength of the Trp fluorescence revealed no populations of any intermediates. An intermediate seems to populate at low denaturant concentrations when observed by Trp fluorescence intensity. However, intrinsic fluorescence intensity is easily affected by numerous environmental factors and is not as reliable as the emission maximum wavelength. Yes, there is an obvious increase in ANS fluorescence. Previously Turoverov and his co-authors have shown that the appearance of off-pathway aggregates, in some cases, can enhance ANS fluorescence.1 It is necessary to avoid the misleading interpretation by identifying the potential intermediate(s) via more methods.\n\nIt is unclear whether all the solid lines are fitted data or not. Moreover, it is better to provide the residuals to facilitate the readers to evaluate whether the fitting is appropriate.\n\nIn Figure 3, the ANS profiles seem to comprise more than one state and are likely to be the sum of at least two states centered at around 1 and 2 M GdnHCl?\n\nCD signals are recommended to report in molar ellipticities. The quality of the far-UV CD spectra is not enough. High concentration of denaturants may affect the signals at low wavelengths, generally below 210 nm. However, the low signal-to-noise seems to be affected by other factors. The authors can try to adjust the measuring parameters such as the slit width and number of repetitions. Furthermore, the authors mentioned that all chemicals were Sigma reagent grade. To my experience, GdnHCl with an ultrapure grade will produce much better signals that the lower grades. Note that the impurities or contaminated solvents will greatly affect spectroscopic measurements. Before spectroscopic measurements, the samples should be filtered and degassed. Detailed guidelines of spectroscopic measurements can be found in the paper by Kelly et al.2\n\nThe units of the fluorescence intensities in Figures 2 and 3 are quite different. Are these data normalized by the same method? Furthermore, the spectra of N in panels E-2 should refer to the state in the absence of denaturant. Why the spectrum of N in Figure 2E-2 is different from that in Figure 3E-2? To me, the spectrum of N in Figure 2E-2 is strange. Please verify that the solutions are not affected by contaminants.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-564
https://f1000research.com/articles/8-1853/v1	05 Nov 19	{ "type": "Case Report", "title": "Case Report: Sarcoidosis in the lymph nodes of a breast cancer patient", "authors": [ "Perwasha Kerio", "Zain Abid", "Masooma Abid", "Desaar Zehra", "Ghulam Haider", "Perwasha Kerio", "Masooma Abid", "Desaar Zehra", "Ghulam Haider" ], "abstract": "Background: Sarcoidosis is an inflammatory disease that affects multiple organs in the body, especially the lungs and lymph nodes. The coexistence of sarcoidosis and breast cancer has been reported, but the coexistence of both diseases in the same patient often leads to misdiagnosis. Case: We report a case of a 36-year-old woman who presented with concerns of a lump in her left breast along with pain and discharge from the nipple. On examination a 3-cm hard and tender mass was noted in the upper medial quadrant of the left breast with no palpable axillary lymph nodes. The patient was diagnosed with an infiltrating ductal cell carcinoma of the left breast with T2N0M1 Stage IV disease, due to positive mediastinal lymphadenopathy on positron emission tomography scan. The biopsy of mediastinal lymph nodes allowed us to diagnose sarcoidosis and correctly stage her disease as T2N0M0 Stage IIA breast cancer. The patient underwent lumpectomy followed by adjuvant chemo radiotherapy and hormonal therapy - corticosteroids given for sarcoidosis up to 1 year. The patient is doing well 18 months later without recurrence of disease. Conclusion: The simultaneous occurrence of both diseases in the same patient is the risk for misdiagnosis and mismanagement, therefore it is of utmost importance to correctly stage the disease with appropriate investigations and histologic confirmation prior to initiate the treatment for breast cancer.", "keywords": [ "breast cancer", "sarcoidosis", "mediastinal lymph adenopathy", "positron emission tomography" ], "content": "Introduction\n\nBreast cancer is the most common cancer in women1. It is a highly curable cancer, and with proper treatment protocols, the five-year survival of Stage IV disease is 22%2. The overall survival of a breast cancer patient depends on the stage of disease with visceral or bony metastasis; therefore at the time of initial treatment planning, it is highly important to determine the intent, which is either curative or palliative. The oncological team exerts the utmost effort to properly stage cancer by giving attention to history and clinical examination for judicious use of staging workup. Usually, on examination of breast cancer patients, the axillary lymph nodes are palpable, but internal mammary lymph node involvement is less commonly seen. We present herein a rare case of breast carcinoma with sarcoidosis. Sarcoidosis is a multisystem disease that has different grades. Most patients remain asymptomatic, and very few require treatment. It is very important to differentiate and diagnose breast cancer with metastasis or breast cancer with sarcoidosis, as there is limited literature available on the coexistence of breast cancer with sarcoidosis.\n\n\nCase presentation\n\nA 36-year-old woman presented with concerns of a lump in her left breast along with pain and discharge from the nipple. Her age of menarche was 12 years. She had no family history of breast or ovarian cancer.\n\nWe noted a 3-cm hard and tender mass in the upper medial quadrant of the left breast. There were no palpable axillary lymph nodes. The breast ultrasound showed a solid lesion with ill-defined margins in the upper medial quadrant of the left breast. The bilateral mammography demonstrated left Breast Imaging Reporting and Data System (BI-RADS) category IV and right breast BI-RADS category I.\n\nThe ultrasound-guided Tru-Cut® biopsy of the mass in the left breast showed Grade 2 infiltrating ductal cell carcinoma. The preoperative chest x-ray showed bilateral hilar lymphadenopathy. The preoperative chest computed tomography (CT) scan showed a small (2.5 cm × 1.6 cm) soft tissue density mass with speculated margins in the upper quadrant of the left breast with possible focal infiltration of the underlying chest wall muscle (Figure 1).\n\nSmall (2.5 cm × 1.6 cm) soft tissue density mass with speculated margins in the upper quadrant of the left breast with possible focal infiltration of the underlying chest wall muscle is observed (shown by yellow arrows).\n\nThe results of the axillary lymphadenopathy were negative bilaterally. Multiple enlarged discrete and confluent lymph nodes were seen in the mediastinum and both hilar regions, which could have been malignant.\n\nPositron emission tomography (PET) scan showed multiple enlarged hypermetabolic lymph nodes in the mediastinum, right paratracheal, carinal, bilateral hilar region, and aortopulmonary window (Figure 1).\n\nThe largest lymph node in the subcranial region was 1.5 cm × 4.0 cm with a maximum standardized uptake value (SUV max) of 8.3 (Figure 2). One of the lymph nodes in the right paratracheal region measured 2.5 cm × 2.1 cm with an SUV max of 9.9, likely representing metastasis.\n\nMultiple enlarged hypermetabolic lymph nodes in the mediastinum, right paratracheal, carinal, bilateral hilar region, and aortopulmonary window are observed (shown by yellow arrows).\n\nThe bronchoscopic biopsy of mediastinal lymph node showed non-caseous necrotic granulomatous inflammation, suggestive of sarcoidosis. This allowed us to correctly stage the disease as T2N0M0 stage IIA. The patient underwent lumpectomy followed by adjuvant chemotherapy, radiotherapy and hormonal therapy for breast cancer, while corticosteroids (prednisone, 1mg/kg for 9 months then tapered off over period of 3 months) were given for sarcoidosis for 1 year. The patient is being followed up with clinical examination every 3 months and breast mammogram done initially at 6 months following completion of radiotherapy (post mastectomy radiation therapy, total dose of 60 Gy: 50 Gy in 25 fractions given to chest wall with scar boost 10 Gy in 5 fractions) then at 1 year. At 18 months, there is no evidence of recurrence of disease and the patient is well.\n\n\nDiscussion\n\nSarcoidosis is a multisystem granulomatous disease of unknown aetiology that manifests as non-caseating granulomas predominantly in the lungs, intrathoracic lymph nodes, and skin. The less commonly affected organs are the eyes, liver, heart, and brain. Sarcoidosis is more common in women; it occurs in patients aged 20 to 50 years3. The incidence rate is higher among African Americans. Many features of sarcoidosis are suggestive of infectious origin4. The diagnosis of sarcoidosis requires radiographic signs (e.g., bilateral hilar lymphadenopathy and pulmonary infiltrations)3 and non-caseating granulomas on histopathology. Chronic inflammation is associated with an increased risk for malignant lymphoma and cancer in the affected tissue. The association between sarcoidosis and cancer is indistinct; although it has been proposed that patients with sarcoidosis are at increased risk for developing cancer of the lung, small intestine, stomach, liver, and skin4. Cancer can produce sarcoid-like reactions in the lymph nodes, and sarcoidosis can occur in patients with cancer. The radiological features of neoplasms can be mistaken for sarcoidosis. Therefore, histologic confirmation is required before making a diagnosis3,5.\n\nThere is increased risk of breast cancer occurrence in patients with sarcoidosis than other inflammatory diseases5. Lungs are common sites for breast cancer metastasis and are detected on chest radiographs in 25% of patients4,5.\n\nCavitation of metastatic nodule is a rare feature on radiography. However, cavitation in metastatic adenocarcinoma including breast cancer is frequently seen on CT5. The axillary, mediastinal, and hilar lymph nodes are commonly involved. PET/CT imaging is superior in detecting distant metastasis in patients with stage II and stage III breast cancer, but false positive 18F-fluorodeoxyglucose (FDG) uptake and negative PET/CT are frequently seen6. The conditions that cause false positive FDG uptake for malignancy include tuberculosis, fungal infection, and sarcoidosis. Initial staging with PET/CT is not recommended in preoperative early stage breast cancer because of high suboptimal sensitivity of the axillary lymph nodes, but specificity is high, and it has excellent positive predictive value confirming immediate axillary dissection instead of sentinel lymph node biopsy in the presence of high FDG uptake. However, sentinel lymph node biopsy is necessary if there is normal or slightly increased FDG uptake6.\n\nSarcoidosis has been reported to increase levels of CA 15-3 in some cases, so the presence of this biomarker might be misleading7.\n\n\nConclusions\n\nIf abnormal FDG uptake is detected on FDG PET in isolated mediastinal lymph nodes, in the absence of axillary lymph node involvement in patients with breast cancer, a thorough preoperative evaluation is warranted with histopathologic confirmation, thereby allowing the choice of correct staging and curative strategy.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394–424. PubMed Abstract \| Publisher Full Text\n\nHowlader N, Noone AM, Krapcho M, et al.: SEER Cancer Statistics Review, 1975-2014. Bethesda: National. Cancer, Institute; 2017; Reference Source\n\nKoo HJ, Kim MY, Shin SY, et al.: Evaluation of Mediastinal Lymph Nodes in Sarcoidosis, Sarcoid Reaction, and Malignant Lymph Nodes Using CT and FDG-PET/CT. Medicine (Baltimore). 2015; 94(27): e1095. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDragoumis DM, Tsiftsoglou AP, Assimaki AS: Pulmonary sarcoidosis simulating metastatic breast cancer. J Cancer Res Ther. 2008; 4(3): 134–6. PubMed Abstract \| Publisher Full Text\n\nKochoyan T, Akhmedov M, Shabanov A, et al.: Sarcoidosis imitating breast cancer metastasis: a case report and literature review. Cancer Biol Med. 2016; 13(3): 396–398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltınkaya M, Altınkaya N, Hazar B: Sarcoidosis mimicking metastatic breast cancer in a patient with early-stage breast cancer. Ulus Cerrahi Derg. 2015; 32(1): 71–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen J, Carter R 3rd, Maoz D, et al.: Breast Cancer and Sarcoidosis: Case Series and Review of the Literature. Breast Care (Basel). 2015; 10(2): 137–40. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "56717", "date": "12 Jun 2020", "name": "Jun Jiang", "expertise": [ "Reviewer Expertise Diagnosis of tumor imaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCT is not the first choice for breast cancer . It should provide ultrasound, X-ray and MRI imaging data.\n\nBreast cancer patients without axillary lymph node metastasis, but found a large number of mediastinal swollen lymph nodes, such cases are very rare.First of all, we need to analyze the relationship between breast tumor and mediastinal enlarged lymph node. If two different diseases are considered, breast cancer is considered as breast cancer, while mediastinal enlarged lymph nodes can be metastatic lymph nodes, sarcoidosis or lymphoma.If breast and mediastinum are considered as the same disease, lymphoma should be considered first.\n\nThe case report has not been discussed in detail in terms of examination method, diagnosis and differential diagnosis, and has limited value in clinical guidance.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "80201", "date": "16 Mar 2021", "name": "Tim L Jansen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI agree on the interest for your readership on this case report. Interesting case report on the simultaneous occurrence of breastca and granulomatous disease so called sarcoidosis.\n\nDiagnostic issues are elegantly depicted.\n\nI think the paper is ready for indexing with a minor improvement on:\nTherapeutic considerations regarding the finding of granulomatous tissue in biopsy versus malignant cells in biopsy only.\n\nPharmacotherapeutic options.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1853
https://f1000research.com/articles/8-1846/v1	04 Nov 19	{ "type": "Brief Report", "title": "Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics", "authors": [ "Sandeep Chakraborty" ], "abstract": "The ability to edit a specific gene within our genomes using guided-nucleases (Cas9/ZFN/TALEN - CaZiTa) presents huge opportunities for curing many genetic disorders. Delivery of this ‘drug’ within cells is a critical step for such therapies. The ability of recombinant adeno-associated virus (rAAV) to enter cells makes it a perfect choice as a vector for gene therapy. A plasmid comprising the rAAV, the CaZiTa, guide RNAs (for CRISPR) is expected to enter the cell, edit the target gene(s), remain episomal, and thus fade away with time. However, the rather obvious danger of integration of the plasmid into the genome, if the episomal hypothesis is incorrect, is under-reported. A recent report has highlighted that bacterial genes from a plasmid were integrated into bovine genomes. Massachusetts General Hospital has recently published data on CRISPR edits (Accid:PRJNA563918), noting ‘high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks’. However, there is no mention of Cas9 integration. Here, the same data from Massachusetts General Hospital shows Cas9 integration in the exact edit sites provided for two genes - TMC1 and DMD. Also, there is a mis-annotation of one sample as ‘no gRNA’, since Cas9 integrations have been detected in that sample. This is an important distinction between AAV and CaZiTa integration: while AAV integration can be tolerated, Cas9 integration is a huge, and unacceptable, danger.", "keywords": [ "Gene-editing", "CRISPR-Cas", "AAV", "plasmid integration" ], "content": "Introduction\n\nNuclease based gene-editing techniques (Cas9/ZFN/TALEN - CaZiTa) introduce a double stranded break at a specified location with the guide of DNA-binding proteins (ZFN/TALEN) or RNA (CRISPR)1. Delivery within cells is a critical step for such gene-therapies2. The ability of recombinant adeno-associated virus (rAAV) to enter cells makes it a perfect choice as a vector for gene therapy3. A plasmid comprising the rAAV, CaZiTa, guide RNAs (for CRISPR), etc. is expected to enter the cell, edit, and remain episomal4. However, the rather obvious danger of integration of the plasmid into the genome is ignored. A recent pre-print5 has highlighted that bacterial genes from the template plasmid (pCR2.1-TOPO) has integrated into bovine genomes6.\n\nRecently, Massachusetts General Hospital published data on CRISPR edits (Accid:PRJNA563918), and concluded that ‘AAV integration should be recognised as a common outcome for applications that utilize AAV for genome editing’7. However, there was no report of Cas9 integration. Here, data showing Cas9 integrating in the exact edit sites is shown. The same caveat applies to all three CaZiTa gene-therapies1.\n\n\nMethods\n\nSequencing data was obtained from BioProject accession number, PRJNA563918, using SRA download tools (https://github.com/ncbi/sra-tools/wiki). This project aims at finding “the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases”7.\n\nThe reads were uniquified, split into open-reading frames using the getorf (version:6.6.0.0) program from the EMBOSS suite (http://emboss.sourceforge.net/download/)8.\n\nCommand: “getorf -find 1 -sequence <infile> -outseq <output> -minsize 10”.\n\nExact 10-kmer were aligned to the Cas9 protein (Accid:AGZ01981.1, 1417aa). Subsequently, these reads were then aligned to the gene of interest - TMC1(Accid:NG_008213.1) or DMD(Accid:NM_001314034.1). The online BLAST interface using the default settings (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to generate images of integration with the genes of interest9. The number of reads with integration is conservative, since reads with less than a 10-kmer match were ignored. The AAV genome used was Accid:MK163936.1. Sequences that integrate Cas9/AAV with TMC1 and DMD are provided in Extended data: TMC1.cas9.fa/TMC1.aav.fa and DMD.cas9.fa/DMD.aav.fa, respectively.\n\n\nResults and discussion\n\nTMC1, a transmembrane protein, is required for proper functioning of cochlear hair cells, and has been implicated in hearing loss and prelingual deafness10. In mice, this gene is located in chr19 (Accid:NG_008213.1)). Table 1 lists the samples sequenced for targeting this gene. As expected, the non-injected controls have no Cas9 reads. The sample SRR10068671 (marked with triple asterisks) has probably been mis-annotated as one with “no gRNA”, since Cas9 integrations are detected in that sample. From the Cas9 integration site, the guide RNA (gRNA) for the TMC1 gene can be deduced to be “CATGGTAATGTCCCTCCTGGGGA”, although this information is not yet available. The sequences for these reads are provided as extended data. As a specific example, the sequence (SRR10068639.63180.1) encodes the 26aa peptide=SPEKLLMYHHDPQTYQKLKLIMEQYG from Cas9 and is merged with the TMC1 gene (Figure 1).\n\nThe paired reads are clubbed into a single file, and then uniquified. Open reading frames from these are then matched to Cas9 proteins for 10 aa, and subsequently these reads are matched to the gene of interest (TMC1 or DMD), helping identify the exact edit site. The sample SRR10068671 (marked with triple asterisks) has probably been mis-annotated as one with “no gRNA”, since Cas9 integrations are detected in that sample. These integrations happen both for SpCas9 and SaCas9. From the Cas9 integration site, the gRNA for the TMC1 gene can be deduced to be “CATGGTAATGTCCCTCCTGGGGA”, although this information is not yet available. CN, cortical neuron; CA: cochlea, adult.\n\nThe sequence (SRR10068639.63180.1) encodes the 26aa peptide=SPEKLLMYHHDPQTYQKLKLIMEQYG from Cas9 and is merged with the TMC1 gene.\n\nDuchenne muscular dystrophy (DMD), a genetic disorder associated with progressive muscle degeneration (dystrophy), is caused by aberrations in the dystrophin protein, located on chromosome X in humans and mice11. According to the data (Accid:PRJNA563918), different exons and introns have been targeted using two different variations of the Cas9 - SpCas9 and SaCas9 - which differ in certain characteristics12. SaCas9 does seem empirically to have more samples without any integration, but that might just be random. The sequences for these reads are provided as extended data. As a specific example, the sequence (SRR10068622.33932.1) encodes the 12aa peptide=LDATLIHQSITG from Cas9 and is merged with the DMD gene (Figure 2).\n\nThe sequence (SRR10068622.33932.1) encodes the 12aa peptide=LDATLIHQSITG from Cas9 and is merged with the DMD gene.\n\nThe integration of AAV into the genome has already been noted7. This has been replicated in this study as well. The AAV genome used was Accid:MK163936.1. The reads can be found in Extended data: TMC1.aav.fa (N=5000) and DMD.aav.fa (N=14000).\n\n\nConclusions\n\nGene-editing based therapies provide revolutionary hope for curing many diseases. However, ensuring safety of any such endeavours must be paramount to avoid doing more harm13,14. Plasmid integration is one such potential hazard5. Recently, Massachusetts General Hospital reported high-levels of in vivo AAV into the genome while providing sequencing data (Accid:PRJNA563918), and concluded that ‘AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing’7. In this study, in addition to AAV, Cas9 integration is shown in the same samples. The same caveat applies to gene-therapies using CaZiTa1. Off-target edits (OTE) are an important aspect of CRISPR-cas gene-editing15–19. Such integrations, found by targeting amplicon sequencing, will only get worse due to OTEs, which are hard to detect20–22. Another problem is pre-existing immunity to Cas9 proteins23,24. This problem can be mitigated by sending the CaZiTa as protein25, but that would seriously restrict the use-cases, and also suffer from OTEs, large deletions, complex rearrangements and translocations26, or even including fragments from exosomes27. This is an important distinction between AAV and CaZiTa integration, since AAV integration can be tolerated (integration at chromosome breakage points28, though there is debate on its role in hepatocellular carcinoma29,30), Cas9 integration is a huge, and unacceptable danger. After integration, individuals may be expressing Cas9, and that is fraught with genotoxic danger.\n\n\nData availability\n\nRaw sequence reads from Massachusetts General Hospital, Accession number PRJNA563918, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA563918/\n\nZenodo: Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics, https://doi.org/10.5281/zenodo.346030531\n\nThis project contains the following extended data:\n\nDMD.aav.fa\n\nDMD.cas9.fa\n\nTMC1.aav.fa\n\nTMC1.cas9.fa\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nGaj T, Gersbach CA, Barbas CF 3rd: ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013; 31(7): 397–405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaFountaine JS, Fathe K, Smyth HD: Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm. 2015; 494(1): 180–194. PubMed Abstract \| Publisher Full Text\n\nPillay S, Zou W, Cheng F, et al.: Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor. J Virol. 2017; 91(18): pii: e00391-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaso MF, Tomkowicz B, Perry WL 3rd, et al.: Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017; 31(4): 317–334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNorris AL, Lee SS, Greenlees KJ, et al.: Template plasmid integration in germline genome-edited cattle. bioRxiv. 2019; 715482. Publisher Full Text\n\nCarlson DF, Lancto CA, Zang B, et al.: Production of hornless dairy cattle from genome-edited cell lines. Nat Biotechnol. 2016; 34(5): 479–81. PubMed Abstract \| Publisher Full Text\n\nHanlon KS, Kleinstiver BP, Garcia SP, et al.: High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat Commun. 2019; 10: 4439. Publisher Full Text\n\nRice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000; 16(6): 276–277. PubMed Abstract \| Publisher Full Text\n\nAltschul SF, Madden TL, Schaffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakanishi H, Kurima K, Kawashima Y, et al.: Mutations of TMC1 cause deafness by disrupting mechanoelectrical transduction. Auris Nasus Larynx. 2014; 41(5): 399–408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffman EP, Brown RH Jr, Kunkel LM: Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987; 51(6): 919–928. PubMed Abstract \| Publisher Full Text\n\nXie H, Tang L, He X, et al.: SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells. Biotechnol J. 2018; 13(4): e1700561. PubMed Abstract \| Publisher Full Text\n\nCouzin J, Kaiser J: Gene therapy. As Gelsinger case ends, gene therapy suffers another blow. 2005; 307(5712): 1028. PubMed Abstract \| Publisher Full Text\n\nSaâda-Bouzid E, Defaucheux C, Karabajakian A, et al.: Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma. Ann Oncol. 2017; 28(7): 1605–1611. PubMed Abstract \| Publisher Full Text\n\nFu Y, Foden JA, Khayter C, et al.: High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9): 822–826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPattanayak V, Lin S, Guilinger JP, et al.: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31(9): 839–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuscu C, Arslan S, Singh R, et al.: Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014; 32(7): 677–83. PubMed Abstract \| Publisher Full Text\n\nChakraborty S: Inconclusive studies on possible CRISPR-Cas off-targets should moderate expectations about enzymes that have evolved to be non-specific. J Biosci. 2018; 43(2): 225–228. PubMed Abstract \| Publisher Full Text\n\nChakraborty S: CRISPR-Cas off-target detection using Oxford Nanopore sequencing - is the mitochondrial genome more vulnerable to off-targets? bioRxiv. 2019; 741322. Publisher Full Text\n\nWienert B, Wyman SK, Richardson CD, et al.: Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science. 2019; 364(6437): 286–289. PubMed Abstract \| Free Full Text\n\nTsai SQ, Nguyen NT, Malagon-Lopez J, et al.: CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017; 14(6): 607–614. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai SQ, Zheng Z, Nguyen NT, et al.: GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015; 33(2): 187–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharlesworth CT, Deshpande PS, Dever DP, et al.: Identification of pre-existing adaptive immunity to cas9 proteins in humans. bioRxiv. 2018; 243345. Publisher Full Text\n\nFerdosi SR, Ewaisha R, Moghadam F, et al.: Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun. 2019; 10(1): 1842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, Kim D, Cho SW, et al.: Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014; 24(6): 1012–1019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKosicki M, Tomberg K, Bradley A: Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018; 36(8): 765–771. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOno R, Yasuhiko Y, Aisaki Ki, et al.: Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing. Commun Biol. 2019; 2: 57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller DG, Petek LM, Russell DW: Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet. 2004; 36(7): 767–73. PubMed Abstract \| Publisher Full Text\n\nNault JC, Datta S, Imbeaud S, et al.: AAV2 and Hepatocellular Carcinoma. Hum Gene Ther. 2016; 27(3): 211–213. PubMed Abstract \| Publisher Full Text\n\nBerns KI, Byrne BJ, Flotte TR, et al.: Adeno-Associated Virus Type 2 and Hepatocellular Carcinoma? Hum Gene Ther. 2015; 26(12): 779–781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChakraborty S: Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics. 2019. http://www.doi.org/10.5281/zenodo.3460305" }	[ { "id": "59005", "date": "06 Feb 2020", "name": "Rakesh Chatrikhi", "expertise": [ "Reviewer Expertise T cell Biology", "RNA Biology", "Cancer Biology", "Molecular Biology", "Regulation of gene expression", "Protein-RNA interactions", "Biochemistry and Molecular Biophysics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChakraborty studied sequencing data on CRISPR-induced DNA edits from Massachusetts General Hospital to show Cas9 integration into the genome. The author analyzed previously published data on CRISPR-induced DNA edits to highlight integration of Cas9 into the genome, which was not reported in the previously published study that generated the sequencing data. This study highlights risks involved in using gene-editing techniques such as CRISPR-Cas9 based system and raises important caveats that need to be considered when using the technology for therapeutics. However, the author needs to address the following points to strengthen the manuscript:\nThe author needs to quantitatively compare the levels of AAV and Cas9 integration and whether it is statistically significant. This is important because the author is using the same samples and sequencing data to discuss Cas9 integration into the genome, which was not reported earlier and thus required to effectively highlight the results in this study. The author needs to make a clear comparison of AAV and Cas9 integration from the data in the Results and Discussion section.\n\nThe author needs to support the argument that Cas9 integration is a serious hazard and is a huge unacceptable danger. This is important because the author argues that Cas9 integration is hazardous throughout the manuscript (title, abstract and conclusion). The author needs to include multiple references to previous work in the literature that have shown or highlighted the toxicity of Cas9 integration into the genome and that it is hazardous.\n\nIn the Conclusions section, the author needs to briefly discuss the alternative approaches that could be used to avoid Cas9 integration. For example, the author needs to elaborate on the use of purified Cas9 ribonucleoproteins as a substitute to plasmids. The author included a sentence on this topic in the Conclusions section, but need to elaborate on the advantages of using Cas9 ribonucleoproteins. For example, in addition to avoiding Cas9 integration, this method has higher efficiency (Farboud B et al., J Vis Exp, 2018), fewer off-targets (Liang X et al., Journal of Biotech, 2015; Kim S et al., Genome Res, 2014 - Ref 25 in this study)1,2.\n\nThe author needs to include the rationale for focusing on the genes TMC1 and DMD. Were those genes studied in the previous study that generated the data (Ref 7)? This needs to be clearly stated. If there are more genes studied in Ref 7, then the author needs to include the rationale of choosing these two genes over other genes in the study.\n\nThe Introduction section could also be strengthened by briefly discussing if there are any similar studies carried out previously in the literature studying the level of Cas9 integration into the genome and their limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "167874", "date": "14 Apr 2023", "name": "Saravanabhavan Thangavel", "expertise": [ "Reviewer Expertise Genome editing", "Hematopoietic stem cell gene therapy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRecombinant adeno-associated virus (rAAV) is one of the tools to deliver Crispr CAS9 into the target cells. The author uses the publicly available dataset containing rAAV delivered Cas9 edited cells to analyse the genomic regions around the cutsite. While it has previously been demonstrated that the AAV genome can integrate into the cutsite, this study detects a 12 to 26 a.a peptide from cas9 integrating into the cutsite.\nThis discovery is significant because integration of the Cas9 sequence has never been demonstrated before, and the consequences of integration and long-term expression of Cas9 are unknown. This emphasizes the importance of carefully selecting a delivery vehicle for Cas9.\nIt’s not clear from the writing that the Cas9 is targeted towards TMC1, DMD.\n\nIt’s not clear that the sequence of saCas9 used for probing\n\nThe authors also miss to cite an important work from Naldini’s lab showing sequestering of AAV genome into the cutsite when AAV6 was used as a HDR donor.\n\nThe possibility of sgRNA or its scaffold integrating into the cutsite should be discussed as well.\n\nThe methodology used for detecting the Cas9 sequence can be more elaborate\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1846
https://f1000research.com/articles/8-1842/v1	01 Nov 19	{ "type": "Study Protocol", "title": "The effectiveness of online parenting programmes in promoting parents’ and adolescents’ mental wellbeing: a systematic review protocol", "authors": [ "Amna Al Falahi", "Cris Glazebrook", "Farhad Shokraneh", "Cris Glazebrook", "Farhad Shokraneh" ], "abstract": "Introduction: Emotional difficulties among young people are debilitating and increasing in prevalence. Parent focused interventions delivered online offer a convenient and potentially effective way to increase young people’s access to support. A systematic review offers the opportunity to assess their effectiveness and to identify characteristic of interventions which are particularly effective. Objective: To assess the existing online interventions for parents of young people that are designed to improve young people’s mental health and wellbeing. Methods: We will conduct a systematic review of randomised controlled trials identified through searching CENTRAL, Embase, MEDLINE, PsycINFO, and PubMed. We will follow Cochrane Handbook and involve at least two people in screening and data extraction. Risk of bias will be assessed using Cochrane risk of bias tool. We will use EndNote, Excel, and Review Manager for managing the studies and data. We will also apply TIDieR checklist to extract and summarise the specific characteristics of interventions. Protocol registration: PROSPERO CRD42018114921; registered on 31 October 2018.", "keywords": [ "Mental Difficulties", "Parenting", "Adolescents", "Systematic Review", "Randomised Controlled Trials" ], "content": "Background\n\nEmotional difficulties among young people are debilitating and a matter of concern for primarily two reasons, one being increases in prevalence rates over the years, while the other pertains to the risk, chronicity and outcomes of mental health during later stages of development. Studies have consistently demonstrated homotypic continuity across individuals who develop emotional problems during their adolescent years (Scholten et al., 2013) and prevalence rates continue to increase with rates for anxiety disorders, reported at 1.52% among children aged 5 to 9 years, 3.71% among children aged 10 to 14 years and 4.36% among adolescents aged between 15 and 19 years; and depression rates residing at 0.13% among children aged 5 to 9 years, 1.24% among children aged 10 to 14 years, and 3.44% among adolescents aged between 15 to 19 years (World Health Organization, 2015). Similar trends have also been reported across ethnic populations (for e.g. Canals et al., 2019: Sandal et al., 2017). Furthermore, the manifestation of such disorders involve a high frequency of comorbidity (Ogundele, 2018). A report by the World Health Organization (2011) cautioned that, per their estimates, mental health issues among young people would reach epidemic proportions by the year 2020 in the absence of preventive measures, timely diagnosis, and early intervention.\n\nThe grim outlook of such research points to the need to explore suitable interventions. In general, interventions catering to emotional difficulties among young people can range from preventive measures, face to face therapy, symptom reduction, guided and unguided self-help tools, and psychotropic medication. In terms of the mental health needs of young people, research has shown considerable support for psychosocial approaches that are transdiagnostic and employ a cognitive behavioural framework (Comer et al., 2013: Ehrenreich-May et al., 2017: Ogundele, 2018: Wehry et al., 2015). A common factor across such treatments is the need to involve the young person’s support system, particularly their caregivers and teachers. Given that the mental and emotional development of young people occur in the context of parental and educational scaffolds, including parents in treatment is found to be an effective strategy either as a standalone intervention (such as parent training programmes) or as part of a comprehensive programme (Comer et al., 2013).\n\nGiven that parents have a key role in shaping and nurturing specific thought patterns and coping strategies throughout an individual’s developmental years, parent-focused early intervention is a befitting avenue for improving the mental wellbeing of young people with emotional difficulties. A national initiative in the UK called the Parenting Early Intervention Pathfinder (PEIP) programme examined the effect of three different programmes with parents of children aged 8 to 13 years (Lindsay et al., 2007). These programmes were developed based on social learning theory. At the start of the programme all the parents who participated in this programme were assessed as having lower mental well-being. They also reported a high level of emotional and behavioural problems in their children. Self-reported improvements following the psychosocial intervention were noted in the areas of prosocial child behaviour, positive parent outcomes (including improved mental health) and improved co-operation in family relationships. These results highlight the potential effectiveness and impact of parent-focused interventions in improving mental health among young people. In another longitudinal study, parental anxiety and depressive symptoms were significant predictors of unemployment, such that, ten years later, their adult children were reported to be using welfare services due to lack of employability (Pape et al., 2012). The authors of this study further concluded that the prevention and treatment of anxiety and depression among young people should be family-orientated and aimed at ensuring an effective work-life integration.\n\nParent-focused interventions are designed to improve the understanding, awareness and skills of parents, so as to improve their relationship with adolescent children and consequently have a positive impact on the young person's well-being. The way in which this works is not well known, though hypotheses have been proposed. The most frequently recognized assumptions often follow either a direct influence in that parents are better able to meet their children’s emotional, psychological and behavioural needs; or that (indirectly) their own emotional, psychological and behavioural needs will be better satisfied therefore enabling them to model and display increased pro-social behaviours (Long et al., 2017).\n\nIn terms of the implementation of parent-focused programmes itself, research has examined the duration, frequency and type of therapy that might be most effective. Among the commonly utilized principles in intensive treatments is emotion focused family therapy. One study by Foroughe et al. (2019) found this type of therapy, when delivered in a two-day intensive period, brought about increased parental self-efficacy and improvement in child symptomology. The study further indicated that this was an effective, intensive short-term mode of treatment that can have a lasting impact at a four-month follow-up assessment. One cost effective method that can reach many parents without a strain on time, resources and services is to deliver online, web-based interventions. These interventions can incorporate mental health principles and theory, are evidence based, and have been shown to positively impact both parental and child mental health. An example of this is a tailored web-based intervention that was aimed to alleviate parental risk of mental health difficulties and to increase protective factors such as coping strategies, improved mood and decision-making ability, for emotional problems among young people (Yap et al., 2018).\n\nWhile the aforementioned research throws light on the forms and effectiveness of parent-training programmes, less is established in terms of potential variations in outcomes owing to contextual factors such as gender, age, socio-economic status and geographic setting/cultural context (Das et al., 2016). Therefore, a systematic review of the effect of parent focused interventions on adolescent mental health and wellbeing allows for more focused future directions for research and is therefore elucidated in the ensuing paragraphs.\n\nAmong the existing parent-focused interventions targeting depression and anxiety, two foundational theories seem to appear at the forefront. Some of these interventions are rooted in Bandura’s (1971) social learning theory, which posits that behaviours are learned through observation of the behaviours of credible and influential models. To this effect, parent-focused interventions are based on the premise that children’s behaviours are learned through their observation of their parents’ behaviour. These interventions therefore emphasize that the way the parents respond to their child’s behaviour is a determinant of continuation and frequency of similar behaviours in the future. While existing evidence supports short-term behavioural improvement among pre-school and primary school children (e.g. Scott & Gardner, 2015), there is also some empirical support for the sustained effectiveness of such programmes. For example, the Incredible Years intervention is found to be useful among young people with conduct disorder (Ryan et al., 2017). Another programme called Triple P, which is also designed using the social learning theory, seems promising in terms of the positive effects on skills and knowledge among the parents and emotional problems among the children and adolescents (Sanders et al., 2014). These are some of the outcomes to be assessed in this systematic review which will also include interventions that target the parents’ skills. For example, training the parents to develop conducive living skills has demonstrated a noticeable effect on positive psychological outcomes in their adolescents (Parmar & Jain, 2019). Taken together, such parenting interventions that are founded on social learning theory can reduce the prevalence of emotional problems among adolescents (Sanders, 2008).\n\nThe second group of parent-focused interventions emphasis the principles of attachment theory which is based on the premise that children intend to build a significant emotional attachment with one or both parents. Such bonds not only support children at times of negative emotions but also empowers the child with crucial social skills, thereby making them confident in facing similar challenges in social environment so that they could develop a social life (Ainsworth et al., 1978). Interventions founded on attachment theory highlight the relational aspect that influences the dynamics and manifestation of emotional difficulties. In this regard, in the wellbeing model discussion, it is also known that the parenting style is inclusive of methods that parents apply on their children that affect the mental health of the adolescents (Arulsubila & Subashree, 2017). Furthermore, the quality of parent-child relationship can predict the psychological wellbeing of young people during their adolescent years (Resnick et al., 1997).\n\nThe main reason to do this review is that there is no systematic effort known to this author that gathers evidence about the effectiveness of online interventions for parents of young people with emotional difficulties. In addition, this review will inform the design of future Randomised Controlled Trials (RCTs) pertaining to this topic.\n\nTo assess the existing online interventions for parents of young people that are designed to improve young people’s mental health and wellbeing.\n\n\nMethods\n\nTypes of studies. This review will include RCTs with no limit to language, publication status, document type, or date of publication.\n\nTypes of participants. Parents of young people (mean age of the child sample will be between 10 and 19 years).\n\nTypes of interventions.\n\n1. This review will consider studies evaluating the following interventions:\n\nParent-based interventions that include parent training and aim to help parents understand, support and improve their child’s mental health and wellbeing.\n\nIf the intervention of study is an integrated intervention consisting of multi-components, they will be included for further assessment.\n\nInterventions that include a young person component, provided that the parent receives at least some intervention.\n\nInterventions that have a digital or online component, including:\n\nWeb-based\n\nMobile application (apps)\n\nSocial media, email, and text\n\n2. This review will consider studies that compare the intervention to:\n\nNo treatment or another active intervention\n\nTreatment as usual\n\nWaitlist control\n\nExclusion criteria. Interventions that will be excluded include those that are entirely for the parent or family where the young person is not the focus, and interventions focused on parents of adolescents with specific physical health conditions.\n\nTypes of outcome measures. Primary outcomes will include parenting behaviour, parenting style, attitudes towards young people’s mental health or knowledge of young people’s mental health including, self-efficacy, expressed emotion, coping style, and care giver’s burden.\n\nSecondary outcomes will include young people’s mental health and wellbeing including depression, anxiety, coping, stress, academic achievement, sleep, school attendance, substance abuse, and self-harm.\n\nElectronic searches. CENTRAL, Embase, MEDLINE, PsycINFO, and PubMed will be searched combining four components: online/digital, parenting, adolescents, and randomised controlled trials. The search will not be limited to language, document type, publication status, or time/date. The following search strategies will be used for searching the databases:\n\nCENTRAL via Cochrane Library. ([mh CD-I] OR [mh CD-ROM] OR [mh \"Compact Disks\"] OR [mh \"Computer-Assisted Instruction\"] OR [mh ^Computers] OR [mh \"Computers, Handheld\"] OR [mh \"Electronic Mail\"] OR [mh Hypermedia] OR [mh Internet] OR [mh \"Mobile Applications\"] OR [mh Smartphone] OR [mh \"Social Media\"] OR [mh ^Software] OR [mh \"Text Messaging\"] OR [mh \"Videodisc Recording\"] OR [mh \"Webcasts as Topic\"] OR (\"Compact Disc\" OR \"Compact Discs\" OR \"Compact Disk\" OR \"Compact Disks\" OR \"Computer Assisted\" OR \"Computer Game\" OR \"Computer Games\" OR \"Computer Program\" OR \"Computer Programme\" OR \"Computer Programmes\" OR \"Computer Programs\" OR \"E-Health\" OR \"Electronic Health\" OR \"Electronic Mail\" OR \"Electronic Mails\" OR \"E-Mail\" OR \"E-Mails\" OR \"Handheld Computer\" OR \"Handheld Computers\" OR \"M-Health\" OR \"Mobile Application\" OR \"Mobile Applications\" OR \"Mobile Health\" OR \"Mobile Phone\" OR \"Palm Pilot\" OR \"Palm Pilots\" OR \"Palmtop\" OR \"Palm-Top\" OR \"Personal Digital Assistant\" OR \"Pocket PC\" OR \"Pocket PCs\" OR \"Short Message Service\" OR \"Smart Phone\" OR \"Smart Phones\" OR \"Social Media\" OR \"Tablet Computer\" OR \"Tablet Computers\" OR \"Text Message\" OR \"Text Messages\" OR App OR Apps OR CD OR CDROM* OR Computerised OR Computerized OR Cyber* OR Digital* OR DVD OR EHealth OR Email* OR Facebook OR Hypermedia OR Hypertext OR Internet OR iPad OR iPhone OR Laptop* OR Messaging OR MHealth OR Online OR PDA OR PDAs OR Podcast* OR Smartphone* OR SMS OR Software* OR Texting* OR Tweet* OR Twitter* OR Virtual* OR Web OR Webcast* OR Website* OR WeChat OR Whatsapp* OR YouTube):ti,ab) AND ([mh Fathers] OR [mh \"Legal Guardians\"] OR [mh Mothers] OR [mh Parenting] OR [mh Parents] OR [mh \"Single Parent\"] OR (Father* OR Guardian* OR Mother* OR Parent):ti,ab) AND ([mh Adolescent] OR (Adolescen OR Teen* OR Young OR Youth):ti,ab)\n\nIn Trials\n\nEmbase 1974 to 2018 week 37 via Ovid SP.\n\n1. CD-I/ OR CD-ROM/ OR Compact Disk/ OR Computer/ OR Digital Computer/ OR Personal Computer/ OR Personal Digital Assistant/ OR E-Mail/ OR Hypermedia/ OR Internet/ OR Mobile Application/ OR Smartphone/ OR Social Media/ OR Software/ OR Text Messaging/ OR Video Disk/ OR Webcast/ OR (\"Compact Dis?\" OR \"Compact Dis?s\" OR \"Computer Assisted\" OR \"Computer Gam\" OR \"Computer Program\" OR E?Health OR \"Electronic Health\" OR \"Electronic Mail?\" OR \"E?Mail?\" OR \"Hand?held Computer?\" OR M?Health OR \"Mobile Application?\" OR \"Mobile Health\" OR \"Mobile Phone?\" OR \"Palm Pilot?\" OR \"Palm?top\" OR \"Personal Digital Assistant?\" OR \"Pocket PC?\" OR \"Short Message Service\" OR Smart?Phone? OR \"Social Media\" OR \"Tablet Computer?\" OR \"Text Messag\" OR App OR Apps OR CD OR CDROM* OR Computeri?ed OR Cyber* OR Digital* OR DVD OR Facebook OR Hypermedia OR Hypertext OR Internet OR iPad OR iPhone OR Laptop* OR Messaging OR Online OR PDA OR PDAs OR Podcast* OR SMS OR Software* OR Texting* OR Tweet* OR Twitter* OR Virtual* OR Web OR Webcast* OR Website* OR WeChat OR Whatsapp* OR YouTube).ti,ab.\n\n2. Father/ OR Legal Guardian/ OR Mother/ OR Parent/ OR Single Parent/ OR (Father* OR Guardian* OR Mother* OR Parent).ti,ab.\n\n3. Adolescent/ OR Adolescence/ OR (Adolescen OR Teen* OR Young OR Youth).ti,ab.\n\n4. Randomization/ OR Crossover-Procedure/ OR Double-Blind Procedure/ OR Randomized Controlled Trial/ OR Single-Blind Procedure/ OR (Randomi?ed OR Randomly OR Factorial OR Cross?over* OR ((Singl* OR Doubl* OR Trebl* or Tripl) adj (Mask OR Blind)) OR Assign OR Allocat* OR Volunteer* OR Groups OR Trial).ti,ab.\n\n5. 1 AND 2 AND 3 AND 4\n\n6. Exp Animals/ OR Exp Invertebrate/ OR Animal Experiment/ OR Animal Model/ OR Animal Tissue/ OR Animal Cell/ OR Nonhuman/\n\n7. Human/ OR Normal Human/ OR Human Cell/\n\n8. 6 AND 7\n\n9. 6 NOT 8\n\n10. 5 NOT 9\n\n11. Limit 10 to Embase\n\nOvid MEDLINE(R) and Epub ahead of print, in-process & other non-indexed citations and daily 1946 to September 07, 2018.\n\n1. CD-I/ OR CD-ROM/ OR Compact Disks/ OR Computer-Assisted Instruction/ OR Computers/ OR Computers, Handheld/ OR Electronic Mail/ OR Hypermedia/ OR Internet/ OR Mobile Applications/ OR Smartphone/ OR Social Media/ OR Software/ OR Text Messaging/ OR Videodisc Recording/ OR \"Webcasts as Topic\"/ OR (\"Compact Dis?\" OR \"Compact Dis?s\" OR \"Computer Assisted\" OR \"Computer Gam\" OR \"Computer Program\" OR E?Health OR \"Electronic Health\" OR \"Electronic Mail?\" OR \"E?Mail?\" OR \"Hand?held Computer?\" OR M?Health OR \"Mobile Application?\" OR \"Mobile Health\" OR \"Mobile Phone?\" OR \"Palm Pilot?\" OR \"Palm?top\" OR \"Personal Digital Assistant?\" OR \"Pocket PC?\" OR \"Short Message Service\" OR Smart?Phone? OR \"Social Media\" OR \"Tablet Computer?\" OR \"Text Messag\" OR App OR Apps OR CD OR CDROM* OR Computeri?ed OR Cyber* OR Digital* OR DVD OR Facebook OR Hypermedia OR Hypertext OR Internet OR iPad OR iPhone OR Laptop* OR Messaging OR Online OR PDA OR PDAs OR Podcast* OR SMS OR Software* OR Texting* OR Tweet* OR Twitter* OR Virtual* OR Web OR Webcast* OR Website* OR WeChat OR Whatsapp* OR YouTube).ti,ab.\n\n2. Fathers/ OR Legal Guardians/ OR Mothers/ OR Parenting/ OR Parents/ OR Single Parent/ OR (Father* OR Guardian* OR Mother* OR Parent).ti,ab.\n\n3. Adolescent/ OR (Adolescen OR Teen* OR Young OR Youth).ti,ab.\n\n4. Controlled Clinical Trial.pt. OR Randomized Controlled Trial.pt. OR (Randomi?ed OR Randomly OR Trial OR Groups).ti,ab.\n\n5. 1 AND 2 AND 3 AND 4\n\n6. Exp Animals/ NOT Humans.sh.\n\n7. 5 NOT 6\n\nPsycINFO 1806 to September Week 1 2018 via Ovid SP.\n\n1. Computer Assisted Instruction/ OR Computers/ OR Computer Applications/ OR Computer Assisted Therapy/ OR Computer Games/ OR Computer Mediated Communication/ OR Computer Software/ OR Digital Computers/ OR Digital Video/ OR Hypermedia/ OR Hypertext/ OR Internet/ OR Online Therapy/ OR Online Community/ OR Online Social Networks/ OR Social Media/ OR Text Messaging/ OR Websites/ OR (\"Compact Dis?\" OR \"Compact Dis?s\" OR \"Computer Assisted\" OR \"Computer Gam\" OR \"Computer Program\" OR E?Health OR \"Electronic Health\" OR \"Electronic Mail?\" OR \"E?Mail?\" OR \"Hand?held Computer?\" OR M?Health OR \"Mobile Application?\" OR \"Mobile Health\" OR \"Mobile Phone?\" OR \"Palm Pilot?\" OR \"Palm?top\" OR \"Personal Digital Assistant?\" OR \"Pocket PC?\" OR \"Short Message Service\" OR Smart?Phone? OR \"Social Media\" OR \"Tablet Computer?\" OR \"Text Messag\" OR App OR Apps OR CD OR CDROM* OR Computeri?ed OR Cyber* OR Digital* OR DVD OR Facebook OR Hypermedia OR Hypertext OR Internet OR iPad OR iPhone OR Laptop* OR Messaging OR Online OR PDA OR PDAs OR Podcast* OR SMS OR Software* OR Texting* OR Tweet* OR Twitter* OR Virtual* OR Web OR Webcast* OR Website* OR WeChat OR Whatsapp* OR YouTube).ti,ab.\n\n2. Fathers/ OR Mothers/ OR Parent Training/ OR Parents/ OR Single Parents/ OR (Father* OR Guardian* OR Mother* OR Parent).ti,ab.\n\n3. (Adolescen OR Teen* OR Young OR Youth).ti,ab.\n\n4. Exp Treatment Effectiveness Evaluation/ OR Clinical Trials/ OR Mental Health Program Evaluation/ OR (Randomi?ed OR Factorial OR Cross?over* OR ((Singl* OR Doubl* OR Trebl* or Tripl) adj (Mask OR Blind)) OR Assign OR Allocat* OR Volunteer* OR Groups OR Trial).ti,ab.\n\n5. 1 AND 2 AND 3 AND 4\n\n6. 1 AND 2 AND 4\n\n7. Limit 6 to Adolescence <13 to 17 Years>\n\n8. 5 OR 7\n\nPubMed. ((CD-I[mh] OR CD-ROM[mh] OR \"Compact Disks\"[mh] OR \"Computer-Assisted Instruction\"[mh] OR Computers[mh:NoExp] OR \"Computers, Handheld\"[mh] OR \"Electronic Mail\"[mh] OR Hypermedia[mh] OR Internet[mh] OR \"Mobile Applications\"[mh] OR Smartphone[mh] OR \"Social Media\"[mh] OR Software[mh:NoExp] OR \"Text Messaging\"[mh] OR \"Videodisc Recording\"[mh] OR \"Webcasts as Topic\"[mh] OR \"Compact Disc\"[tiab] OR \"Compact Discs\"[tiab] OR \"Compact Disk\"[tiab] OR \"Compact Disks\"[tiab] OR \"Computer Assisted\"[tiab] OR \"Computer Game\"[tiab] OR \"Computer Games\"[tiab] OR \"Computer Program\"[tiab] OR \"Computer Programme\"[tiab] OR \"Computer Programmes\"[tiab] OR \"Computer Programs\"[tiab] OR \"E-Health\"[tiab] OR \"Electronic Health\"[tiab] OR \"Electronic Mail\"[tiab] OR \"Electronic Mails\"[tiab] OR \"E-Mail\"[tiab] OR \"E-Mails\"[tiab] OR \"Handheld Computer\"[tiab] OR \"Handheld Computers\"[tiab] OR \"M-Health\"[tiab] OR \"Mobile Application\"[tiab] OR \"Mobile Applications\"[tiab] OR \"Mobile Health\"[tiab] OR \"Mobile Phone\"[tiab] OR \"Palm Pilot\"[tiab] OR \"Palm Pilots\"[tiab] OR \"Palmtop\"[tiab] OR \"Palm-Top\"[tiab] OR \"Personal Digital Assistant\"[tiab] OR \"Pocket PC\"[tiab] OR \"Pocket PCs\"[tiab] OR \"Short Message Service\"[tiab] OR \"Smart Phone\"[tiab] OR \"Smart Phones\"[tiab] OR \"Social Media\"[tiab] OR \"Tablet Computer\"[tiab] OR \"Tablet Computers\"[tiab] OR \"Text Message\"[tiab] OR \"Text Messages\"[tiab] OR App[tiab] OR Apps[tiab] OR CD[tiab] OR CDROM[tiab] OR Computerised[tiab] OR Computerized[tiab] OR Cyber[tiab] OR Digital[tiab] OR DVD[tiab] OR EHealth[tiab] OR Email[tiab] OR Facebook[tiab] OR Hypermedia[tiab] OR Hypertext[tiab] OR Internet[tiab] OR iPad[tiab] OR iPhone[tiab] OR Laptop[tiab] OR Messaging[tiab] OR MHealth[tiab] OR Online[tiab] OR PDA[tiab] OR PDAs[tiab] OR Podcast[tiab] OR Smartphone[tiab] OR SMS[tiab] OR Software[tiab] OR Texting[tiab] OR Tweet[tiab] OR Twitter[tiab] OR Virtual[tiab] OR Web[tiab] OR Webcast[tiab] OR Website[tiab] OR WeChat[tiab] OR Whatsapp[tiab] OR YouTube[tiab]) AND (Fathers[mh] OR \"Legal Guardians\"[mh] OR Mothers[mh] OR Parenting[mh] OR Parents[mh] OR \"Single Parent\"[mh] OR Father[tiab] OR Guardian[tiab] OR Mother[tiab] OR Parent[tiab]) AND (Adolescent[mh] OR Adolescen[tiab] OR Teen[tiab] OR Young[tiab] OR Youth*[tiab]) AND (\"Controlled Clinical Trial\"[pt] OR \"Randomized Controlled Trial\"[pt] OR Groups[tiab] OR Randomised[tiab] OR Randomized[tiab] OR Randomly[tiab] OR Trial[tiab]) NOT (Animals [mh] NOT Humans [mh])) NOT MEDLINE[sb]\n\nWe will inspect the references of all included studies within this review to identify further relevant studies.\n\nSelection of studies. Identified citations will be collected and imported into EndNote X8 (27) and duplicates removed. Titles and abstracts will be screened by the first researcher for assessment against the inclusion criteria for the review and her work was double-checked by the second researcher. Disagreement between these two researchers and a random selection of excluded papers will be reviewed by the team leader. Full texts of remaining articles will be obtained and reviewed against the inclusion criteria by AF and FS and their work will be checked and verified by the team leader.\n\n1. Extraction. A Microsoft Excel-based data extraction form designed for the purpose of this study will be used to extract the data such as details of population of the study, interventions, outcomes, and study design.\n\n2. Management. The data will be managed and summarized in Microsoft Excel (for Office 365) as a data entry and management tool to create the relevant tables and graphs. Review Manager 5.3 will be used for creating Risk of Bias tables and figure. Template for Intervention Description and Replication (TIDieR) will also be used to check for extracted of reported interventions from the trials.\n\nAssessment of risk of bias in included studies. The data related to the quality of each study will be extracted using Cochrane’s Risk of Bias Tool (Higgins et al., 2017). This data will then be double-checked by the second researcher. Disagreements between these two reviewers and a random sample of the data extraction were checked and verified by the team leader.\n\nAnalysing/synthesising the data. If we find enough homogeneous studies, we will consider conducting meta-analysis for the relevant outcomes. We will also consider that, because of the possible heterogeneity of the interventions, we might not be able to analyse the data; then we will report a summary of the studies beside their quality based on the assessment of risk of bias.\n\nIf the data meet the criteria for meta-analysis, we might consider running sensitivity analysis for the studies with higher contribution to the weight of the analysis and for the studies with higher heterogeneity. We may also consider subgroup analysis for this review to evaluate key features of parent-based interventions associated with most effective treatment outcomes.\n\nThis study has been registered with PROSPERO on 31st October 2018 (CRD42018114921).\n\nDissemination of information. We will publish the results of this review and will make the data accessible openly in re-usable format.\n\nStudy status. Search and screening for the study has been done and the data extraction, synthesis and writing the final report is ongoing.\n\n\nData availability\n\nNo data are associated with this article\n\nOpen Science Framework: PRISMA-P checklist for “The effectiveness of online parenting programs in promoting parents’ and adolescents’ mental wellbeing: a systematic review protocol”. https://doi.org/10.17605/OSF.IO/KZNQE (Shokraneh, 2019).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAinsworth MDS, Blehar MC, Waters E, et al.: Patterns of attachment: A psychological study of the strange situation. Oxford: Lawrence Erlbaum; 1978. Reference Source\n\nArulsubila M, Subasree R: Parenting and Psychological Wellbeing of Adolescents: An Intervention Study. IOSR Journal of Humanities and Social Science (IOSR-JHSS). 2017; 1–9. Reference Source\n\nBandura A: Social learning theory. New York (NY): General Learning Press. 1971. Reference Source\n\nCanals J, Voltas N, Hernández-Martínez C, et al.: Prevalence of DSM-5 anxiety disorders, comorbidity, and persistence of symptoms in Spanish early adolescents. Eur Child Adolesc Psychiatry. 2019; 28(1): 131–143. PubMed Abstract \| Publisher Full Text\n\nComer JS, Chow C, Chan PT, et al.: Psychosocial treatment efficacy for disruptive behavior problems in very young children: a meta-analytic examination. J Am Acad Child Adolesc Psychiatry. 2013; 52(1): 26–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDas JK, Salam RA, Lassi ZS, et al.: Interventions for Adolescent Mental Health: An Overview of Systematic Reviews. J Adolesc Health. 2016; 59(4S): S49–S60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhrenreich-May J, Rosenfield D, Queen AH, et al.: An initial waitlist-controlled trial of the unified protocol for the treatment of emotional disorders in adolescents. J Anxiety Disord. 2017; 46: 46–55. PubMed Abstract \| Publisher Full Text\n\nForoughe M, Stillar A, Goldstein L, et al.: Brief Emotion Focused Family Therapy: An Intervention for Parents of Children and Adolescents with Mental Health Issues. J Marital Fam Ther. 2019; 45(3): 410–430. PubMed Abstract \| Publisher Full Text\n\nHiggins JPT, Altman DG, Sterne JAC (editors): Chapter 8: Assessing risk of bias in included studies. In: Higgins JPT, Churchill R, Chandler J, Cumpston MS (editors). Cochrane Handbook for Systematic Reviews of Interventions version 5.2.0 (updated June 2017), Cochrane. 2017. Reference Source\n\nLindsay G, Band S, Cullen MA, et al.: Evaluation of the parent early intervention pathfinder 2nd Interim report. DCSF-RW035. London: DCSF. 2007; 59. Reference Source\n\nLong N, Edwards MC, Bellando J: Parent training interventions. In Handbook of Childhood Psychopathology and Developmental Disabilities Treatment. Springer, Cham. 2017; 63–86. Publisher Full Text\n\nOgundele MO: Behavioural and emotional disorders in childhood: A brief overview for paediatricians. World J Clin Pediatr. 2018; 7(1): 9–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPape K, Bjørngaard JH, Holmen TL, et al.: The welfare burden of adolescent anxiety and depression: a prospective study of 7500 young Norwegians and their families: the HUNT study. BMJ Open. 2012; 2(6): pii: e001942. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParmar K, Jain K: Enhancing Well Being Through Life Skills Training Among Tribal Youths. Indian Journal of Applied Research. 2019; 9(5). Reference Source\n\nResnick MD, Bearman PS, Blum RW, et al.: Protecting adolescents from harm. Findings from the National Longitudinal Study on Adolescent Health. JAMA. 1997; 278(10): 823–32. PubMed Abstract \| Publisher Full Text\n\nRyan R, O'Farrelly C, Ramchandani P: Parenting and child mental health. London J Prim Care (Abingdon). 2017; 9(6): 86–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSandal RK, Goel NK, Sharma MK, et al.: Prevalence of Depression, Anxiety and Stress among school going adolescent in Chandigarh. J Family Med Prim Care. 2017; 6(2): 2249–4863. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanders MR: Triple P-Positive Parenting Program as a public health approach to strengthening parenting. J Fam Psychol. 2008; 22(4): 506–17. PubMed Abstract \| Publisher Full Text\n\nSanders MR, Kirby JN, Tellegen CL, et al.: The Triple P-Positive Parenting Program: a systematic review and meta-analysis of a multi-level system of parenting support. Clin Psychol Rev. 2014; 34(4): 337–357. PubMed Abstract \| Publisher Full Text\n\nScholten WD, Batelaan NM, van Balkom AJ, et al.: Recurrence of anxiety disorders and its predictors. J Affect Disord. 2013; 147(1–3): 180–185. PubMed Abstract \| Publisher Full Text\n\nScott S, Gardner F: Parenting programs. In: Rutter M, Bishop D, Pine D, editors. Rutter’s child and adolescent psychiatry. Oxford: Wiley. 2015; 483–95. Publisher Full Text\n\nShokraneh F: PRISMA-P checklist for ‘The effectiveness of online parenting programs in promoting parents’ and adolescents’ mental wellbeing: a systematic review protocol. 2019. http://www.dx.doi.org/10.17605/OSF.IO/KZNQE\n\nWehry AM, Beesdo-Baum K, Hennelly MM, et al.: Assessment and treatment of anxiety disorders in children and adolescents. Curr Psychiatry Rep. 2015; 17(7): 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Comprehensive Action Plan: 2013-2020. 2011. Reference Source\n\nWorld Health Organization: Global Burden of Disease Study 2015. 2015. Reference Source\n\nYap MBH, Mahtano S, Rapee RM, et al.: A Tailored Web-Based Intervention to Improve Parenting Risk and Protective Factors for Adolescent Depression and Anxiety Problems: Postintervention Findings From a Randomized Controlled Trial. J Med Internet Res. 2018; 20(1): e17. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77911", "date": "01 Mar 2021", "name": "Sophie Bennett", "expertise": [ "Reviewer Expertise Mental health interventions for children and young people", "low intensity mental health interventions", "systematic reviews" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a protocol for a systematic review (with possibility for meta-analysis) of the effectiveness of online parent-based interventions that include parent training and aim to help parents understand, support and improve their adolescent child’s mental health and wellbeing. The methodology appears strong and is well written and the protocol has been registered on PROSPERO.\nOverall, my main comment is that I did not find that the background and aim of the systematic review were clear. I think that the review is of interventions that include a parenting element, but this wasn't clear until the methods.\nDescription of the condition I was unclear whether the authors were referring to 'parenting' programmes (typically used for the treatment of disruptive behaviour disorders), interventions delivered to parents that were aimed to treat anxiety and depression, or interventions for child mental health problems that included some parent component (e.g. intervention for child anxiety that include a session on psychoeducation for anxiety delivered to parents). Similarly, was the primary aim to understand the effect of the interventions on parent wellbeing, child wellbeing, or both and if so, why? If the focus is both, then the need to assess parent mental health should perhaps be explained in the description of the condition.\nDescription of parent interventions The description of parent interventions suggests that parent-focused interventions 'improve their relationship with adolescent children', which may not be the case for an anxiety intervention psychoeducation session, for example. The methods suggest that only interventions 'that include parent training' will be included and this seems an important point to clarify and expand on in the introduction.\nThe example intervention provided is of emotion focused family therapy and I was unclear why this one intervention was described in detail. This section ends with a suggestion that 'less is established in terms of potential variations in outcomes owing to contextual factors such as gender, age, socio-economic status and geographic setting/cultural context' but it is unclear why these factors relate to this specific review, which appears to focus on the online element?\n\nHow the intervention might work Again, this section discusses theories related to different interventions than those described in the section above (i.e. IY/Triple P vs. emotion focused family therapy described above).\nWhy it is important to do this review I was not clear from the previous sections why a review of online interventions specifically was needed. It would be helpful if previous sections provided a summary of existing related reviews so that it is clear why this review is needed and the gap it intends to fill.\n\nMethods These are comprehensive, with detailed search strategies.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "153274", "date": "04 Nov 2022", "name": "Carlo Schuengel", "expertise": [ "Reviewer Expertise parent-child attachment", "developmental psychopathology", "parenting", "theory of interventions", "research synthesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction\nWhile including parents in prevention and intervention aimed at children's psychosocial difficulties makes a lot of sense, the literature review that forms the basis for this argument needs strengthening. Under the heading of \"Description of the condition\", a rather broad conception of \"emotional difficulties\" is given, which is further described on the basis of epidemiological figures for anxiety and depression. Whether emotional difficulties may also include problems, such as post-traumatic stress, remains unclear, which is pertinent given the purpose of including the wider family in interventions. It is also unclear how the concept of emotional difficulties relates to the concept of mental wellbeing from the title.\n\nThe third paragraph under this heading cites findings and conclusions from some earlier studies, but it is unclear why these studies were selected for review here. For example, the paper by Pape et al. (2012) appears to be only tangentially relevant to the current study, as this study related to welfare factors. What rather is missing from the Introduction, is a developmental conceptualization of the central concept (mental wellbeing or emotional difficulties or anxiety and/or depression). Various developmental frameworks have been formulated that might be helpful for positioning the current review in the wider scientific literature (e.g., Sameroff's dialectical model of development or Bronfenbrenner's model).\n\nThe structure of the Introduction also needs work in the 5th paragraph (\"in terms of implementation...\"). While starting out to address implementation in relation to intervention characteristics, the paragraph then veers into discussing results of emotion focused therapy, after which the option of online intervention is raised. In all, this latter option appears out of the blue, again lacking a wider framework within which issues of intervention design and implementation can be conceptualized. This paragraph also contains factual statements that are not backed up by references (e.g., \"These interventions can incorporate...mental health\"). Also, following paragraph (\"while the aforementioned research...\") lists seemingly unrelated contextual factors, without any rationale why these factors (and not others) may be important to study.\n\nUnder the heading \"How the intervention might work\", two theories are described that have been the source of rationales for parent-focused interventions against depression and anxiety. I expect that when the conceptual framework for the field is described, the explanations for the contribution of parenting-focused components will also need to be revised. For instance, both social learning theory and attachment theory are discussed as underlying a causal role of parenting behaviors in the etiology of emotional difficulties. However, the evidence for a direct causal effect is quite weak. Not only do behavior genetic studies of internalizing problems in children show a moderate role of heritability and weak or absent roles of shared environmental factors. But meta-analyses have shown that the quality of attachment relationships with parents is only weakly or not at all predictively associated with internalizing problems (in contrast to their moderately predictive role for externalizing problems). See e.g., Groh et al (3). Although, see also Dagan et al. (2)).\n\nRather than or in addition to removing parenting behavior that causes anxiety/depression, parent interventions may also work as parents may mediate or moderate the effects of intervention components. For instance, by supporting their child with the homework exercises, motivating their child to engage with therapy. Given that the evidence for a direct involvement of parenting in the etiology of anxiety/depression is scant, it might be especially helpful to also consider the role of parenting in recovery or desistance (e.g., Scholten et al., (1)).\n\nRegarding the main research question of the review, it is unclear whether effectiveness regards the absolute efficacy or relative efficacy of such interventions, with research on e-mental health often focusing on non-inferiority rather than superiority trials.\nMethods\nIt is rather surprising, given the study objective, that the primary outcomes relate to the parents whereas the outcomes related to the mental health problems of the youth are only secondary. Should this not be the other way around if the review is to inform practice?\n\nHow were the search strings developed? Were these based on combining search strategies in other systematic reviews of online interventions and interventions in youth emotional difficulties? Was a thesaurus used? Were the strings piloted and then refined?\n\nThe data to be extracted are left rather open. However, further conceptualization of the study might allow for more concrete, a priori selection of study characteristics, such a intervention components, purpose of the parent-focused intervention components.\n\nHow large are the random samples of papers on which the reliability of study selection is estimated? How will the in/exclusion criteria be adapted on the basis of persistent differences in inclusion? Similarly for the reliability of the risk of bias rating.\n\nWhat are the criteria for conducting a quantitative synthesis (meta-analysis) of the retrieved studies? What meta-analytic approach will be used?\nMinor points:\nReference is missing for Emotion Focused Therapy (5th paragraph).\n\nWhy is inclusion limited to studies with young people between 10 and 19?\n\nWhy are studies with youth with health conditions excluded?\n\nWhy are Web of Science and Scopus not searched? Their coverage only partly overlaps with the databases now selected, where MEDLINE and PubMed appear redundant.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/8-1842
https://f1000research.com/articles/8-1836/v1	31 Oct 19	{ "type": "Research Article", "title": "Voiding profile in recipients post renal transplant: A prospective observational study", "authors": [ "Dyandra Parikesit", "Indra Wicaksono", "Muhamad Iqbal Tawfid", "Fina Widia", "Harrina Erlianti Rahardjo", "Dyandra Parikesit", "Indra Wicaksono", "Muhamad Iqbal Tawfid", "Fina Widia" ], "abstract": "Background: Renal transplantation (RTX) is thought to have high survival rates. However, patients with long-term dialysis have decreased bladder function due to disuse. High urine production after RTX surgery might cause patients to have urinary symptoms, thus decreasing their quality of life. The aim of this study was to evaluate voiding characteristics of patients after RTX surgery. Methods: All patients were diagnosed with chronic kidney disease (CKD) and underwent kidney transplantation from a living donor. Anthropometric parameters, physical examinations, cause of CKD, daily urine production, types and duration of dialysis, and basic laboratory examination were collected before transplant surgery. Post-operative examinations included laboratory examination, international prostate symptom score (IPSS; for male patients only), overactive bladder symptom score (OABSS), uroflowmetry, and post void residue (PVR). Results: 71 patients were evaluated with a mean age of 46 ± 17.9 years, with male and female ratio of 52:19. Significant negative correlation was seen between duration of dialysis and daily urine production (r: -0.68, p<0.01). Majority of patients had a maximum flow rate of >15 cc/s (70.4 %) with average flow of 22 ± 9.8 cc/s. The majority of patients had PVR <100 cc (91.5%) with median PVR of 33.5 cc (range, 2.3 – 142 cc). IPSS result showed that frequency [2 (0 – 5)] and nocturia [2 (0 – 5)] are the main problem in these patients (n = 52). OABSS questionnaire also showed that frequency (OABSS 1; score 1 [1-2]) and nocturia (OABSS 2; score 2 [0-3]) was the main symptoms reported by patients. Conclusion: After RTX, the majority of patients experienced urinary frequency and nocturia problems due to various causes such as increased daytime and nighttime urine production, urinary tract infection, changes in bladder capacity (both small and large), and a decrease in bladder compliance.", "keywords": [ "renal transplant", "uroflowmetry", "international prostate symptom score", "overactive bladder symptom score" ], "content": "Introduction\n\nChronic kidney disease (CKD) is a health burden affecting the global population, which has a high economic cost to health care, increases the risk of cardiovascular morbidity and decreases overall quality of life (QoL)1. The global prevalence of the five stages of CKD is between 11 – 13% with the majority of patients being stage 3, while stages 3–5 (end stage renal disease) is 10.6% (9.2 – 12.2%)1. Diabetes mellitus (DM) and hypertension (HT) is known to be significantly associated with CKD, with global prevalence of 8.5% in 20142 and 31% in 20103, respectively. According to the Basic Health Research in Indonesia (Riskesdas) in 20184, prevalence of DM and HT was 10.9% and 34.1%, however both non-communicable disease contribute to around 52% and 24% of people with CKD, respectively5. Previously, dialysis was thought to be the best treatment for patients with CKD, nowadays transplantation is a more attractive treatment due to its higher survival rates, QoL, cost effectiveness, and better clinical results6,7. On the other hand, patients with CKD who experienced prolonged oliguria and or anuria may develop bladder function complications and high intravesical pressure, known as neurogenic bladder, which may lead to renal impairment8. QoL might also be impacted due to obstruction and an overactive bladder, thus evaluation of bladder function is paramount after RTX9.\n\nThe aim of this study is to evaluate voiding characteristics of patients that have undergone RTX surgery in Jakarta, Indonesia.\n\n\nMethods\n\nIn this prospective observational study, 71 patients diagnosed with CKD and had undergone a kidney transplantation from a living donor between January and December 2018 in Cipto Mangunkusumo General Hospital, Jakarta were examined. Total sampling method were implemented to include all potential subjects. Inclusion criteria for participants were consenting adults (> 18 years old) with sterile urine. Patients with neurogenic bladder and history of urinary obstruction were excluded.\n\nRTX procedures were performed according to standardized techniques including a Gibson incision, extraperitoneal dissection, and end-to-side or side-to-side anastomosis of the donor to the recipient iliac vessels. Extravesical ureteral implantation (ureteroneocystostomy) was performed in an antireflux manner according to the Lich-Gregoir technique10. In all patients, a double-J stent was placed for 4 weeks and a transurethral catheter for the duration of hospital stay after operation.\n\nPatients were informed regarding data collection before undergoing transplantation surgery. Pre and post-operative parameters were collected from medical record data. Voiding data (uroflowmetry), IPSS, and OABSS questionnaire was collected directly from the patients during follow up 1 month after the surgery.\n\nBefore transplant, anthropometric parameters, physical examinations, cause of CKD, daily urine production, types and duration of dialysis, and basic laboratory examination were collected (Table 1). Anuria and oliguria were defined as urine production of <100 cc/day and between 100 and 400 cc/day, respectively11.\n\nBMI: body mass index; CKD: chronic kidney disease; HD: hemodialysis; CAPD: continuous ambulatory peritoneal dialysis; FBG: fasting blood glucose; RBG: random blood glucose. * Preemptive dialysis\n\nPost-transplant, laboratory examination, international prostate symptom score (IPSS; male patients only), overactive bladder symptom score (OABSS), uroflowmetry, and post void residue (PVR) was gathered (Table 2).\n\nPVR: post-void residual; IPSS: international prostate symptom score; QOL: quality of life; OABSS: overactive bladder symptom score. Wilcoxon signed rank test was used for statistical analysis. ⊗ Normally distributed, paired T test was used for statistical analysis. * p <0.05, compare to previous value; ** p <0.001, compare to previous value.\n\nFrequency, urgency, and nocturia were added and categorized as storage score, while the sum of incomplete emptying, intermittency, weak stream and straining were considered to be voiding score. Total IPSS score were classified into two groups: mild (score 0 – 7), and moderate-severe (score 8 – 35). A QoL question was also included as part of the IPSS questionnaire. The IPSS questionnaire was translated into Indonesian, reviewed and validated by the Indonesian Urological Association12. The sum score of daytime frequency, nocturia, urgency and urge incontinence made up the OABSS questionnaire with a maximum (worst) score of 2, 3, 5, and 5, respectively. A higher score indicated more severe symptoms with a maximum score of 15. A validated Indonesian version of this questionnaire was used13.\n\nThe maximum flow rate (Qmax) was measured with stationary uroflow recorder (Medtronic Urodyn® 1000 system), where <15 cc/s were associated with lower bladder outlet obstruction (BOO) (specificity of 38%, positive predictive value of 67%, and sensitivity of 82%)14. Consecutive urine voided >150 cc was accepted as valid14. PVR was measured after uroflowmetry examination with ultrasonography in supine position using an elliptical formula15. This examination was taken 4 weeks after RTX.\n\nSpearman’s correlation was used to analyze the association between all continuous variables. Wilcoxon signed rank test was used for comparing medians in abnormally distributed and paired T test was used for comparing means in normally distributed continuous values. A P value of less than 0.05 and 0.001 were considered statistically significant and very statistically significant, respectively. All analyses were performed with commercial statistical software (SPSS version 23 for Macintosh; SPSS, Chicago, IL).\n\nThis research was conducted under ethical review and approval from The Ethics Committee of Faculty of Medicine, University of Indonesia corresponding to protocol number 17-07-0631 and ethical approval number 602/UN2.F1/ETIK/2017.\n\nAll participants included in this research provided written informed consent according to ethical guideline from The Ethics Committee of Faculty of Medicine, University of Indonesia.\n\n\nResults\n\nBetween January and December 2018, there were 107 RTX surgeries performed in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. A total of 36 patients were excluded from this study due to various reasons (15 had previous surgeries for urinary tract obstruction, 2 had previous RTX surgeries, 4 had acute rejection, 2 had hyper-acute rejection and immediate allograft nephrectomies, and 13 patients did not give consent). Therefore, a total of 71 patients were included in this study (Table 1). The mean age of patients were 46 ± 17.9 years old with male and female ratio of 52:19, respectively.\n\nThe major cause of CKD was HT (59.2%), followed by a combination of HT and DM (26.8%), other cause such as glomerulonephritis, nephrotic syndrome, polycystic kidney disease, and autoimmune disease (12.7%), and DM alone (1.4%). The mean preoperative creatinine was 5.86 ± 1.3 mg/dL. The majority of diabetic patients had controlled glucose level with median fasting blood glucose of 87 (72 – 242) mg/dL and HbA1c of 5.1% (4 – 8.2), according to the American Diabetes Association guidelines16. A total of 28% and 26.8% of patients had anuria (urine production <100 cc per day) and oliguria (urine production <400 cc per day), respectively. The main renal replacement therapy (RRT) used by patients was hemodialysis (98.6%) with median duration of 10 months [range, 0.3 (preemptive dialysis) – 120 months]. Significant negative correlation was seen between duration of dialysis and daily urine production (r: -0.68, p<0.01), as shown in Figure 1.\n\nAll postoperative variables are shown in Table 2. There are trends of decreased creatinine from the first day of 4.8 ± 2.27 mg/dL to 1.2 mg/dL (0.6 – 7.9) at day 5-post operation. The increase in urine production was also seen before and after the operation. During the first day, urine production varied between patients with a median of 6150cc (1530 – 16150) per day. However, from the second day onwards, urine production started to decrease from 6428.3 ± 3035.7 cc per day to 4800.2 ± 1705 cc per day at day 5. On uroflowmetry examination, the majority of patients had Qmax >15 cc/s (70.4%) with average flow of 22 ± 9.8 cc/s. Median voided volume was 208cc (25 – 800); 19 patients (26.8%) had insignificant voided volume (<150cc), which made the examination invalid. However, only 2 out of 19 patients had urinary retention (PVR >100 cc). The majority of patients had PVR <100 cc (91.5%) with median PVR of 33.5 cc (2.3 – 142). IPSS result shows that frequency [2 (0 – 5)] and nocturia [2 (0 – 5)], components of irritative symptoms, were the main problem in these patients with a total number of 20/52 (38.5%) and 40/52 (76.9%) patients, respectively. In total, 35 patients (67.3%) had only mild symptoms, with a median total IPSS score of 6 (0 – 21). Nevertheless, the majority of patients were “pleased” [1 (0 – 3)] on their QoL assessment. Similar to IPSS, the OABSS questionnaire showed that frequency (OABSS 1) and nocturia (OABSS 2) was the main symptoms reported by patient, with scores of 1 (0 – 2) and 2 (0 – 3), respectively.\n\n\nDiscussion\n\nRTX is thought to be a better alternative RRT compared to dialysis in terms of QoL, long-term survival, and financial burden6,7. However, a sudden increase in urine volume inside the bladder, a smaller bladder capacity, and bladder smooth muscle atrophy might cause irritative and/or obstructive symptoms, which in the long term might lead to decrease of graft function17–21. Low-pressure urine storage, effective bladder emptying, and minimal residual volume is necessary for maintaining sufficient and prolonged renal function.\n\nMedian daily urine production in our study was 400cc (0 – 4000) with 28.8% and 26.8% of patients having anuria and oliguria, respectively. Median duration of dialysis, 98.6% used hemodialysis, was 10 months (preemptive – 120). There was significant negative correlation between daily urine production and duration of dialysis (r: -0.68, p<0.01). This shows that longer duration of dialysis may decrease urine production, resulting in smaller bladder capacity8,17,19–24. Mean Qmax was 22 ± 9.8 cc/s, with 70.4% of patients exceeding 15 cc/s and valid examination (voided volume > 150 cc) in only 73.2% of patients. This result was not associated with gender (p>0.05). There was no definitive normal value of Qmax in women to determine BOO. However, a study by Mahfouz et al. shows that normal range of Qmax in asymptomatic women was 13 to 25 cc/s25. Therefore, in the present study we set the same threshold for Qmax between men and women to define BOO. To the authors knowledge, there are not many studies evaluating Qmax in patients after RTX; however, a study by Gratzke et al., concluded that in male patients with a long history of dialysis, early operative intervention due to urinary retention must be anticipated after RTX26. In our study, 6 patients (3 male and 3 female) had urinary retention (PVR > 100 cc) after uroflowmetry. Two patients had HT alone and 4 patients had both HT and DM as the cause of CKD. In both animal models and human, HT is shown to cause bladder dysfunction by altering beta-adrenergic receptor-mediated detrusor relaxation and alpha(1)-adrenergic receptor-mediated contraction of the bladder neck27.\n\nIn the present study, the main symptoms patients experienced after RTX was frequency of urination and nocturia with a total of 20/52 (38.5%) and 40/52 (76.9%) patients, respectively. This result was similar to other studies by Zermann et al.21 (frequency 87% and nocturia 94%), Van der Weide et al.20 (frequency 54% and nocturia 60%), and Lebadi et al.28 (nocturia 71.1%). Frequency might be due to thick-walled uncompliant high-pressure bladders, which might be caused by increased daytime production of urine, urinary tract infection (UTI) (common condition in post-transplant patients), and small bladder capacity (due to bladder atrophy)20. On the other hand, nocturia is mainly caused by increase in nighttime urine production (due to dysfunctional diurnal rhythm in urine production), large bladder capacity (which might lead to increase PVR and UTI), and decreased bladder compliance due to aging28. Both symptoms might also be caused by increased oxidative stress, which damages nerves, epithelium and smooth muscle of the bladder; however more studies are warranted. Similar results were shown with the OABSS questionnaire in the current study, which suggests patients after RTX have symptoms of mild-moderate overactive bladders. Regardless of this fact, the majority of patients’ QoL were not affected; 94.2% of all patients were pleased after RTX due to the fact that they do not need RRT, and a similar result was found by another study21.\n\nIn this study, one male older patient had decreased urine production (910 cc/day at day 3 to 375 cc/day at day 5) and increased creatinine (12.2 mg/dL at day 3 to 13.1 mg/dL at day 4, decreased to 7.9 mg/dL at day 5) starting from the third day after RTX, which suggested delayed graft function. This patient had left ventricular ejection fraction (LVEF) of 56%, chronic heart failure, and long term controlled HT. According to Rusinaru et al., renal dysfunction was found in around 14% of cases with heart failure (LVEF ± 50%), which is caused by intrinsic nephropathy, poor renal perfusion, vasoconstriction, and renal venous congestion29. Renal dysfunction is increased considerably with the degree of heart failure30. One month after surgery the patient had creatinine level of 1.9 mg/dL with normal Qmax and PVR of 33 cc/s and 35.4 cc, respectively. This proves that patients with decreased LVEF had increased risk for delayed graft and worse long-term graft function31.\n\nSeveral limitations of this study were: 1) urodynamic evaluation was not used due to the limited financial and technical resource, 2) short duration of follow-up (1 month) was due to the variety of origin of transplant patients across Indonesia’s archipelago (difficult to maintain long-term follow up). In the future, prospective long-term follow up studies may be needed to precisely confirm our findings.\n\n\nConclusion\n\nDaily urine production significantly correlates to duration of dialysis which, in the long-term, might alter lower urinary tract function. After RTX, the majority of patients experienced frequency and nocturia problems due to various causes, such as increased daytime and nighttime urine production, UTI, bladder capacity (both small and large), and decreased bladder compliance. Regardless of this fact, the majority of patients’ QoL were not affected; 94.2% of all patients were pleased after RTX due to the fact that they do not need RRT.\n\n\nData availability\n\nHarvard Dataverse: Voiding Profile in Recipients Post Renal Transplant, https://doi.org/10.7910/DVN/XGKFWV32\n\nThis project contains the following underlying data:\n\nData File 4. SPSS tabular data\n\nHarvard Dataverse: Voiding Profile in Recipients Post Renal Transplant, https://doi.org/10.7910/DVN/XGKFWV32\n\nThis project contains the following extended data:\n\nData File 1. Ethical approval from The Ethics Committee The Ethics Committee of Faculty of Medicine, University of Indonesia\n\nData File 2. Scatter plot and Spearman Correlation Coefficient of duration of dialysis to daily urine production.\n\nHarvard Dataverse: STROBE checklist for ‘Voiding Profile in Recipients Post Renal Transplant’, https://doi.org/10.7910/DVN/XGKFWV32\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nHill NR, Fatoba ST, Oke JL, et al.: Global Prevalence of Chronic Kidney Disease - A Systematic Review and Meta-Analysis. PLoS One. 2016; 11(7): e0158765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoglic G: WHO Global report on diabetes: A summary. Int J Noncommun Dis. 2016; 1(1): 3–8. Publisher Full Text\n\nBloch MJ: Worldwide prevalence of hypertension exceeds 1.3 billion. J Am Soc Hypertens. 2016; 10(10): 753–4. PubMed Abstract \| Publisher Full Text\n\nHealth Reseach and Development: Basic Health Research (RISKESDAS)2018. Reference Source\n\nIndonesian Society of Nephrology: 9th Report Of Indonesian Renal Registry. 2016. Reference Source\n\nRoodnat JI, Zietse R, Mulder PG, et al.: The vanishing importance of age in renal transplantation. Transplantation. 1999; 67(4): 576–80. PubMed Abstract \| Publisher Full Text\n\nSilva SB, Caulliraux HM, Araujo CA, et al.: Cost comparison of kidney transplant versus dialysis in Brazil. Cad Saude Publica. 2016; 32(6): pii: S0102-311X2016000605005. PubMed Abstract \| Publisher Full Text\n\nAsakura H, Nakamura K, Tachibana M, et al.: Evaluation of lower urinary tract function in renal transplant recipients by urodynamic study. Transplant Proc. 1998; 30(1): 119–21. PubMed Abstract \| Publisher Full Text\n\nYanishi M, Kawa G, Nakamoto T, et al.: [Lower Urinary Tract Symptoms and Functions after Renal Transplantation at Our Hospital]. Nihon Hinyokika Gakkai Zasshi. 2015; 106(4): 249–54. PubMed Abstract \| Publisher Full Text\n\nRiedmiller R, Gerharz EW: Antireflux surgery: Lich-Gregoir extravesical ureteric tunneling. BJU Int. 2008; 101(11): 1467–1482. PubMed Abstract \| Publisher Full Text\n\nBellomo R, Ronco C: Indications and criteria for initiating renal replacement therapy in the intensive care unit. Kidney Int Suppl. 1998; 66: S106–9. PubMed Abstract\n\nMonoarfa R, Mochtar C: Validation of Indonesian version of IPSS. Indonesian Journal of Urology. 2014; 21(1): 15–9. Publisher Full Text\n\nSumardi R, Mochtar CA, Junizaf, et al.: Test - retest reliability of the Indonesian version of the Overactive Bladder Symptom Score (OABSS) and its correlation with standard assessment tools. Acta Med Indones. 2012; 44(3): 214–21. PubMed Abstract\n\nGratzke C, Bachmann A, Descazeaud A, et al.: EAU Guidelines on the Assessment of Non-neurogenic Male Lower Urinary Tract Symptoms including Benign Prostatic Obstruction. Eur Urol. 2015; 67(6): 1099–109. PubMed Abstract \| Publisher Full Text\n\nChan E, Smith K, Mohamoud G, et al.: Tri-Axial Ellipsoid Volume Calculation: A Method for Bladder Volume Estimation. J Med Imaging Radiat Oncol. 2008; 44(1): 46–47. Publisher Full Text\n\nAmerican Diabetes A: 6. Glycemic Targets: Standards of Medical Care in Diabetes-2018. Diabetes Care. 2018; 41(Suppl 1): S55–S64. Publisher Full Text\n\nMitsui T, Moriya K, Morita K, et al.: Risk Factors for Lower Urinary Tract Dysfunction and Symptoms After Successful Renal Transplantation. Ann Transplant. 2015; 20: 757–63. PubMed Abstract \| Publisher Full Text\n\nSarier M, Tekin S, Duman I, et al.: Results of transurethral resection of the prostate in renal transplant recipients: a single center experience. World J Urol. 2018; 36(1): 99–103. PubMed Abstract \| Publisher Full Text\n\nSerrano DP, Flechner SM, Modlin CS, et al.: Transplantation into the long-term defunctionalized bladder. J Urol. 1996; 156(3): 885–8. PubMed Abstract\n\nVan der Weide MJ, Van Achterberg T, Smits JP, et al.: Causes of frequency and nocturia after renal transplantation. BJU Int. 2008; 101(8): 1029–34. PubMed Abstract \| Publisher Full Text\n\nZermann DH, Janitzky A, Hohne M, et al.: Frequency and nocturia after successful renal transplantation: a normal situation? BJU Int. 2006; 97(3): 555–8. PubMed Abstract \| Publisher Full Text\n\nChen JL, Lee MC, Kuo HC: Reduction of cystometric bladder capacity and bladder compliance with time in patients with end-stage renal disease. J Formos Med Assoc. 2012; 111(4): 209–13. PubMed Abstract \| Publisher Full Text\n\nHotta K, Miura M, Wada Y, et al.: Atrophic bladder in long-term dialysis patients increases the risk for urological complications after kidney transplantation. Int J Urol. 2017; 24(4): 314–9. PubMed Abstract \| Publisher Full Text\n\nSchmaelzle JF, Cass AS, Hinman F Jr: Effect of disuse and restoration of function on vesical capacity. J Urol. 1969; 101(5): 700–5. PubMed Abstract \| Publisher Full Text\n\nMahfouz W, Al Afraa T, Campeau L, et al.: Normal urodynamic parameters in women: part II--invasive urodynamics. Int Urogynecol J. 2012; 23(3): 269–77. PubMed Abstract \| Publisher Full Text\n\nGratzke C, Pahde A, Dickmann M, et al.: Predictive factors for urinary retention following kidney transplantation in male patients. Scand J Urol Nephrol. 2012; 46(1): 44–7. PubMed Abstract \| Publisher Full Text\n\nMichel MC, Barendrecht MM: Physiological and pathological regulation of the autonomic control of urinary bladder contractility. Pharmacol Ther. 2008; 117(3): 297–312. PubMed Abstract \| Publisher Full Text\n\nLebadi M, Monfared A, Atrkar Roshan Z, et al.: The Prevalence of Nocturia and Nocturnal Polyuria After Renal Transplantation and Associated Factors. Nephro-Urol Mon. 2018; 10(1): e60128. Publisher Full Text\n\nRusinaru D, Buiciuc O, Houpe D, et al.: Renal function and long-term survival after hospital discharge in heart failure with preserved ejection fraction. Int J Cardiol. 2011; 147(2): 278–82. PubMed Abstract \| Publisher Full Text\n\nSarnak MJ: A patient with heart failure and worsening kidney function. Clin J Am Soc Nephrol. 2014; 9(10): 1790–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzález Monte E, Mora MT, Polanco N, et al.: Impact of left ventricular dysfunction on renal transplant survival: study of paired kidneys from the same donor. Transplant Proc. 2015; 47(1): 70–2. PubMed Abstract \| Publisher Full Text\n\nParikesit D: Voiding Profile in Recipients Post Renal Transplant. Harvard Dataverse, V1, UNF:6:/AP2A2KeUpyVddKO4irntA== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/XGKFWV" }	[ { "id": "86192", "date": "10 Jun 2021", "name": "Marcelo Lopes de Lima", "expertise": [ "Reviewer Expertise Kidney Transplant." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a study that is very interesting and properly conducted.\nI suggest some changes with the intention of improving the understanding of the results:\nIf the aim of this study was to evaluate voiding characteristics of patients after RTX surgery, it would be better concluded only with the phrase “After RTX, the majority of patients experienced frequency and nocturia problems”; all the other affirmations are pertinent in the discussion of the paper, but not in the conclusion.\n\nI think that urinary infection can not be labeled as cause of symptoms based on the results of this paper, because inclusion criteria for participants were adults with sterile urine.\n\nPatients with neurogenic bladder and history of urinary obstruction were excluded, but urodynamic evaluation was not performed. How do the authors explain this?\n\nI agree that the most influential of limitations of this study were absence of urodynamic evaluation and short follow-up, but the findings may guide early drug control of symptoms in kidney transplant patients. This may be discussed.\n\nGood job, I hope I’ve helped.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "87269", "date": "10 Jun 2021", "name": "Doaa M Salah", "expertise": [ "Reviewer Expertise Pediatric Nephrology and Transplantation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments to the authors: This study is interesting, well written and informative about voiding profile after RTX. The authors well-addressed the issue having impact on graft function and patient QoL and found that prolonged dialysis duration negatively impacted urine flow after RTX, they also reported frequency and nocturia as the most prevalent symptoms. Nevertheless; patient`s QoL is still very satisfactory.\nMore specific comments:\nThe term CKD is better to be replaced in certain places (as when authors mention need for RRT or RTX or stage 5) by ESKD (end stage kidney disease).\nIntroduction:\nToo many details were mentioned about CKD epidemiology, prevalence and causes while only the last paragraph focused on the importance of this study.\n\nIt would be better to summarize CKD data in one small paragraph than to mention in more details the hypothesis of voiding dysfunction (and its pattern) after RTX and its impact on renal graft and patient QoL.\n\nMethod:\nIt is not clear what the authors meant by inclusion of adults with sterile urine. Do you mean those with no history of UTI before RTX or those who did not experience UTI after RTX until they were enrolled into the study? Please clarify.\n\nResults:\n26.8% of included patients had DM. Why are the authors sure that diabetic neuropathy is not a confounding factor of interpretation of voiding disorders during evaluation? This issue has to be clarified in the methods and discussion sections.\n\nIn Tables 1 & 2; the authors have to change its title or to mention the number of patients (n=71). It`s impressive from the current title of the table that all transplanted patients were described. While it was mentioned that 36 transplanted patients were excluded from this analysis.\n\nThe authors did not mention UTI incidence/frequency after RTX among included patients, taking into account that UTI is one of the most common causes of urine retention after RTX.\n\nDiscussion:\nThe authors mentioned in the 2nd paragraph that \"Median duration of dialysis was 10 months (preemptive – 120)\". What do you mean by 'preemptive - 120'?\n\nThe authors stated \"In both animal models and human, HT is shown to cause bladder dysfunction by altering beta-adrenergic receptor-mediated detrusor relaxation and alpha(1)- adrenergic receptor-mediated contraction of the bladder neck\". The explanation is perfect however it does not rule out that HT is a confounding factor for incidence of urine retention (PVR > 100 cc) after RTX - I think this should be mentioned in limitations of your study.\n\n\" The majority of patients’ QoL were not affected due to the fact that they do not need RRT, and a similar result was found by another study21\" - Too many studies (old and recent ones) reported this fact. The authors can use at least more than one study to prove this fact and ensure that RTX still carries better QoL than RRT despite some voiding abnormalities.\n\n\"In this study, one male older ... This proves that patients with decreased LVEF had increased risk for delayed graft and worse long-term graft function\" - I wonder how this story is relevant to your main aim and focus of the study?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1836
https://f1000research.com/articles/8-1834/v1	31 Oct 19	{ "type": "Software Tool Article", "title": "Profile Comparer Extended: phylogeny of lytic polysaccharide monooxygenase families using profile hidden Markov model alignments", "authors": [ "Gerben P. Voshol", "Peter J. Punt", "Erik Vijgenboom", "Gerben P. Voshol", "Peter J. Punt" ], "abstract": "Insight into the inter- and intra-family relationship of protein families is important, since it can aid understanding of substrate specificity evolution and assign putative functions to proteins with unknown function. To study both these inter- and intra-family relationships, the ability to build phylogenetic trees using the most sensitive sequence similarity search methods (e.g. profile hidden Markov model (pHMM)–pHMM alignments) is required. However, existing solutions require a very long calculation time to obtain the phylogenetic tree. Therefore, a faster protocol is required to make this approach efficient for research. To contribute to this goal, we extended the original Profile Comparer program (PRC) for the construction of large pHMM phylogenetic trees at speeds several orders of magnitude faster compared to pHMM-tree. As an example, PRC Extended (PRCx) was used to study the phylogeny of over 10,000 sequences of lytic polysaccharide monooxygenase (LPMO) from over seven families. Using the newly developed program we were able to reveal previously unknown homologs of LPMOs, namely the PFAM Egh16-like family. Moreover, we show that the substrate specificities have evolved independently several times within the LPMO superfamily. Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification.", "keywords": [ "LPMO", "HMM", "Hidden Markov Model", "Lytic Polysaccharide Mono-oxygenase", "phylogeny" ], "content": "Introduction\n\nRenewable feedstocks, such as wheat straw, rice straw and other agricultural waste residues are used by the bioindustry for the production of sugars and value-added products. One of the first steps in this process is the enzymatic breakdown of these raw materials into smaller building blocks. For this, hydrolytic enzyme cocktails are extensively used. However, some biopolymers are resistant to complete enzymatic degradation by available enzyme cocktails. Lytic polysaccharide monooxygenases (LPMOs) are a relatively new class of metalloenzymes that can perform oxidative cleavage and aid breakdown by conventional hydrolytic enzymes (Harris et al., 2010; Vaaje-Kolstad et al., 2010).\n\nCurrently there are seven families of LPMOs defined in the Carbohydrate–Active Enzymes database (CAZy) (Lombard et al., 2014), namely the auxiliary activity families AA9 (formerly GH61), AA10 (formerly CBM33), AA11 (Hemsworth et al., 2014), AA13 (Vu et al., 2014), AA14 (Couturier et al., 2018), AA15 (Sabbadin et al., 2018; Voshol et al., 2017) and AA16 (Filiatrault-Chastel et al., 2019; Voshol et al., 2017). Although identifying members belonging to these known families is relatively easy, it is more difficult to identify members belonging to potentially novel LPMO families (Lo Leggio et al., 2015), given the very low level of overall sequence similarity between LPMO families. Therefore, we developed a profile hidden Markov model (pHMM) and used it to mine several genomes for new LPMO families (Voshol et al., 2017). pHMM-sequence searches are sensitive enough to identify putative LPMOs, but they are not suitable to establish the evolutionary relationship between these LPMOs. For example, a pHMM build from an alignment of AA13s was only able to identify AA13s (Lo Leggio et al., 2015) indicating that a more sensitive approach is necessary to build a phylogeny for all LPMOs.\n\npHMM-pHMM alignments are the most sensitive for this purpose (Sadreyev & Grishin, 2008; Söding, 2005). In 2017, Huo and colleagues developed a pHMM phylogentic tree approach and used it to study the evolutionary relationship of CAZy protein families with pHMM-pHMM alignments (pHMM-tree; Huo et al., 2017). Unfortunately, due to the exponential time required for generating the distance matrix and the tree, the number of pHMMs which can be included in the phylogenetic tree is limited (max 500). Therefore, this program is not applicable to study the relationship of proteins within large families.\n\nIn this study we apply both pHMM-sequence searches and pHMM-pHMM alignments to gain a deeper understanding of LPMO domain organization and phylogeny. To overcome the limitations of pHMM-tree, we extended the original Profile Comparer program (PRC; Madera, 2008) for the construction of large pHMM phylogenetic trees (>1800 HMMs) and added several additional capabilities. The resulting program, named PRCx (PRC eXtended) is several orders of magnitude faster than pHMM-tree and was used to reveal both the inter- and intra-family LPMO evolutionary relationship. Moreover, using PRCx, we were also able to reveal a previously unknown distantly related member of the LPMO superfamily.\n\n\nMethods\n\nTo create the initial LPMO dataset (See Figure 1), the UniprotKB database (downloaded on 18-10-2017) was searched for 10 iterations using a truncated version (containing only the “core” LPMO domain, see Figure 2) of the previously published pHMM (Voshol et al., 2017). This core LPMO pHMM has a total model length of 165, starting at the N-terminal histidine, that makes up part of the histidine brace, up to a relatively well conserved threonine. With the aim to analyze proteins related to LPMOs an E-value of 1 was used. It is possible to extend the dataset with another ~20% using an E-value of 1000 at the expense of increasing the number of unrelated hits (Wistrand & Sonnhammer, 2005).\n\nThe steps are as follows. Create a sequence database, cluster sequences and construct alignments from them. Convert these alignments to pHMMs and construct the tree. The resulting tree can be used for example to identify relatives, extract sequences from a clade and mine determine their accessory domains or perform structural alignments to identify important residues.\n\nThe height of the letter indicates the information content (level of conservation and the number indicates the position in the pHMM.\n\nAfter generating the initial dataset, the taxonomic distribution and the presence of accessory domains were analyzed using the HMMER web server (Potter et al., 2018). The sequences were retrieved and a non-redundant dataset was created by clustering sequences at a 100% sequence identity using the CD-HIT toolset (Fu et al., 2012; Li & Godzik, 2006). The non-redundant dataset was subsequently clustered at 70% sequence identity and sequences contained within those clusters were grouped into their respective fasta files. Fasta files containing two or more sequences where aligned using the kalignP alignment program (Lassmann et al., 2009; Shu & Elofsson, 2011) and pHMMs were built using HMMer 3.0 (Eddy, 2011). This resulted in 1828 pHMMs and 2296 singletons (sequences which did not cluster at 70% identity with any other sequence). PHMMs from dbCAN2 and PFAM protein families were downloaded from their respective web servers (El-Gebali et al., 2019; Yin et al., 2012; Zhang et al., 2018). PRCx was used to search for distantly related LPMO PFAM protein families that were used as an outgroup during the tree building stage (see Results for more details).\n\nSeveral new features were added to the original PRC program (Madera, 2008), including the ability to (i) use HMMer3.0 pHMM files, (ii) build pHMM using single or aligned fasta files, (iii) speed up pHMM-pHMM searches using prefiltering and (iv) generate a PHYLIP compatible distance matrix and associated UPGMA Newick formatted phylogenetic tree (Felsenstein, 1989).\n\nThe original PRC program has the ability to, amongst others, load SAM3, HMMer2 and PSI-Blast profile files (Madera, 2008). However, since the release of the original PRC program in 2008, a new version of HMMer was released in 2011 (Eddy, 2011). Soon thereafter, public databases such as PFAM and dbCAN updated to the newer HMMer version. Since this format is used so extensively, we added support for HMMer3.0 pHMM files to PRC.\n\nTo facilitate both pHMM building and fast prefiltering, support for sequence context-specific pseudocounts was added. The idea behind context-specific pseudocounts is that the local environment around an amino acid determines what mutations can occur at that particular amino acid location (Overington et al., 1992). This rationale has been applied in numerous programs to increase the sensitivity of protein-protein alignments (Gambin et al., 2002; Huang & Bystroff, 2006; Jung & Lee, 2000). For PRCx we implemented the context-specific pseudocount method for the context-specific BLAST program (Biegert & Söding, 2009).\n\nAn additional advantage of implementing support for context-specific libraries is the ability to reduce the amino acid probability vectors of a pHMM to a discretized alphabet. This was achieved by the same method as used by HHblits to translate the amino acid profiles to 219 distinct letters (Remmert et al., 2011). Subsequently a mutational substitution matrix was calculated and used together with a fast implementation of the Single-Instruction-Multiple-Data Smith-Waterman algorithm (Zhao et al., 2013; Remmert et al., 2011).\n\nThe final noteworthy feature is the ability to create a distance matrix by comparing all the pHMMs in a library of pHMMs against each other and determining the simple co-emission score (Madera, 2008). This score is converted to a distance score identical to the algorithm as used by the pHMM-tree program (Huo et al., 2017). The resulting distance matrix is saved in a PHYLIP-compatible file and used to build an unweighted pair group method with arithmetic mean (UPGMA)-based phylogenetic tree. This means that given identical input pHMMs, trees generated using pHMM-tree and PRCx are identical. This was manually validated for a tree generated using the top 248 pHMMs out of the total 1828 pHMMs generated using both PRCx and pHMM-tree. In our implementation, the most time-consuming step was the UPGMA clustering. Therefore, we adapted the fast O(n2) algorithm as implemented in the MUSCLE and Clustal Omega alignment programs (Edgar, 2004; Sievers et al., 2011).\n\nThe PRCx program was developed and tested using both GNU/Linux (Ubuntu version 18.04) and MacOSX (version 10.14.5). The computer system used for testing contained an Intel Core i5 with 8 GB of memory.\n\n\nResults\n\nThe initial sequence dataset was created by iteratively searching the UniprotKB database using the Jackhmmer program and our previously published LPMO pHMM (Johnson et al., 2010; Voshol et al., 2017). After 10 iterations, 12819 non-redundant putative LPMO sequences were identified. The resulting refined pHMM (Figure 2) clearly shows several residues that have a high informational content (i.e. conserved residues). Not surprisingly, these residues include the two histidines that form the essential copper binding histidine brace (Aachmann et al., 2012; Chaplin et al., 2016; Gudmundsson et al., 2014; Hemsworth et al., 2013). Another conserved feature is the N/Q/E-x-F/Y/(W) motif, which was previously used to mine for novel starch active LPMOs (Vu et al., 2014). Finally, there are two conserved cysteines and a proline. The proline is located distal from the active site therefore it is most likely important for structural reasons (Voshol et al., 2017).\n\nAfter the initial dataset was created, the taxonomic occurrence and domain organization were analyzed using the HMMER web server (Potter et al., 2018). The dataset mainly contains sequences belonging to the domains of Eukaryota and Bacteria (98%) (Figure 3). Within the domain of Eukaryota, Fungi are by far the largest contributor of LPMO sequences (84%). This is in line with the hypothesis that Fungi play a major role in the global carbon cycle and contain a large repertoire of carbohydrate-degrading enzymes (Benocci et al., 2017). Actinobacteria, proteobacteria and Firmicutes contribute most of the LPMO sequences (99%) within the domain of Bacteria. The sequences identified in viruses are predominantly from the Baculoviridae (65%) and Phycodinaviridae (28%). The only two Archaeal LPMO sequences that were found, both belong to the Euryarchaeota. Out of all the LPMO sequences identified, only 19% have known accessory, mainly carbohydrate binding, domains (Figure 4).\n\nFrom left to right, the first bar shows the distribution of the sequences according to their Domain, Eukaryota, Bacteria, Viruses, Others, indicated in percentages of total sequences. The following three bars indicate the distribution (in percentages) of sequences as a function of the total number of sequences in the Domain (indicated below the bar).\n\nIndicated is the percentage of LPMO sequences that have a known accessory domain (left, green). Those with a known accessory domain are indicated in more details (right) with their occurrence in percentage of total LPMO sequences and rounded to the nearest full percentage.\n\nTo gain a better understanding of LPMO evolution, Book et al. (2014) created two phylogenetic trees, one for the AA10s and one for the AA9s. With their approach, they were able to show that there are different clades within these two families and each clade has evolved a specific substrate and oxidation preference (e.g. C1, C4, C1/C4). However, their approach is not sensitive enough to show the relation between the different families of LPMOs, therefore we undertook the construction of a comprehensive phylogenetic tree using the sensitivity of pHMM alignments.\n\nBefore building the LPMO tree, we searched PFAM for related families of the core LPMO HMM to find an appropriate outgroup (starting point of the tree). As expected, the PFAM LPMO_10 (PF03067) and GH61 (PF03443) families were identified as close relatives. Surprisingly, we were also able to identify one distantly related family, namely the PFAM Egh16-like family, formerly known as DUF3129 (PF11327; available from http://pfam.xfam.org). The homology between the Egh16-like family and the LPMO family is in part due to the histidine located at the third position of the PFAM HMM, which in the LPMO family is part of the histidine brace. It should be noted that the Egh16-like family HMM is presumably based on an incorrectly predicted signal peptide cleavage site, resulting in the conserved histidine not being the first residue of the PFAM model. When examining several sequences within the Egh16-like family, the latest version of SignalP predicts the signal peptide cleavage site right before the histidine (Almagro Armenteros et al., 2019). Unlike the LPMO family however, the Egh16-like family does not appear to have a second histidine (forming the histidine brace), but instead contains a conserved aspartic acid. The Egh16-like family is restricted to Fungi and proteins within this family might play an important role in pathogenic fungi in the early stages of plant and insect infection (Xue et al., 2002).\n\nAfter the outgroup was identified, the LPMO phylogenetic tree was built as follows. The original nonredundant dataset of 12,819 sequences was clustered at 70% homology (leaving 2296 sequences as singletons) and sequences contained within where aligned and used to build HMMs. Initially a small tree was constructed, containing a subset of 248 HMMs, using the pHMM-tree program (Huo et al., 2017). This process took 7.5 hours. Extrapolating this amount of time to the time required to make the entire tree (>1800 HMMs), would result in a tree construction time of 14 years. This is in line with the original paper describing pHMM-tree and its algorithm (Huo et al., 2017). As an alternative, it was decided to extend PRC to be able to make simple UPGMA phylogenetic trees. This resulted in PRCx, which was able to build the small tree (248 HMMs) in 0.5 hours and the final tree in approximately 20 hours. Which is a 15-6000x speed improvement versus the original pHMM-tree method (Figure 5).\n\nThe X-axis indicates the number of pHMMs in the tree and the Y-axis is the runtime in seconds. For example, building a tree containing 248 pHMMs with pHMM-tree took 27,059 seconds (~7.5 hours), while building the same tree with PRCx took 1504 seconds (~25 minutes). The blue and grey lines are the estimated trend lines that best fits the data for pHMM-tree and PRCx, respectively.\n\nThe resulting tree was rooted using the Egh16-like family as an outgroup. A simplified representation is shown in Figure 6 and the entire tree is available as a searchable PDF (Figure S1) with sequence data (Table S1) (see Extended data; Voshol et al., 2019a). As can be seen from the tree, the AA9s are by far the largest family (41%), followed by AA10s (27%), AA11s (14%), AA15s (7%), AA16s (4%), LPMO16s (4%), AA13s (1%) and AA14s (<0.5%). An additional 2% of HMMs branch off early in the LPMO tree before any of the known or putative LPMO families. The earliest branch splits into two branches, namely one strictly containing Egh16-like members and another which splits further and contains PFAM DOMON/EGF and LPMO_10 domain-containing sequences. The DOMON domain might play a role in metal or sugar binding and is often associated with redox enzymes (Iyer et al., 2007). A more detailed biochemical understanding of what the Egh16-like family does will shed a better light upon the possible relation of the Egh16-like, LPMO_10, DOMON and EGF domains.\n\nThe initial non-redundant dataset was clustered at 70% sequence homology and each cluster resulted in a single alignment. PHMMs were build and a UPGMA tree was constructed using PRCx. The phylogenetic tree was subsequently rooted using the Egh16-like family as an outgroup.\n\nWhen moving up the tree the first large branch contains the LPMO16s which were previously identified as putative LPMOs while mining genomes of filamentous Fungi (represented by An07g08250 in Aspergillus niger) (Voshol et al., 2017). This family is related to the AA16s (Filiatrault-Chastel et al., 2019; Voshol et al., 2017), AA14s (Couturier et al., 2018) and AA11s ((Hemsworth et al., 2014). This suggests that the common ancestor of this branch evolved not only to oxidize cellulose (AA16s), but also xylan (AA14s) and even chitin (AA11s). A similar observation can be made for the next branch, which contains the AA15s and the AA13s. The AA15s were first identified in 2017 and later it was shown that they have the ability to cleave cellulose or chitin (Sabbadin et al., 2018; Voshol et al., 2017). The AA13s were identified and characterized in 2014 and can cleave starch (Vu et al., 2014). Taken together, this suggests that ancestral LPMOs have evolved multiple times to oxidize a diverse range of substrates. The tree is completed with the large AA10 and AA9 family of LPMOs. The AA10 contains LPMOs which can cleave both cellulose and chitin, while the AA9 family contains members which can cleave cellulose or xylan. Similar to the observations by Book et al. (2014), clades within the AA9 and AA10 family appear to have a specific substrate and oxidation preference. However, only a tiny percentage of LPMOs have been characterized and even in these cases the measured enzyme activity may have been misinterpreted (Eijsink et al., 2019). This makes drawing general conclusions on functionality somewhat preliminary.\n\nOn closer examination, the AA9 clade also contains LPMOs which have either an arginine or a lysine instead of the N-terminal histidine (Yakovlev et al., 2012). An arginine containing LPMO has recently been characterized, but no activity was identified (Frandsen et al., 2019). The place of these LPMOs present in node 726 and 650 suggest that these LPMOs evolved relatively recent from “normal” histidine-containing AA9 LPMOs. It would therefore be interesting to see whether restoring the arginine or lysine to a histidine will result in active LPMOs.\n\nTaxonomically, the LPMO subfamilies as we have classified them with PRCx, have a peculiar distribution different from either their substrate or taxonomic based classification (see Table S1). The subfamilies, AA9, AA11, AA13 and AA14 are mostly found in Fungi (>90% of LPMO sequences), the AA16 are found in both Fungi (82%) and Oomycetes (12%), while the AA10 are almost exclusively bacterial (99%) and the AA15 are mainly found predominantly in Metazoa (95%). The recently discovered LPMO16 are mostly found in Fungi (78%), but are also found in Metazoa (4%) and Oomycetes (6%). This observation suggests that LPMOs have found their true functional diversity in the fungal kingdom.\n\n\nUse cases\n\nAfter constructing the phylogenetic tree, it is possible to use it in several ways. For example, it is possible to search an unknown sequence against the pHMMs used for the tree building and discover to which LPMO subfamily and specific branch this protein belongs. This might give an indication of substrate specificity and oxidation preference that the newly discovered protein has.\n\nIt is also possible to extract sequences or pHMMs from the tree that belong to a specific LPMO subfamily or clade. These can subsequently be analyzed for the presence of specific accessory domains or domains of unknown function. This might also give an indication of localization or substrate preference. For example, after extracting all the AA15 pHMMs and searching them against the PFAM database using PRCx, it appears that some of the members have a fasciclin domain. This domain may be involved in cell adhesion, suggesting that some of these proteins are targeted to the cell membrane (Huber & Sumper, 1994).\n\nLastly it is possible to take sequences belonging to one or several subtrees and align them using structural alignments. Using this approach, it is possible to get an indication of residues involved in substrate specificities or oxidation preference.\n\n\nConclusions\n\nThis is the first time that a phylogenetic tree showing both the intra- and inter-family relations of LPMOs is constructed. We believe that the new PRCx program will help researchers to determine where their LPMO is located in the phylogenetic tree, what the putative substrate specificities are and identify LPMOs with a yet unknown substrate specificity (e.g. the LPMO16s). Moreover, the PRCx program can also be applied to other large proteins families in which it can aid in discovering long distance evolutionary relations.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Profile Comparer Extended: phylogeny of LPMO families using profile hidden Markov model alignments. http://doi.org/10.5281/zenodo.3518352 (Voshol et al. 2019a).\n\nThis project contains the following extended data:\n\nFigure S1 (searchable phylogenetic tree).\n\nTable S1 (sequence data used in this study).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code for the PRCx program is available from: https://github.com/gerbenvoshol/PRCx.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.3518337 (Voshol et al, 2019b).\n\nLicense: GNU General Public License version 2.", "appendix": "References\n\nAachmann FL, Sørlie M, Skjåk-Bræk G, et al.: NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "56318", "date": "19 Nov 2019", "name": "Miaomiao Zhou", "expertise": [ "Reviewer Expertise Bioinformatics", "Biotechnology", "Machine learning", "High dimensional statistics", "Comparative genomics", "Computational Biology", "Evolutionary biology", "High performance computing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have developed a program to study the phylogeny of large groups of proteins. This new method, named PRC Extended (PRCx), was applied on the lytic polysaccharide monooxygenase (LPMO) from over seven families. Conventional pHMM-pHMM methods failed to perform such a large construction due to the overwhelming calculation requirements. In this study, the authors used both pHMM-sequence searches and pHMM-pHMM alignments, they extended the original Profile Comparer program (PRC) and added several capabilities. It is plausible that the authors updated the popular original PRC program with the ability to load HMMer3.0 pHMM files, this is a very desired feature among the users of the original PRC program.\nThe authors applied PRCx on the large LPMO superfamily to study the inter and intra-family enzyme evolutionary relationship. This study was not feasible without the newly constructed PRCx program due to the large size of the superfamily. The PRCx program was able to build the phylogenetic tree for hundreds of HMMs within a few days, which is a huge improvement comparing to the original pHMM-based method.\n\nDuring the preparation of the phylogenetic studies, the authors identified the PFAM LPMO_10 (PF03067) and GH61 (PF03443) families to be the in-groups. They also found PFAM Egh16-like family to be a distant relative of the LPMO family. They found the evidence to correct the mis-predicted cleavage site of the Egh16-like family PFAM HMM and used this family as the out-group for the study. The close examination of the resulting tree revealed several interesting features of the LPMO family. Many of these features point to potential experimental targets, for example, altering arginine, lysine and histidine in AA9 LPMOs might give impact on the activity level of the enzyme.\nLast but not the least, it is interesting to notice that the core LPMO pHMM has a model length of merely 165. Would a much bigger pHMM model compromise the performance of PRCx? Considering the fact that the authors tested the PRCx program on a moderate computer of Intel Core i5 with 8 GB of memory, a high performance computer with more RAM might solve the problem?\n\nAs a researcher in the fields of applied Bioinformatics in biotechnology, I would recommend PRCx, together with the phylogenetic analysis of LPMO, to be published. The PRCx program will be a very useful tool to study big enzyme groups beyond the LPMO superfamily.\n*availability: The authors have made their program available via github, the supporting data is all accessible via Zenodo.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5182", "date": "30 Jan 2020", "name": "Erik Vijgenboom", "role": "Author Response", "response": "Dear Dr. Zhou,Thank you for taking the time to review our manuscript.You are correct in your observation that increased model length leads to decreased performance. Since HMMs tend to be short, the longest HMM in PFam is ~2000 match states long.Memory in general is not the bottleneck. The HMM-HMM calculation is the limiting step. For tree building a relatively simple solution could be to implement multi-threading support using for example OpenMP and calculate the distance scores in parallel." } ] }, { "id": "57829", "date": "18 Dec 2019", "name": "Stjepan Krešimir Kračun", "expertise": [ "Reviewer Expertise Analytical Chemistry", "Bioconjugation Chemistry", "Enzyme Screening", "Enzyme Characterization", "Chemoenzymatic Synthesis", "Data Analysis", "High-Throughput Screening", "High-Throughput Data Analysis", "Assay Development", "Method Development" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have developed a UNIX-based program to study the phylogeny of large groups of proteins focusing on LPMOs. The program is novel, performs faster and contains features that no other program currently contains.\nThe manuscript is written well, everything is clearly, accurately and sufficiently explained.\n\nThe methods used, prior art and references are addressed appropriately.\n\nSpecific comments and questions:\nLPMOs are a very interesting group of enzymes that have emerged as essential in degradation of biomass.\n\nThe authors mention this briefly in their Introduction section (first paragraph).\nHow do the authors envisage the utility and impact of their program for people who work with LPMOs in the wet-lab (enzyme discovery and screening, for example)?\nThis should be reflected upon in the Conclusions section.\nThe authors mention development of the program for UNIX-based platforms such as Linux and MacOS. Have they considered, or plan to implement an online solution? Without an online solution - the usefulness of the program will be severely limited. In addition, informing readers about how and when such a tool will be implemented, with a provisional online link (even if the implementation is not entirely completed) - will certainly increase the impact of the article.\nI understand that most Linux code can be run on virtual machines, but from my experience - this is not necessarily accessible to people that are not bioinformaticians. Therefore, in order to make this tool useful for the scientific community - a user-friendly online solution would be highly recommended.\nCould the authors comment, as well as include in the manuscript, their plans regarding such implementation?\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5183", "date": "30 Jan 2020", "name": "Erik Vijgenboom", "role": "Author Response", "response": "Dear Dr. Kračun,Thank you for taking the time to read and comment on our manuscript.We expect this tree to provide guidance to enzyme discovery by allowing an easy means to identify novel LPMOs and placing those with known activity into their respective subbranch. Other possible use cases are indicated in the “Use cases” section. This is actually nicely illustrated by a recent paper by Labourel et al. (2020, https://doi.org/10.1038/s41589-019-0438-8), describing one of the early LPMO branches showing in our tree.We ourselves are interested in using the tree to get a better understanding of substrate evolution and stability and applying this knowledge for the industrial application of LPMOs.We agree that having an online implementation of PRCx will increase its impact. However, we currently do not have the resources for such an endeavor. We would be willing to collaborate with interested parties to help to put PRCx into operation online." } ] }, { "id": "57830", "date": "08 Jan 2020", "name": "Mirjam Kabel", "expertise": [ "Reviewer Expertise Enzyme conversion of plant biomass for food and biorefinery", "LPMOs", "lignin chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research describes the development of a sequence-model (profile hidden Markov model), which can be used to give insights into the inter- and intra-family relationships of protein families – based on their structure. The provided tool is, in particular, faster than the conventional tools to assess phylogenetic relationships.\nA phylogenic tree of over 10000 LPMO sequences was made to illustrate the usefulness of the LPMO-model developed. The authors show that their model indeed shows the expected AA families as listed in the CAZy database. But, what is less clear for me, is how could this LPMO-model be used to make phylogenic trees within one AA family? The authors mention that their model has only a length of 165, starting from the N-terminal histidine. Others (various papers, please check) have shown that LPMO-segments important for substrate recognition, all neighboring the active site, are spread over the total sequence. Hence, using the 165 amino acids only would lack information of certain binding-segments. Do I understand correctly that such information cannot be picked up? I would appreciate if the authors can better highlight for which applications their pHMM-approach could be used and for which it would not be suitable.\nAs far as I can judge, still, the tool is an asset to make complex and more complete phylogenetic trees (more sequences) in a relatively short time. The research shown is relevant.\nOverall, the manuscript is clearly written, although several choices made could have been explained better (see itemized points below).\nAbstract: please specify \"speeds several orders of magnitude\" to be more specific.\n\nAbstract: remove the last sentence, it is not relevant. Or explain.\n\nIntro: \"aid breakdown by conventional enzymes\". Not absolutely clear, please reformulate.\n\nIntro: \"sequence similarity between LPMO families\". The sequence similarities within for example the AA9 family is also rather low even. Add?\n\nResults: phylogenetic tree: The authors refer to a paper in which phylogenetic trees per AA family are described, and subsequently write that this approach was not sensitive enough to show the relationship between the different families. Are these two not based on completely different research questions? Instead of being not sensitive enough, could the authors rewrite this section to better highlight the value of trees within and between AA families and which tools/models are best to use?\n\nFigure 5: for clarity, the y-axes could be converted to minutes.\n\nFigure 6: the model seems to pick up well the large AA families (AA9 and AA10), but in some of the others extra branches appear (i.e. AA16 & LPMO16). Is this a ‘real’ branching or the result of using the short 165 amino acid model? Please discuss and explain the consequences in the manuscript.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5184", "date": "30 Jan 2020", "name": "Erik Vijgenboom", "role": "Author Response", "response": "Dear Dr. Kabel,Thank you for taking the time to read and comment on our manuscript.You are correct that the initial LPMO model contains only 165 match states. These states contain several aromatic amino acids which seems conserved within all LPMOs, but there is also for example a conserved proline distal from the active site necessary to maintain LPMO structure. This initial 165 state model was only used to mine LPMO sequences in the Uniprot database. For phylogenetic tree construction, sequences with 70% identity were clustered together and approximately 1800 HMMs were constructed from these sequences. These HMMs were subsequently used for phylogenetic tree construction. Therefore, conserved binding segments are present within the constructed HMMs.The speed gain depends on the amount and the length of HMM sequences used. When comparing PRCx against pHMM-tree the small (248 HMMs) tree generation using PRCx is ~15x faster, while the final tree construction using ~1800 HMMs is roughly 6000x faster.With the line “Furthermore, the LPMO phylogenetic tree, does not seem to follow taxonomy-based classification.”, we tried to indicate that different LPMO families are not restricted to a specific taxonomical class. For example, the AA15 LPMO family contains Fungi, Oomycetes and also Metazoa.I’m afraid that converting from seconds to minutes does not improve Figure 5.With \"aid breakdown by conventional enzymes\", we are referring to the synergy between LPMOs and hydrolytic enzymes.You are correct in the observation that the sequence similarities within for example the AA9 family, is rather low. This is true when looking at all the sequences contained within these large families. However, a more detailed look at the subfamily sequences shows far less divergence.We agree that the tools used depend on the research questions. The approach using pHMM-pHMM alignments is a good way to get a bird’s eye view of protein families and their inter-connectedness. However, it is not suitable for comparison of more closely related sequences. Therefore, sequences clustered closely in the tree should subsequently be analyzed with structural alignments to identify defining residues between these subfamilies.As mentioned earlier, the initial 165 state model was only used to mine LPMOs. For the tree construction new HMMs were constructed (see earlier comments). Therefore, the distinct location of LPMO16 and AA16 are well supported. Whether the LPMO16 family actually contains active LPMOs still needs to be validated experimentally." } ] } ]	1	https://f1000research.com/articles/8-1834
https://f1000research.com/articles/8-1828/v1	30 Oct 19	{ "type": "Research Article", "title": "Phylogenetic groups and antimicrobial susceptibility patterns of uropathogenic Escherichia coli clinical isolates from patients at Mulago National Referral Hospital, Kampala, Uganda", "authors": [ "Paul Katongole", "Daniel Bulwadda Kisawuzi", "Henry Kyobe Bbosa", "David Patrick Kateete", "Christine Florence Najjuka", "Daniel Bulwadda Kisawuzi", "Henry Kyobe Bbosa", "David Patrick Kateete", "Christine Florence Najjuka" ], "abstract": "Introduction: Uropathogenic Escherichia coli (UPEC) remains the most common cause of urinary tract infections (UTIs). They account for over 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. E. coli strains have been found to belong to evolutionary origins known as phylogenetic groups. In 2013, Clermont classified E. coli strains into eight phylogenetic groups using the quadruplex PCR method. The aim of this study was to identify the phylogenetic groups of UPEC strains in Uganda using Clermont’s quadruplex PCR method and to assess their antibiotic susceptibility patterns in Uganda. Methods: In this cross-sectional study, 140 stored uropathogenic E. coli isolates from the Clinical Microbiology Laboratory, Department of Medical Microbiology, College of Health Sciences Makerere University were subjected to phylogenetic typing by a quadruplex PCR method. Antimicrobial susceptibility testing was performed by disk diffusion method according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Phenotypic detection of extended-spectrum beta-lactamase, AmpC and carbapenemases was done according to CLSI guidelines and Laboratory SOPs. Results: Phylogenetic group B2 (40%) was the most predominant, followed by A (6.23%), clade I and II (5%), D and E (each 2.14%), B1 (1.43%) and F and C (each 0.71%). The most common resistant antibiotic was trimethoprim-sulphamethoxazole (90.71%) and the least was imipenem (1.43%). In total, 73.57% of isolates were multi-drug resistant (MDR). Antibiotic resistance was mainly detected in phylogenetic group B2 (54%). Conclusions: Our findings showed the high prevalence of MDR E. coli isolates, with the dominance of phylogenetic group B2. About 9% of E. coli isolates belonged to the newly described phylogroups C, E, F, and clade I and II.", "keywords": [ "Uropathogenic E.coli", "Phylogenetic groups", "Clermont’s", "Quadruplex PCR", "Antimicrobial resistance." ], "content": "Introduction\n\nUrinary tract infections (UTIs) remain a major cause of morbidity, with over 1.5 million annual cases reported worldwide (Lee & Neild, 2007)(Neild, 2003). Escherichia coli is the major cause of UTIs accounting for over 80–90% and 30–50% of all community-acquired and hospital-acquired UTIs respectively(Foxman, 2010). Phylogenetic (evolutionary) groups of E. coli strains have been shown to differentiate between pathogenic and commensal strains depending on their fitness landscapes and virulence characteristics. Although multi-locus sequence typing (MLST) and ribotyping are the gold standard methods for phylogenetic typing of E.coli strains, these methods are expensive, time-consuming and require the collection of typed strains (Clermont et al., 2000).\n\nIn 2000, Clermont et al (Clermont et al., 2000). developed a triplex PCR assay that classified E.coli strains into four different phylogenetic groups, A, B1, B2 and D based on the presence or absence of two genes, namely chuA, yjaA, and one DNA fragment TspE4.C2. Since 2000, growing knowledge of MLST for E.coli from different habitats has made it possible to validate the triplex PCR method (Gordon et al., 2008). The validation studies found that only 80–85% of all E. coli phylogenetic groups were assigned correctly(Gordon et al., 2008)(Clermont et al., 2013).\n\nIn 2013 Clermont et al. (Clermont et al., 2013) added an additional gene target, arpA, to the three candidate markers (chuA, yjaA and TspE4.C2) and developed a quadruplex PCR assay to classify E. coli isolates into eight phylogroups: A, B1, B2, C, D, E, F, and clade I/II. The use of this quadruplex PCR phylotyping method has been found to correctly assign 95% of all E. coli strains. Phylogenetic analysis has demonstrated a relationship between different E. coli phylogenetic groups, antimicrobial resistance and other virulence characteristics (Iranpour et al., 2015). Different studies have shown that the most virulent and antimicrobial-resistant extra-intestinal E. coli strains belong mainly to group B2 and, to a lesser extent, to group D (Iranpour et al., 2015)(Bashir et al., 2011)(Liu et al., 2014). In contrast, most of the commensal strains are associated with group A or group B1 (Clermont et al., 2000). In this study, we aimed to determine the prevalence of the different phylogenetic groups of Uropathogenic E. coli strains using the new Clermont quadruplex PCR phylotyping method and their antimicrobial susceptibility patterns.\n\n\nMethods\n\nThis was a cross-sectional laboratory-based study carried out in the Clinical Microbiology and Molecular Biology Laboratories in the Department of Medical Microbiology, College of Health Sciences, Makerere University (Kampala, Uganda).\n\nA total of 140 Uropathogenic E. coli strains that belonged to the bacterial collection of the Department of Medical Microbiology, College of health sciences Makerere University were studied. These strains had been isolated and stored at -80°C during a 12-month period (between January and December 2016). These isolates had been recovered from samples with a bacterial count of 105 CFU/ml of midstream urine samples of patients with a suspected UTI. The samples were selected by consecutive sampling and all samples that were poorly labeled and lacked traceable laboratory request form were excluded from the study. All isolates were plated on MacConkey agar and incubated at 37°C aerobically for 18–24 hours to obtain pure growth.\n\nAntimicrobial susceptibility testing was done using the Kirby Bauer disk-diffusion method. Briefly, between one and five colonies of the isolate from the MacConkey agar were emulsified in saline and adjusted for turbidity to obtain a 0.5 McFarland standard. A sterile cotton swab on a stick was dipped in the colony-saline mixture, excess saline was then squeezed out by pressing the swab against the test tube, and the cotton swab gently applied onto the surface of the Mueller-Hinton-2- agar to obtain a uniform lawn. Up to six antibiotic impregnated discs were gently placed on the agar surface, at a minimum distant of 25 mm from each other, and the plates incubated at 37°C aerobically for 18–24 hours. The zones of inhibition diameters around each disc were measured using a ruler and compared against the zone diameter interpretative standards according to CLSI (2014). A panel of 12 antibiotics were used: ampicillin (30 μg), cefuroxime (30 μg), amoxicillin/clavulanic acid (20/10 μg), gentamycin (10 μg), trimethoprim/sulphamethoxazole (1.25/23.5 μg), chloramphenicol (5 μg), ciprofloxacin (5 μg), ceftriaxone (30 μg), ceftazidime (30 μg), imipenem (10 μg), nalidixic acid (30 μg) and nitrofurantoin (300 μg). E. coli ATCC 25922 was used as a control strain.\n\nAll isolates that showed an area of inhibition of diameter <22 mm for ceftazidime and <25 mm for ceftriaxone, which were selected for phenotypic detection of The test isolate was mixed in saline to obtain a suspension of 0.5 McFarland standard. The suspension was later swabbed to make a uniform lawn on Mueller-Hinton agar (MHA) plate. An amoxicillin-clavulanate (Augmentin) (20 μg/10 μg) disk was placed in the center of the plate with a 30-μg disk of a third-generation cephalosporin (ceftazidime and cefotaxime) at a distance of 20 mm from center to center on a Mueller Hinton agar (MHA) plate on opposite sides. The plate was later incubated aerobically at 37°C for 18–24 hours. All isolates that showed a clear extension of the edge of inhibition zone of the third-generation cephalosporin toward the augmentin disk were interpreted as positive for ESBL production.\n\nDetection of carbapenemases was determined by a positive Modified Hodge test (MHT) as described by CLSI (2014); In brief, E. coli ATCC 25922 was streaked for confluent growth on Mueller-Hinton II agar plates. A disk saturated with 10 μg of imipenem was placed in the center of the plate, and each sample was then subsequently streaked from the disk to the edge of the plate. The presence of a distorted inhibition zone after overnight incubation was interpreted as a positive result. Klebsiella pneumoniae ATCC BAA-1705 and K. pneuomniae ATCC BAA- 1706 were used as positive and negative controls, respectively. Phenotypic detection of Metallo-beta-lactamases (MBL), K. pneuomniae producing carbapenems (KPC) and AmpC was carried out as described by Andrea Bartolini et al and Tsakris et al (Bartolini et al., 2014)(Tsakris et al., 2008).\n\nDNA for amplification was extracted from whole cells by the boiling lysis method, as explained briefly below. A full loop of pure colonies from fresh pure cultures was suspended in 1 ml of sterile distilled water. The cells were lysed by heating at 95°C for 10 minutes. The cells were then vortexed for 5 seconds. Centrifugation was later done at 13,000 rpm for 5 minutes at room temperature and the sample kept at -20°C to harvest the supernatant containing the DNA. The supernatant was subsequently used for PCR as template DNA. The integrity of extracted DNA was evaluated by electrophoresis on a 1% agarose gel. The purity of DNA was also determined by the ratio A260/A280 using a spectrophotometer.\n\nThe distribution of phylogenetic groups amongst E. coli isolates was determined by the New Clermont Quadruplex PCR phylotyping method of 2013 (Clermont et al., 2013). Briefly, a single reaction mixture contained 2 μL of 10x buffer (supplied with Taq polymerase), 2 μL of DNA (approximately 100 ng), 20 pmol of each appropriate primer (except for AceK.f (40 pmol), ArpA1.r (40 pmol), trpBA.f (12 pmol), and trpBA.r (12 pmol)) (Shanghai Generay Biotech Co., Ltd.), 2mM of each dNTP, and 2U of Taq DNA polymerase (Fermentas, Lithuania) in a total volume of 20 μL. Primer sequences for the new Clermont’s quadruplex PCR phylogroup assignment method are as shown in Table 1. PCR amplifications were carried out on a thermal cycler Master-cycler gradient (Eppendorf, USA) under the following conditions: initial denaturation at 94°C for 4 min and 30 cycles for each denaturation at 94°C for 5 sec, annealing at 57°C for 20 sec (group E) or 59°C for 20 sec (quadruplex and group C), amplification at 72°C for 1 min, and final extension at 72°C for 5 min. PCR products were analyzed by electrophoresis with Qiaxel machine and a 2% agarose gel, stained with DNA safe stain (CinnaGen, Tehran, Iran) and visualized usingGelDoc 2000 transilluminator (Bio-Rad Laboratories, Milan, Italy). A molecular weight standard (100-bp ladder, Fermentas, Lithuania) was used.\n\nThe data was entered in an excel spreadsheet, cleaned and double-checked for missing variables duplicate entries and values out of range. The data was then exported to STATA version 14 for statistical analysis. Means and proportions were obtained. Chi-square test or the Fisher exact test was applied to compare categorical variables. P values < 0.05 were considered to be statistically significant.\n\nEthical approval (SBS-HDREC- 487) and a waiver of consent were sought to use stored clinical isolates from the higher degrees Research and Ethics Committee of the School of Biomedical Sciences, College of Health Sciences Makerere University and the Uganda National Council of Science and Technology.\n\n\nResults\n\nOf the 140 E. coli isolates, 102 (72.9%) were females and 38 (27.1%) were males. The age of the patients was ranging from 2 to 91 years. The mean age was 36.27 with a standard deviation of 18.98.\n\nResistance was highest to trimethoprim-sulphamethoxazole 127/140 (90.71%) followed by ampicillin 122/140 (87.14%) while resistance to nitrofurantoin 16/140 (11.43%) and imipenem 2/140 (1.43%) was minimal. Resistance to other antibiotics were as follows; cefuroxime 72/140 (51.43%), amoxicillin-clavulanic acid 46/140 (32.86%), gentamycin 52/140 (37.14%), chloramphenicol 35/140 (25%), ciprofloxacin 80/140 (57.14%), ceftriaxone 69/140 (49.29%), ceftazidime 50/140 (35.71%) and nalidixic acid 94/140 (67.14%). Figure 1 contains a bar chart depicting percentage resistance to different antibiotics. Data from disc-diffusion assays are available as Underlying data (Katongole, 2019).\n\nShown are bands for different genes for phylogenetic typing\n\nIn the study, 103/140 isolates (73.57%) were found to be MDR. In addition, 4/140 isolates (2.85%) were resistant to all antibiotics tested.\n\nIn this study, 61/140 (43.57%) were positive for ESBL production. Out of all the ESBL positive isolates, (57/61) 93.44% were MDR. All isolates were negative for carbapenem production. Only 2/140 (1.43%) were AmpC producers.\n\nPhylogenetic group B2, in 56/140 of samples (40%), was the most prevalent, followed by group A in 9/140 samples (6.23%), Clade I/II in 7/140 samples (5%). The least prevalent were groups F (1/140 samples, 0.71%) and C (1/140 samples, 0.71%) (Table 2). Of the total isolates, 41.43% were unknown. Phylogenetic groupings for each patient are available as Underlying data (Katongole, 2019).\n\nPhylogenetic group B2 was the most frequently resistant (38.57%). Phylogenetic groups B1 and F were the least resistant with a recorded highest resistance rate of 0.71% (Table 3).\n\n\nDiscussion\n\nThe clinical management of UTIs is becoming a major burden due to the emergence of MDR uropathogens (Shabbir et al., 2018)(Flores-Mireles et al., 2015). Currently, third-generation cephalosporins are the most commonly used drugs in the management of complicated and uncomplicated UTIs (Stiller et al., 2017)(Bonkat et al., 2017). This study aimed to identify the phylogenetic groups of UPEC clinical isolates based on the Clermont quadruplex PCR method and to assess the relationship between these phylogroups and antibiotic susceptibility patterns in Uganda. In this study majority of the E. coli strains belonged to B2 (40%). This was similar to other studies worldwide (Iranpour et al., 2015)(Basu et al., 2013)(Moreno et al., 2008)(Takahashi et al., 2006). Other studies have, however, the most predominant phylogenetic group was group A (Ejrnæs et al., 2011)(Zhao et al., 2015). Phylogenetic group B2 has been associated with MDR-UPEC strains and increased expression of virulence factors (Ochoa et al., 2016)(Molina-López et al., 2011)(Nüesch-Inderbinen et al., 2017). Persistent and recurrent UTIs have also been associated with phylogenetic group B2 and this has been implicated in the pathogenesis of pyelonephritis(Ejrnæs et al., 2011)(Kudinha et al., 2013)(Luo et al., 2012). A similar study by Ramos et al. of pregnant mothers at Mulago National Referral Hospital found B1 to be the most predominant phylogenetic group (Ramos et al., 2012). The difference could be explained by the different study populations. In our study we used isolates from significant bacteriuria while in the study by Ramos et al., they used urine with asymptomatic bacteriuria(Ramos et al., 2012). Other studies have also demonstrated phylogenetic group B1 to be associated with commensal and less virulent strains of E. coli (Clermont et al., 2013)(Tenaillon et al., 2010). We also noticed the existence of phylogenetic groups A, C, E, F and clade I/II, which could not be detected using the triplex PCR method. This was similar with other previous studies(Iranpour et al., 2015)(Clermont et al., 2013)(Clermont et al., 2015)(Massot et al., 2016). The study also recorded 41.3% of the isolates that belonged to the unknown group; this could be due to new strains or the un-typable by PCR as explained by Clermont et al. (Clermont et al., 2013).\n\nThis study demonstrated very high rates of resistance to commonly used antibiotics, especially ampicillin, trimethoprim/sulphamethoxazole, and ciprofloxacin. This finding was similar to that of other studies(Gupta et al., 2001)(Kothari & Sagar, 2008). This could also be explained by the poor antimicrobial use policy in the country evidenced by the over the counter antimicrobial prescriptions(Mukonzo et al., 2013). We demonstrated increased prevalence of ESBL producing UPEC strains (43.57%). This was similar to other studies in the region(Kateregga et al., 2015)(Ampaire et al 2017). This could be explained by the increased pressure on third-generation cephalosporins in the management of UTIs(Ullah et al 2009)(Brosh-Nissimov et al., 2018).\n\nIn this study, we did not find any carbapenemases on phenotypic and genotypic screening. This was, however, different from other studies conducted in a similar setting(Okoche et al., 2015)(Kateete et al., 2016).\n\nThis study showed that group B2 isolates were the most resistant to most antimicrobials (38.57%). This was similar to other similar studies (Iranpour et al., 2015)(Massot et al., 2016). In addition, groups D, B1, and F were least resistant, again similar to other studies(Iranpour et al., 2015)(Massot et al., 2016).\n\nIn conclusion, our findings showed that group B2 (40%) were the most predominant and most resistant phylogenetic group among UPEC clinical isolates. About 9 % of E. coli isolates belonged to the newly described phylogroups C, E, F, and clade I. We recommend routine surveillance of antibiotic resistance patterns in the region to help clinicians make the treatment options for patients with UTIs. We recommend a longitudinal study employing whole-genome sequencing of E. coli strains in relation to UTI acquisition this will provide more insight on the role of Phylogenetic groups in the pathogenesis Of UTIs.\n\n\nData availability\n\nFigshare: Supplemental files_Paul Katongole et al_v2. https://doi.org/10.6084/m9.figshare.10006418.v1 (Katongole, 2019).\n\nThis project contains the disc-diffusion antibody sensitivity data and phylogenetic group for samples taken from each patient in this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAmpaire L, Nduhura E, Wewedru I: Phenotypic prevalence of extended spectrum beta-lactamases among Enterobacteriaceae isolated at Mulago National Referral Hospital: Uganda. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamos NL, Sekikubo M, Dzung DT, et al.: Uropathogenic Escherichia coli isolates from pregnant women in different countries. J Clin Microbiol. 2012; 50(11): 3569–3574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShabbir M, Iman NU, Shah MZ: Multidrug resistant uropathogens in urinary tract infections and their antibiotic susceptibility patterns. J Med Sci. 2018; 26(1): 24–27. Reference Source\n\nStiller RJ, Hicks C, Saul Z: Treating UTIs in the age of antibiotic resistance. Contemporary OB/GYN. 2017; 62(2): 30.\n\nTakahashi A, Kanamaru S, Kurazono H, et al.: Escherichia coli isolates associated with uncomplicated and complicated cystitis and asymptomatic bacteriuria possess similar phylogenies, virulence genes, and O-serogroup profiles. J Clin Microbiol. 2006; 44(12): 4589–4592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTenaillon O, Skurnik D, Picard B, et al.: The population genetics of commensal Escherichia coli. Nat Rev Microbiol. 2010; 8(3): 207. PubMed Abstract \| Publisher Full Text\n\nTsakris A, Kristo I, Poulou A, et al.: First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests. J Antimicrob Chemother. 2008; 62(6): 1257–1260. PubMed Abstract \| Publisher Full Text\n\nUllah F, Malik SA, Ahmed J: Antibiotic susceptibility pattern and ESBL prevalence in nosocomial Escherichia coli from urinary tract infections in Pakistan. Afr J Biotechnol. 2009; 8(16): 3921–3926. Reference Source\n\nZhao R, Shi J, Shen Y, et al.: Phylogenetic Distribution of Virulence Genes Among ESBL-producing Uropathogenic Escherichia coli Isolated from Long-term Hospitalized Patients. J Clin Diagn Res. 2015; 9(7): DC01–4. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63986", "date": "07 Aug 2020", "name": "Juan Xicohtencatl-Cortes", "expertise": [ "Reviewer Expertise Bacterial pathogenesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments to the manuscript\n\nAbstract This is a study of frequencies in UPEC strains, recovered from patients with UTIs, whose purpose was the association of phylogenetic groups (PG) with antimicrobial resistance, using the Quadruplex-PCR suggested by Clermont et al., 2013.\n\nAlthough the approach to PG and resistance has been widely discussed, the information from other countries can support treatment and the epidemiological studies. However, I believe that more information can be extracted from the data already presented, which supports these results and an adequate discussion.\n\nIntroduction The introduction is clear and contains enough information for the article to be read clearly.\n\nMaterial and Methods It is important to indicate the origin of the strains since it is not indicated what type of UTI they are related to, that is, if they are community UTIs, hospital UTIs, complicated or uncomplicated UTIs.\n\nThe authors describe the presence of UPEC-MDR strains; however, it is important to define the significance of an MDR strain.\n\nAs a suggestion for the authors, it is important to add the identification of UPEC-XDR and PDR strains. Based on these data, it is necessary to add 2 more categories of antibiotics, such as monobactams and tetracycline.\n\nCheck the name of the authors.\n\nThe purpose of the work was: \"to typify the PGs, in a collection of UPEC clinical strains, by the new scheme of Clermont et al., 2013 and to relate it to antibiotic susceptibility\". However, the results showed that 58/140 (They are many) strains were not typed by this method. This is a result that needs to be discussed in more detail.\n\nAccording to the analysis made by the authors, does it mean that the Clermont system implemented does not work on Ugandan strains?\n\nRegarding the DNA extraction method? Raw or boiled extracts are generally not suitable for multiplex PCR assays. Is DNA quantification better with this scheme? I suggest that in untyped strains, PCR be performed again using purified DNA (manual or with a commercial kit), quantified and adjusted to a standard concentration. This experiment will validate the negative results.\n\nAs a suggestion to the authors, it would be interesting to integrate an analysis of PG, resistance, and the age of the patients (pediatric and adult) for the improvement of the manuscript.\n\nConsidering the enzyme production, GF, age, characteristic of UTI as related to the 4 strains that were characterized as R to all antibiotics, is it an XDR or PDR strain?. Under this same context, how were the phenotypic AmpC-producing strains?\n\nIn various populations, the phylogenetic group D is highly prevalent, why is it not mentioned in the article and it is not discussed either?\n\nHow is the MDR profile (73%) in relation to the PG? This information is also important for inclusion in the manuscript.\n\nFig. 2 may be supplemental material. I suggest adding a caption, indicating the following: 1) The corresponding to each lane, 2) the presence of molecular size, 3) the presence of results of the clinical strains, and 4) the presence of positive control strains.\n\nIn the discussion, the authors justify the presence of strains that did not typify, data that correlate with the article by Clermont et al., 2013, in addition to this, that other studies show this same trend.\n\nThe manuscript requires a more solid conclusion, regarding whether the new PG scheme of Clermont et al., 2013, is optimal or not for this study.\n\nAlthough many results were disputed, I consider that this manuscript requires a more detailed analysis.\n\nIn the discussion, the authors described: “In this study, we did not find any carbapenems on phenotypic a genotypic screening”. The methodology and results, only indicate that they carried out the phenotypic screening, they do not indicate how they carried out the genetic typing. Correct or add this information in the methodology and results section.\n\nIn the results section, I suggest that the authors describe in which groups the statistical test was performed, for example, if the statistics were performed only for PG vs antibiotic resistance or considering the MDR groups, or the presence of ESBL, carbapenems, and AmpC.\n\nIt is not clear whether the identification of AmpCs was chromosomal or plasmid. Please indicate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "67743", "date": "19 Aug 2020", "name": "Lizziane Kretli Winkelstroter", "expertise": [ "Reviewer Expertise Microbiology and molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study evaluated the Phylogenetic groups and antimicrobial susceptibility patterns of uropathogenic Escherichia coli clinical isolates from patients at Mulago National. The topic is very interesting, However, the manuscript needs to be reviewed and there are also some important questions that require consideration.\n\nSpecific Comments:\n\nAbstract:\n\nIt was not clear the results about ESBL, AmpC, and carbapenem detection. Also, it should be emphasized that 41.3% of the isolates belonged to the unknown Phylogenetic group.\n\nIntroduction:\nIt is clear. However, I would suggest include more recent references.\n\nMethods:\nWhy CLSI (2014) was used? There is probably a more recent edition of CLSI.\n\nIt was not included how the identification/ classification of MDR was done. Also, it is necessary to include the Demographics data analyses (gender, age..).\n\nResults:\n\nIn case, 41.3% of the isolates that belonged to the unknown Phylogenetic group it is an impressive result. Is there E. coli control for each Phylogenetic group? It deserves more attention and discussion. It seemed to be a high number considering the fact to be new strains or the un-typable by PCR\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1828
https://f1000research.com/articles/8-1827/v1	30 Oct 19	{ "type": "Research Article", "title": "Efficacy of Curcuma longa in treatment of postprandial distress syndrome: An open-label randomized-controlled trial", "authors": [ "Nicharat Sawangroj", "Jiratha Budkaew", "Bandit Chumworathayi", "Nicharat Sawangroj", "Jiratha Budkaew" ], "abstract": "Background: Proton pump inhibitors are effective for functional dyspepsia but ineffective in relieving postprandial distress syndrome. Curcuma longa might be effective for postprandial distress syndrome. The objective of this study was to compare the efficacy of Curcuma longa and simethicone for postprandial distress syndrome in an open-label randomized-controlled trial. Methods: This trial was conducted between July 2018 and February 2019. In total, 78 patients were randomly assigned to receive 4 weeks of treatment with 750 or 1,500 mg oral Curcuma longa per day or 240 mg simethicone per day. The patients assessed their symptoms using the dyspepsia Global Overall Symptom scale at baseline, week 2, and week 4. After stopping medication for 2 weeks, the patients assessed recurrent symptoms and day of recurrence by themselves at the end of week 6. Results: In total, 78 patients underwent randomization (27 in 750 mg Curcuma longa, 26 in 1500 mg Curcuma longa, and 25 in simethicone groups). After 2 weeks, there were no significant differences in all mean changes of symptoms scores (95%CI) of postprandial distress syndrome [-4.1 (-4.5, -2.6) vs -4.3 (-5.2, -3.3) vs -4.2 (-4.8, -3.5), P=0.954]. Over a period of 4 weeks, the reduction in mean scores was greater among participants receiving simethicone (although not statistically significant) compared with two intervention groups [-4.6 (-5.7, -3.6) vs -5.4 (-6.6, -4.1) vs -6.2 (-7.2, -5.2), P=0.122]. The rate of recurrence was significantly lower in simethicone than the two Curcuma longa groups (42.9 vs 45.5 vs 13.6%, P=0.047). There was no serious adverse event reported in all three groups. Conclusions: Curcuma longa had a similar effect on treatment outcomes to simethicone after 2 and 4 weeks, but the recurrence rate of symptoms was significantly higher without serious adverse events. Registration: Registered with the Thai Clinical Trials Registry on 31 January 2018; TCTR20180131001.", "keywords": [ "Functional dyspepsia", "postprandial distress syndrome", "global overall symptom scale", "Curcuma longa", "simethicone" ], "content": "Introduction\n\nDyspepsia is a common functional gastrointestinal disorder which affects 20% of the global population1. Although dyspepsia is not a life-threatening condition, it disrupts the quality of life and also socioeconomic impaction for suffering patients2–4. The United States population spends $18 billion annually on dyspepsia management4. In Thailand, the prevalence of dyspepsia is higher than global prevalence, affecting more than half of the Thai population5.\n\nRome IV criteria for diagnosis classifies functional dyspepsia (FD) into two groups based on symptoms; (1) postprandial distress syndrome (PDS), consisting of postprandial fullness and early satiety, and (2) epigastric pain syndrome (EPS)6. Currently, proton pump inhibitors (PPI) are regarded as an effective treatment for FD but ineffective in relieving PDS symptoms6. Therefore, physicians frequently consider prescribing other agents for these patients.\n\nPathogenesis of FD is likely complex and multifactorial. The factors that cause PDS comprise delayed gastric emptying time, impaired gastric accommodation, and gut inflammation6. In addition, psychosocial factors such as anxiety, depression and psychiatric disorders also induce pathogenesis6.\n\nSimethicone is a defoaming agent. Foam, formed by gas in the gastrointestinal tract (GI) and gastric mucous, is a cause of fullness if it accumulates in GI tract7. Therefore, foam reduction can increase gastric emptying time and relieve postprandial fullness8–13. Many studies have found that simethicone has efficacy for treatment of dyspepsia and no serious adverse reaction14–17.\n\nCurcuma longa is a Thai herb that effectively relieves flatulence18. Previous rodent studies19–21 documented that Curcuma longa can decrease gut inflammation via its active ingredient curcumin (R = OCH3, R' = OCH3). Curcumin inhibits many proinflammatory enzymes such as cyclooxygenase-2, 5-lipoxygenase, and inducible nitric oxide synthase enzymes etc. Not only does it have an anti-inflammatory effect, but curcumin also increases gastric emptying time and reduces depressive symptoms via the brain-gut axis22. Thus, Curcuma longa is commonly used for FD treatment.\n\nMany human studies supported the efficacy of Curcuma longa compared with other agents for treatment of dyspepsia19,22,23.A trial in 2007 found that taking a 2-g Curcuma longa capsule daily for four weeks indicated no significant difference with ranitidine for dyspepsia relief23. A later study in 2016 showed that addition of curcumin on top of the standard anti-helicobacter regimen in patients with peptic ulcers was safe and improved symptoms of dyspepsia but did not enhance effect on the eradication of Helicobacter pylori infection24.\n\nHowever, no current evidence of the efficacy of Curcuma longa as compared with simethicone for PDS symptoms. Thus, the aim of this present study was to assess the efficacy of Curcuma longa compared with simethicone in patients with PDS.\n\n\nMethods\n\nAdults (age 20–60 years) with FD, diagnosed during a routine clinical appointment by physicians working at any Social Medicine clinic of Khon Kaen Hospital (Khon Kaen, Thailand), on the basis of Rome IV criteria6, were screened by nurse officers for participation then enrolled in the study by the principal investigator. Inclusion criteria included postprandial distress syndrome, no alarm features, and discontinuation of all GI drugs at least one week before randomization. Patients with a history of either simethicone or Curcuma longa allergy, gastric malignancy, gallstone or biliary obstruction, pregnancy, and on breastfeeding period were excluded. Written informed consent was obtained from all patients.\n\nThe Khon Kaen Hospital Institute Review Board in human research approved the study protocol. This trial was registered with the Thai Clinical Trials Registry on 31st January 2018; registration number, TCTR20180131001. This randomized, active-comparator, open-label trial was conducted at primary care clusters of Khon Kaen Hospital, and Nam Pong Community Hospital between July 2018 and February 2019. All the authors were involved in the design and performance of the study, which was conducted according to the Declaration of Helsinki. First research assistant (CT) used computer-generated simple randomization and sequentially labeled the number on opaque drug containers for concealment. After the principle investigator (NS) enrolled participants, the second research assistant (MJ) then assigned the concealed interventions in order. There was no deviation from the original trial protocol.\n\nPatients were randomly assigned to take 750 mg Curcuma longa capsule per day, 1500 mg of Curcuma longa capsule per day or 240 mg of simethicone per day for four weeks. All patients were educated on lifestyle modification, such as stopping drinking and smoking, decreasing spicy foods and the volume eaten per meal, and trying to choose foods with softer consistentcy. Female participants were given a urine pregnancy test, and were excluded if result indicated positive. Baseline characteristics were measured together with BMI and global overall symptom (GOS) scale (described below). Medication was administered orally 30 to 60 minutes after each meal (250 mg (one Curcuma longa capsule per meal), 250 mg (two Curcuma longa capsule per meal), or 80 mg (one simethicone tablet per meal) three times per day). Patient's visits were scheduled at the start of treatment and at the end of 2, 4, and 6 weeks (2 weeks after stopping treatment). All patients discontinued medication after week 4 and reported for recurrence of symptoms at week 6. Patients also completed daily logs of symptoms and adverse events during the week preceding each visit. At each visit, the patient, with the principle investigator (NS), completed the seven-point GOS scale for dyspepsia (which ranges from 1 to 7, with 1 indicating no problem, 2 indicating minimal problem (can be easily ignored without effort), 3 indicating mild problem (can be ignored with effort), 4 indicating moderate problem (cannot be ignored but does not influence my daily activities), 5 moderately severe problem (cannot be ignored and occasionally limits my daily activities), 6 indicating severe problem (cannot be ignored and often limits my concentration on daily activities), and 7 indicating very severe problem (cannot be ignored and markedly limits my daily activities and often requires rest)25.\n\nAt the last visit, patients were asked to report their recurrence of symptoms and the date of recurrence after discontinuation of treatment.\n\nWe focused on PDS symptoms, measured using the GOS scale, so we combined the early satiety score and postprandial score as the composite outcome. The two primary endpoints were the comparison of mean changes of composite outcome between groups from baseline to week 2 and week 4. Secondary end points were rates and durations of recurrences at week 6, and also adverse effectsas assessed by a daily log of adverse events. Post-hoc analyses included the changes from baseline in each group at week 2 and week 4.\n\nWe calculated that a sample of 69 patients would provide adequate power for the proposed tests in this three-group study using the formula for sample size calculation to compare k means by one-way ANOVA pairwise, 2-sided equality26. By substitution of mean in treatment group (µtrt) = -1.8624, mean in control group (µcon) = -1.3024, SD in each group = 0.6524, α = 0.05 and β = 0.20, r = 1, n = 22 per group was be derived.\n\nSPSS version 24.0 was used. Mean changes from baseline to week 2 and week 4, in GOS scale, were analyzed with one-way ANOVA. Dichotomous endpoints (recurrent rates) were compared among the groups with the use of Chi-squared and Z-test. The duration of symptoms was compared using Kruskall-Wallis test. Means among the groups were also analyzed using one-way ANOVA. To compare means in each group (before and after), a paired t-test was used. Bonferroni post-hoc test was used for post-hoc analysis.\n\n\nResults\n\nA total of 94 patients with functional dyspepsia were assessed for eligibility. There were 16 patients excluded from study due to not meeting inclusion criteria (n= 14) and declined to participate (n=2). A total of 78 patients underwent randomization. There were 27 in the 750 mg Curcuma longa group, 26 in 1500 mg Curcuma longa group and 25 in the simethicone group; there were 6, 4, and 2 patients lost to follow-up in each group, respectively. Intention-to-treat was used for data analysis, with n=21 in 750 mg Curcuma longa, n=22 in 1500 mg Curcuma longa, and n=23 in simethicone (Figure 1). The characteristics of the patients at baseline were similar across study groups (Table 1). However, in both Curcuma longa groups, patients were slightly overweight (BMI, 23.0-24.9 kg/m2), while in the simethicone group, patients were normal weight (BMI, 18.5-22.9 kg/m2). Participant characteristics, alongside all variables assessed, are available as Underlying data27,28.\n\nPlus minus values are means ± SDs.\n\nValues are medians (IQRs).\n\nAfter 2 weeks, there was no significant difference in mean change of PDS symptoms among three groups [-4.1 (-4.5, -2.6) vs -4.3 (-5.2, -3.3) vs -4.2 (-4.8, -3.5), P=0.954]. Over a period of 4 weeks, patients who received simethicone, as compared with those who received Curcuma longa, had a greater reduction (improvement) in the composite outcomes of PDS symptoms, but there was no statistically significant difference [-4.6 (-5.7, -3.6) vs -5.4 (-6.6, -4.1) vs -6.2 (-7.2, -5.2), P=0.122] (Table 2).\n\nFigure 2 shows the mean differences in GOS between three groups at the end of 2 and 4 weeks. When calculating mean differences of treatment effect between Curcuma longa groups and simethicone, there was no significant difference of treatment effect among two pair-wise comparisons (group 3 vs group 1 and group 3 vs group 2) at weeks 2 and 4 (Table 3).\n\naBy one-way ANOVA.\n\nbPairwise comparison of mean differences by Bonferroni post-hoc test.\n\nComparison of before and after treatment using the GOS scale at the end of 2 weeks. In Table 2, the 750 mg Curcuma longa group showed a significant reduction in all items of GOS scale except nausea which similar in simethicone group. However, in the 1500 mg Curcuma longa group indicated a significant reduction in almost all items of GOS scale except excessive belching and nausea.\n\nComparison of before and after treatment using the GOS scale at the end of 4 weeks. Over a period of 4 weeks, the participants among three groups showed significant improvement of their symptoms unless heartburn in 750 mg Curcuma longa, excessive belching and nausea in 1500 mg Curcuma longa, and nausea in simethicone (Table 2).\n\nRate of recurrence. After discontinuing treatment for 2 weeks (washout period), The patients with 1500 mg Curcuma longa reported the highest rate of recurrence, 45.5%, followed by the patients with 750 mg Curcuma longa, 42.9% and the lowest rate was in simethicone group, 13.6% (Table 4). In addition, the rate of symptom recurrence was found statistically significant among three groups (P=0.047).\n\nProportion differences when compared to simethicone with its 95%CI (by Z-test)\n\n**The day after day 28th\n\naOne-way ANOVA\n\nbChi-square\n\nDuration of recurrence. There was no significant difference in the duration of recurrence between groups (Table 4).\n\nAdverse events. There was no any patient who needed to discontinue treatment due to the serious adverse events. Non-serious adverse events were reported in 8 cases (11.9%) from the patients who receive Curcuma longa, including nausea, diarrhea, fever, dizziness and headache (Table 5).\n\n\nDiscussion\n\nThe main limitation of this study is that it is an open-label trial, in which blinding was not performed. There was no co-intervention, but few attrition biases. Although this is an open-label trial, we performed the allocation concealment and a good randomization that the results of similar characteristics among three treatment groups. Due to validity of the outcomes measured with precise 95% CIs in Table 3, our findings are summarizable and generalizable to all similar settings and populations.\n\nThe efficacy of Curcuma longa showed non-inferiority to simethicone according to the composite outcome of PDS symptoms among three treatment groups had no significant difference at week 2 and week 4. Our findings were similar to the findings of Sirijarugul and Pongchaidecha23 and Khonche et al24.\n\nThe study data also provide evidence of four important aspects of dyspepsia treatment. First, from baseline characteristics, there were more female participants. This is similar to the most recent meta-analysis in 20141. Data from this prior study indicated a greater prevalence of dyspepsia in the women from 312,415 samples (OR 1.24; 95% CI 1.13 to 1.36)1. The other characteristics were also accordant with previous studies23,24.\n\nSecond, our findings showed that Curcuma longa groups were also effective in different doses. Suprisingly, the 1500 mg group developed a higher symptom recurrence rate. Therefore, 750 mg Curcuma longa per day should be the recommended dose for FD.\n\nThird, the simethicone group developed significant lower rate of symptom recurrence. To explain this phenomenon, these patients were normal weight from obesity Asian criteria (BMI, 18.5-22.9 kg/m2)29. On the other hand, in both Curcuma longa groups, patients were slightly overweight (BMI 23.0-24.9 kg/m2) that associated with the greater prevalence of GI symptoms30,31.\n\nFinally, the three treatment groups were safe for all participants, similar to previous studies19,22–24, indicating that Curcuma longa can be used generally.\n\nStrengths of this study were; this was a randomized controlled trial that had a high quality of evidence, we studied a washout period, and we are the first who compare the efficacy of Curcuma longa and simethicone. On the other hand, our limitations were; having lower sample size than calculated due to loss to follow up patients that might have less power of study and dyspepsia associated with the multifactorial factor such as environment, various types of food, and participant behaviors. Despite we randomly assigned the treatments, it could not eliminate all confounders. So the outcomes could be imprecise.\n\n\nConclusion\n\nIn conclusion, Curcuma longa had significant effects on reduction of FD, similar to simethicone after 2 and 4 weeks, but the recurrence rate (i.e. the proportion of reappearance) of dyspeptic symptoms was slightly significantly higher without serious adverse events.\n\n\nData availability\n\nFigshare: CurcumaUnderlyingData. https://doi.org/10.6084/m9.figshare.9962723.v127.\n\nThis project contains all de-identified variables assessed in this study.\n\nFigshare: CurcumaDataDictionary. https://doi.org/10.6084/m9.figshare.1000062528.\n\nThis project contains the data dictionary for the underlying data, described above.\n\nFigshare: CONSORT checklist for ‘Efficacy of Curcuma longa in treatment of postprandial distress syndrome: An open-label randomized-controlled trial’. https://doi.org/10.6084/m9.figshare.9962723.v127.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nAuthors appreciate all supports from Khon Kaen Hospital, patients, volunteers, first research assistant (Chutarat Tanchonnang) second research assistant (Manipa Jamsuwan) and all the staffs of all clinics involving in this study.\n\n\nReferences\n\nFord AC, Marwaha A, Sood R, et al.: Global prevalence of, and risk factors for, uninvestigated dyspepsia: a meta-analysis. 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PubMed Abstract \| Publisher Full Text\n\nLecuyer M, Cousin T, Monnot MN, et al.: Efficacy of an activated charcoal-simethicone combination in dyspeptic syndrome: Results of a placebo-controlled prospective study in general practice. Gastroenterol Clin Biol. 2009; 33(6–7): 478–84. PubMed Abstract \| Publisher Full Text\n\nCoffin B, Bortolloti C, Bourgeois O, et al.: Efficacy of a simethicone, activated charcoal and magnesium oxide combination (Carbosymag®) in functional dyspepsia: Results of a general practice-based randomized trial. Clin Res Hepatol Gastroenterol. 2011; 35(6–7): 494–9. PubMed Abstract \| Publisher Full Text\n\nThamlikitkul V, Bunyapraphatsara N, Dechatiwongse T, et al.: Randomized double blind study of Curcuma domestica Val. for dyspepsia. J Med Assoc Thai. 1989; 72(11): 613–20. PubMed Abstract\n\nLiu L, Liu YL, Liu GX, et al.: Curcumin ameliorates dextran sulfate sodium-induced experimental colitis by blocking STAT3 signaling pathway. Int Immunopharmacol. 2013; 17(2): 314–20. PubMed Abstract \| Publisher Full Text\n\nZeng Z, Zhan L, Liao H, et al.: Curcumin improves TNBS-induced colitis in rats by inhibiting IL-27 expression via the TLR4/NF-κB signaling pathway. Planta Med. 2013; 79(2): 102–9. PubMed Abstract \| Publisher Full Text\n\nAli T, Shakir F, Morton J: Curcumin and inflammatory bowel disease: biological mechanisms and clinical implication. Digestion. 2012; 85(4): 249–255. PubMed Abstract \| Publisher Full Text\n\nPatcharatrakul T, Gonlachanvit S: Chili Peppers, Curcumins, and Prebiotics in Gastrointestinal Health and Disease. Curr Gastroenterol Rep. 2016; 18(4): 19. PubMed Abstract \| Publisher Full Text\n\nSirijarugul S, Pongchaidecha M: Comparative efficacy of curcuma longa and ranitidine in patients with uninvestigated dyspepsia. Proceedings of The Eighth Joint Sminar Innovative Research in Natural Products for Sustainable Development. 3rd-4th December 2008. Bangkok: Faculty of Pharmaceutical Sciences, Chulalongkorn University; 2008; 261–262. Reference Source\n\nKhonche A, Biglarian O, Panahi Y, et al.: Adjunctive Therapy with Curcumin for Peptic Ulcer: a Randomized Controlled Trial. Drug Res (Stuttg). 2016; 66(8): 444–8. PubMed Abstract \| Publisher Full Text\n\nVeldhuyzen van Zanten SJ, Chiba N, Armstrong D, et al.: Validation of a 7-point Global Overall Symptom scale to measure the severity of dyspepsia symptoms in clinical trials. Aliment Pharmacol Ther. 2006; 23(4): 521–9. PubMed Abstract \| Publisher Full Text\n\nChow SC, Shao J, Wang H: Sample Size Calculations in Clinical Research, 2nd edition. New York: Chapman and Hall/CRC; 2008. Reference Source\n\nSawangroj N, Budkaew J, Chumworathayi B: CurcumaUnderlyingData and CurcumaCONSORTchecklist. Figshare. 2019. http://www.doi.org/10.6084/m9.figshare.9962723\n\nChumworathayi B: CurcumaDataDictionary. Figshare. 2019. http://www.doi.org/10.6084/m9.figshare.10000625\n\nInternational Obesity Task Force: Asia-Pacific regional obesity guidelines. Sydney: International Obesity Task Force; 1999.\n\nBernal-Reyes R, Monzalvo López A, Bernal-Serrano M: [Prevalence of gastrointestinal symptoms in overweight and obese subjects: an epidemiologic study on a Mexican population]. Rev Gastroenterol Mex. 2013; 78(1): 28–34. PubMed Abstract \| Publisher Full Text\n\nTrujillo-Benavides OE, Rojas-Vargas EE: [Influence of obesity on dyspepsia symptoms]. Rev Gastroenterol Mex. 2010; 75(3): 247–52. PubMed Abstract" }	[ { "id": "62820", "date": "12 May 2020", "name": "Jie-ying Bai", "expertise": [ "Reviewer Expertise Molecular epidemic", "Immunogenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the effection of Curcuma Longa vs simeticone to postprandial distress was described. Two dose, 750mg/day and 1500mg/day, of C. longa were employed, the dose of simeticone was 240mg/day. Totally, the study design is appropriate, and enough literature was cited. According the data, we can find the similar effect between C. longa and simethicone. While, simethicone displayed less recurrence in this investment, which maybe show the tranditional Thai medicine, C. longa, is not a good alternative in treatment of postprandial distress syndrome.\nIn addition, there are so many people suffered from dyspepsia in Thai according introduction of this article. There were only 94 patients with functional dyspepsia were assessed for eligibility in this open-label trial. The patient samples were deficiency. This study should employ an cohort research in Thailand.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5505", "date": "14 May 2020", "name": "Bandit Chumworathayi", "role": "Author Response", "response": "Thank you very much for your comments. Further research with larger sample size will be conducted." } ] }, { "id": "74786", "date": "14 Jan 2021", "name": "Somsook Santibenchakul", "expertise": [ "Reviewer Expertise Epidemiologist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery nice work!\nJust one issue: I was curious about the following sentence.\nThe patients with 1500 mg Curcuma longa reported the highest rate of recurrence, 45.5%, followed by the patients with 750 mg Curcuma longa, 42.9% and the lowest rate was in simethicone group, 13.6% (Table 4).\n\nThe percentages stated here were different from those in table 4. Could you please explain?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1827
https://f1000research.com/articles/7-1832/v1	21 Nov 18	{ "type": "Research Article", "title": "Association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan: A cross-sectional, retrospective study", "authors": [ "Loly R.D. Siagian", "Vera M. Lumbantoruan", "Nurul Hasanah", "Fransiska A. Sihotang", "Carta A. Gunawan", "Vera M. Lumbantoruan", "Nurul Hasanah", "Fransiska A. Sihotang", "Carta A. Gunawan" ], "abstract": "Background: Malaria still presents as a major health problem in Indonesia and specifically in East Kalimantan. One common finding in malaria is thrombocytopenia, the mechanism of which is still unclear. Several studies have suggested some mechanisms, one of which is splenomegaly. This study aimed to discover the association between thrombocytopenia and splenomegaly of malaria patients in East Kalimantan. Methods: This study was a descriptive retrospective study with clinical and laboratory data obtained from the medical records of malaria patients in four major public hospitals from January 2015 to July 2018. The association between thrombocytopenia with splenomegaly was analysed using Chi-Square test. Results: A total of 215 patients were included; 189 male (87.9%) and 26 female (12.1%). The most common aetiology were Plasmodium vivax (43.2%), P. falciparum (42.8%), and mixed infection (P. falciparum and P. vivax) (4.6%). The distribution of thrombocyte count were normal in 28 patients (13%) and decreased in 187 patients (87%). Among patients with thrombocytopenia, the percentage of mild, moderate and severe thrombocytopenia was 18.2%, 43.8% and 33%, respectively. Splenomegaly was found in only 11 patients (5.1%). We found no association between thrombocytopenia with splenomegaly (p=0.61). Conclusions: We conclude that thrombocytopenia is not associated with splenomegaly in these malaria patients.", "keywords": [ "thrombocytopenia", "splenomegaly", "malaria", "East Kalimantan" ], "content": "Introduction\n\nMalaria is still a serious health problem in Indonesia; the 2007 and 2013 Basic Health Surveys of the Health Ministry show that the prevalence of malaria in Indonesia increased from 2.9% to 6.0%1.\n\nData from the East Kalimantan Provincial Health Office showed that there were 7,045 cases of malaria in 2010. This number fluctuated in the following years, with 3,021 cases in 2011, 9,966 cases in 2012 and 2,603 cases in 2013. In terms of Annual Parasitic Incidence (API), in 2014 East Kalimantan was still above the national average with an API of 2,04 per 1000 population, leading to it being categorized as a low cumulative incidence area2.\n\nOne common finding in malaria is decreased platelet count or so-called thrombocytopenia. This laboratory finding is often confused with other infectious diseases, especially dengue infection in which thrombocytopenia is a major diagnostic parameter. There are many studies that demonstrate thrombocytopenia in malaria patients3–5, and this is found in both infection with Plasmodium falciparum and Plasmodium vivax. The results from our previous study demonstrated that from 1041 malaria patients in East Kalimantan, 85% presented with thrombocytopenia of varying degrees4. Therefore, thrombocytopenia has been suggested as one important diagnostic parameter in malaria. The mechanism of thrombocytopenia in malaria is still unclear. Several theories, such as mechanical trapping of thrombocytes inside the spleen and immune response that attacks thrombocytes, has been proposed5. A study by Coelho et al. found that platelet phagocytosis may contribute to thrombocytopenia in vivax malaria6.\n\nSplenomegaly is also one common clinical finding in malaria patients. The spleen is part of the reticuloendothelial system, which becomes active in order to get rid of plasmodium-infected erythrocytes. Splenomegaly can also contribute to increased destruction of thrombocytes due to mechanical trapping. Therefore, the aim of the present study was to determine the association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan.\n\n\nMethods\n\nThis study was a cross-sectional retrospective study. This study was approved by the Ethical Committee for Health Research at Abdul Wahab Syahranie Hospital Samarinda, East Kalimantan (approval number 124/KEPK-AWS/V/2018). This ethical approval also covers the research conducted in all hospitals included in our study. Patient consent for the use of their data records was waived by the ethical committee due to the retrospective nature of the study.\n\nData were collected between June and August 2018 from the medical records of patients with malaria during the period of January 2015 to July 2018. Clinical and laboratory data of both outpatients and inpatients diagnosed with malaria from four major hospitals in East Kalimantan was collected. The hospitals were: Abdul Wahab Sjahranie Hospital in Samarinda, Aji Putri Botung Hospital in Penajam Paser Utara, Abdul Rivai Hospital in Tanjung Redeb, and Panglima Sebaya Hospital in Tanah Grogot.\n\nAll patients with malaria, both paediatric and adult patients, were included in the study. Patients were excluded from the study if the necessary data were incomplete or patients discharged themselves during treatment.\n\nIn order to collect data, first, the hospital’s database was searched for patients diagnosed with malaria. Second, after identifying these patients, the relevant medical records were retrieved which contained age, gender, type of Plasmodium, thrombocyte count, presence of splenomegaly.\n\nFor descriptive data, we described patients’ characteristics that include age, sex, type of Plasmodium, and thrombocyte count on the first day admission. The association between thrombocytopenia and splenomegaly was analyzed by Chi-square test using SPSS 23.0 software. Results were considered statistically significant if p<0.05.\n\n\nResults\n\nOur study identified a total of 215 malaria patients from January 2015 to July-2018 in five hospitals in East Kalimantan. There were 189 male patients (87.9%) and 26 female patients (12.1%). From 215 malaria patients (Table 1).\n\nThe association of thrombocytopenia with splenomegaly in malaria patients shown in Table 2. There were 11 patients with splenomegaly and 204 patients without splenomegaly. There was no association between the presence of splenomegaly and thrombocytopenia in these malaria patients (p=0.661).\n\n\nDiscussion\n\nWe found that the incidence of malaria was higher in males than females in this group of patients, and the parasite infecting the majority of patients was Plasmodium vivax (42.0%). This study found that thrombocytopenia affected the majority of malaria patients; thrombocytopenia occurred in 87% of patients, with moderate thrombocytopenia (43.8%) as the majority. This result is in concordance with another study by Arif et al., in India, that found that 79% of patients had moderate thrombocytopenia7, while Ansari et al. found 69.18%8. In the present study, the degree of thrombocytopenia classified to mild, moderate and severe was 18.2%, 43.8%, 33.0%, respectively. If we compare to study of Arif et al. that found 33.96%, 51.15% and 14.89%, respectively, we find that the moderate thrombocytopenia was the highest percentage in both studies7.\n\nA study by Hanson et al. in Vietnam found that thrombocytopenia was a marker of disease severity in adults with Plasmodium falciparum infection, but has limited utility in prognostication, triage and management9. On the other hand, Rao et al. in India found that severe thrombocytopenia showed positive correlation with complicated malaria and become a good predictor for poor prognosis10.\n\nIndeed, splenomegaly is a hallmark of malaria, but the result of this study found that only 11 patients (5.1%) showed splenomegaly. In India, Gupta et al. found that only 20% malaria patients had splenomegaly, but there are no information about thrombocyte count in those malaria patients with splenomegaly11.\n\nIn our study, we describe the thrombocyte count in malaria patients with splenomegaly. We found that there was no association between thrombocytopenia with splenomegaly (p=0.611). Therefore, we propose that thrombocytopenia is not caused by the mechanical trapping of thrombocytes in the spleen. This result suggests another mechanism of thrombocytopenia that involves immune process, as proposed by Coelhoe et al. in 20136. Until now, the definitive mechanism of thrombocytopenia in malaria was unclear; however, some factors that contribute to thrombocytopenia have been reported, such as decreased thrombopoiesis, peripheral destruction induced by P. falciparum and disseminated intravascular coagulation12.\n\nThis study has its limitations. Different types of automatic haematology analysers were used in the hospitals and this might contribute to variations in thrombocyte count. Therefore, we suggest the use of a single haematology analyser across hospitals to improve accuracy. In addition, we did not exclude other causes of thrombocytopenia that could coexist with malaria infection, such as viral infection and autoimmune diseases, which would be require to further validate our findings. Finally, the examination of splenomegaly was conducted by different physicians, which might contribute to subjective factors in determining splenomegaly, such as physician’s expertise and thoroughness, especially in cases with minor or subclinical splenomegaly.\n\nOverall, we conclude that thrombocytopenia is not associated with splenomegaly in this set of malaria patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Data retrieved from the medical records of malaria patients in East Kalimantan, Indonesia, including age, gender, type of Plasmodium, thrombocyte count, and presence of splenomegaly. , https://doi.org/10.5256/f1000research.16606.d22512413", "appendix": "Grant information\n\nThe Islamic Development Bank funded this study.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBasic Health Survey: Health Ministry of Indonesia. 2013.\n\nAnnual Report East Kalimantan Provincial Health Office. 2013.\n\nGill MK, Makkar M, Bhat S, et al.: Thrombocytopenia in Malaria and Its Correlation with Different Types of Malaria. Annals of Tropical Medicine and Public Health. 2013; (6): 197–200. Publisher Full Text\n\nSiagian LRD, Asfirizal V, Toruan VDL, et al.: Thrombocyte counts in malaria patients at East Kalimantan. IOP Conf. Series: Earth and Environmental Science. 2018; 144(1): 012007. Publisher Full Text\n\nDel Portillo HA, Ferrer M, Brugat T, et al.: The role of the spleen in malaria. Cell Microbiol. 2012; 14(3): 343–35. PubMed Abstract \| Publisher Full Text\n\nCoelho HC, Lopes SC, Pimentel JP, et al.: Thrombocytopenia in Plasmodium vivax malaria is related to platelets phagocytosis. PLoS One. 2013; 8(5): e63410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArif M, Jelia S, Meena SR, et al.: A Study of Thrombocytopenia in Malaria and Its Prognostic Significance. International Journal of Research in Medical Sciences. 2016; 4(6): 2373–2378. Publisher Full Text\n\nAnsari S, Khoharo H, Abro A, et al.: Thrombocytopenia in plasmodium falciparum malaria. J Ayub Med Coll Abbottabad. 2009; 21(2): 145–7. PubMed Abstract\n\nHanson J, Phu NH, Hasan MU, et al.: The clinical implications of thrombocytopenia in adults with severe falciparum malaria: a retrospective analysis. BMC Med. 2015; 13: 97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao BS, Vano MS, Latha GJ, et al.: Incidence, Severity, Prognostic Significance of Thrombocytopenia in Malaria. International Journal of Research in Medical Sciences. 2015; 3(1): 116–121. Publisher Full Text\n\nGupta NK, Bansal SB, Jain UC, et al.: Study of thrombocytopenia in patients of malaria. Tropical Parasitology. 2013; 3(1): 58–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbro AH, Ustadi AM, Younis NJ, et al.: Malaria and Hematological Changes. Pak J Med Sci. 2008; 24(2): 287–291. Reference Source\n\nSiagian LRD, Lumbantoruan VM, Hasanah N, et al.: Dataset 1 in: Association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan: A cross-sectional, retrospective study. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16606.d225124" }	[ { "id": "40937", "date": "05 Jun 2019", "name": "Jontari Hutagalung", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is important and has a great impact for clinicians in the subject, however I found many lacks in the assessment methodologies, especially:\nHow to define the confirmed cases of this study (microscopic ELISA or PCR).\n\nThe methodologies to count the thrombocytopenia and splenomegaly.\n\nIt is important to separate the cases by species of Plasmodium and the day of infection.\n\nAdditional comments - there are some important things missing in this manuscript that need to be explained by the authors:\nThe 1st paragraph; Please use the national Annual Parasite Incidence (API) below 1.00 per 1.000 population or the prevalence < 1%.\n\nThe methodologies need to be addressed, because this is the most important; a) how did the authors use the definition of malaria cases in this study (giemsa 3%)? and b) for the methods of counting the parasite and thrombocytes (after treatment or before treatment, or before and after treatment), did the authors make QA for the test?\n\nWhich prevalence of P. vivax is correct – 42.0% (from page 3 in the discussion) or 43.2% (from Table 1)?\n\nIn the discussion I need more explanation about the aims of this study; \"Discover the association between thrombocytopenia & splenomegaly”, please write about the following: a) the main function of thrombocytes and the kinds of situation risks decreased and for which patient conditions (acute or chronic diseases) and b) the authors should be addressing why their hypothesis was not considered significant.\n\nPlease find an annotated copy of the article here for my full comments.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-1832
https://f1000research.com/articles/8-1601/v1	06 Sep 19	{ "type": "Method Article", "title": "A new statistical method to analyze Morris Water Maze data using Dirichlet distribution", "authors": [ "Marianne Maugard", "Cyrille Doux", "Gilles Bonvento", "Marianne Maugard", "Cyrille Doux" ], "abstract": "The Morris Water Maze (MWM) is a behavioral test widely used in the field of neuroscience to evaluate spatial learning memory of rodents. However, the interpretation of results is often impaired by the common use of statistical tests based on independence and normal distributions that do not reflect basic properties of the test data, such as the constant-sum constraint. In this work, we propose to analyze MWM data with the Dirichlet distribution, which describes constant-sum data with minimal hypotheses, and we introduce a statistical test based on uniformity (equal amount of time spent in each quadrant of the maze) that evaluates memory impairments. We demonstrate that this test better represents MWM data and show its efficiency on simulated as well as in vivo data. Based on Dirichlet distribution, we also propose a new way to plot MWM data, showing mean values and inter-individual variability at the same time, on an easily interpretable chart. Finally, we conclude with a perspective on using Bayesian analysis for MWM data.", "keywords": [ "Morris Water Maze", "Statistical analysis", "Dirichlet distribution" ], "content": "1 Introduction\n\nThe Morris Water Maze (MWM) was first described by Richard Morris in the 80’s 1 and is still one of the most commonly used tasks to evaluate spatial learning in rodents, including normal and genetically modified mice. It is validated as an assay for hippocampus-dependent spatial navigation and reference memory. The maze consists of a large circular tank filled with opaque water in which rodents can escape onto a platform hidden just beneath the surface. During a training phase animals perform repeated blocks of 60 second-long trials to find the location of a fixed platform using distant visual cues from semi-random start locations and the escape latency is recorded. Since data are right-truncated at 60 seconds, in contradiction with a normal distribution and causing potentially biased results, statistical guidelines have been published to properly characterize learning behaviors using survival data 2.\n\nDuring a probe test session, the platform is removed and animals freely navigate into the pool from the same start location and for the same fixed amount of time (e.g. 60 seconds). The path of the animal is recorded using a video camera and an automatic tracking system. Data collected during the probe test session can be classified into three categories: time spent per zone, which can be theoretical quadrants defined on the pool or a theoretical annulus drawn around the platform location; number of crossings of the platform area; or total proximity to the platform center 3. Creating a large database using several published tests from their institute and simulated data, Maei et al. have shown that total proximity allows the best detection for small samples, whereas time spent in quadrants is of great interest for bad performers 4. Since this test is often used to characterize memory loss, time spent in theoretical quadrants is mostly found in the literature.\n\nSeveral hypotheses can be tested using data obtained from time spent in quadrants: ’Can one group of rodent remember the platform location?’ or ’Is there any difference of memory abilities between several groups of rodents?’. In both cases, the statistical analysis of the data often focuses on the target quadrant (e.g. that where the platform was placed during the learning phase) using parametric tests like ANOVAs and t-tests1. These tests are based on normal distributions and independence, which cannot be accurately assumed in this context since 1) variables are defined on a finite interval and 2) variables corresponding to the time spent in the four quadrants are necessarily anti-correlated. Moreover, these tests neglect the time spent in the three other quadrants inducing a loss of information and hiding the aspect of preference for one quadrant that is supposed to reflect efficient spatial memory. Some authors used non-parametric alternatives, but even if their use may be preferable with the sample size of behavioral studies, they still do not fully describe the experiment. These observations suggest that a better characterization and a more suitable statistical analysis of data obtained through the MWM could significantly improve the accuracy of the results.\n\nFocusing on the question ’Can one group of rodents remember the platform location?’ to evaluate memory abilities, we suggest to use the Dirichlet distribution, a distribution that describes several variables with a constant sum, to collectively describe the fraction of time spent in the four quadrants of the maze. This test would provide a unique p-value allowing determination of whether the rodents spent the same amount of time in the four quadrants or not, a primary indication of significant spatial memory. In the case of differences between the four quadrants, this test can be followed by four post-hoc Student t-tests to identify preference or aversion for some quadrants. In comparison, the currently used method (i.e. directly using the Student t-test on the target quadrant) does not allow to identify memory loss and may hide some bias (Figure 1).\n\nTo determine whether one group of animals has a preference for a quadrant one usually uses t-tests or ANOVAs assuming normal distribution and independence. Those assumptions do not describe correctly the dataset obtained from a MWM. In comparison, the Dirichlet distribution allows to answer the same question but describes properly the constant sum constraints and interedependence of the variables.\n\nWe will first describe the methodology we developed with the Dirichlet distribution and the correction required to fit with the sample size of behavioral experiments. Using simulated data we will show that beyond the better description of the results, using Dirichlet distribution allows reducing the number of false positives and false negatives, significantly improving the reliability of the analysis. We then applied this test on in vivo data to validate its use in experimental conditions, also providing a way to graphically present the data that takes into account interindividual variability. Finally, we will discuss the advantages and limits of the application of the Dirichlet distribution on behavioral studies, broaching the major inputs that using Bayesian inferences could bring in this field of research.\n\n\n2 Methods\n\nThe Dirichlet distribution is a multivariate generalization of beta distributions. It describes the distribution of K-dimensional vectors p for which the sum of all the coordinates is fixed, i.e. ∑k=1Kpk=1. It is parametrized by a K-dimensional vector α of positive reals αk > 0, 1 ≤ k ≤ K, such that its probability density function is given by\n\n\n\nwhere B(α)=∏k=1KΓ(αk)/Γ(s) is the multivariate beta function and s=∑k=1Kαk is the precision. The marginal distributions are beta distributions with parameters (αk, s –αk) with expectation values mk = αk/s, variance mk (1 – mk)/(s + 1) and covariance between coordinates pi and pj given by – mimj/(s + 1). Therefore, the higher the precision s, the less diffuse coordinates are around their means. The Dirichlet distribution is the most general distribution for fixed-sum variables, motivating its use to describe compositional or fractional data such as MWM data, where K = 4. The likelihood of a sample of N independent observations D = {p1, . . . , pN} is given by\n\n\n\nDescription of the test To reflect memory abilities, we would like to test whether the fraction of time spent in the four quadrants significantly differs from a uniform distribution, thus showing preference for one or several quadrants. To do so, we propose a likelihood-ratio test based on the Dirichlet distribution to distinguish between the null hypothesis of uniformity H0 : {∃α > 0, ∀k, αk = α} (implying that all means mk are equal to 1/K but the precision is not constrained), and the general hypothesis H1 where the αk’s are unconstrained. The likelihood-ratio statistic reads\n\n\n\nIn order to fit the distribution parameters to their maximum likelihood values, we refer to the numerical schemes developed in 5 and we used the open-source Python module dirichlet implemented by Eric Suh2 and run with Python 3.6. In particular 5, proposes a technique to alternatively fit the means mk and precision s, faster than fitting directly the αk’s. The maximum likelihood parameters under the null hypothesis are thus estimated by setting the means to their uniform value, mk = 1/K, and fitting the precision s. We provide a slightly modified version of the dirichlet package, forked from that of Eric Suh, which is publicly available3. Under the null hypothesis, the likelihood-ratio statistic Λ asymptotically follows a χ2-distribution with K – 1 degrees of freedom.\n\nBartlett correction Biological samples are usually limited and for small samples the statistic’s distribution deviates from a χK−12, as can be seen in Figure 2. We propose an approximate Bartlett-type correction 6 for small samples, which amounts to rescale the likelihood-ratio statistic to match its asymptotic mean, which is K – 1 in this test. Such a correction has been shown to correctly reproduce the first three moments of the asymptotic χ2-distribution 6. In order to derive the scaling factor, we needed to compute the expected value of the statistic as a function of the number of samples N and the precision s. To do so, we drew random samples of N observations from uniform Dirichlet distributions with precision s, varying N between 2 and 100 and s between 1 and 1000 (with logarithmically-spaced values), and measured the mean of the statistic Λ4. We found that the mean value of the likelihood-ratio statistic Λ depends very little on s in the probed range, and that the difference to the asymptotic value of K – 1 is well-fitted with a power law in N, i.e. 〈Λ〉 – (K – 1) ~ aKNbK (data not shown). We found the approximate values aK = 5.9 and bK = –1.4 for K = 4. We therefore propose to use a corrected statistic\n\nHistograms of the likelihood-ratio statistic Λ are represented in different colors according to the N value. Means are represented by vertical lines of the same color. The χ32-distribution is represented in blue. For small samples, the distribution of the likelihood ratio slightly deviates from a χ32 distribution and the mean is significantly greater than the theoretical value of 3.\n\n\n\nValidation of the test To validate this correction, we compared the distribution of the uncorrected statistic Λ and the corrected statistic Λ˜ from our simulated samples to a χK−12-distribution with probability-probability plots. As shown in Figure 3, the uncorrected statistic yields p-values significantly different from the theoretical ones while the corrected p-values are in perfect agreement with the χ32-distribution. Therefore, this correction significantly improves the reliability of the test for small samples.\n\nThe uncorrected and corrected statistics are compared with a χK−12 distribution for several sample sizes N. Grey lines represent equality between Λ percentiles and χK−12 percentiles. Blue lines correspond to the uncorrected statistics and green lines to the corrected one. There is a difference between the p-values from the uncorrected statistics and the theoretical ones that disappears after correction, especially for small sample size.\n\nWe also computed the number of false negatives on the simulated data to evaluate the rate of type 1 error (Figure 4). We found that using the p-value from the corrected statistic leads to a consistent rate of type 1 error (i.e. α =5%), independent on the number of samples N or the precision s. On the contrary, using the non-corrected statistic leads to more false negatives, especially when the number of samples is small.\n\nThe rate of false-negative (i.e. type 1 error) using the corrected version of the statistic is represented by the blue line whereas the rate of false-negative using the uncorrected version of the statistic is represented by the blue dotted line. We found that the correction results in a consistent rate of false negatives, independent of the sample size N and precision s in the range tested. The red line represents the rate of false-negative using a single-sample t-test on the target quadrant.\n\nComparison with the one-sample Student t-test In order to compare the type 2 error obtained with the Dirichlet distribution with the results obtained using a one-sample t-test on the target quadrant as often done in the literature, we simulated data from a non-uniform Dirichlet distribution with the parameters α = (40; 20; 20; 20). We found that the p-value from the corrected statistic of the Dirichlet distribution is mainly lower than the one obtained with a single t-test on the target quadrant (75% of the p-values are lower for s = 30). This means that for some cases where the target quadrant is preferred, using a one-sample t-test on the target quadrant would not detect this preference whereas the test based on Dirichlet distribution would detect the divergence from uniformity. Beyond improving the description and the interpretation of data from the MWM, the test we propose extracts more information from the same experiment as it is based on a larger dataset and then decreases the number of false-positives.\n\nPost-hoc analysis Using the test based on Dirichlet distribution, we can determine whether the fraction of time spent in the quadrants is uniformly distributed. In the case of a divergence from uniformity, we would like to evaluate what are the quadrants responsible for this divergence as a post-hoc analysis.\n\nThis can be performed by comparing the marginal distributions of each quadrant, that are Beta distributions, to a theoretical Beta distribution with parameters α = 0.25s and β = 0.75s. However, we noticed that in the range of inter-individual variability we have in this kind of study (given by the parameter s of the Dirichlet distribution, usually found between 20 and 50), the marginal distribution are fairly close to a normal distribution. Seeking for simplification, we advise to apply single sample t-tests for a post-hoc characterization of the preference for a quadrant in groups showing a divergence from uniformity.\n\nBayesian analysis can be used to infer constraints on the parameters αi’s of the Dirichlet distribution used to model the data (and subsequently the means mi’s). Specifically, we performed nested sampling of the parameter space of the Dirichlet distribution using the PyMC3 package with Python 3.6. For simplicity, we used the Jeffreys prior5 π(α) which does not depend on the model parametrization (e.g., sampling over α or (m, s)) and leave the discussion about this choice for future work. The output is a sample of vectors α distributed as the posterior given the data, i.e. 𝒫 (α \| D) ∝ 𝒫 (D \| α)π(α), which enables us to compare confidence regions for different groups and visualize the consistency with uniformity.\n\n\n3 Results\n\nWe used a dataset obtained comparing memory abilities of female wild-type mice to female 3xTg AD mice, a model for Alzheimer’s Disease 7. All information related to experimental and ethical procedures are available in 8.\n\nWe compared the distribution obtained for each group to a uniform distribution and we found that the Dirichlet distribution obtained for wild-type mice was significantly different from a uniform distribution (p = 0.0021), whereas the one obtained for 3xTg mice did not differ from a uniform distribution (p = 0.26). We also propose a module, included in the Dirichlet package, to draw charts showing at the same time mean values with uncertainties6 and inter-individual variability according to Dirichlet distribution (Figure 5). This result shows that 3xTg AD mice display long term memory deficits, which is in accordance with previous observations 9.\n\nIn this plot, each column represents a sample and each color represents a quadrant. Mean values for the fraction of time spent in each quadrant is represented by a dotted line and the error bars on the means are approximated with the inverse Fisher information. For 3xTg mice the fraction of time spent in each quadrant is approximately similar leading to a uniform distribution (p = 0.26) whereas for wild-type mice the time spent in the target quadrant is significantly higher leading to a non-uniform distribution (p = 0.0021).\n\nTo better characterize long term memory in wild-type mice, we applied single sample t-tests on the four quadrants as a post-hoc analysis. We performed a one-tailed single sample t-test to assess whether the fraction of time spent in the target quadrant by wild-type mice is greater than the theoretical value 25%. Conversely, we performed a one-tailed single sample t-test to assess whether the fraction of time spent in the opposite quadrant by wildtype mice is lower than the theoretical value 25%. For adjacent quadrants we performed two-tailed single sample t-tests. We observed that the fraction of time spent in the target and opposite quadrants were respectively significantly higher (p = 0.025) and lower (p = 0.013) than 25%. The fraction of time spent in the adjacent quadrants did not differ from 25%.\n\nUsing this dataset with usual sample sizes for behavioral studies (N=7), we confirmed that our test is able to discriminate efficient and deficient memory abilities on real data.\n\nWe inferred constraints on the parameters of the Dirichlet distribution for wild-type and 3xTg mice. Figure 6 indicates compatibility of the data with uniformity for 3xTg mice and shows a clear preference for the target quadrant for wild-type mice suggesting memory deficits in 3xTg mice compared with wild-type.\n\nCorner plot representing constraints on the mean fractions of time mi’s for the two data sets wild-type (blue) and 3xTg (green). The diagonal plots show the marginal distributions of mi’s (with shaded 68% confidence interval) and off-diagonal plots show the two-dimensional distributions of pairs of these variables (inner and outer contours represent the 68% and 95% confidence levels). The black dashed lines represent the case of uniformity (25%) and the red lines correspond to equal time spent in both considered quadrant. Constraints on m1 (leftmost column) indicate that wild-type mice favor the target quadrant.\n\n\nDiscussion\n\nWe proposed a statistical approach for the analysis of MWM data based on the Dirichlet distribution as a model for the fraction of time spent by rodents in the quadrants of the maze. In the context of behavioral experiments that usually generate a lot of data with high inter-individual variability, a lot of parameters can be taken into account to extract evidence of memory abilities 3. In the literature, the time spent in quadrants – the target quadrant, but sometimes also the opposite quadrant 9,10 – is commonly used to assess long-term memory. Even if the focus on the time spent in quadrants is broadly accepted as a good index to evaluate reference memory, there is no consensus about the processing of these data. In this context, the Dirichlet distribution has the great advantage to simultaneously take into account the four quadrants and to correctly account for the constant-sum constraint of such data, which implies both deviation from the normal distribution and interdependence. That way, it gives a correct description of the data obtained from MWM probe tests and provides meaningful plots representing mean performances and inter-individual variability.\n\nWe showed that the corrected test based on the Dirichlet distribution gives a consistent rate of false-negative, even for small sample size. This indicates that this test can be safely used even in the context of behavioral studies with sample size smaller than 10 individuals, as we confirmed using the results previously obtained on wild-type and 3xTg AD mice.\n\nBeyond the great improvement in the description of MWM data, we also showed that this test gives less false-negatives than its inaccurate but commonly used alternative, the Student t-test. Therefore, using Dirichlet distribution is the best option to extract reliable information from a MWM probe test.\n\nHowever, there are two main limitations in the use of the likelihood-ratio test based on the Dirichlet distribution: 1) it cannot directly identify the preferred quadrant and 2) it cannot compare memory abilities between several groups of animals. We proposed to overtake the first limitation by performing a post-hoc analysis to determine which quadrants are responsible for divergence from uniformity. We showed that performing single-sample t-tests as a post-hoc analysis (instead of ad-hoc) is satisfying. However, more interesting results can be obtained using Bayesian statistics, a method that can also permit comparison between groups. Deriving informative p-values on binary tests from such analysis remains challenging but represents an active field of research that could soon provide a great opportunity to improve MWM statistical analyses.\n\n\nConclusion\n\nWe propose here a new way to analyze MWM probe test data that accurately and simultaneously describes the four variables of time spent in the quadrants and allows to extract more information from the same experiments than the currently used method. All the packages required to perform the statistical test and to draw the corresponding chart are publicly available7 and can be easily run with R using the reticulate package 11. Minor modifications of this test would allow to apply the same methodology on other behavioural tests facing the same constraints like H-Maze or Y-Maze.\n\n\nData availability\n\nA Python notebook with the code to reproduce the simulations and figures is available at https://github.com/xuod/dirichlet 12. In vivo dataset available from: https://github.com/xuod/dirichlet/tree/master/example 12. (Experimental procedures and data acquisition are detailed in 8).\n\n\nSoftware availability\n\nDirichlet package from Eric Suh (Fitting the parameters of a Dirichlet distribution) available from: https://github.com/ericsuh/dirichlet\n\nLicense: MIT\n\nDirichlet package used in the present study (Likelihood-ratio test based on Dirichlet distribution) available from: https://github.com/xuod/dirichlet/tree/master/dirichlet\n\nArchived package as at time of publication: http://doi.org/10.5281/zenodo.3373955 12\n\nLicense: MIT", "appendix": "Footnotes\n\n1Among the 30 most recent articles using the Morris Water Maze test and published at the end of October 2018, 25 used time spent in quadrants as a criterion. 23 used a parametric test (Student t-test or ANOVA) to analyze their data, whereas only 2 used a non parametric alternative. 24 out of 25 articles presented data as bar charts without presenting inter-individual variability.\n\n2https://github.com/ericsuh/dirichlet\n\n3https://github.com/xuod/dirichlet\n\n4For each tuple (N, s), the number of samples is increased until the means is measured with relative error below 0.01.\n\n5For the K-dimensional Dirichlet distribution, the Fisher information is I (α) = diag(Ψ1 (α)) − Ψ1 (s)J4, where J4 is a K × K matrix of ones, and the Jeffreys prior is π(α)∝\|I(α)\|.\n\n6We approximate the variance by the inverse Fisher information, which is a lower bound, given by σ^2(αi)=1/(ψ1(αi)−ψ1(s))n where ψ1 is the trigamma function. A full bayesian analysis can be performed to obtain those error bars, as suggested in Section 2.3.\n\n7https://github.com/xuod/dirichlet\n\n\nReferences\n\nMorris R: Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984; 11(1): 47–60. PubMed Abstract \| Publisher Full Text\n\nJahn-Eimermacher A, Lasarzik I, Raber J: Statistical analysis of latency outcomes in behavioral experiments. Behav Brain Res. 2011; 221(1): 271–275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVorhees CV, Williams MT: Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006; 1(2): 848–858. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaei HR, Zaslavsky K, Teixeira CM, et al.: What is the Most Sensitive Measure of Water Maze Probe Test Performance? Front Integr Neurosci. 2009; 3: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinka TP: Estimating a Dirichlet distribution. Annals of Physics. 2003; 2000(8): 1–13. Reference Source\n\nBarndorff-Nielsen OE, Cox DR: Bartlett adjustments to the likelihood ratio statistic and the distribution of the maximum likelihood estimator. J R Stat Soc Series B Stat Methodol. 1984; 46(3): 483–495. Publisher Full Text\n\nOddo S, Caccamo A, Shepherd JD, et al.: Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003; 39(3): 409–421. PubMed Abstract \| Publisher Full Text\n\nMaugard M: Role of Astrocytic Serine in Learning and Memory and its Implications in Alzheimer’s Disease. PhD thesis, Université Paris Saclay. 2018; 6. Reference Source\n\nClinton LK, Billings LM, Green KN, et al.: Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice. Neurobiol Dis. 2007; 28(1): 76–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBillings LM, Oddo S, Green KN, et al.: Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice. Neuron. 2005; 45(5): 675–688. PubMed Abstract \| Publisher Full Text\n\nAllaire JJ, Ushey K, Tang Y, et al.: reticulate: R Interface to Python. 2017. Reference Source\n\nSuh EJ, Doux C, Braem N: xuod/dirichlet 0.8 (Version 0.8). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3373955" }	[ { "id": "53541", "date": "23 Sep 2019", "name": "Richard GM Morris", "expertise": [ "Reviewer Expertise Neurobiology of learning and memory." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMaugard et al. identify a major difficulty in analyzing probe test data from the watermaze which typically consists of time (in sec) or proportions of time (%) spent in each of four quadrants of the pool. While it is straightforward to suppose that more time should be spent in the training quadrant, leading some to analyze only that quadrant, a better method would be to use a mathematical approach that recognizes that the time in all 4 quadrants must add to 100% but their distribution is of interest. Some authors have attempted (upon advice) to address the quadrants problem by reducing the numerator degrees of freedom by 1 (e.g. Morris), but this is unlikely to be statistically adequate, and anyway suffers from issues associated with the normality of the data etc. What is proposed here is something called the Dirichlet distribution which explicitly recognizes the summation to 100% problem and provides a mathematical way of looking at more than just the training quadrant.\n\nThis innovation seems valuable, but I add a qualification. This is that the watermaze is, essentially, no more than a pool of water with a hidden platform in which a large variety of different tasks can be run. That most users of the watermaze use only the standard reference memory task does not mean that other variants are not of interest - reversal learning (Hans-Peter Lipp and David Wolfer, Univ Zurich), the delayed match to place task (Morris, e.g. Steele and Morris Hippocampus 19991), and procedures for dissociating encoding and retrieval (e.g. Rossato et al. Current Biology 20182). These other procedures are of analytical interest. However, they may still require probe test data (e.g. Rossato et al. 20182).\n\nMy recommendation is that the paper be provisionally accepted - it makes a very valuable point - but attention must be paid to the fact there is not just one single task that can be run in a watermaze. There is greater potential.\n\nTo add also, there are a large variety of measures of performance including latency, path-length, directionality, proximity index and all the measures associated with a probe test. Given this, the thrust of the paper is arguably less novel than the authors surmise, albeit this being a very clever solution to an unsolved problem associated with probe tests.\n\nRecommendation: index subject to small revisions (as advised above).\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4987", "date": "28 Oct 2019", "name": "Gilles Bonvento", "role": "Author Response", "response": "We would like to thank the reviewer for his positive comments. We appreciate the explicit acknowledgment regarding our valuable test. We have now mentioned in the text that a large variety of different tasks can be run using modifications of the basic protocol." } ] }, { "id": "53542", "date": "30 Sep 2019", "name": "Avgoustinos Vouros", "expertise": [ "Reviewer Expertise Machine Learning", "Behavioural Neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors suggest a new statistical procedure to analyze in more depth a common performance measurement in the Morris Water Maze experimental procedure.\nThe manuscript is well-written and all the methods explained in detail. The new proposed method is well-justified and shows much potential in the field of data analytics for behavioural procedures similar to the Morris Water Maze. Code of all the methods that the author describe is open source and publicly available. Instructions on how to run the code are clear.\nI would like to point out some aspects that could have been addressed/discussed more:\n\nFor the post-hoc analysis the authors suggest the usage of t-tests on the four quadrants. Have the authors consider, if applicable, alternative tests of hypothesis in case the assumptions of the t-test (e.g. the data are not normally distributed) are not fulfilled?\n\nThe authors focus on the probe trials but couldn't analysis on other types of trials (e.g. reversal learning) also been benefit from their method? Can the authors comment if their method is also applicable to other scenarios where there are mutually exclusive possibilities?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4988", "date": "28 Oct 2019", "name": "Gilles Bonvento", "role": "Author Response", "response": "We thank the reviewers for their positive comments regarding our statistical test. For the post-hoc analysis the authors suggest the usage of t-tests on the four quadrants. Have the authors consider, if applicable, alternative tests of hypothesis in case the assumptions of the t-test (e.g. the data are not normally distributed) are not fulfilled? As far as we know, there is no test available to compare non-normal distribution with a theoretical one. The authors focus on the probe trials but couldn't analysis on other types of trials (e.g. reversal learning) also been benefit from their method? Can the authors comment if their method is also applicable to other scenarios where there are mutually exclusive possibilities? Our method can be used each time a probe test is performed with the sum spent in the 4 quadrants totalling 100%, such as for reversal learning." } ] } ]	1	https://f1000research.com/articles/8-1601
https://f1000research.com/articles/7-1644/v1	16 Oct 18	{ "type": "Study Protocol", "title": "Assessing service and treatment needs of young people who use illicit and non-medical prescription drugs living in Northern Ontario, Canada", "authors": [ "Thepikaa Varatharajan", "Pamela Sabioni", "Cayley Russell", "Joanna Henderson", "Benedikt Fischer", "Sarah Miles", "Jürgen Rehm", "Pamela Sabioni", "Cayley Russell", "Joanna Henderson", "Benedikt Fischer", "Sarah Miles", "Jürgen Rehm" ], "abstract": "Background: The use of illicit and prescription drugs for non-medical purposes among youth and young adults living in Northern Ontario communities is a major public health concern. This problem is amplified in that there is insufficient knowledge on the types of services and treatment centers available for and utilized by young people with substance use issues in Northern Ontario. This needs assessment study aims to examine the service and treatment needs of youth and young adults who use drugs in Northern Ontario communities. Methods/Design: A mixed-methods study design will be used to assess the service and treatment needs of youth and young adults (aged 14-25) who have used one or more illicit drug (excluding cannabis) and/or psychoactive prescription drug for non-medical purposes for at least 3 months and on at least 10 days in the last month. Participants will be recruited from approximately ten Northern, remote and rural communities across Northern Ontario using a mobile research lab. Eligible study candidates from each community will be asked to partake in a focus group and questionnaire exploring service and treatment utilization and needs. We will additionally collect basic socio-demographic information as well as examine patterns of problematic drug use. Interviews with service providers and community organizers will also be conducted in each community. Discussion: Findings from our study will highlight the availability, accessibility and utilization of existing services; identify the gaps and barriers in current service provision; and provide insight into the service and treatment needs of youth and young adults who use drugs in Northern Ontario communities. Assessing the needs of young people who use drugs will allow service providers, community organizers and health policymakers to improve addiction-related services and treatment centers in Northern Ontario.", "keywords": [ "Canada", "drug use", "needs assessment", "Northern Ontario", "services", "treatment", "youth", "young adults" ], "content": "Introduction\n\nThe high prevalence of illicit drug (such as heroin, cocaine, and amphetamines) and non-medical prescription drug (such as oxycodone, barbiturates and anti-depressants) use has become a major global health concern, with the non-medical use of prescription opioids (POs) especially prevalent in North America1–4. According to the Canadian Tobacco, Alcohol and Drug Survey (2015), approximately 2% of Canadians in the past-year used at least one of the five following illicit drugs: cocaine or crack, ecstasy, speed or methamphetamines, hallucinogens or heroin5. Moreover, approximately 60% of people who use illicit drugs are between the ages of 15 to 24, and this age group is more likely to use illicit drugs heavily, and in more risky ways6,7.\n\nIn Ontario, the most commonly used illicit drugs among young people after cannabis are cocaine, ecstasy and hallucinogens8. The most frequently used prescription drugs for non-medical purposes among youth and young adults are opioid pain relievers, drugs prescribed for attention deficit hyperactivity disorder and tranquilizers5,8. In 2015, 21.5% of Ontario students from grades 7–12 reported using any drug other than cannabis in the last year, including 12% who used a prescription drug (e.g. Percodan, Tylenol #3, codeine) for non-medical purposes8. Among young adults (18–29 years old), approximately 5% used POs for non-medical purposes in the past year9.\n\nSubstance use patterns among youth and young adults living in Ontario vary with geographical setting (e.g.; northern vs. southern jurisdictions and rural vs. urban communities)10–12. These differences could be due to economic, cultural, social and educational factors11. For example, opioid dependence among young people has become a widespread issue in Northern Ontario10,13,14. Findings from a study by Kurdyak et al., (2017) indicate that PO misuse is significantly higher among youth and young adults in Northern Ontario than the rest of the province15. As an indicator, the rate of methadone maintenance treatment patients per 100,000 between the ages of 15 to 24 in 2014 was approximately 2-fold (118 per 100,000) and 6-fold (325 per 100,000) higher among the North East and North West Local Health Integrated Networks (LHINs), respectively, compared to the other LHINs in Ontario (50 per 100,000)15.\n\nSurvey findings suggest that the past-year use rates of illicit and non-medical prescription drugs among youth and young adults living in the Northern LHINs (North West, North East and North Simcoe Muskoka) are generally similar to the rest of Ontario8,9. Approximately 13% and 11% of youth attending schools in the North East and North West LHINs respectively, used a non-prescription medical drug in the past year, while the provincial estimate was 12%8. However, use rates in the Northern LHINs may be underestimated due to sampling procedures and survey inclusion and exclusion criteria (e.g. youth and young adults not attending schools; young people residing in hospitals, and prisons; First Nation communities; and transient populations such as the homeless or marginally housed are excluded)8–10.\n\nIn addition, Northern Ontario is home to a large number of Indigenous communities16,17. Indigenous youth are considered particularly vulnerable to illicit substance misuse and abuse due to the historical and systemic impacts of colonization, inter-generational violence/trauma and cultural genocide18. Some First Nations youth begin using drugs, such as ecstasy and POs, at ages as young as nine years old19,20. In the Nishnawbe Aski Nation, which represents 49 First Nation communities within Northern Ontario with a total population of 45,000 people, up to 50% of the youth misuse POs (mainly Oxycontin)21,22. Furthermore, solvent abuse (especially inhaling gasoline) was found to be a major concern among First Nation and Inuit youth living in Northern Ontario communities10,23,24.\n\nAcross Northern Ontario, there are a variety of community health and social services and treatment centers available to individuals with mental health and addiction issues10,25–27. Approximately 34 and 44 of these mental health and addiction service providers are funded by the North West and North East LHINs, respectively17,28. However, it is unclear whether these services and treatment centers are being accessed and utilized by young people who use drugs. For example, in 2017 it was reported that children and youth (aged 0–24 years) living in the North West LHIN had the highest rates of emergency department visits (37.7 per 1000 population) for mental health and addiction problems compared to the rest of Ontario (16.3 per 1000 population)29,30. Although more children and youth are seeking help in these socially disadvantaged areas, the use of emergency departments for mental health and addiction problems may indicate a lack of timely access to primary care or community-based mental health and addictions services and treatment centers29,30. Furthermore, high rates of emergency department visits may also indicate a low awareness of community-based services in Northern Ontario communities29,30.\n\nMoreover, there is currently limited knowledge with regards to how well the existing services and treatment centers in Northern Ontario are addressing the needs of young people with substance use issues. An addictions service needs assessment study conducted in Ontario-based First Nations communities - of which a majority were located in Northern Ontario - reported that the addiction-related education, promotion and prevention programs in their communities mainly targeted adults19. For Indigenous youth and young adults who use drugs, there have been calls for culturally-based and culturally safe services, particularly those that are informed by the institutional and intergenerational trauma experienced by Canada’s First Nations, Métis and Inuit peoples19,31. Previous studies have also emphasized the importance of providing adequate support to transitional-aged youth (ages 18–24); Lesbian, Gay, Bisexual, Trans and Queer (LGBTQ)-competent services; and gender-specific services in Northern, rural and remote regions26,27,32,33. A more thorough understanding of the service and treatment needs of youth and young adults who use drugs in Northern Ontario is essential in identifying and addressing the gaps in current service provision and utilization.\n\n\nMethods/design\n\nThis study aims to determine the service and treatment needs of youth and young adults (aged 14–25) who regularly use illicit and non-medical prescription drugs in communities across Northern Ontario. The specific objectives of this community-based needs assessment are to:\n\n1. Examine the service and treatment needs of young people who use drugs in Northern Ontario.\n\n2. Assess the availability, accessibility and utilization of existing services and treatment centers in Northern Ontario for youth and young adults with addiction and substance use issues.\n\n3. Identify the gaps and key barriers to service and treatment utilization among young people who use drugs in Northern Ontario communities.\n\nThis study will be conducted in approximately ten remote and rural communities across Northern Ontario, including: North Bay, Thunder Bay, Sudbury, Sault Ste. Marie, Timmins, Kenora, Fort Frances, Dryden, Smooth Rock Falls and Kapuskasing. Other communities might be added to the study according to availability and feasibility.\n\nThis study will use a convergent parallel mixed-methods approach, which includes conducting: 1) a self-administered questionnaire and a focus group with young people between the ages of 14–25 years; and 2) one-on-one interviews with service providers who directly work with youth and young adults who use drugs in their communities34,35.\n\nTo pilot the questionnaire and focus group questions and the study procedures in the mobile research lab, we contacted community organizers from addiction and substance use related services in the Greater Toronto Area and surrounding area to help with participant recruitment. Youth and young adults (aged 14–25) who use illicit and non-medical prescription drugs were eligible to participate and were compensated with a $20 gift card for their participation. The data collected from our pilot sessions will not be used for data analysis. At the end of the pilot study sessions, we asked participants to provide feedback on our focus group and questionnaire questions.\n\nParticipant recruitment. A collaborative approach will be used to recruit young participants, where study personnel will aim to network and build relationships with health and social service organizations within each community prior to entering the field. Key community collaborators will be asked to inform their colleagues, community partners, and young people about the study. Posters and handouts containing study details (such as date, time, contact information and location of data collection procedures) will be sent to community collaborators to distribute and post in their services. If possible, community collaborators will also organize focus groups (4–5 participants per group) in advance for study investigators.\n\nResearchers will also attempt to directly recruit participants by informing individuals who approach the mobile research lab about the study. Participants who have completed the study will also be encouraged to ‘spread the word’ about the study to their peers using a respondent-driven sampling technique, which has been approved by the Centre for Addiction and Mental Health (CAMH) Research Ethics Board (REB)36. Participants who would like to recommend the study to their peers will be given five coupons. For every eligible participant that is recruited, a $5.00 gift card will be given to the individual who made the recruit. An individual can earn up to a maximum of $25.00 for participant recruitment. Research staff will keep track of the coupons distributed by recording the coupon code under each participant’s unique study code.\n\nStudy procedures. Two study personnel will be travelling to each of the communities, using the CAMH mobile research lab [designed for an earlier study: Wells et al., 201137]. The mobile research lab is a custom-built cargo van that allows researchers to conduct data collection procedures in communities across Northern Ontario, including remote and rural communities37. The mobile lab will be parked in a location that is easily accessible and visible to participants in each community.\n\nAll study candidates will be screened for eligibility either in-person or over the phone by study staff using a screening form. The screening process should take less than 5 minutes. Eligible participants must meet the following criteria: a) be between 14 and 25 years of age at the time of consent; b) have used illicit drugs (including prescription drugs used for non-medical purposes and illicit psychotropic drugs, but not including cannabis) for at least 3 months; and c) used one or more of these drugs on at least ten days within the past month. Youth aged 14–16 years old will be eligible to participate as the risks associated with this study are low. Participants will be excluded if they are: a) unable to provide informed consent or b) unable to speak English. Eligible participants, who have been screened over the phone and have verbally agreed to participate in the study, will be asked to meet research personnel at a specified date/time/location to partake in research activities. A unique and confidential code will be assigned to participants to confirm their identity on the day of data collection. Eligible study candidates who were screened in-person will be asked to immediately partake in study activities.\n\nAll eligible participants who are interested in partaking in the study will be asked to sign a consent form (Supplementary File 1). We will not seek parental consent for youth between the ages of 14 to 16 years old because they may be street-involved, disconnected from their parents, or not want to reveal their drug use to their parents. To ensure that all participants (including those who are under 16 years of age or may not be capable of consent) understand the study and the possible consequences of participation, study staff will review the consent form with the participants and clarify any questions the participants may have. Research staff working with participants will be trained by a CAMH clinical physician to judge capacity by assessing acute intoxication and other relevant impairments that may interfere with the participant’s ability to consent.\n\nOnce the consent forms have been signed, participants will be asked to partake in a 30-min, semi-structured focus group, which will be audio-recorded (Supplementary File 2). In each focus group, there will be two moderators and 2–5 participants. If only one participant is available, a one-on-one interview will be conducted using the same questions in the focus group to capture the thoughts and perceptions of the participant. Both moderators will facilitate the discussion using open-ended questions and maintain an audit trail of emerging themes through memo writing and reflections during focus group sessions.\n\nAfter completing the focus group session, participants will partake in a 20-min self-administered questionnaire (Supplementary File 3). Depending on their preference, participants can choose to complete the questionnaire on paper or using a tablet (off-line Qualtrics Survey software)38. Study staff will offer to administer the questionnaire verbally to accommodate participants who might have low literacy skills and/or prefer verbal completion. Both the focus group and questionnaires will explore the participants’: 1) drug use; 2) knowledge of and experience using the available services and treatment centers in their community for their drug use; 3) personal barriers to accessing and utilizing addiction-related services and treatment centers; and 4) service and treatment needs. At the end of the study session, participants will be compensated with a $20 gift card and will receive a listing of available addiction and mental health services and treatment programs in their community.\n\nParticipants and recruitment. In each community, we will recruit 3–5 service providers to participate in a semi-structured, one-on-one interview. Eligible participants will hold a formal position in the community in which they serve or interact with young people who use drugs (e.g., social worker, addictions counsellor), or hold a position in which they influence services, decisions or policies that affect young people who use drugs (e.g., police chief, town councilor).\n\nStudy procedures. Interviews with service providers will either be conducted in-person while visiting each community or over the phone. Prior to starting the 30-minute interview, participants will provide written informed consent. The interview, which will be audio-recorded, will comprise of open-ended questions that will allow service providers to express themselves freely on the following topics: 1) their role in the community; 2) patterns of drug use among young people in their community; 3) the availability, accessibility and utilization of addiction-related services and treatment centers in their community and 4) the service and treatment needs of youth and young adults with addiction and substance use issues (Supplementary File 4). Service providers will not be compensated for their participation in this study.\n\nDuring the eligibility screening session, youth and young adults will be asked to provide their full name and telephone number. If the participant is eligible, they will receive a unique participation study code that cannot be associated with their personal information. To maintain confidentiality and anonymity, the eligibility screening forms will be kept separate from all other data collection materials and eligible study candidates will sign the consent forms using only their initials. At no point during the questionnaire or focus group sessions will we require a participant to report any personal identifiers (such as name, phone number, address, and date of birth). Service providers who agree to participate in the study will also be assigned a unique study code to protect their confidentiality and anonymity.\n\nThe unique study codes assigned will be used to link study data for the individual participant. Only members of the research team will have access to this code. Paper documents will be de-identified and stored in a locked filing cabinet in the CAMH mobile lab or in CAMH offices. Electronic files will be saved in encrypted devices in secure files that are password protected/locked. Hard copy files of data and electronic files will be held and destroyed according to usual research standards.\n\nWe anticipate recruiting at least 90 youth and young adults and 30 service providers to participate in this study. The total number of participants required for the qualitative portion of this study will be based on the principle of saturation sampling. We will continue to recruit participants until a sufficient saturation of responses or information (themes or categories) is achieved from the focus group and interviews.\n\nFor the quantitative portion of the study, we determined a sample size of at least N=90 for youth and young adults fulfilling the inclusion criteria. Such a minimal sample size would allow us to detect the following effect size for associations: a sample size of 90 achieves 80% power to detect a difference of 0.29 between the null hypothesis of zero and the alternative hypothesis correlation of 0.29 using a two-sided hypothesis test with a significance level of 0.0539–41. As for mean differences of subgroups, group sample sizes of 45 and 45 achieve 80% power to reject the null hypothesis of zero effect size when the population effect size is 0.60, and the significance level (alpha) is 0.05, using a two-sided two-sample equal-variance t-test. Effect Size was defined as d = (μ1 - μ2) / σ. According to the classification of Cohen, this corresponds to sufficient power to detect medium effect sizes42–45.\n\nFor the qualitative portion, audio-recorded data from the focus groups and interviews will be transcribed by research staff. A constant comparative method will be used to compare and contrast the data and an inductive qualitative thematic analysis approach will be used to identify key issues and themes regarding service use and needs46,47. Themes identified in the data will be coded into categories and then interpreted46,48. A qualitative computer software package, NVivoTM (version 11.0), will be used to store and organize the various codes derived from the data49,50.\n\nFor the quantitative portion of the study, a basic descriptive analysis will be conducted to assess the demographic characteristics and drug use patterns of young participants (e.g., age, gender, education level, income level, prevalence of drug use, etc.). A multivariable analysis using the statistical analysis software SAS (version 9.4) will be conducted to distinguish the relationship between characteristics (e.g. regression, analysis of variance)51.\n\nData from the qualitative and quantitative portion will be merged, analyzed and interpreted to form final conclusions34. To ensure that the needs and experiences of the youth and young adults are appropriately captured and reported, we will seek engagement with service providers, community organizers, individuals with lived experiences and First Nations, Inuit and Métis community partners and youth whenever possible to help analyze and interpret the data. Before finalizing all relevant reports and publications, all collaborators will have the opportunity to review research findings.\n\nStudy methods in this protocol have been reviewed and approved by the CAMH REB (protocol reference #133/2015). Ethics approval has also been granted by the Lake of the Woods District Hospital REB in Kenora and Sault Area Hospital REB in Sault Ste. Marie. Further ethics approval is not required.\n\nEach community partner will receive a final report with overall and community-specific study results. Study results will be published in a peer-reviewed journal and widely disseminated to service providers, agencies and systems relevant to Northern Ontario communities.\n\nWe have completed data collection and are currently in the process of writing the results.\n\n\nDiscussion\n\nFindings from this study will add to our limited knowledge of the drug use patterns of youth and young adults across Northern Ontario. This study will also identify both the availability and utilization gaps for addiction and substance use related services and treatment centers for young people who use illicit and non-medical prescription drugs living in Northern Ontario communities. This study may also be valuable for future community-based research studies that intend to use a mobile research lab in Northern, remote and rural communities.\n\nWe anticipate that the results from this study will help service providers, community organizers and health policymakers make informed decisions on how to improve the current provision of addiction-related services for young people who use drugs across Northern Ontario communities. As a result, youth and young adults between the ages of 14 and 25 who have substance use problems may find that their needs are being addressed. The young individuals who participated in the study may find the list of community specific services and treatments centers provided to them at the end of the study to be a useful resource for their addiction and substance use issues. We also anticipate that research participants will benefit from the gift cards provided at the end of the study for participation and/or participant recruitment.\n\nIn this study, there are no inherent physical risks for participants (youth, young adults and service providers). Prior to starting data collection activities, we will let participants know that they may withdraw from the study at any time without prejudice or penalty and that the research staff will be willing to answer any questions or concerns that might arise during the study session. We will also remind participants that they always have the right to decline to answer any question that makes them feel uncomfortable or they find difficult to answer. Furthermore, study staff will inform all participants that all quotes used in this study will remain anonymous and will be reported in such a way that the identity of the participant is not revealed. Quantitative data will only be reported in aggregate.\n\nFor youth and young adults, there are no risks associated with completing the questionnaire. There may be some risks associated with partaking in the focus groups. For example, a participant may experience strong feelings about disclosing their drug use or a participant's drug use could possibly be revealed by other fellow focus group participants. However, we believe these risks of disclosure are no greater than those caused by usual standard of care (e.g., attending addictions treatment or support group). In addition, we anticipate that since all focus group participants are using drugs, the youth will be less hesitant to share their experiences. To ensure the privacy of all participants, we will remind all focus group members to maintain confidentiality post research activities. We will also phrase focus group questions to elicit responses about drug use in the community, rather than about a participant’s own drug use.\n\nThere are a few limitations to this study. First, we believe that despite our efforts, fewer youth may participate in our study than anticipated. This could be due to multiple reasons including, but not limited to, lack of awareness of study, lack of interest, forget to attend study sessions, not enough motivation to complete data collection procedures, and/or fear of being stigmatized as a drug user by a family member or friend. Second, we anticipate that we will not be able to target as many youth and remote communities as planned if there is insufficient support from community providers and outreach workers. Third, the external validity of the results may be jeopardized due to self-selection bias. This is because participants may only volunteer to participate in the study to receive the gift card incentive52. Fourth, we foresee both respondent and interview biases in this study. Richness of the questionnaire data may be limited as participants may skip answers or falsify their responses. This may be because participants are limited in time, have low literacy or feel uncomfortable answering questions about their drug use. Focus group participants may have poor memory, speak dishonestly, or alter their responses to fit in with the popular response or due to the presence of an opinionated or dominating participant. Finally, we anticipate that participant recruitment and data collection procedures may be hindered due to time and weather constraints36,52,53.\n\nA follow-up study assessing the specific service needs of Indigenous youth and young adults who use drugs in Northern Ontario is imperative for future research. This study should adopt a culturally-appropriate and culturally-informed collaborative approach and should attempt to reach rural and remote Northern Ontario communities, including the Treaty 9 First Nation reserves. Future needs assessment studies conducted in Northern Ontario should also focus on addressing the service gaps experienced by specific subgroups of youth (e.g., Francophone youth, homeless youth and LGBTQ youth) who use drugs.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe authors acknowledge funding from Canadian Institutes of Health Research (CIHR) for the Ontario CRISM Node Team grant (#SMN-139150) in supporting the present work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank Cynthia Trayling for her contribution towards organizing and facilitating necessary project logistics. We would like to thank Dr. Andriy Samokhvalov for training the research staff on how to assess capacity to consent for youth who use drugs. We would also like to thank Dr. Maria Zhang for training staff on how to identify drug overdose and how to administer naloxone in response to opioid overdose.\n\n\nSupplementary material\n\nSupplementary File 1: Youth consent form.\n\nClick here to access the data\n\nSupplementary File 2: Youth focus group guide.\n\nClick here to access the data\n\nSupplementary File 3: Youth questionnaire.\n\nClick here to access the data\n\nSupplementary File 4: Key informant interview guide.\n\nClick here to access the data\n\n\nReferences\n\nHammond D, Ahmed R, Yang WS, et al.: Illicit substance use among Canadian youth: trends between 2002 and 2008. Can J Public Health. 2011; 102(1): 7–12. PubMed Abstract\n\nUnited Nations Office on Drugs and Crime: World Drug Report 2016. New York: United Nations publication (Sales No. E.16.XI.7); 2016. Reference Source\n\nUnited Nations Office on Drugs and Crime: The non-medical use of prescription drugs: Policy direction issues. 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Cambridge, MA: QSR International Pty Ltd.; 2012.\n\nSAS Institute: Statistical Analysis System Software for Windows. 9.4 ed. Cary, NC: SAS Institute Inc; 2004.\n\nRothman KJ, Greenland S, Lash TL: Modern epidemiology. Lippincott Williams & Wilkins; 2008. Reference Source\n\nEffective Interventions Unit: Needs Assessment: A practical guide to assessing local needs for services for drug users. Edinburgh, SCT; Scottish Executive Effective Interventions. Unit; 2004. Reference Source" }	[ { "id": "44607", "date": "14 Mar 2019", "name": "Marilyn Clark", "expertise": [ "Reviewer Expertise Addiction Studies", "Youth Studies", "Criminal Career Research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study protocol provides a clear and realistic research design.\nThe protocol should clearly indicate whether or not parental consent is required by law (in Ontario) to be sought for the participation of minors in addition to consent from the minors themselves. Some indication of what the legal requirements in this regard are in Canada would be useful. Provincial legislation and other applicable legal and regulatory requirements related to legal age of consent should be included in the protocol.\nThe ethical principle of respect for persons recommends that parental permission and minor consent function together to protect the child. The researchers' decision to not seek parental permission because it would disclose the behaviour of the minors to their parents is understandable but it needs to be pitted against the possible risks for the minor and the rights of the parents.\n\nAcceptance recommended if the issue of parental consent is addressed in more detail with reference to the attendant legislation.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4599", "date": "29 Apr 2019", "name": "Thepikaa Varatharajan", "role": "Author Response", "response": "Thank you for reviewing our protocol and providing your comments. According to Health Canada and the Public Health Agency of Canada's Research Ethics Board (REB), in Ontario parental/guardian consent is required for children under the age of 16. However, youth assessed and determined as “mature minors” may also provide consent. A “mature minor” is a person under 16 years of age who can demonstrate adequate understanding and decision-making capacity (i.e. being able to fully understand the nature, risks and benefits of a proposed study). In line with legal requirements, we did not seek parental consent for individuals aged 14-16. This research approach was discussed exclusively before REB approval (Centre for Addiction and Mental Health’s REB protocol reference #133/2015): “Given the low risk nature of the study, youth 14-16 years old will be eligible for the study, but only if they have the capacity to consent (i.e. able to understand the purpose of the research, what is involved and potential consequences). Study staff will be trained by clinical staff to judge capacity by gauging whether the potential participant has an understanding of the study and the consequences of participation, for example, by asking the participant to summarize the study and to answer questions about the study during the eligibility screener. Youth who are not capable of providing informed consent are not eligible for the study.” In addition, we made sure that no false decisions were taken with respect to the ability to give informed consent. The following procedures were introduced (cited from our protocol): “To ensure that all participants (including those who are under 16 years of age or may not be capable of consent) understand the study and the possible consequences of participation, study staff will review the consent form with the participants and clarify any questions the participants may have. Research staff working with participants will be trained by a CAMH clinical physician to judge capacity by assessing acute intoxication and other relevant impairments that may interfere with the participant’s ability to consent.”" } ] }, { "id": "43269", "date": "10 Jul 2019", "name": "Tess K. Drazdowski", "expertise": [ "Reviewer Expertise Young adults", "adolescents", "substance use", "prescription drug misuse", "vulnerable populations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents a study protocol for a needs assessment research project aimed at determining the substance use treatment needs of youth and young adults who currently use illicit substances (including the non-medical use of prescription drugs) in Northern Ontario. Overall, the study protocol is set to examine an important public health problem in areas that are typically under served (remote and rural communities). An innovative feature of the protocol includes a mobile research lab. Other strengths are the mixed method design, plans to interview service providers and community organizers as well as the youth and young adults, and to conduct 1-on-1 interviews with youth and adults if not enough are available for a focus group.\nSome points of consideration that may improve the study protocol are listed below:\nFirst, given that the areas of interest are likely not densely populated the inclusion criteria may be too stringent to get a large enough sample of youth and young adults to be representative of the substance using population in these areas. Specially, needing to have misused substances for at least 10 days in the last month (especially for prescription drug misuse where only 5-12% report misuse in the past year) seems as though it may eliminate a lot of individuals who would otherwise qualify if the threshold was lower. Information for why these thresholds were chosen would be helpful.\n\nRationale for why there is no compensation for service providers for their participation is recommended.\n\nThe authors mention solvent abuse in the introduction as a problem, however it is not clear if that was included in the screening of participants. It is noted that it was included in the data collection.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4981", "date": "28 Oct 2019", "name": "Thepikaa Varatharajan", "role": "Author Response", "response": "Thank you for reviewing our article. Please see our responses below. This article presents a study protocol for a needs assessment research project aimed at determining the substance use treatment needs of youth and young adults who currently use illicit substances (including the non-medical use of prescription drugs) in Northern Ontario. Overall, the study protocol is set to examine an important public health problem in areas that are typically underserved (remote and rural communities). An innovative feature of the protocol includes a mobile research lab. Other strengths are the mixed method design, plans to interview service providers and community organizers as well as the youth and young adults, and to conduct 1-on-1 interviews with youth and adults if not enough are available for a focus group. Some points of consideration that may improve the study protocol are listed below. Thank you for your comments. First, given that the areas of interest are likely not densely populated the inclusion criteria may be too stringent to get a large enough sample of youth and young adults to be representative of the substance using population in these areas. Specially, needing to have misused substances for at least 10 days in the last month (especially for prescription drug misuse where only 5-12% report misuse in the past year) seems as though it may eliminate a lot of individuals who would otherwise qualify if the threshold was lower. Information for why these thresholds were chosen would be helpful. The criteria for participants to have misused substances for at least 10 days in the last month allows us to capture youth who are frequently misusing drugs (i.e., on daily/more than weekly basis). Youth who are misusing drugs regularly are more likely to seek services and treatment centres for their drug use than youth who are experimenting with drugs. Rationale for why there is no compensation for service providers for their participation is recommended. We saw service providers as key informants. Key informants in this study either held a formal position in the community that allowed them to directly serve or interact with young people who use drugs (e.g., social worker, addictions counsellor), or held a position in which they influence services, decisions or policies that affect young people who use drugs (e.g., police chief, town councillor). These individuals were not only recruited to participate in the study, but also played a key role in helping us recruit participants and planning study logistics, i.e. they were part of the study team. We considered paying an honorarium, and informally asked about this, and received advice that this was not seen appropriate, since our key informants were participating in a professional capacity and were keen to help in any way they can. Service providers did, however, show a lot of interest in contributing to and/or receiving a community specific report with study findings. The authors mention solvent abuse in the introduction as a problem, however it is not clear if that was included in the screening of participants. It is noted that it was included in the data collection. During the screening process participants were asked if they had used any illicit or non-medical prescription drugs which includes inhalants such as gases, solvents and aerosol sprays." } ] } ]	1	https://f1000research.com/articles/7-1644
https://f1000research.com/articles/8-1798/v1	25 Oct 19	{ "type": "Case Report", "title": "Case Report: Nodular colloid degeneration of the skin", "authors": [ "Nasim Niknezhad", "Farahnaz Bidari-Zerehpoosh", "Nakisa Niknejad", "Nikoo Mozafari", "Nasim Niknezhad", "Farahnaz Bidari-Zerehpoosh", "Nakisa Niknejad" ], "abstract": "In this report, we described a 58-year-old man, presenting with multiple plaques and nodules over the nose and forehead resembling sarcoidosis or lepromatous leprosy. The histologic study revealed deposition of the mass of amorphous, eosinophilic-cleaved, colloid materials in the papillary dermis and deep dermis. The periodic acid-Schiff (PAS) stain was positive, whereas the Congo red stain was negative. Based on the clinical and pathologic findings, the patient was diagnosed with nodular colloid degeneration(NCD). To treat the patient, lesions were flattened using a Co2 laser. The patient did not return for follow-up. NCD is a rare disease, with only 12 cases having been previously reported. Here, we present a case of NCD that occurred on the face and discuss the topic of how to discriminate between NCD and other similar entities, emphasizing that nodular colloid degeneration should be considered in the differential diagnosis of asymptomatic facial plaques and nodules.", "keywords": [ "Nodular colloid degeneration", "Colloid milium", "Nodular amyloidosis" ], "content": "Introduction\n\nNodular colloid degeneration (NCD), also named paracolloid, is a rare subtype of colloid deposition disorders. It presents as a single or multiple soft to rubbery nodules or plaques usually on chronically sun-exposed skin. Histologically, there are masses of amorphous, eosinophilic material expanding the papillary dermis and extending into the deep dermis1. Nodular colloid degeneration is a rare disease. Only 12 cases have been previously reported in the literature. Here, we present a case of NCD that occurred on the face and discuss the topic of how to discriminate between NCD and other similar entities such as nodular amyloidosis.\n\n\nCase presentation\n\nA previously healthy 58-year-old farmer from Hamedan province, Iran, presented to the dermatology clinic with a three year history of multiple papular and nodular lesions on his face. The lesions had been gradually enlarging during last six months. An examination of the skin carried out by a dermatologist revealed multiple skin-colored to yellow papules and nodules of different sizes (10–25mm) located on the forehead, cheeks, and nares (Figure 1 and Figure 2). The nodules were painless and mobile, they were rubbery-soft in consistency and were attached to the overlying skin. Findings of the systemic physical examination were otherwise unremarkable.\n\nAfter local anesthesia using 2–3ml of intralesional lidocaine (2%), an excisional biopsy was obtained from forehead lesions. The specimen was put in formalin and was sent to the lab for routine hematoxylin-eosin stain and special stains (periodic acid-Schiff (PAS) staining, crystal-violet and Congo red staining).\n\nHistopathologic examination revealed hyperkeratosis, flattened epidermis, and amorphous eosinophilic material deposits in the upper dermis, which were separated from the epidermis by a Grenz zone of collagen fibers and extended into the deep dermis. The hair follicles and sebaceous glands were well preserved. There were some irregular fissures and clefts in the hyaline material dividing this material into smaller islands. Scattered nuclei of fibroblast were aligned along the line of fissuring (Figure 3).\n\nThe amorphous colloid material shows numerous irregular clefts. Scattered nuclei of fibroblasts are observed in the colloid material (haemotoxylin and eosin staining, ×40).\n\nThe special stains revealed positive reactivity of materials with periodic acid-Schiff (PAS) staining but negative reactivity with crystal-violet and Congo red staining. According to clinical and histopathological findings, nodular lesions on sunexposed areas with colloid material deposition in the dermis, a diagnosis of nodular colloid degeneration was made.\n\nFor treatment, forehead lesions were excised and for the lesions on the nose, resurfacing using a Co2 laser (Jeisys Medical Korea) was applied with a frequency of 50kHz and pulse duration of 500μs. Unfortunately, the patient did not return for follow-up and we could not obtain data on treatment efficacy, cosmetic results or recurrence.\n\n\nDiscussion\n\nColloid milium and colloid degeneration are rare degenerative cutaneous disorders, with at least four distinct clinicopathological variants. The four variants of this disease are as follows: adult colloid milium, juvenile colloid milium, pigmented colloid milium, and nodular colloid degeneration (para colloid)2.\n\nIn both the adult and juvenile forms, numerous yellow-brown, semitranslucent dome-shaped papules are developed in areas of chronic sun exposure; for example, the cheeks, ears, neck, and dorsum of the hands. In pigmented colloid milium, gray to black grouped or confluent papules are present on the face, following the excessive use of topical hydroquinone for skin bleaching2.\n\nNCD clinically presents with solitary or multiple asymptomatic nodules or plaque-like lesions usually in chronically sun-exposed areas, particularly the face. The lesions have been rarely reported on sun protected areas such as penile skin3. Although the exact etiologic role of chronic sun exposure remains uncertain, it is suggested that photo-induced damage to dermal elastic fibers, as in solar elastosis, produce the colloid4.\n\nClinically, the differential diagnosis of NCD includes nodular amyloidosis, nodular mastocytosis, nodular histiocytosis, steatocystoma multiplex, sarcoidosis, lepromatous leprosy, leishmaniasis, cutaneous lymphoma, and pseudolymphoma1,2. A histopathologic workup is a helpful tool in establishing the appropriate diagnosis. Upon histopathologic examination, an amorphous eosinophilic material with small fissure and clefts is observed in the dermis, separated from the epidermis by a Grenz zone of narrow collagen tissue2,5.\n\nHistologically, NCD must be distinguished from lipoid proteinosis, different types of colloid milium, and amyloidosis. In cutaneous lesions of lipoid proteinosis, there are masses of amorphous or laminated hyaline-like PAS-positive deposits in the dermis that have the striking perivascular pattern and also can surround the eccrine glands as well as the hair follicles and sebaceous glands6.\n\nIn juvenile colloid milium, amorphous eosinophilic deposits are found in the dermis next to basal layer without Grenz zone. In adult colloid milium, nodules of homogenous eosinophilic colloid material expand the papillary dermis and extend into the mid dermis. Fissures and clefts divide this material into smaller islands and evidence of solar elastosis are commonly seen beneath the nodules. The Grenz zone is often present in the dermis. A pigmented colloid milium is similar to adult colloid milium but contains an area of yellow-brown islands of colloid material in the upper dermis. Nodular colloid degeneration is distinguished from adult colloid milium and juvenile colloid milium by more deeply extended and less conspicuous clefting of amorphous materials within the dermis2,7.\n\nThe main histologic differential diagnosis of nodular colloid degeneration includes nodular amyloidosis. In nodular amyloidosis, there are large masses of amyloid in the dermis and subcutaneous fat, accentuate around deep vascular channels, adnexal structures, and infiltrate blood vessel walls8. Infiltration of plasma cells are usually present in and around the amyloid deposition7.\n\nThere is a considerable resemblance between colloid milium and amyloidosis, as both may stain positively for Congo red, PAS and crystal violet, and produce fluorescence with Thioflavin-T. In contrast to colloid, amyloid reacts with Pagoda-red and other cotton dyes8. Moreover, infiltrate of plasma cells and positive immunostaining for light chain deposition can aid in the differentiation of amyloidosis from NCD8.\n\nTo improve the appearance of the lesions, cryotherapy, dermabrasion, erbium:YAG or carbon dioxide laser resurfacing have been reported as effective methods, although lesions may recur. Considering the pathogenesis of NCD, sun protection can be effective in preventing NCD2. In this case, we used excision and Co2 laser resurfacing to flatten the lesions. Unfortunately, the patient did not return for follow-up and we could not obtain data on treatment efficacy, cosmetic results or recurrence.\n\nIn summary, we encountered a rare case of nodular colloid degeneration arising on the face, which was diagnosed by histopathological examination. Although it is rare, the clinical diagnosis of nodular colloid degeneration should be considered in differential diagnosis of any cases presenting with facial nodules.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nChu H, Kim HJ, Lee MG: Xanthoma-like multiple yellowish nodular colloid degeneration on the face and scalp. J Eur Acad Dermatol Venereol. 2017; 31(4): e195–e196. PubMed Abstract \| Publisher Full Text\n\nChoi WJ, Kim BC, Park EJ, et al.: Nodular colloid degeneration. Am J Dermatopathol. 2011; 33(4): 388–391. PubMed Abstract \| Publisher Full Text\n\nPreston PW, Orpin SD, Muc R, et al.: Penile colloid degeneration. Clin Exp Dermatol. 2006; 31(5): 674–676. PubMed Abstract \| Publisher Full Text\n\nHashimoto K, Black M: Colloid milium: a final degeneration product of actinic elastoid. J Cutan Pathol. 1985; 12(2): 147–156. PubMed Abstract \| Publisher Full Text\n\nPatterson JW, Wilkin JK, Schatzki PF: Nodular colloid degeneration: distinctive histochemical and ultrastructural features. Cutis. 1985; 36(4): 355–358. PubMed Abstract\n\nMcGrath JA: Lipoid proteinosis. Handb Clin Neurol. 2015; 132: 317–322. PubMed Abstract \| Publisher Full Text\n\nPatterson J: Weedon's Skin Pathology. 4th ed:Elsevier. 2016. Reference Source\n\nLai KW, Lambert E, Coleman S, et al.: Nodular amyloidosis: differentiation from colloid milium by electron microscopy. Am J Dermatopathol. 2009; 31(5): 472–474. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56166", "date": "08 Nov 2019", "name": "John J. Dávila‐Rodríguez", "expertise": [ "Reviewer Expertise Dermatology", "Melanoma", "Laser", "Cutaneous Oncology", "Dermatologic Surgery", "Aesthetic dermatology", "Dermoscopy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a very interesting case of nodular type milium colloid associated to UV radiation. It affirms the association demonstrated in several studies. The authors have made an appropriate review of the topic. I consider that after some minimum suggested changes the article should be indexed. Please make the following corrections in these sections:\nAbstract\nP.1: Please change the text “deposition of the mass…” to deposition of an amorphous, eosinophilic-cleaved, colloid material.\n\nCase Presentation\nP.2: The text “After local anesthesia using 2–3ml of intralesional lidocaine (2%), an excisional biopsy was obtained from forehead lesions. The specimen was put in formalin and was sent to the lab for routine hematoxylin-eosin stain and special stains (periodic acid-Schiff (PAS) staining, crystal-violet and Congo red staining) ” is not relevant. Please remove it.\n\nP.3: Please delete the text “of collagen fibers”.\n\nP.5: Please indicate the CO2 laser fluences used.\n\nFigure 3: Please delete the text “The biopsy specimen from the forehead area showing”.\n\nDear authors, it would be interesting to create a comparative table of the 12 cases reported in the literature. It should include possible causes, clinical features, histopathology, treatments performed and evolution of the cases.\n\nPlease include the PAS staining photograph. It could be done in the figure 3 or adding a new figure.\nPlease include the following two current references on this topic:\nDávila-Rodríguez J, Aguilar K, García L. Colloid milium, an expression of excessive sun exposure in Ecuadorian patients. Int J Dermatol. 2019: 58 (4); e80-e82. https://doi.org/10.1111/ijd.143841\n\nGhanadan A, Kamyab K, Daneshpajouh M, et al. Nodular colloid degeneration of the skin: report of three cases with review and update. Int Dermatol J. 2014; 5: 36. https://doi.org/10.4103/2229-5178.1445272\n\nWith these references please try to broaden the knowledge about the pathophysiology of the disease, for example \"UV exposure could cause degeneration of keratinocytes and elastic and collagen fibers with secondary accumulation of protein products and fibroblast secretions\".\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "55748", "date": "18 Nov 2019", "name": "Zahra Azizian", "expertise": [ "Reviewer Expertise Dermatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease mention other case reports of nodullar colloid millium.\n\nExplain about the difference of your case with other case reports of this disease\n\nExplain about the method of ruling out other differential diagnosis?\n\nPlease mention the period of time that the patient was free of disease.\n\nExplain about the common age and gender of this disease. Is your case different in any point of view from other cases of this disease?\n\nMore up to date references are required.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1798
https://f1000research.com/articles/8-1797/v1	25 Oct 19	{ "type": "Research Article", "title": "Clinico-pathology of newly diagnosed breast cancer with expression of ER, PR, and HER/2neu in cell blocks: An observational prospective study", "authors": [ "Mazin Judy Ibrahim Albaldawy", "Ahmed Abdulshahed Mubark", "Ahmed Abdulshahed Mubark" ], "abstract": "Background: Breast cancer is a worldwide problem, and early positive diagnosis is critical for establishing the optimal therapeutic strategy. Following a preliminary diagnosis, fine-needle aspirate cytology (FNAC) may be used to obtain cells for immunohistochemical (IHC) analysis and histopathological examination. This study aimed to assess the FNAC method combined with embedding samples in paraffin blocks (cell blocks) and comparing this with core biopsies (tissue blocks). Methods: This observational, prospective study was performed at our hospital and involved 50 female participants who presented with breast masses and were subsequently evaluated for high-risk status by FNAC and IHC. Tests for estrogen receptor (ER), progesterone receptor (PR), and human EGF receptor 2 (HER2/neu) were performed and the sensitivity, specificity, and discrepancy rates between methodologies were calculated using correlation analysis and agreement tests. Results: The correlation analysis between immuno-staining of sections from cell blocks and histopathological examination of sections from tumor-tissue blocks revealed a high concordance for HR and HER2/neu. Conclusion: IHC of cell-block sections was found to be better for the determination of HR status and HER2/neu levels. It is very important to obtain high-quality cell blocks with strict quality control for their clarification.", "keywords": [ "ER", "PR", "HER2neu", "CISH", "breast carcinoma", "FNAC", "cell block", "IHC" ], "content": "Introduction\n\nBreast cancer is a national health problem in Iraq and a serious health concern in all countries1. The early detection of breast cancer represents a major challenge for oncologists, pathologists, and surgical oncologists1. Fine-needle aspiration biopsy (FNAB) is less invasive than a core needle biopsy but may provide insufficient tissue for identifying specific markers and phenotypes1. As an advanced molecular technique, immunohistochemistry (IHC) can be used to assess tumor subtypes based on histo-chemical markers2 and is routinely used in the work-up of breast cancer3. The FNAB method allows the retrieval of small tissue fragments from a fluid specimen that can be embedded in a paraffin block, sectioned, and analyzed4,5. Immunohistochemical detection of hormone receptors (HR) is part of the regular work-up of breast cancer management6. Human EGF receptor 2 (HER2/neu) was one of the first oncogenes identified in samples of invasive cancer and is found in 10%–20% of women with breast cancer. It is an indicator for sensitivity to Herceptin (trastuzumab)7,8. The presence of Her2 can be detected by IHC using either chromogenic in situ hybridization (CISH) or fluorescence in situ hybridization (FISH). CISH resembles FISH in that they are both in situ hybridization techniques used to identify the presence or absence of specific DNA sequences; however, CISH is a much more practical technique in diagnostic laboratories as it requires a bright-field microscope in contrast to the more costly and complex fluorescence microscope required for FISH9.\n\nThe present study was conducted to assess the use of FNABs embedded in cell blocks to determine their reliability in assessing HR status and HER2/neu status by IHC compared with standard histological staining and examination of tissue block sections.\n\n\nMethods\n\nWe enrolled 67 female participants, of whom 50 completed the study. These were women who attended our clinic for a consultation. They had been diagnosed with breast cancer at our hospital clinic and then visited our institute for a further diagnosis of detected breast masses.\n\nThe patients were referred from the Primary Breast Cancer Detection Clinic (PBCDC) screening program in Medical City to our clinic. Although this program is offered at the clinic, it is mainly carried out at our center because we can provide a more accurate diagnosis of breast cancer and offer further assurances about a diagnosis, being oncologists. The primary focus of the screening program is the early diagnosis of breast cancer. Each participant had a file containing records of dates and times of visits, investigations performed, and the date of their next appointment. Detection of breast masses was either due to a patient finding a lump and presenting at the clinic themselves or due to a lump being found by clinic staff during a routine visit. Further assessments included bilateral breast and axilla ultrasonography, mammography, and magnetic resonance imaging (MRI) of the breasts. Following this, FNAC or FNAB was performed on the masses and samples were sent for histopathology. All participants were women diagnosed with breast cancer for the first time. Some patients required further assessment in the form of IHC analysis to determine their eligibility for hormonal or anti-Her2 therapy. Once all diagnoses were complete, we recruited women who consented to inclusion in the study. All patients who presented from 1 April 2017 to 30 November 2017 who met the study criteria and voluntarily gave informed consent were included.\n\nWritten, informed consent from all study participants was obtained in accordance with the Helsinki Declaration of 1975, as revised in 2000, at the time of their visit to the clinic. The Medical Ethical Committee of the Iraqi Board for Medical Specializations approved this work (No. 2015; date: 24/03/2017).\n\nEach of the 50 (initially 67) women, aged 42 to 66 years, who participated in the study frequently visited our clinic for either hormonal or chemotherapy treatment. Of the 17 patients lost to follow-up, the reasons included: Patients changed their place of clinical management (n=4), patients refused treatment at our hospital (n=5), patients decided to complete their treatment outside of Iraq (n=4), and patients changed oncologist (n=4).\n\nThe inclusion criteria were: Women aged 18-years or more, novel breast cancer diagnosis, provided written, informed consent, and had breast cancer independent of any other primary tumors. (The presence of other primary tumors could lead to the misinterpretation of clinical findings and mismanagement of cases). The exclusion criteria were: Other primary tumor(s), male breast cancer (due to its rarity and its need for special interventions), metastatic breast cancer, and inflammatory breast cancer (due to the need for urgent management).\n\nFollowing a diagnosis of breast cancer by FNAC by a histopathologist, patients were sent for surgery. This involved either a mastectomy or a lumpectomy based on the stage of the tumor. Women with early-stage breast cancer (stage I or II) they were sent first for surgery, then chemotherapy was planned, and they were given an appointment for radiotherapy. Women with locally advanced breast cancer, or stage III patients, received neoadjuvant chemotherapy before being sent for surgery and then underwent radiotherapy.\n\nFine-needle aspirate (FNA) samples were obtained from participants upon presentation once the clinical and radiological examinations had been performed. Following the initial diagnosis, participants were sent to our laboratory for FNA sample collection. The samples were analyzed by a histopathologist who also wrote a histopathology report about each patient undergoing FNA. Needle core samples were taken for more accurate staging of breast cancer or if the FNA sample was insufficient. Needle core samples can be obtained either before or after treatment to confirm the diagnosis and staging or after treatment has begun in patients with advanced-stage cancer when they start neoadjuvant chemotherapy. All patients underwent FNA because this analysis process represents the main part of the diagnostic process, and thus happens once a woman has attended our clinic. This protocol was not changed (a patient was not started on any type of treatment if no histopathology analysis had been undertaken). In cases of advanced inflammatory breast cancer neoadjuvant chemotherapy is required immediately, in these cases FNA samples may need to be taken post-treatment. We did not observe any effects of neoadjuvant treatment on the histochemical properties of tumors, since the benefits of neoadjuvant treatment (which is mainly used in patients with locally advanced stages) are to delay the development of local recurrence and to decrease treatment failure rates. Such treatment will also result in the downstaging of a tumor, getting close to the resection negative margin, and making a tumor mass more operable for radical surgery. All histopathology was performed by experienced histopathologists, and we also received written reports from them which contained both patient and tumor data, including a patient’s age, address, stage, grade, number of samples, and the gross and microscopic appearance of the tumor. Cell blocks were prepared using the collodion bag technique5.\n\nThis study depended on histopathology reports and/or IHC results and extracted HR and Her/2neu information. The authors did not collect any demographic data. FNAC was performed by routine immune-staining of paraffin block sections for HR and HER2/neu and the results were compared with diagnoses made using stained tissue block sections.\n\nBased on the biopsy results of histopathology findings in reports and the tumor work-up following clinical examination of the breast and axilla of the affected and healthy side, ultrasonography, mammography, and breast magnetic resonance imaging (MRI) were also performed if required to confirm the diagnosis and to evaluate and assess each patient for any locoregional spreading or metastasis elsewhere in the body (this is routine workup that is carried out for each patient), which is a routine steps perform for each patients.\n\nThe decision to use either FNAC, core biopsy, or both was influenced by different factors, including the following: lesion size; palpable clinical features of masses; lesion features identified from imaging, such as mass, architectural distortion, asymmetric density, or micro-calcifications; the clinician performing the procedure; the availability of a histopathologist; the need for a hormone receptor assay or tumor marker studies in inoperable cases; consideration for management protocols.\n\nFNA was performed using a 23-gauge needle. After use, the needles and syringes were rinsed with 10 mL 50% (v/v) ethanol (Science Ltd., Catalogue No.: C15K30002-00RE200-L3), and the cell suspension was placed in a 10-mL disposable centrifuge tube and pelleted by centrifugation at 4,000 rpm for 6 min. The supernatant was removed and pellets were fixed and stained with 10% buffered formalin (Sigma-Aldrich, Catalogue No.: F-10NB1G-123456) containing hematoxylin (Sigma-Aldrich, Catalogue No.: 685042) and eosin (Science Ltd., Catalogue No.: E10022661208) for 30–45 minutes at room temperature. The fixed material was filtered, and the fragments were processed for tissue sampling10. Collodion reagent (Sigma-Aldrich, Catalogue No.: E68562K31) was poured into 15-mL glass tubes until they were full. Then, the collodion was left to stand in the tubes for 10–15 min. After that, we poured the collodion back into the reagent bottle, rotating the tubes while pouring. The tubes were placed upside-down in a test-tube rack and left to stand for a further 30 min until the collodion had dried. Once the collodion had dried, the tubes were filled with distilled water. We discarded the distilled water just before use. The collodion bag can be stored for up to one-week5.\n\nTissue blocks were prepared by embedding samples in paraffin wax (BDH Ltd., Catalogue No.:0008-GFSD), Xyline (BDH Ltd., Catalogue No.:0009-HTCR) and water soluble glycol’s solution (Sigma-Aldrich, Catalogue No.: H-50M1H-01859) with Resin B compound (Sigma-Aldrich, Catalogue No.: H-629110AA-00505) to ensure a good specimen matrix for cryo-state sectioning by CRYOCUT 1800 (YU JIE / Japan/ Model:120ZB BB, Catalogue No.: 11S0B66090ZV). The aims for FNA slide preparation is to get a thin smear that is not subject to crush artifacts, and to allow rapid drying of air-dried slides and quick fixation of wet-fixed slides10. A drop of the aspirated fluid is placed onto two of the pre-labelled glass slides (ATACO / China, Catalogue No.:202441). The slides are then immediately placed into a Coplin jar (JACK Med./ China, Catalogue No.:ME-60166RS) containing 95% (v/v) ethanol (Science Ltd., Catalogue No.: C20K30004-11RE210-L5) for five minutes, to avoid excessive cell shrinkage. The slides were then dried by gently waving them in the air or by using a hair dryer (CROUN/MARTA TRADE INC., Catalogue No.:195220) on a cold or low heat setting. The slide then placed in 25 mL 15% (v/v) methanol (Science Ltd., Catalogue No.: C30K40004-22RE400-L8) for at least 30 seconds. Core biopsy slides were fixed in 10% buffered formalin (Sigma-Aldrich, Catalogue No.: F-10NB1G-123456) for 3–4 hours. Tissue was routinely processed. While practices vary, more common practices include cutting approximately 10 serial ribbons and staining several ribbons at regular intervals, for example, 1, 5 and 10, or staining at least three levels with haematoxylin (Sigma-Aldrich, Catalogue No.: 685042) and eosin (Science Ltd., Catalogue No.: E10022661208) (H&E) stains.\n\nThe cell block process was started by pipetting a specimen into a collodion coated test tube, then centrifuging it for 10 min at 2,500 rpm. The supernatant was removed using a pipette, and the edge of the collodion bag was placed on the lip of the tube using forceps. The bag was then lifted from the tube with forceps and tied with cotton string just above the pellet. Excess collodion bag and string was cut away, the remaining bag was placed in a tissue cassette, and then the cassette was placed in a specimen cup filled with 10% neutral buffered formalin (Sigma-Aldrich, Catalogue No.: F-10NB1G-123456). The specimen was then subjected to routine processing in the histopathology laboratory5,10.\n\nSample analysis was performed by a histopathologist doctor in Lab by comparing to IHC control slides (Cell Signaling Technology, Danvers, USA) by fixing and embedding the tissue, then cutting and mounting the section, followed by de-paraffinizing and rehydrating the section.\n\nAir-dried slides were fixed in methanol (Science Ltd., Catalogue No.: C30K40004-22RE400-L8) for 2 minutes, allowed to dry and then immersed in a stain. Immunohistochemical staining was performed using a rapid Romanovsky stain (Sigma-Aldrich, Catalogue No.: 890601), Papanicolaou stain (Sigma-Aldrich, Catalogue No.: 110399) or haematoxylin (Sigma-Aldrich, Catalogue No.: 685042) and eosin (Science Ltd., Catalogue No.: E10022661208) (H&E) stains. IHC antibodies used in the analysis process were:\n\n1. Anti-estrogen receptor (ER) rabbit monoclonal primary antibody (Roche Medical Systems, Inc., Catalogue No.: 790-4325 SP1) It was diluted as 1:100 in 0.05 M Tris-HCl (GoldBio com., Catalogue No.: T-099) with 0.10% ProClin 300 (Sigma-Aldrich, Catalogue No.: 669400R).\n\n2. Anti-progesterone receptor (PR) rabbit monoclonal primary antibody (Roche Medical Systems, Inc., Catalogue No.: 790-4296 1E2) It was diluted as 1:200 in 0.1M phosphate buffered saline (Sigma-Aldrich, Catalogue No.: 980221M) with 0.05% ProClin 300 (Sigma-Aldrich, Catalogue No.: 659011Y).\n\n3. Anti-HER-2/neu rabbit monoclonal primary antibody (Roche Medical Systems, Inc., Catalogue No.: 790-2991 4B5) It was diluted as 1:100 in 0.05 M Tris buffered saline (Sigma-Aldrich, Catalogue No.: 782330L), 0.01 M EDTA (RPI Ltd., Catalogue No.: E68820-372-2), 0.05% Brij-35 (Sigma-Aldrich, Catalogue No.: 433067H) with 0.05 % sodium azide (Sigma-Aldrich, Catalogue No.: 551108K).\n\nExcess stains were discarded, and the slides rinsed in buffered water (BDH Ltd., Catalogue No.:0011-PODR). The slides were left to drain thoroughly in vertical position and allow them to dry. After that the dehydrating and stabilizing apply. Finally, viewing the staining under the microscope. Reagents:\n\nPeroxidase-blocking reagent (Sigma-Aldrich, Catalogue No.: 559012)\n\nDAB buffered substrate (Sigma-Aldrich, Catalogue No.: 859331)\n\n5% di-amino-benzidine tetra-hydrochloride chromogen solution (Sigma-Aldrich, Catalogue No.: 859221)\n\nEpitope retrieval solution (Bio Mark Pte. Ltd., Catalogue No.: PS003-KD)\n\nWash buffer (Azure Biosystems crop., Catalogue No.:AC211300)\n\nAmmonium hydroxide (Science co., Catalogue No.: CSA1033-32610051822)\n\nEquipment:\n\nSlides (ATACO / China, Catalogue No.:99111A0350)\n\nCoverslips (ATACO / China, Catalogue No.:98111A8044)\n\nOven (Memmert/ Germany, Catalogue No.: 1505456300123)\n\nCoplin jars (JACK Med./ China, Catalogue No.:ME-60166RS)\n\nTimer (YU JIE / Japan, Catalogue No.:A1H118DD)\n\nWater bath (Gemmy industrial corp. /Taiwan, Catalogue No.:8996406933110)\n\nData tabulation and input was performed using IBM© SPSS© V 22. Sensitivity, specificity, and discrepancy rates were analyzed using Spearman’s rank-correlation analysis (ρ) and test of agreement using Cohen’s kappa coefficient (κ).\n\n\nResults\n\nThe participants were aged from 42 to 66 years, with women of different ages presenting with different tumor phenotypes (Table 1 and underlying data11). Tissue biopsies, examined either directly or embedded in paraffin blocks, sectioned, and analyzed for breast cancer markers, revealed that the most common type of cancer identified by either technique was ductal carcinoma (Figure 1). Using the cell block method for IHC, 11 cases were diagnosed as HER2/neu-positive, while 39 cases were diagnosed as HER2/neu-negative. Five patient biopsies gave false-negative results when compared with the results from IHC of the tissue block sections (Table 2). In total, 31 cases were diagnosed as ER-positive, and 19 cases were diagnosed as ER-negative, with two false-positive and three false-negative results, when compared with the results of tissue-block histopathology (Table 2). Immunostaining of cell-block sections revealed 12 PR-positive cases and 38 PR-negative cases, with four false-negative results and no false positives. The correlation between cell-block sectioning/IHC and tissue-block staining is shown in Table 3. For HER2/neu, there were 11 patients positive for both cell- and tissue-block, while 34 were negative. For HR, 29 were positive for ER and 16 were negative, whereas PR was positive in 15 and negative in 34 stains. Different H & E stain histopathology images are shown in Figure 2 (raw images are available as underlying data12).\n\nER - estrogen receptor, PR - progesterone receptor, HER2/neu - human EGF receptor 2\n\nρ: Spearman’s rank-correlation coefficient PR\n\nk: Cohen’s Kappa\n\nER - estrogen receptor, PR - progesterone receptor, HER2/neu - human EGF receptor 2\n\nHaemotoxylin and Eosin stain histopathology images: IDC on cell block H&E stain A) [10x], B) [20x], C) [20x clear infiltration to the adipose tissue], D) [40x clear infiltration to the adipose tissue], E) [IDC with desmoplasia], F) [Metastatic ductal carcinoma to the axillary LN], G) [ER score 6/8], H) [PR score 6/8], I) [PR score 4/8], J) [ER score 4/8], K) [ER moderate staining 4/8 on 20x], L) [PR weak staining 3/8 on 40x], M) [Her2/Neu score +++ 20x], N) [Her2/Neu score +++ 40x], O) [Her2/Neu score +++ 20x], P) [Her2/Neu score +++ 40x], Q) [Her2/Neu score ++ 20x], R) [Her2/Neu score ++ 40x]. ER - estrogen receptor, PR - progesterone receptor, HER2/neu - human EGF receptor 2.\n\n\nDiscussion\n\nThe cell block results showed a total of 74% of tumors were ductal carcinoma, 16% were lobular, and 4% were mixed, with 6% being mucinous. The tissue block results differed: 64% were ductal, 16% were lobular, and 14% were mixed. This difference in results may be related to the disturbance of morphological features, which has been noted in previous studies such as the one by Makki6. The disparity may also be a result of the disturbance of morphological features and rearrangement of the malignant cells of lobular carcinoma by FNAC. The differentiation of in situ carcinoma from the invasive type cannot be differentiated by cell-block preparation, since the cells and basement membranes of the ducts cannot be differentiated. Our results are consistent with results from studies in various Western countries, which have indicated that invasive ductal carcinoma is the most common variant of invasive breast cancer9,13. The patients’ ages ranged from 42 to 66 years (mean ± SD = 52.94 ± 5.51 years). Patients with lobular breast cancer tended to be younger. These results were different from those obtained in Western countries7, where in situ breast cancer is detected more frequently than the invasive form because of more accurate and reliable screening. Developed countries generally have better screening programs compared with developing countries such as Iraq.\n\nFNA cytology and core biopsy are the two main ways to diagnose and determine the optimal management of breast disease. Accurate procedures depend on an awareness of available techniques, utilization of better tests for a particular clinical setting, and the sensitivity, specificity, and interpretation of results in clinical practice.\n\nThese two procedures, FNA cytology and core biopsy, are sufficient for making a diagnosis and deciding whether surgery or other treatments should be applied8.\n\nSome pathologists prefer to histologically evaluate core biopsies because they can be analyzed relatively quickly and easily, and they allow IHC to be applied. Cell blocks of breast FNAB offer these same advantages, while combining FNAB with core biopsies has been found to increase diagnostic accuracy9. Pinder et al.13 were among the first to confirm the value of analyzing multiple prognostic markers for breast cancer. Our data showed good concordance for HER2/neu and PR, with only moderate concordance for ER expression when comparing two blocks. The false-negative rate for HER2/neu amplification did not differ significantly between the two blocks. There was a strong, statistically significant positive correlation (p= 0.0419; ρ = 0.774) between the two blocks regarding HER2/neu status, and with moderate agreement (k = 0.749), 68.75% sensitivity and 100.0% specificity. Five cases were labeled as false-negatives according to the cell-block results.\n\nSimilar results were obtained by Bueno Angela et al.14 and Vohra et al.15. There was a strong, positive, statistically significant correlation (p value = 0.022; ρ = 0.786) between both blocks for the detection of ER status with moderate agreement (k = 0.786), 90.62% sensitivity and 88.89% specificity. Our findings differed slightly from the Bueno Angela study11, in which it was concluded that cell blocks provide a useful method for assessing HR and HER-2/neu mainly in inoperable and recurrent cases, but consideration should be given to carrying out FISH analysis on 1+ as well as 2+ HER-2/neu findings14; our results were similar, however, to those of Vohra et al12. There was a strong, positive, statistically significant correlation (p= 0.0408; ρ = 0.803) and strong agreement (k = 0.819) between the two blocks with respect to the detection of PR status, with 75.0% sensitivity and 100.0% specificity. These results were similar to those reported by Bueno Angela et al.14 but differed slightly from Vohra et al.15. Vohra et al.15 reported ER, PR, and HER2 determination on FNA-acquired cell blocks which showed excellent agreement with ER and HER2, and moderate agreement for PR with the corresponding tissue block. These findings support the equivalency of ER and HER2 evaluation performed on FNA cell blocks compared with surgical tissue blocks15.\n\nIt is possible that a small number of ER-positive, PR-negative cases could be missed by IHC when using a cell block, and therefore further research is needed to evaluate the significance of this possibility. One of the limitations of this study was its small sample size.\n\nOther limitations included the time consumed in performing IHC analyses at the histopathology laboratory due to a lack of materials necessary for completing the procedures (most of the time these materials are lacking in our clinic and we send samples to a private clinic for analysis, which is more expensive and patients often cannot afford it). This causes delays in the completion of the IHC analysis we require, and sometimes the private laboratory loses samples, so we suggest that they are done outside of the country. Other challenges include the small number of pathologists available, poor facilities for the accurate recording of data, and ineffective screening programs. All of the above points may cause changes in the sequence of obtaining optimal work-up data for our patients, which is necessary if they are to have better outcomes from the management of newly diagnosed breast cancer. All above points may cause alteration in sequences of obtain optimal work-up for our patients to have a better results from management of newly diagnosed breast cancer.\n\n\nConclusion\n\nThe combined use of FNAC smears and cell blocks can be useful for establishing a more definitive diagnosis of breast cancer. HR and HER2/neu determination by IHC on sections from cell blocks was in close agreement with HER2/neu and PR, and in partial agreement with ER. These findings support the equivalency of these markers on cell blocks compared with tissue blocks.\n\n\nData availability\n\nZenodo: Cell block vs tissue block. http://doi.org/10.5281/zenodo.350735611\n\nThis project contains the following underlying data:\n\nDr. Mazin Data (2).xlsx (Excel file of Patients age, breast cancer types, and results of cell and tissue block for ER, PR, and Her2neu)\n\nZenodo: Histological staining of samples [Clinico-pathology on newly diagnosed breast cancer with expression of ER, PR and HER 2neu in the cell block: observational study]. http://doi.org/10.5281/zenodo.333814612\n\nThis project contains the following underlying data:\n\n10X.png (Raw microscope image, Invasive ductal carcinoma (IDC) on cell block H&E stains)\n\n20 x clear infiltration to the adipose tissue.png (Raw microscope image, IDC cells infiltrated the adipose tissue on H&E stains)\n\n20X.png (Raw microscope image, IDC on cell block H&E stains)\n\n40 x clear infiltration to the adipose tissue.png (Raw microscope image, IDC cells infiltrated the adipose tissue on H&E stains)\n\nER moderate staining 4–8 on 20 x.png (Raw microscope image, IHC staining using the Anti-ER antibody showed distinct nuclear positivity in glandular cells in tumor cells in breast cancer)\n\nER score 4–8.png (Raw microscope image, IHC staining using the Anti-ER antibody showed distinct nuclear positivity in glandular cells in tumor cells in breast cancer)\n\nER score 6–8.png (Raw microscope image, IHC staining using the Anti-ER antibody showed strong nuclear positivity in glandular and stromal cells in tumor cells in breast cancer)\n\nHer2score ++.png (Raw microscope image, IHC staining using the Anti-Her-2/neu antibody showed HER2-negative ductal carcinoma with no membranous positivity)\n\nHer2Neu score +++ 20x.png (Raw microscope image, IHC staining using the Anti-Her-2/neu antibody showed strong membranous (combined with moderate cytoplasmic) positivity in tumour cells in HER2-positive ductal carcinoma)\n\nHer2Neu score +++ 20x.png (Raw microscope image, IHC staining using the Anti-Her-2/neu antibody showed strong membranous positivity in tumour cells in HER2-positive ductal carcinoma)\n\nHer2Neu score +++ 40x.png (Raw microscope image, IHC staining using the Anti-Her-2/neu antibody showed strong membranous positivity in tumour cells in HER2-positive ductal carcinoma)\n\nHer2neu score ++.png (Raw microscope image, IHC staining using the Anti-Her-2/neu antibody showed HER2-negative ductal carcinoma)\n\nIDC with desmoplasia.png (Raw microscope image, IDC cells grouped as desmoplasic fashion on H&E stains)\n\nMetastatic ductal carcinoma to the axillary LN.png (IDC cells spreading to lymphatic tissue)\n\nPR score 4–8.png (Raw microscope image, IHC staining using the Anti-PR antibody showed weak nuclear positivity in glandular cells in breast cancer)\n\nPR score 6–8.png (Raw microscope image, IHC staining using the Anti-PR antibody showed strong nuclear positivity in glandular cells in breast cancer)\n\nPR weak staining 3–8 on 40 x.png (Raw microscope image, IHC staining using the Anti-PGR antibody showed very weak nuclear positivity in glandular cells in breast cancer)", "appendix": "References\n\nParkin DM, Fernández LM: Use of statistics to assess the global burden of breast cancer. Breast J. 2006; 12(Suppl 1): S70–80. PubMed Abstract \| Publisher Full Text\n\nBassett L, Winchester DP, Caplan RB, et al.: Stereotactic core-needle biopsy of the breast: a report of the Joint Task Force of the American College of Radiology, American College of Surgeons, and College of American Pathologists. CA Cancer J Clin. 1997; 47(3): 171–172. PubMed Abstract \| Publisher Full Text\n\nBöcker W, Bier B, Freytag G, et al.: An immunohistochemical study of the breast using antibodies to basal and luminal keratins, alpha-smooth muscle actin, vimentin, collagen IV and laminin. Part II: Epitheliosis and ductal carcinoma in situ. Virchows Arch A Pathol Anat Histopathol. 1992; 421(4): 323–330. PubMed Abstract \| Publisher Full Text\n\nVarsegi GM, Shidham V: Cell block preparation from cytology specimen with predominance of individually scattered cells. J Vis Exp. 2009; (29): pii: 1316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarga Z, Noske A: Impact of Modified 2013 ASCO/CAP Guidelines on HER2 Testing in Breast Cancer. One Year Experience. PLoS One. 2015; 10(10): e0140652. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMakki J: Diversity of Breast Carcinoma: Histological Subtypes and Clinical Relevance. Clin Med Insights Pathol. 2015; 8: 23–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnster VL, Ballard-Barbash R, Barlow WE, et al.: Detection of ductal carcinoma in situ in women undergoing screening mammography. J Natl Cancer Inst. 2002; 94(20): 1546–1554. PubMed Abstract \| Publisher Full Text\n\nNational Breast Cancer Centre: Breast fine needle aspiration cytology and core biopsy: a guide for practice. National Breast Cancer Centre, Camperdown, NSW 2004. Reference Source\n\nLiao J, Davey DD, Warren G, et al.: Ultrasound-guided fine-needle aspiration biopsy remains a valid approach in the evaluation of nonpalpable breast lesions. Diagno Cytopathol. 2004; 30(5): 325–31. PubMed Abstract \| Publisher Full Text\n\nNational Breast Cancer Centre: Breast fine needle aspiration cytology and core biopsy: a guide for practice. National Breast Cancer Centre, Camperdown, NSW. 2004. Reference Source\n\nJody M: Cell block vs tissue block. F1000research. 2019. http://www.doi.org/10.5281/zenodo.3507356\n\nJody M: histological staining of samples [Clinico-pathology on newly diagnosed breast cancer with expression of ER, PR and HER 2neu in the cell block: observational study]. 2019. http://www.doi.org/10.5281/zenodo.3338146\n\nPinder SE, Wencyk PM, Naylor HE, et al.: The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations. Cytopathology. 1995; 6(5): 316–324. PubMed Abstract\n\nBueno Angela SP, Viero RM, Soares CT: Fine needle aspirate cell blocks are reliable for detection of hormone receptors and HER-2 by immunohistochemistry in breast carcinoma. Cytopathology. 2013; 24(1): 26–32. PubMed Abstract \| Publisher Full Text\n\nVohra P, Buelow B, Chen Y, et al.: Estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression in breast cancer FNA cell blocks and paired histologic specimens: A large retrospective study. Cancer Cytopathol. 2016; 124(11): 828–835. PubMed Abstract \| Publisher Full Text" }	[ { "id": "95803", "date": "15 Nov 2021", "name": "Anil Mukund Limaye", "expertise": [ "Reviewer Expertise Gene expression", "estrogen receptor signaling", "breast cancer", "phytoestrogens", "mechanisms of endocrine resistance", "breast cancer initiation and progression." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work is a comparison of paraffin embedded FNABs versus tissue blocks for the assessment of HR, and HER2/neu expression. This is an important piece of work. However, the study lacks depth. I have the following comments and suggestions.\nThe manuscript requires language correction. Some sections need to be re-written as they were not clear to me.\n\nRoutine immunohistochemical method for assessment of HR, and HER2/neu has been used for a long time now. It appeared to me that invasive nature of core biopsies is a problem and the authors seek to evaluate the performance of paraffin embedded FNAB. The authors could have given an account of similar work upfront in the introduction. That could be followed by what this study particularly has to offer to provide new or additional insights. The literature on others' work could have been discussed.\n\nSample size is small. Hence the authors should be cautious in making conclusions about the outcome of the study, with particular reference to the data in tables 2 and 3.\n\nThe scoring method for HR and HER2/neu expression should be clearly stated.\n\nDiscrepancy between Table 1 and data described in the first paragraph of the Discussion section. Figure 1 shows 78% ductal carcinoma, 16% lobular, 4% mixed and 2% mucinous. However, in the cell block data discussed in the first paragraph of the discussion mentions- 74% ductal, 16% lobular, 4% mixed and 6% mucinous.\n\nSimilar data for the tissue block results were 64% ductal, 16% lobular and 14% mixed. However, I don't see any raw data for the same.\n\nThe expression of HR and HER2/neu was concluded as false positive or false negative based on the assumption that the data from the tissue blocks provided the true picture. However, the tissue block method may itself have a small but a certain rate of false positives and false negatives. Although, I do understand that specificity and sensitivity are evaluated based on certain truth, which the tissue block data is assumed to represent.\n\nThe authors have mentioned that \" the patients with lobular breast cancer tended to be younger\". What is the statistical basis for that? The F value (0.356) and the p value (0.785) does not suggest that this is true.\n\nI did not find the relevance or utility of the second and third paragraphs in the discussion part of the manuscript.\n\nHow is the discrepancy (Table 3) calculated?\n\nThe authors have determined the specificity and sensitivity of the cell block method in reference to the tissue block method. However, there is no clear picture as to how the levels of expression been scored. In table 2, what is the cut-off score for each marker (HRs and HER2/neu) for categorizing the samples as positive or negative.\n\nHaving used the Cohen's kappa for assessment of the agreement between the data from two methods, what is the need for Spearman's correlation coefficient? Having good correlation is one thing, but whether the methods are agreeing well with the levels of expression of a particular marker is another. In this context the protocol for scoring is important. These issues need to be scrutinized by an expert statistician.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "122982", "date": "21 Feb 2022", "name": "Torill Sauer", "expertise": [ "Reviewer Expertise Cytopathology", "mainly Breast FNAC", "immunocytochemistry and methodology", "Breast Pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study assess the FNAC method combined with cell blocks for immunostaining of ER, PgR and HER-2 in breast cancer cell material compared with CNB. As such I consider it a validation study and the number of cases as sufficient.\nThe introduction part about the cell block technique and immunostaining is brief, and could be expanded. There is quite a number oF articles on the topic, both in cytology journals and others. There are also a number of articles on ER, PgR and HER-2 on FNAC materiale from breast cancer, AND you should confer with and refer to chapters 8 and 9 in \"The International Academy of Cytology Yokohama System for Reporting Breast Fine Needle Aspiration Biopsy Cytopathology\" by Andrew Field and coworkers. ISBN 978-3-030-268824.\nA validation study should tell us if the two methods we validate are equal. As such the concordance should be high, > 90 %. About 1/3 of your HER-2 positives were missed by the cell block method. ER and PgR have divergent results in both directions: positive and negative. Your results will have treatment implications, and divergent results must be minimal.\nYou use the same IHC protocol both for cell block and CNB. That is common, BUT they are not equal. The preanalytical handling of FNAC material is not the same as for CNB.\nFrom your description it seems that all your aspirated material is an ethanol cell suspension. Ethanol is a good fixative, but it is a precipitating/coagulating type of fixative that changes the tertiary structure of the cell molecules in a quite a different way as the cross-linking formalin. The time in alcohol is the primary fixation. Your material is brought to the laboratory when you have finished your out patient clinic, which can be from 30 minutes up to more than one hour. Your cells are fully fixed in ethanol when they reach the lab. You use formalin as post-fixation, which is a good thing, but your epitope presentation will be determined by the alcohol fixation. That means you need to modify your protocol, because the demasking should not be equal to a primary formalin fixed tissue. I suggest that this is the reason for the significant discrepancy of HER-2 positivity. I disagree with your conclusion then, that the two are equal, but with a protocol modification and optimisation, I think you could achieve it.\nThe subtype of BC is hardly relevant as a cause of discrepancy, nor the number of cells as long as the number is sufficient for evaluation.\nYou mix methodology, screening and clinical issues in your discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1797
https://f1000research.com/articles/8-1792/v1	23 Oct 19	{ "type": "Research Article", "title": "Malaria management in children with fever in rural Sierra Leone. Has anything changed after the Ebola outbreak?", "authors": [ "Joseph Bangalie Sesay", "Olga Denisiuk", "Katrina Hann", "Rony Zachariah", "Francis Lionel Moses", "Umaru Dumbuya", "Olga Denisiuk", "Katrina Hann", "Rony Zachariah", "Francis Lionel Moses", "Umaru Dumbuya" ], "abstract": "Background: Sierra Leone is one of the highest malaria burden countries in the world and was severely affected by the 2014-15 Ebola outbreak. As fever is a common symptom of both malaria and Ebola, it might have affected the management of fever in children. Among under-fives in Koinadugu district, Sierra Leone, we determined fever cases that had malaria diagnostic testing and treated with Artemisinin-based Combination Therapy (ACT) during pre-Ebola, intra-Ebola and post-Ebola periods. Methods: The study population included all children under five with fever who presented to 68 primary healthcare facilities in Koinadugu district. Malaria management was in line with national guidelines. All individuals presenting with fever should be subjected to a malaria diagnostic test, which may involve a Rapid Diagnostic Test (RDT) or microscopy. Only confirmed malaria cases should receive ACTs. The study spanned pre-Ebola (June 1, 2013 – April 30, 2014), intra-Ebola (June 1, 2014 – April 30, 2015) and post-Ebola (June 1, 2016 – April 30 ,2017) periods. Data were sourced directly from routine morbidity registers available at each health facility. Results: In the 68 health facilities, fever cases increased from 43,245 pre-Ebola to 74,367 post-Ebola (1.7-fold increase). Diagnosed malaria ranged between 66% and 75%. Only 47% of malaria cases were treated during Ebola. ACT use was 95% pre-Ebola, 99% intra-Ebola and dropped to 71% post-Ebola. Post-Ebola, an average of 40 (59%) facilities had monthly stock-outs of ACT (range 28-45). Conclusion: What has changed since the Ebola outbreak is the increased utilisation of services for malaria. However, ACT stockouts are of concern, and this requires attention in order to ensure compliance with national malaria treatment guidelines.", "keywords": [ "Health Systems Strengthening", "SORT IT", "Sustainable Development Goals", "Artemisinin Combination Therapy" ], "content": "Introduction\n\nA cross-sectional study conducted in 20171 involving 68 primary health facilities in Koinadugu district of rural Sierra Leone compared the management of children with fever for malaria for a period before, during and after the Ebola outbreak. There were two key findings. First, less than half of all confirmed malaria cases were treated for malaria during the Ebola outbreak. As fever is a common symptom of both malaria and Ebola, health workers may have “played safe” by simply referring such children to Ebola management sites2. Second, monthly utilization of malaria diagnostics closely matched the number of reported fever cases, implying that fever cases were being routinely subjected to malaria testing3.\n\nAlthough the post-Ebola period was included in this evaluation, it was for a relatively short period (six months) which was probably too soon to gauge health system recovery. At the primary healthcare level in the same district and among children under five, we thus performed a new analysis with a longer post-Ebola period and compared these data for similar periods before and during the Ebola outbreak. Our specific objectives were to determine: a) numbers of reported fever cases and malaria tests done, and b) numbers treated for malaria with artemisinin combination treatment (ACT) and within 24 hours of fever onset\n\n\nMethods\n\nThis was a cross-sectional study using routine program data. The setting has been previously described1. The study population included all children under five with fever who presented to 68 primary healthcare units. Malaria management was in line with national guidelines and has been described previously4. In brief, all individuals presenting with fever to any given health facility were subjected to a malaria diagnostic test, which may involve a Rapid Diagnostic Test (RDT) or microscopy. Only confirmed malaria cases should receive ACT.\n\nThe study spanned a pre-Ebola (June 1 2013 – April 30 2014), intra-Ebola (June 1 2014 – April 30 2015) and post Ebola (June 1 2016 – April 30 2017) periods.\n\nData were sourced directly from routine morbidity registers available at each health facility. A data manager was responsible for data quality, and a dedicated data entry clerk performed data entry. We used EpiData software for data entry and analysis (version 4.1 for entry and version 2.2.2.182 for analysis; EpiData Association, Odense, Denmark).\n\nEthics approval was obtained from the Sierra Leone Ethics and Scientific Review Committee (dated 18 December 2018) and the Union Ethics Advisory Group (International Union against Tuberculosis and Lung Disease, Paris, France; EAG number 70/18). As we used aggregated data, the need for informed consent was waived by the ethics committees.\n\n\nResults\n\nTable 1 shows the numbers of fever cases reported and malaria tests done during the pre-, intra- and post-Ebola periods (see underlying data5). Fever cases increased from 43,245 pre-Ebola to 74,367 post-Ebola (1.7-fold increase). Diagnosed malaria among fever cases ranged between 66% and 75%. During Ebola, all diagnosed malaria cases had malaria tests, while in the pre- and post-Ebola periods, 99% received testing and 1% was diagnosed on clinical grounds.\n\n1 Pre-Ebola (1st June, 2013 to 30th April, 2014); Intra-Ebola (1st June, 2014 to 30th April, 2015); Post-Ebola (1st June 2016 to 30th April, 2017)\n\n2 Using Rapid Diagnostic Test or Laboratory-based Microscopy\n\n3 Without Rapid Diagnostic Test or Laboratory-based Microscopy\n\nTable 2 shows numbers treated for malaria in relation to ACT and timing of fever onset. While all diagnosed malaria cases received treatment in the pre- and post-Ebola periods, only 47% were treated during Ebola. ACT use was 95% pre-Ebola, 99% intra-Ebola and dropped to 71% post-Ebola. In the post-Ebola period, out of 68 health facilities, an average of 7.5 facilities per month had 7 days stockouts of ACT.\n\n1ACT: Artemisinin-based Combination Therapy. Percentage of all treated with ACT is calculated out of total positive Malaria cases\n\n2Injectable artesunate, rectal artesunate, quinine tablets, injectable quinine. Percentage treated with other antimalarials is calculated out of total treated for malaria\n\n3Percentage treated within 24 hours of fever onset is calculated out of total treated for malaria\n\n\nDiscussion\n\nThis study shows that despite the Ebola outbreak, the number of reported fever cases progressively increased and was 1.7 times higher post-Ebola compared to the pre-Ebola period.\n\nReassuringly, while less than half of malaria cases received treatment with ACT drugs during Ebola, in the post-Ebola period, all cases received treatment (which included ACT and other anti-malarial drugs). ACT use declined from 99% during Ebola to 71% post-Ebola, and this was accompanied with ACT stock-outs. This could be explained by the increase in numbers needed to be treated for malaria in the post-Ebola period (2.8-times that during Ebola), which might have caused pressure on available ACT stocks and supply chains.\n\nAs part of Sierra Leone’s post-Ebola health recovery strategy6, more health personnel were trained in surveillance and reporting, incentives were introduced, and community involvement was promoted. Operationally, this seems to have increased health service utilisation for malaria, but the health system seemed unable to cope with the increased ACT demand. This needs to be addressed in order to ensure compliance with malaria treatment guidelines and the rational use of antimalarial drugs.\n\nThe main study strength is that we used data from all health facilities in the district and compared similar periods of time before, during and after the Ebola outbreak. A study limitation was the lack of data on complicated and uncomplicated malaria cases. Availability of such information would help to justify (or not) the use of other antimalarial drugs apart from ACT which was seen in the post-Ebola period. A short-coming in the district health information system (DHIS2) is that it does not capture severity of malaria (complicated and uncomplicated malaria). Adding this variable would improve monitoring of rational use of antimalarials.\n\nIn conclusion, what has changed since the Ebola outbreak is the increased utilisation of services for malaria. However, ACT stockouts are of concern, and this requires attention in order to ensure compliance with national malaria treatment guidelines.\n\n\nData availability\n\nOriginal, hard-copy data is accessible through consultation with the Ministry of Health and Sanitation’s District Health Management Team, Kabala, Koinadugu, Sierra Leone, led by Dr Kwame O’Neill, District Medical Officer (shakoneill@yahoo.com).\n\nOpen Science Framework: Dataset 1. Sesay_JosephB_SORTIT2_malaria_data 2019. https://doi.org/10.17605/OSF.IO/GS2CP5\n\nThis project contains the following underlying data:\n\nSesay_F_malaria_data.csv (CSV file contain Koinadugu district child malaria data)\n\nSesay_F_malaria_datadictionary.csv (Data dictionary for Sesay_F_malaria_data.csv)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) and implemented with partners. The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: WHO/TDR, the Sierra Leone Ministry of Health and Sanitation, WHO Sierra Leone and the Centre for Operational Research, The Union, Paris, France; Alliance for Public Health, Ukraine; Institute of Tropical Medicine, Antwerp, Belgium; and Sustainable Health Systems, Freetown, Sierra Leone.\n\n\nReferences\n\nMoses FL, Tamang D, Denisiuk O, et al.: Management of malaria in children with fever in rural Sierra Leone in relation to the 2014-2015 Ebola outbreak. Public Health Action. 2017; 7(1): S22–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDallatomasina S, Crestani R, Sylvester Squire J, et al.: Ebola outbreak in rural West Africa: epidemiology, clinical features and outcomes. Trop Med Int Health. 2015; 20(4): 448–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHennessee I, Guilavogui T, Camara A, et al.: Adherence to Ebola-specific malaria case management guidelines at health facilities in Guinea during the West African Ebola epidemic. Malar J. 2018; 17(1): 230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health and Sanitation: Malaria strategic plan 2016-2020. Freetown, Sierra Leone. 2016; (Accessed 20th February 2019). Reference Source\n\nHann K, Sesay JB: Sesay_JosephB_SORTIT2_malaria_data. 2019. http://www.doi.org/10.17605/OSF.IO/GS2CP\n\nGovernment of Sierra Leone: National Ebola Recovery Strategy for Sierra Leone, 2015-2017. (Accessed 20th February 2019). Reference Source" }	[ { "id": "55647", "date": "20 Nov 2019", "name": "Khin Thet Wai", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors used recent references to synthesize the research gap and the generation of new knowledge. However, scientifically strong justification is essential to clarify why the existing records needed a comparative analysis (pre, intra and post Ebola periods). Please include why this research is necessary.\n\nThe study design used is appropriate in addressing the research question.\n\nSufficient details of the methods used were noted except the list of variables used for analyses. The authors can improve the methods section by adding the type of variables extracted from the morbidity register so as to allow replication by others.\n\nFor the analysis, in Table 1, the percentages of total cases diagnosed as malaria either by RDT or microscopy out of all fever cases are to be included and interpreted carefully. “Diagnosed malaria among fever cases ranged between 66% and 75%” is not enough. In fact, in post Ebola period, diagnosed malaria among fever cases was lower than pre and intra Ebola outbreak period (66%). This indicates less need for diagnostics despite increased utilization of health services for children with fever.\n\nIn Table 2, during Ebola period, cases treated for malaria dropped to 47% which should be included in the discussion part.\n\nConclusion part is weak and needs to rewrite and add what needs to be done further.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "64791", "date": "03 Jul 2020", "name": "Martina Oneko", "expertise": [ "Reviewer Expertise Pediatrician with experience in neuropediatrics and clinical trials", "especially malaria vaccines" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript expands on previously published data regarding the management of malaria before, during, and after the Ebola outbreak in Sierra Leone. While the conclusions seem more sound than in the first article published by the same group, this manuscript cannot be interpreted without the first article and is, therefore, more of an addendum than an original article. This should be made clearer in the introduction, including a more elaborate description of why this additional analysis was necessary. While the first article describes a deterioration of malaria reporting in the post-Ebola period, this new analysis claims a higher utilization of health services for malaria is responsible for the higher number of fever cases. However, they could have mentioned some details about the 'other' fever cases, which were higher in the post-Ebola period. Overall the article is a useful addition to the previous article and in combination, the two articles give valuable advice for improvement of the management of febrile illness in under-fives in this area of Sierra Leone. Whether they are generalizable to a wider region is not known to me.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1792
https://f1000research.com/articles/8-1784/v1	22 Oct 19	{ "type": "Software Tool Article", "title": "SNPnotes: high-throughput tissue-specific functional annotation of single nucleotide variants", "authors": [ "Shraddha Pai", "Michael J. Apostolides", "Andrew Jung", "Matthew A. Moss", "Andrew Jung", "Matthew A. Moss" ], "abstract": "A key challenge in the application of whole-genome sequencing (WGS) for clinical diagnostic and research is the high-throughput prioritization of functional variants in the non-coding genome. This challenge is compounded by context-specific genetic modulation of gene expression, and variant-gene mapping depends on the tissues and organ systems affected in a given disease; for instance, a disease affecting the gastrointestinal system would use maps specific to genome regulation in gut-related tissues. While there are large-scale atlases of genome regulation, such as GTEx and NIH Roadmap Epigenomics, the clinical genetics community lacks publicly-available stand-alone software for high-throughput annotation of custom variant data with user-defined tissue-specific epigenetic maps and clinical genetic databases, to prioritize variants for a specific biomedical application. In this work, we provide a simple software pipeline, called SNPnotes, which takes as input variant calls for a patient and prioritizes those using information on clinical relevance from ClinVar, tissue-specific gene regulation from GTEx and disease associations from the NHGRI-EBI GWAS catalogue. This pipeline was developed as part of SVAI Research's \"Undiagnosed-1\" event for collaborative patient diagnosis. We applied this pipeline to WGS-based variant calls for an individual with a history of gastrointestinal symptoms, using 12 gut-specific eQTL maps and GWAS associations for metabolic diseases, for variant-gene mapping. Out of 6,248,584 SNPs, the pipeline identified 151 high-priority variants, overlapping 129 genes. These top SNPs all have known clinical pathogenicity, modulate gene expression in gut tissues and have genetic associations with metabolic disorders, and serve as starting points for hypotheses about mechanisms driving clinical symptoms. Simple software changes can be made to customize the pipeline for other tissue-specific applications. Future extensions could integrate maps of tissue-specific regulatory elements, higher-order chromatin loops, and mutations affecting splice variants.", "keywords": [ "bioinformatics", "genomics", "genetics", "GWAS", "variant annotation", "SNPs", "software", "clinical genetics", "epigenetics", "epigenomics" ], "content": "Introduction\n\nGenome sequencing has become an invaluable tool for clinical diagnostics. Several methodologies exist to look at the human genome, each with own benefits and pitfalls. Whole exome sequencing has been a cost-effective technique to identify structural and nucleotide variants in protein-coding regions of the genome, and has identified variants associated with a number of diseases, including psoriasis1, Factor V Leiden thrombophilia2, and Miller Syndrome3. Genome-wide associations studies, originally based on SNP microarrays, have found that roughly half of genetic associations with disease are located outside gene bodies; this fraction approaches 90% with the inclusion of intronic regions4. With dropping costs for DNA sequencing, whole genome sequencing (WGS) promises to extend the ability to identify disease-associated single and structural nucleotide variants in the clinic, to non-coding regions of the genome, including gene and chromatin regulatory sequences. However, the increased size and complexity of the data creates a parallel challenge of annotating non-coding variants, as it requires knowledge of tissue-specific gene regulation (or epigenetics), including regulatory elements such as promoters and enhancers, and features of higher order chromatin organization such as Topological Associated Domains.\n\nSeveral large-scale epigenomics projects have catalogued tissue-specific regulatory elements. This includes genetic modulation of gene expression (GTEx project)5, chromatin state (Roadmap Epigenomics)6, and enhancer-promoter loops for mapping of distal regulatory elements to genes (FANTOM and individual studies)7,8. A computational workflow that integrated these functional annotation maps to annotate variants from WGS assays would be a valuable resource to prioritize variants with potential functional impact in tissues of interest. Popular high-throughput variant annotation tools, such as BioMart and Variant Effect Predictor9,10, do not provide tissue-specific annotation. While FUMA11 integrates comprehensive epigenetic annotation, it is a web-based service used to annotate top-ranking variants from GWAS studies, rather than being a standalone tool for variant annotation.\n\nIn this work, we describe and provide variant annotation software that starts with output from a WGS assay and prioritizes variants based on epigenomic resources described above, as well as clinical genetic and GWAS catalogs of variant-disease association. This tool will allow users to capture functional and clinical information and to analyze variants simply by providing the commonly-used VCF file format. We demonstrate the software's functionality by prioritizing variants from WGS data for a single patient.\n\n\nMethods\n\nThis work was undertaken as part of SVAI Undiagnosed-1 (https://sv.ai/undiagnosed-1), which was a collaborative event with the goal of diagnosing a patient with an unknown genetic condition. As data, participant groups were provided with detailed medical history, genotyping and metabolic data from a 33-year old Caucasian male patient, JCM. The event was hosted in June 2019 by the not-for-profit organization SVAI (http://sv.ai), with participants located in the San Francisco Bay Area (USA) as well as in Toronto, Canada.\n\nFigure 1 shows the workflow for the pipeline. Patient genotypes are provided in Variant Call Format as input. Conceptually, the tool compiles prior knowledge about the functional significance of variants from the perspective of tissue-specific regulatory information, known genetic disease associations in the literature, tissue-specific genetic modulation of gene expression, and clinical pathogenicity. The annotation sources are integrated with the genotype calls in the VCF file, and this integration results in a single output table with available annotation for the variant. The user can then prioritize variants based on the combination of known or predicted functional consequences.\n\nThe NIH Roadmap Epigenomics project performed comprehensive mapping of noncoding DNA in 111 epigenomes to identify putative regulatory elements in diverse human tissue and cell types (http://www.roadmapepigenomics.org/)6. The presence of a variant overlapping a tissue-specific promoter or enhancer element signifies that variant alteration could change the regulation of a tissue-specific gene, i.e. that it has regulatory impact on gene expression. Tissue-specific 15-state chromatin state models were downloaded from the Roadmap Epigenomics Portal (downloaded from https://egg2.wustl.edu/roadmap/data/byFileType/chromhmmSegmentations/ChmmModels/coreMarks/jointModel/final/all.mnemonics.bedFiles.tgz). As the symptoms for John M, hereafter referred to as “the Patient”, included impaired function of the gastrointestinal tract, we limited our annotation to tissues and organs of the digestive system (e.g. esophagus, stomach, intestine). Chromatin states for digestive system tissues were used, including fetal stomach, small and large intestine, sigmoid colon, colonic mucosa, mucosa from stomach, duodenum and rectum, esophagus, rectal mucosa, and stomach mucosa. Regions with open chromatin were included for variant annotation (Table 1; states ≤ 1–7).\n\nThe GTEx project identified variants that significantly modulate gene expression in each of 44 human tissues5. Variants that modulate transcription in gut tissues (gut eQTLs) were downloaded from the GTEx portal (downloaded from https://storage.googleapis.com/gtex_analysis_v7/single_tissue_eqtl_data/GTEx_Analysis_v7_eQTL.tar.gz; Table 2).\n\nGenome-wide SNP-disease associations were downloaded from the NHGRI-EBI GWAS catalog12, using the TargetValidation.org API13; only those associations mapped to metabolic disorders were included (EFO:0000589). In addition, information on known clinical pathogenicity was downloaded from the ClinVar database14 (downloaded from ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/tab_delimited/variant_summary.txt.gz).\n\nThe pipeline software was implemented in bash and R 3.4.4. VCFtools v0.1.1515 was used to filter SNPs from the VCF input file. SNP locations were downloaded from the dbSNP 151 database ((downloaded from ftp://ftp.ncbi.nih.gov/snp/organisms/human_9606_b151_GRCh37p13/VCF/common_all_20180423.vcf.gz). All coordinates are in GRCh37/hg19 build. SNP coordinates were converted to bed format using awk. dbSNP identifiers were converted to genomic coordinates using dbSNP 151 reference (see above). Bedtools v2.28.016 was used to identify overlap of SNP coordinates with individual annotation sources. Finally, R merge was used to join tables by variant location.\n\nThe output file (\"final_table.txt\") contains SNP coordinates, positionally-overlapping genes, associated clinical significance from ClinVar, associated GWAS trait and p-value, coordinates and state name of overlapping open chromatin states in gut tissues, and name of tissue and genes for significant eQTL associations. The pipeline then filters this file to report only those SNPs with GWAS hits that achieve genome-wide significance (p < 5×10-8); this file is titled \"GWASsignficant.txt\", and creates a third file with the list of unique genes that meet this criterion (\"GWASsignificant_genes.unique.txt\"). The user may filter this data still further to identify SNPs with known clinical pathogenicity, as well as SNPs in functionally annotated non-coding regions.\n\n\nResults\n\nFor our test case, we used the SVAI Undiagnosed-1 Patient whole genome sequencing data provided for the Undisclosed-1 hackathon event, hosted by SVAI/Research to the People. We applied our pipeline to chromosomes 1 to 22 and X, Y chromosomes. Out of 6,248,584 SNPs, we identified 151 high-priority variants, overlapping 129 genes, which demonstrate strong evidence for functional significance (Table 3). Therefore, this pipeline allows the user to prioritize certain variants for further downstream analyses, or clinical follow up.\n\nInformation includes mapped genes, disease associations from genetic studies, eQTL associations from GTEx, and overlap with open chromatin regions in gut tissues.\n\n\nConclusions and next steps\n\nThis pipeline will allow easy integration of several epigenomic functional annotation maps that could assist in SNV prioritization for clinical and basic research applications. Our provided software can be customized by someone with basic bioinformatics or scripting expertise to generalize to other tissues profiled in the GTEx and NIH Roadmap Epigenomics project.\n\nOne beneficial extension would be the identification of variants predicted to affect gene splicing. Such predictions are available in databases of splicing variants, such as dbscSNV17,18, or simple splice site prediction algorithms such as MaxEntScan19,20; the latter uses sequence motifs and maximum entropy calculations for its predictions. This approach is limited by the quality of variant databases, and by models that are limited to predicting only in instances that follow canonical rules for splice site regulation. Another promising avenue to predict aberrant splicing is SpliceAI21, a deep learning-based model that predicts splice junctions from an arbitrary pre-mRNA transcript sequence. Another valuable addition would be that of using higher-order chromatin interaction maps, which allow the mapping of distal regulatory elements, such as enhancers, to genes (e.g. 8).\n\n\nEthical statement and consent\n\nThis article is based on research that occurred at the Undisclosed-1 hackathon event, hosted by SVAI/Research to the People.\n\nThe patient provided written informed consent for data release of their medical records, including genetic results, blood work and clinical laboratory reports, to the organisers of the event (SVAI/Research to the People). This consent included data release to participants of Undisclosed-1 during the event, and subsequently for this data to be hosted by SVAI/Research to the People in an online data repository with restricted access (see details in “Data Availability”).\n\nThe patient provided written informed consent for the publication of all articles based on the research that was carried out at Undisclosed-1 and any accompanying images.\n\nSince the medical records are the patient’s property, the patient was fully informed of what data release would entail and written informed consent was obtained for the release of the medical records. No ethical approval was sought for the Undisclosed-1 event or publication of articles relating to this event.\n\n\nData availability\n\nSynapse repository: SVAI, Undiagnosed-1 (syn:20554923), https://doi.org/doi:10.7303/syn2055492322.\n\nLicense information: Since the data contains detailed medical records, access is restricted in order to protect the identity of the patient. Intermediary data is provided throughout the article. In order to access the data, applicants must be registered users of synapse.org and must provide a proposal detailing what the data will be used for. Applicants will also be required to sign a statement that ensures that the data are not shared with others who have not applied to use the data from the SVAI. Please submit applications for data access to hello@sv.ai.\n\n\nSoftware availability\n\nSoftware for this pipeline is available at: https://github.com/shraddhapai/SNPNotes\n\nArchived release at time of publication is located at: http://doi.org/10.5281/zenodo.335227623.\n\nLicense: MIT\n\nData in this project contains:\n\ndata/final_table.txt.informative.txt contains the list of informative genes obtained by running this pipeline on WGS data from the patient at the hackathon.\n\ndata/NHGRI_GWAS contains precompiled GWAS associations by disease category.", "appendix": "References\n\nZuo X, Sun L, Yin X, et al.: Whole-exome SNP array identifies 15 new susceptibility loci for psoriasis. Nat Commun. 2015; 6: 6793. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKujovich JL: Factor V Leiden thrombophilia. Genet Med. 2011; 13(1): 1–16. PubMed Abstract \| Publisher Full Text\n\nNg SB, Buckingham KJ, Lee C, et al.: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 2010; 42(1): 30–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHindorff LA, Sethupathy P, Junkins HA, et al.: Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A. 2009; 106(23): 9362–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGTEx Consortium; Laboratory, Data Analysis &Coordinating Center (LDACC)—Analysis Working Group; Statistical Methods groups—Analysis Working Group, et al.: Genetic effects on gene expression across human tissues. Nature. 2017; 550(7675): 204–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoadmap Epigenomics Consortium, Kundaje A, Meuleman W, et al.: Integrative analysis of 111 reference human epigenomes. Nature. 2015; 518(7539): 317–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersson R, Gebhard C, Miguel-Escalada I, et al.: An atlas of active enhancers across human cell types and tissues. Nature. 2014; 507(7493): 455–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmitt AD, Hu M, Jung I, et al.: A Compendium of Chromatin Contact Maps Reveals Spatially Active Regions in the Human Genome. Cell Rep. 2016; 17(8): 2042–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLaren W, Gil L, Hunt SE, et al.: The Ensembl Variant Effect Predictor. Genome Biol. 2016; 17(1): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmedley D, Haider S, Durinck S, et al.: The BioMart community portal: an innovative alternative to large, centralized data repositories. Nucleic Acids Res. 2015; 43(W1): W589–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatanabe K, Taskesen E, van Bochoven A, et al.: Functional mapping and annotation of genetic associations with FUMA. Nat Commun. 2017; 8(1): 1826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuniello A, MacArthur JAL, Cerezo M, et al.: The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. Nucleic Acids Res. 2019; 47(D1): D1005–D12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarvalho-Silva D, Pierleoni A, Pignatelli M, et al.: Open Targets Platform: new developments and updates two years on. Nucleic Acids Res. 2019; 47(D1): D1056–D65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandrum MJ, Lee JM, Riley GR, et al.: ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014; 42(Database issue): D980–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDanecek P, Auton A, Abecasis G, et al.: The variant call format and VCFtools. Bioinformatics. 2011; 27(15): 2156–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Jian X, Boerwinkle E: dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional predictions. Hum Mutat. 2011; 32(8): 894–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Wu C, Li C, et al.: dbNSFP v3.0: A One-Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice-Site SNVs. Hum Mutat. 2016; 37(3): 235–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEng L, Coutinho G, Nahas S, et al.: Nonclassical splicing mutations in the coding and noncoding regions of the ATM Gene: maximum entropy estimates of splice junction strengths. Hum Mutat. 2004; 23(1): 67–76. PubMed Abstract \| Publisher Full Text\n\nYeo G, Burge CB: Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol. 2004; 11(2–3): 377–94. PubMed Abstract \| Publisher Full Text\n\nSpliceAI. Reference Source\n\nSVAI Research: \"SVAI Undiagnosed-1: WGS\". 2019. http://www.doi.org/10.7303/syn20554923\n\nPai S, Apostolides MJ, Moss MA, et al.: SNPnotes - initial release (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3352276" }	[ { "id": "56414", "date": "27 Nov 2019", "name": "Deepti Jain", "expertise": [ "Reviewer Expertise Molecular biology and Human genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report the workflow and pipeline code they developed to annotate variants. The authors were motivated to develop this pipeline specifically so that a user could use tissue- and disease-specific resources of annotation and annotate a large set of variants, such as from a whole genome sequencing (WGS) analysis, provided in a VCF format. As a use case, their provided code is suppose to download annotations linked with gut related tissues and metabolic disorders, format them and use them to annotate and filter variants provided in a VCF format. The authors applied the provided code to annotate 6,248,584 variants from a patient with history of gastrointestinal symptoms and generated a filtered set of 151 variants using the annotations.\nThe manuscript reports the pipeline-code for a workflow which is very important and useful for generating a filtered set of variants that likely have biological function and which could be followed-up in more detail after a WGS study. However I have major concerns about the practical feasibility and interests of others using this code because it is tailored for a very specific use case and lacks documentation. For these reasons I hesitate to endorse its acceptance at the present stage.\nMajor concerns:\nThe features for which the authors developed the pipeline i.e ability to use tissue- and disease-specific annotations, and to provide VCF as an input is already available in the Whole Genome Sequence Annotator (WGSA, https://sites.google.com/site/jpopgen/wgsa)1. In addition, WGSA has a large selection of a recently updated annotation resources that a user can choose to annotate the single nucleotide variants as well as indels.\n\nThe code provided is very specific to a use case. A user wanting another set of annotations or more than one set of annotations might end up having multiple versions of the code. In general, such an approach in not considered a good practice as it leads to duplication of lot of code. This can be avoided if the code is updated to handle user specifications provided through a config file.\n\nA user's ability to use tissue specific annotations in a format different than the currently used resources (example annotations from long range chromatin interactions experiments ) will be limited.\n\nI did not come across any documentation associated with the code. A documentation and vignette will be very helpful so that user can use the code as is, as well as modify it if they desire to use other resources.\n\nIt would be helpful to add a description about how the code handles and reports multiple annotations for a given variant from a given resource. For example, if a variant was found as an eQTL for two different tissues is that variant reported twice or the information from the two tissues combined and reported in a specific format\n\nMinor suggestion:\nAnnotation of a large set of variants can have a huge computational burden. A user would find it useful if the authors could provided software performance benchmarks.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "60483", "date": "04 Mar 2020", "name": "Pavel P. Kuksa", "expertise": [ "Reviewer Expertise Algorithms", "bioinformatics", "genomics", "sequence modeling and analysis", "high-throughput sequencing data analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this contribution authors propose SNPnotes pipeline for high-throughput tissue-specific functional annotation of single-nucleotide variants.\nThis work is motivated by a need for a stand-alone software capable of high-throughput annotation of custom WGS variant data. Several types of data are integrated into the pipeline to enable functional annotation including known genetic associations (GWAS catalog), data on clinical significance of variants (ClinVar), expression QTL (GTEx) and others. The pipeline is made available as a Github repository.\nMajor comments:\nFunctionality of the pipeline overlaps with existing tools such as WGSA1 and FAVOR (http://favor.genohub.org). The authors make no comparisons with these other tools. The manuscript will benefit greatly from clear comparisons of features, highlighting novel functionalities, benefits, etc.\n\nNo data on the pipeline performance (running time, scalability) is provided. It is also not clear if pipeline can benefit from multi-core or HPC cluster environments to speed-up annotation.\n\nCurrent implementation of the pipeline requires modification of the source code in order to apply to the custom user data and specify parameters. The pipeline scripts should be modified in order to accept custom data, target output directory, analysis parameters, etc as command-line arguments. e.g., main script could be parametrized vcfFile=${1:-input.vcf}, outdir=${2:-snpnotes_output} instead of using hard-coded absolute paths to the data and output directories. Same applies to the scripts that set up/preprocess annotation data repository.\n\nPipeline documentation is very limited and should be expanded and provide description of the command-line interface (see also point 3).\n\nMinor comments:\nOne suggestion is to include a small test data, corresponding reference output along with a test script that executes the pipeline on the test data. This can help prospective users 1) with making sure their set up/settings are correct if they can reproduce results on their systems, and 2) with making progress in using pipeline on their own data.\n\nAnother suggestion is to include in the documentation a description of software requirements (dependencies, versions, etc) and brief instructions on installing them.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1784
https://f1000research.com/articles/8-258/v1	05 Mar 19	{ "type": "Study Protocol", "title": "A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans", "authors": [ "Claire S. Byrne", "Dominic Blunt", "James Burn", "Edward Chambers", "Aygul Dagbasi", "Georgia Franco Becker", "Glenn Gibson", "Lilian Mendoza", "Kevin Murphy", "Carlos Poveda", "Anya Ramgulam", "Martina Tashkova", "Gemma Walton", "Chaiwat Washirasaksiri", "Gary Frost", "Claire S. Byrne", "Dominic Blunt", "James Burn", "Edward Chambers", "Aygul Dagbasi", "Georgia Franco Becker", "Glenn Gibson", "Lilian Mendoza", "Kevin Murphy", "Carlos Poveda", "Anya Ramgulam", "Martina Tashkova", "Gemma Walton", "Chaiwat Washirasaksiri" ], "abstract": "Introduction: A major component of the digesta reaching the colon from the distal ileum is carbohydrate. This carbohydrate is subject to microbial fermentation and can radically change bacterial populations in the colon and the metabolites they produce, particularly short-chain fatty acids (SCFA). However, very little is currently known about the forms and levels of carbohydrate in the ileum and the composition of the ileal microbiota in humans. Most of our current understanding of carbohydrate that is not absorbed by the small intestine comes from ileostomy models, which may not reflect the physiology of an intact gastrointestinal tract. Methods: We will investigate how ileal content changes depending on diet using a randomised crossover study in healthy humans. Participants will be inpatients at the research facility for three separate 4-day visits. During each visit, participants will consume one of three diets, which differ in carbohydrate quality: 1) low-fibre refined diet; 2) high-fibre diet with intact cellular structures; 3) high-fibre diet where the cellular structures have been disrupted (e.g. milling, blending). On day 1, a nasoenteric tube will be placed into the distal ileum and its position confirmed under fluoroscopy. Ileal samples will be collected via the nasoenteric tube and metabolically profiled, which will determine the amount and type of carbohydrate present, and the composition of the ileal microbiota will be measured. Blood samples will be collected to assess circulating hormones and metabolites. Stool samples will be collected to assess faecal microbiota composition. Subjective appetite measures will be collected using visual analogue scales. Breath hydrogen will be measured in real-time as a marker of intestinal fermentation. Finally, an in vitro continuous fermentation model will be inoculated with ileal fluid in order to understand the shift in microbial composition and SCFA produced in the colon following the different diets. Registration: ISRCTN11327221.", "keywords": [ "Dietary Fibre", "Carbohydrate", "Gastrointestinal tract", "Ileum", "Colon", "Gut microbiota", "Nasoenteric" ], "content": "Introduction\n\nObesity is a chronic health problem that has reached epidemic proportions globally1. Obesity increases the risk of developing a range of non-communicable diseases including type 2 diabetes, cardiovascular disease, certain cancers and osteoarthritis2. It has been projected that obesity rates could double by 2050, which would add £5.5 billion to the total annual expenses of the National Health Service (NHS)3. The Foresight report highlighted appetite regulation as a major target in the dietary treatment of obesity3. However, it is not yet fully understood how the gastrointestinal (GI) tract senses dietary content in order to suppress subsequent food intake. This knowledge could inform the development of foods or dietary regimes that increase fullness and prevent weight gain4.\n\nThe GI tract is the largest endocrine organ in the body and is responsible for digesting and absorbing dietary components. The GI tract is also the host to a microbiota, which ferment undigested material. The GI tract senses changes in the luminal nutrient content and modulates neuronal and hormonal signals from the GI tract in order to help regulate appetite and food intake5. The anorexigenic gut hormones peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), released from enteroendocrine cells (EEC)5, are examples of such GI signals secreted following a meal in a two-phase process. The first phase is thought to be mediated primarily by neural mechanisms and may help drive satiation (the sum of processes that cause meal termination). The second phase is thought to be mediated by direct nutrient sensing in the lower parts of the GI tract and to be an important long-term satiety signal. The peripheral administration of PYY or GLP-1 has previously been shown to reduce food consumption in animal models and humans, highlighting the importance of these gut hormones6–10. It has therefore been suggested that the incorporation into the diet of foods that stimulate a greater gut hormone secretion could suppress appetite to a greater extent and therefore help control body weight in the long term.\n\nThere is an increasing amount of evidence to suggest that consumption of dietary fibre is beneficial to human health. Dietary fibre refers to carbohydrates that cannot be digested by mammalian enzymes, and thus remain in relatively intact form when they reach the caecum, where they are subsequently available for fermentation by the gut microbiota. Epidemiological evidence suggests that diets high in non-digestible fibres are associated with lower body weight gain in humans11,12. In addition, fermentable fibres have been shown to protect against weight gain and fat mass development in rodents fed a high-fat diet13. Dietary fibre is thought to aid in weight management through a number of mechanisms including the promotion of satiation, increased GI transit time and stimulation of gut hormone secretion14. Short-chain fatty acids, products of microbial fermentation, have also been shown to stimulate the secretion of GLP-1 and PYY. Together, this evidence suggests that fibre may be beneficial in the management of obesity.\n\nThe cellular structure of foods can also be an important determinant of the subsequent impact of a food on appetite. In order for macronutrients within foods to be digested, they need to be in contact with digestive enzymes of the GI tract. In plant tissues, cell wall rupture can lead to a release of macronutrients into the extracellular environment, or enzymes can diffuse through a permeable cell wall in order to digest the encapsulated macronutrients15. However, cell wall matrices or individual cell wall polysaccharides of plant foods can behave in a variety of ways during digestion. For example, some cell walls are highly impermeable and less susceptible to rupture, which leads to a reduction in the rate and extent of nutrient release and digestion. Thus, the cell walls of plants can behave as a physical barrier to digestion in the upper GI tract. The degree of domestic and industrial processing (e.g. milling, blending) of plant foods and ingredients also affects macronutrient bioaccessibility and digestion by modifying the structural integrity of the plant tissue. The importance of cell wall integrity has previously been highlighted in determining the effect of plant foods on physiological functions. These studies reported that structurally intact plant tissues tend to be digested to a lesser extent and at a slower rate, which attenuated the postprandial rise in glycaemia and/or lipaemia16–18, and may also trigger the release of lower concentrations of anorexigenic gut hormones within the small intestine, compared to foods in which the nutrients are bioaccessible. However, upon reaching the colon from the distal ileum, undigested plant material serves as a substrate for microbial fermentation, which leads to the production of SCFA and triggers the release of anorexigenic gut hormones. This process is thought to result in a more long-term satiety signal following the consumption of minimally processed plant-based foods.\n\nWithin the GI tract, the colon contains the highest density of EEC19. These cells express a large array of G protein coupled receptors (GPCRs) that sense nutrients and metabolites in the colonic lumen20. This gives the colon the ability to regulate appetite and the GI tract in response to the nutrient environment in the colonic lumen. A major component of the digesta reaching the colon is carbohydrate, which is a major fuel source of the gut microbiota21. The flow of carbohydrate into the colon from the distal ileum can radically change populations of bacteria in the colon and metabolites they produce, particularly SCFAs21. However, very little is known about the type and amount of carbohydrate present in the ileum as well as the composition of the ileum microbiota in humans. Most of our current understanding of carbohydrate that is not absorbed by the small intestine comes from ileostomy models22. These models may not reflect the physiology of an intact GI tract, as the gut alters following the surgery. In the present study we will use an ileum intubation method in healthy humans, which will allow us to gain a ‘real-time’ perspective on how the actual ileum content changes in response to different diets and the subsequent effect on measures of appetite and gut hormone secretion. We will also conduct an in vitro study in order to understand how ileum content affects the colonic gut microbiota and the production of SCFA in the colon, as this is important in determining how dietary carbohydrate drives colonic gut hormone release.\n\n\nStudy objectives\n\nWe hypothesise that the consumption of minimally-processed high-fibre foods will result in the presence of a greater number of intact cellular structures in the ileum, which will subsequently be fermented in the colon resulting in the production of SCFAs and greater release of PYY and GLP-1.\n\nThere are a number of objectives to the present study: to identify the impact of dietary carbohydrate on (1) the forms and levels of carbohydrates in, and (2) the microbiological profile of, the ileum, and to (3) to determine whether the carbohydrate content of the ileum relates to appetite responses and gut hormone release.\n\nThe co-primary outcome measures for this study are:\n\n1. Metabolic profiling of ileal samples\n\n2. Microbiological profiling of ileal samples\n\nThe secondary outcome measures include:\n\n1. Microscopy of ileal samples for plant structures, cellular structures and starch granules\n\n2. Metabolic and hormonal profiling of blood samples\n\n3. Metabolic and microbiological profiling of faecal samples\n\n4. Subjective appetite measurements\n\n5. Breath H2 concentrations\n\n6. Ileum samples for the inoculation of an in vitro continuous fermentation model\n\n\nProtocol\n\nThis study will be a randomised crossover study consisting of three separate 4-day inpatient study visits at the National Institute for Health Research (NIHR) Imperial Clinical Research Facility (CRF), Hammersmith Hospital, London, United Kingdom.\n\nHealthy humans will be recruited using existing healthy volunteer databases (e.g. the Healthy Volunteer Panel at the NIHR Imperial CRF) and by advertisement in public buildings, on the internet and social media. Participants who express an interest in taking part in the study will be provided with the Patient Information Sheet (PIS). Following an initial telephone screening, potential participants will be invited to attend a health screening visit at the NIHR Imperial CRF in order to further assess their eligibility and provide written, informed consent to a member of the study team. The consent form and participant information sheet are available on Figshare23. During health screening, the study protocol and the risks and benefits of participating will be explained in full and any questions that the participant may have will be answered. Participants will be asked questions about their medical history, anthropometric measurements will be collected, blood pressure (BP) will be measured, an echocardiogram (ECG) will be performed and a blood sample taken. The collected blood sample (1 x 20 ml) will be used to assess HbA1c, full blood count, liver function, renal function and blood lipids. A pregnancy test will be performed on women of childbearing age. Inclusion and exclusion criteria will be assessed as described.\n\nHealthy humans aged between 18–65 years (inclusive) with a body mass index (BMI) of 18.5-30 kg/m2 will be recruited for this study. They must also show a willingness and ability to give written informed consent and to understand, to participate and to comply with the study requirements. Exclusion criteria includes: abnormal echocardiograph (ECG), screening blood results outside of normal reference values, weight change of ≥5kg in the preceding 2 months, current smokers, history of substance abuse and/or excess alcohol intake in the last 2 years, pregnancy, diabetes, cardiovascular disease, cancer, gastrointestinal disease, kidney disease, liver disease, pancreatitis, started new medication within the last 3 months likely to interfere with energy metabolism/appetite regulation/hormonal balance, antibiotic use within the last months, participation in a research study in the 12 week period prior to entering this study, any blood donation within the 12 week period prior to entering this study.\n\nFollowing the health screening, eligible participants will be randomised into the study. Randomisation will be performed by an independent internet and telephone randomisation company (sealedenvelope.com). Due to the different physical appearance of the diets, participants will not be blinded.\n\nWe aim to recruit 15 subjects. This is a pilot study in a new area of research and therefore a formal power calculation is not possible. However, a recent study in ileostomy patients investigating differences in the carbohydrate output in ileal effluent depending on the quality of carbohydrate consumed was able to detect significant differences between outcome measures in a similar number of subjects (n=9)17.\n\nDietary calculations. All three diets are tailored to meet the energy requirements (TEE, total energy expenditure) of each participant. Using the participant’s weight and age at screening, Schofield equations will be used to calculate each participant’s basal metabolic rate (BMR) as shown in Table 1. This will then be multiplied by 238.85 to convert MJ/d to kcal/d and then by 1.2 to account for their low physical activity level (PAL) while being inpatients at the CRF.\n\nBMR, basal metabolic rate.\n\nPAL, physical activity level.\n\nTEE, total energy expenditure.\n\nDietary overview. During the 3 separate study visits, volunteers will be provided with diets differing in carbohydrate quality, which have been designed using DietPlan 6 software. In a randomised order, volunteers will receive:\n\nDiet 1 (low fibre (LF)-refined): This diet contains highly refined and processed carbohydrate. Foods are low in fibre and intact cell structures.\n\nDiet 2 (high fibre (HF)-whole): This diet is high in fibre providing a high intake of intact cellular structures. Foods have resistant cell structures such as beans, nuts, fruit and vegetables.\n\nDiet 3 (HF-disrupted): This diet is also high in fibre but with disrupted cellular structures. It is the same as the HF-whole diet, but foods will be milled or blended to disrupt the cellular structure.\n\nAll the diets have a similar macronutrient content (55% energy from carbohydrate, 30% energy from fat, 15% energy from protein). However, the quality of carbohydrate present is significantly different. For example, the HF-whole and HF-disrupted diets contain 46.5 g and 47.7 g dietary fibre/2000 kcal, respectively, while the LF-refined diet contains only 16.3 g dietary fibre/2000 kcal.\n\nBreakfast, lunch and dinner will each provide 30% of the participant’s prescribed caloric intake and an evening snack will provide the final 10%. The dietary plan for each diet is outlined in Table 2. All food provided to the participants will be prepared by the study team in the CRF Diet Kitchen according to food hygiene/food safety standards.\n\n*Chickpea and chickpea humus recipes will be energy matched. E, energy, HF, high fibre; LF, low fibre.\n\nDiet preparation. For the LF-refined diet, breakfast will be prepared the night before serving but the bread will be toasted and the milk will be added to the cornflakes immediately before serving. The meals served at 13:00 and 17:00 will be ready meals, which will be cooked in the microwave according to the manufacturer’s instructions and the required portion will be weighed out before serving.\n\nFor the HF-whole diet, the breakfast will be prepared the day before serving and the oats will be soaked in milk from approximately 16:30 the evening before. The lunch will be prepared in the hour before serving; the apples will be cut into chunks that are easier to eat and the soup will be weighed out and microwaved just before serving. The dinner will be prepared in the hour before serving. The potatoes will be boiled for 20 min and the peas microwaved according to the manufacturer’s instructions. Both will be drained and weighed after cooking. All other foods will be served cold. The carrot, beetroot and tomatoes will be cut up into smaller pieces that are easier to eat. The evening snack will be prepared at dinner-time.\n\nFor the HF-disrupted diet, the oats will be blended in advance into a flour-like consistency. The rye bread and humous will be made in advance in-house, frozen and defrosted as needed. The breakfast will be prepared the day before serving. The oranges will be blended to a smoothie. The oats will be soaked in milk from approximately 16:30 the evening before and heated in the microwave immediately before serving. The lunch will be prepared in the hour before serving. The apples will be blended to a puree. The can of soup will be blended, the required portion will be weighed out and microwaved just before serving. The dinner will be prepared in the hour before serving. The beetroot and tomatoes will be blended to a smoothie. The potatoes and carrots will be boiled for 20 min and the peas will be microwaved according to the manufacturer’s instructions. The vegetables will then be drained, blended and weighed. The potato, carrot and peas will then be re-heated in the microwave to comply with food safety standards. All other foods will be served cold. The snack is prepared at dinner-time and the bananas and oranges will be blended.\n\nAll foods that are heated or cooked by the study team will be temperature probed before serving.\n\nAn overview of the study visit protocol is outlined in Figure 1. Participants will attend their first study visit within 12 weeks of their screening visit. There will be a minimum wash out period of 7 days between study visits.\n\nAbbreviations: BL, baseline; CX, Charing Cross Hospital; HH, Hammersmith Hospital; Pt, patient; VAS, visual analogue scale.\n\nDay 1: Nasoenteric tube insertion. Participants will be asked to arrive at the Imaging Department in Charing Cross Hospital, London, having fasted overnight and having avoided intense exercise, caffeine and alcohol the day before their study visit. Females of child-bearing age will be asked to provide a urine specimen in order to perform a pregnancy test. A custom-made nasoenteric tube (Figure 2) will then inserted through the nose. Once it has passed the stomach and the first part of the small intestine, the balloon at the terminal end will be inflated to approximately 6 ml and will be used to carry the tube through the small intestine by peristalsis. The tube position will be confirmed by fluoroscopy throughout the tube insertion process. Dilute barium sulphate may also be administered in order to help confirm the positioning. Once the tube has reached the terminal ileum, the balloon will be deflated and the tube will be restrained from additional movement for the rest of the 4-day visit. Participants will be provided with food and drinks during the tube insertion process. However, the food provided will not be standardised to allow for flexibility to aid tube insertion. Following the tube insertion, participants will travel back to the NIHR Imperial CRF, Hammersmith Hospital, accompanied by a trained medical professional. The final position of the tube will be recorded in the participant’s notes.\n\nPublished with permission from MUI Scientific.\n\nDay 1 – Start of dietary intervention. Participants will be fed one of the three diets over the 4-day study period according to their randomisation. The dietary intervention will start with dinner on Day 1 and end with lunch on Day 4. Meals will be provided at set times; breakfast at 09:00, lunch at 13:00, dinner at 17:00 and an evening snack at 21:00. During their stay, participants will be asked to eat all of the food that is provided and to not eat any other food including chewing gum. Participants will have free access to water, except for during sampling periods on Day 3 and 4 where water will be limited. Participants will not be allowed to leave the CRF during their visit but will have access to WiFi and TV. Participants will be asked to collect a stool sample each time they pass stool during their inpatient stay for metabolomic and microbiological analysis.\n\nDay 2 – Acclimatisation day. Day 2 will allow participants to acclimatise to their new diet and environment. Meals will be provided at set meal times and participants will have free access to water. The only samples collected on Day 2 will be stool samples.\n\nDay 3 – Ileal sampling. On day 3, ileal samples will be collected via the nasoenteric tube for metabolomic and microbiological analysis. For the 30 minutes before breakfast, the participant’s water jug will be removed. Two baseline ileal samples will be collected >10 min apart. Breakfast will be served at 9:00 am (±10 min) with 500 ml water. The time at which each participant starts eating breakfast will be considered to be 0 min. Further ileal samples will be collected every 60 min for 480 min. Lunch will be served directly after the 240 min sample with 500 ml water. Dinner will be served at 17:00, after the 480 min sample and participants will have free access to water for the rest of the day.\n\nDay 4 – Ileal and blood sampling. On the morning of day 4, participants will have an intravenous cannula inserted to allow for blood sampling and body composition will be assessed by bio-impedance analysis. For the 30 minutes before breakfast, the participant’s water jug will be removed, as on day 3. Two baseline samples will be collected >10 min apart before breakfast. At each timepoint, subjective feelings of appetite (‘how hungry/full do you feel?’) and mood (‘how nauseous do you feel?’) will be collected using a series of 100 mm visual analogue scales (VAS). The left extremity of the VAS is labelled with ‘not at all’ and the right-hand extremity is labelled with ‘extremely’. Participants will be asked to draw a vertical line on the VAS depending on how intensely they are experiencing each feeling. Baseline breath H2 measurements will be collected in real-time using a handheld monitor (Gastro+ Gastrolyser Breath Hydrogen Monitor, Bedfont Scientific) and will be used as a marker of intestinal fermentation24. Baseline blood samples will be collected to assess blood hormones and metabolites and ileal samples will be collected via the nasoenteric tube as on Day 3. Ileal samples will be collected across both Day 3 and 4 in order to gain a full understanding of the dynamic change of the ileum environment over the study period. Once both baseline samples have been collected, breakfast will be served at 9:00 am (±10 min) with 500 ml water. The time at which the participant starts eating breakfast will be considered to be 0 min. Further samples will be collected every 60 min for 480 min. Lunch will be served directly after the 240 min sample with 500 ml water. Following the final sample at 480 min, the cannula and nasoenteric tube will be removed and participants will be discharged.\n\nStool samples. Stool samples will be frozen at -80°C immediately following collection and the time of collection will be recorded.\n\nIleal samples. In order to collect ileal samples, the aspiration channel of the tube will first be flushed with a volume of water that is equal to the deadspace leading to the lumen. The air channel will be opened and the volume of water that the tube was flushed with will be taken back using a 50 ml syringe. Once the flush has been collected, the aspiration channel will be clamped using a medical clamp and the flush will be discarded. Up to 5 ml ileal sample will be collected at each sampling timepoint. Samples will be put on ice immediately following collection and frozen at -80°C. Following sampling, the aspiration channel of the tube will be flushed with a volume of water that is equal to the deadspace and both the aspiration and air channels will be closed until the next sample.\n\nBlood samples. A total of 10 ml blood will be collected at each time point and aliquoted into vacutainers: 1 ml blood will be added to a BD Fluoride Ethylenediaminetetraacetic acid (EDTA) Vacutainer; 2 ml blood will be added to a BD Lithium Heparin Vacutainer containing 20 μl/ml whole blood Aprotinin pancreatic protease inhibitor (Nordic Pharma UK Ltd, Reading, UK), 2ml blood will be added to a BD Lithium Heparin Vacutainer and 5 ml blood will be added to a BD Serum SST Vacutainer. Plasma tubes will be centrifuged immediately at 2,500 relative centrifugal force (RCF) for 10 min at 4°C. Serum tubes will be allowed to clot before centrifugation. Resulting plasma and serum will be separated and frozen at -80°C until analysis.\n\nSamples will be analysed upon completion of the study. Metabolic and microbiological profiling of ileal and faecal samples will be performed using 1H-NMR spectroscopy and 16S sequencing, respectively. The metabolic and hormonal profiling of blood samples will be assessed using 1H-NMR spectroscopy, radioimmunoassays, Ultraperformance Liquid Chromatograpy coupled to Mass Spectrometry (UPLC-MS) and Gas Chromatography coupled to Mass Spectrometry (GC-MS). Subjective appetite will be assessed using visual analogue scales (VAS) and breath H2 will be measured in real-time using a handheld gastrolyser.\n\nA one-stage continuous fermentation model system will be established using ileal samples from the human study to monitor in vitro the effect of carbohydrate quality on ileal microbiota composition and their subsequent metabolites. The system will be adapted from the three-stage colonic model described by Gibson et al.25. Ileal samples will be collected via a nasoenteric tube (Figure 2) as previously described and anaerobically transferred into hungate tubes, which will then be used to inoculate the in vitro system. Samples will be obtained from the system at baseline (t=0) and 24 hours after (t=24).\n\nThe diets from the human study will then be tested in the in vitro system. Diets will be digested by an in vitro simulation of upper gut digestion as per Mills et al.26. The remaining products will be dialysed using a membrane of 100–200 Dalton cut off (Biotech CE Dialysis Tubing, Spectrum Europe, Netherlands). The products will be freeze-dried before their addition to the in vitro system. Samples will be obtained from the system 24 hours after the addition of the diets (t=48) to measure main bacterial groups using fluorescent in situ hybridisation (FISH) and for microbial produced metabolites using 1H-NMR spectroscopy.\n\n\nData management and dissemination\n\nParticipants will be given an anonymised personal study code number once randomised, which will be used throughout the study and in the analysis of data. The study codes will be kept on departmental databases. All personal data will be stored in locked filing cabinets in the Section of Investigative Medicine, Imperial College London. Only members of the Section of Investigative Medicine will have access to these. The Principal Investigator and the study team will have access to the final trial dataset.\n\nA formal data analysis plan will be drawn up upon completion of the study. However, assuming the data is parametric and conforms to similar studies we have conducted, we would expect our outcome measures to be analysed using a variety of statistical methods. These will include parametric statistics and multivariate analysis using pattern recognition techniques such as principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA) and non-metric multidimensional scaling (NMDS) plots, using a number of statistical packages including SPSS, MATLAB and R.\n\nAs this is a pilot study, there are too few participants to have a data monitoring committee.\n\nImperial College is the main research sponsor for this study.\n\nOnce data analysis is complete, participants will receive a lay summary of the study’s findings. In addition, the results of the study will be presented at scientific meetings and conferences, and published in relevant high-impact journals. Research papers will be written by the study team. Authors will be included according to the International Committee of Medical Journal Editors (ICMJE) Recommendations. An anonymised dataset will be made available to the scientific community.\n\nThis study has been approved by the London – Bloomsbury Research Ethics Committee (REC) and Health Research Authority (HRA; REC Reference Number: 17/LO/0354). This study will be conducted in accordance with the recommendations for physicians involved in research on human subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions. Any substantial amendments made to the protocol, PIS or consent form will be submitted to the REC and HRA. Once these changes have been approved, these modifcations will be explained to participants and implemented with their consent.\n\nParticipants will have the choice during the consent process to agree to their samples being stored and used for future analyses. Samples will be kept in the Section of Investigative Medicine and will only be used for other research purposes that have been ethically approved.\n\nAny significant adverse event as assessed by the researchers will halt the study and the research ethics committee and sponsor will be informed as per standard protocol. All adverse events will be recorded and investigators will review each adverse event as it arises.\n\nImperial College London holds negligent harm and non-negligent harm insurance policies, which apply to this study.\n\n\nDiscussion\n\nThe maintenance of body weight throughout life is important for metabolic homeostasis. Over the last 15 years, both epidemiological and experimental evidence have highlighted the importance of carbohydrate fermentation within the gut to appetite regulation. However, our current investigative tools are not capable of characterising the complex signalling that mediates these effects in the gut. In the current study, we will investigate how the quality of dietary carbohydrate consumed affects the form and type of carbohydrate in the ileum. This will allow us to gain a deeper understanding of the composition of the digesta reaching the colon from the ileum following such diets. In addition, we will determine how this subsequently affects subjective appetite measures and gut hormone secretion. We will use an in vitro model, inoculated with ileal fluid, to provide a deeper understanding of how ileum contents, following diets differing in carbohydrate quality, affects the colonic gut microbiota and the production of SCFAs in the colon. This is important in identifying how dietary carbohydrate drives colonic gut hormone release and is critical in understanding the relationship between the colon and the maintenance of energy homeostasis. This knowledge may facilitate the development of food products designed for body weight maintenance.\n\nThe first study visit was completed in October 2017. Eight participants have successfully completed the study so far.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans. https://doi.org/10.6084/m9.figshare.7752131.v123.\n\nThis project contains the following extended data:\n\nParticipant Information Sheet - Version 5 – 111017.doc (information sheet for research participants)\n\nConsent Form - Version 1 - 060217.doc (consent form given to each participant)\n\nFigshare: SPIRIT checklist for “A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans”. https://doi.org/10.6084/m9.figshare.7752131.v123.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis article presents independent research funded by the UK Biotechnology & Biological Sciences Research Council (BBSRC) (BB/N016847/1) and Nestec Ltd, and is supported by the NIHR CRF and BRC at Imperial College Healthcare NHS Trust. The views expressed are those of the authors and not necessarily those of our funders, the NHS, the NIHR or the Department of Health. The Section of Endocrinology and Investigative Medicine is funded by grants from the MRC, BBSRC, NIHR, an Integrative Mammalian Biology (IMB) Capacity Building Award, an FP7- HEALTH- 2009- 241592 EuroCHIP grant and is supported by the NIHR Biomedical Research Centre Funding Scheme. GF holds an NIHR Senior Investigator Award.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to acknowledge the support and help of our colleagues in the NIHR Imperial CRF and the Charing Cross Imaging Unit.\n\n\nReferences\n\nWHO: Obesity and overweight - Fact sheet at World Health Organisation Media Centre 2016. Reference Source\n\nPeeters A, Barendregt JJ, Willekens F, et al.: Obesity in adulthood and its consequences for life expectancy: a life-table analysis. Ann Intern Med. 2003; 138(1): 24–32. PubMed Abstract \| Publisher Full Text\n\nButland B, Jebb S, Kopelman P, et al.: Tackling Obesities: Future Choices - Project Report, 2nd Edition. 2007. Contract No.: 17 March. Reference Source\n\nHill JO, Peters JC: Biomarkers and functional foods for obesity and diabetes. Br J Nutr. 2002; 88 Suppl 2: S213–S8. PubMed Abstract \| Publisher Full Text\n\nMurphy KG, Bloom SR: Gut hormones in the control of appetite. Exp Physiol. 2004; 89(5): 507–16. PubMed Abstract \| Publisher Full Text\n\nAbbott CR, Monteiro M, Small CJ, et al.: The inhibitory effects of peripheral administration of peptide YY3-36 and glucagon-like peptide-1 on food intake are attenuated by ablation of the vagal-brainstem-hypothalamic pathway. Brain Res. 2005; 1044(1): 127–31. PubMed Abstract \| Publisher Full Text\n\nFlint A, Raben A, Astrup A, et al.: Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest. 1998; 101(3): 515–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBatterham RL, Cohen MA, Ellis SM, et al.: Inhibition of food intake in obese subjects by peptide YY3–36. N Engl J Med. 2003; 349(10): 941–8. PubMed Abstract \| Publisher Full Text\n\nBatterham RL, Cowley MA, Small CJ, et al.: Gut hormone PYY3-36 physiologically inhibits food intake. Nature. 2002; 418(6898): 650–4. PubMed Abstract \| Publisher Full Text\n\nChallis BG, Pinnock SB, Coll AP, et al.: Acute effects of PYY3-36 on food intake and hypothalamic neuropeptide expression in the mouse. Biochem Biophys Res Commun. 2003; 311(4): 915–9. PubMed Abstract \| Publisher Full Text\n\nLiu S, Willett WC, Manson JE, et al.: Relation between changes in intakes of dietary fiber and grain products and changes in weight and development of obesity among middle-aged women. Am J Clin Nutr. 2003; 78(5): 920–7. PubMed Abstract \| Publisher Full Text\n\nLudwig DS, Pereira MA, Kroenke CH, et al.: Dietary fiber, weight gain, and cardiovascular disease risk factors in young adults. JAMA. 1999; 282(16): 1539–46. PubMed Abstract \| Publisher Full Text\n\nByrne CS, Chambers ES, Morrison DJ, et al.: The role of short chain fatty acids in appetite regulation and energy homeostasis. Int J Obes (Lond). 2015; 39(9): 1331–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlavin JL: Dietary fiber and body weight. Nutrition. 2005; 21(3): 411–8. PubMed Abstract \| Publisher Full Text\n\nGrundy MM, Edwards CH, Mackie AR, et al.: Re-evaluation of the mechanisms of dietary fibre and implications for macronutrient bioaccessibility, digestion and postprandial metabolism. Br J Nutr. 2016; 116(5): 816–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerry SE, Tydeman EA, Lewis HB, et al.: Manipulation of lipid bioaccessibility of almond seeds influences postprandial lipemia in healthy human subjects. Am J Clin Nutr. 2008; 88(4): 922–9. PubMed Abstract \| Publisher Full Text\n\nEdwards CH, Grundy MM, Grassby T, et al.: Manipulation of starch bioaccessibility in wheat endosperm to regulate starch digestion, postprandial glycemia, insulinemia, and gut hormone responses: a randomized controlled trial in healthy ileostomy participants. Am J Clin Nutr. 2015; 102(4): 791–800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGidley MJ: Hydrocolloids in the digestive tract and related health implications. Curr Opin Colloid Interface Sci. 2013; 18(4): 371–8. Publisher Full Text\n\nMurphy KG, Bloom SR: Gut hormones and the regulation of energy homeostasis. Nature. 2006; 444(7121): 854–9. PubMed Abstract \| Publisher Full Text\n\nSpreckley E, Murphy KG: The L-Cell in Nutritional Sensing and the Regulation of Appetite. Front Nutr. 2015; 2: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlint HJ, Scott KP, Louis P, et al.: The role of the gut microbiota in nutrition and health. Nat Rev Gastroenterol Hepatol. 2012; 9(10): 577–89. PubMed Abstract \| Publisher Full Text\n\nCummings JH, Pomare EW, Branch WJ, et al.: Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut. 1987; 28(10): 1221–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nByrne C, Blunt D, Burn J, et al.: A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7752131.v1\n\nLevitt MD: Production and excretion of hydrogen gas in man. N Engl J Med. 1969; 281(3): 122–7. PubMed Abstract \| Publisher Full Text\n\nGibson GR, Cummings JH, Macfarlane GT: Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Appl Environ Microbiol. 1988; 54(11): 2750–5. PubMed Abstract \| Free Full Text\n\nMills DJ, Tuohy KM, Booth J, et al.: Dietary glycated protein modulates the colonic microbiota towards a more detrimental composition in ulcerative colitis patients and non-ulcerative colitis subjects. J Appl Microbiol. 2008; 105(3): 706–14. PubMed Abstract \| Publisher Full Text" }	[ { "id": "45303", "date": "05 Sep 2019", "name": "Ellen Blaak", "expertise": [ "Reviewer Expertise metabolic interorgan crosstalk in insulin resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study design investigating the role of the ileum and its microbiota in the processing of dietary fibers on 3 different diet, a low fiber diet and 2 high fiber diets, one with intact cellular structures and the other one with disrupted cell structures. This study gives a real time perspective how the ileum content changes in response to different diets and is therefore unique.\n\nThe hypothesis may be formulated a little bit sharper, the idea is that minimal processed fiber may lead to a higher fermentation in the colon with a more beneficial metabolic profile, how exactly is this question answered since fermentation and SCFA production is not directly measured in the colon?\nWould an increased fermentation in the ileum also be beneficial?\nHow much time does the sampling of the ileum take? Is also the total microbial content determined in ileal and fecal samples, expressed per what?\nWhat is the period between the visits i.e. the washout period?\nThe in vitro approach may be an interesting addition. Sampling takes place at baseline and at 24 h, why are these time points chosen and is more frequent sampling a possibility?\nLooking at the effect of whole diets in the 'in vitro' system, diets are first freeze dried, to what extent are the cellular structures maintained in this procedure and to what extent are the outcomes still relevant for the in vivo conditions?\nOverall, a challenging and interesting study protocol.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4968", "date": "11 Oct 2019", "name": "Aygoul Dagbasi", "role": "Author Response", "response": "Thank you for your time to review our article and provide valuable comments.We agree with all your comments.We will be changing our hypothesis as per your advice to fit the study design better.We do think that an increased fermentation in the ileum would also be beneficial in addition to the colonic fermentation. This is because GLP-1 and PYY secreting L-cells increase in number along the GI tract with the highest numbers found in distal ileum and colon (Suzuki et al., 2018). Therefore the composition of the ileal microbiota and their subsequent metabolites including SCFAs can be sensed by the dense population of ileal L-cells and potentially have a profound effect on appetite hormone secretion and host metabolism. We are hoping to answer this question better with our study results.We had a trial run of ileal sampling which took around 5-10 minutes per sample. The microbial content will be expressed as Log 10 numbers per ml of ileal/faecal content as measured by flow-FISH (fluorescent in-situ hybridisation) and as ratios when measured by 16s rRNA sequencing.The washout period will be minimum of 7 days.The in-vitro system takes 24 hours to stabilise after inoculation and after the addition of diets therefore any samples taken in between would be inaccurate. Sampling can continue after 24 hours however since the system is stabilised, no change is anticipated after this period.Microscopy pictures were taken after the diets were freeze dried. These revealed that this method is not disruptive to the cellular structures. The slides were stained with iodine which showed carbohydrate still present in intact cellular structures for Diet 2 (Whole Diet).Thank you again for your valuable comments." } ] }, { "id": "47113", "date": "23 Sep 2019", "name": "Marcel van de Wouw", "expertise": [ "Reviewer Expertise Microbiota-gut-brain axis in metabolism and eating behaviour" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study protocol submitted by Byrne et al. is an exciting and well-thought out experiment, aimed at investigating the impact of diets with different carbohydrate quality (i.e. low fibre, whole high fibre and disrupted high fibre) on the gastrointestinal microbiota and appetite in humans. The repeated ileal sampling provides for a real time analysis of the response of the microbiota to these different diets.\nMinor comments:\nSome lines of research indicate that different dietary fibres (e.g. inulin, rhamnose) differentially impact the gut microbiota and subsequent SCFA production (Baxter et al. 20191; Reichardt et al. 20182). This is well-controlled for when comparing the HF-whole and HF-disrupted groups, but not so much for the LF-refined group. It might be good to, if possible, analyse dietary fibre profile of the diets. This will increase the interpretability of the data and allow for a better comparison between the LF and HF groups, as LF will likely have a different dietary fibre profile.\n\nIt is likely that the food intake patterns of participants prior to the study will influence their microbiota and therefore how their microbiota will respond to the different diets. Performing a food frequency questionnaire to assess their diet prior to the visit date might therefore provide valuable insight into how day-to-day food intake patterns can influence the microbiota and its response to these diets.Or any other measure to have an record of baseline food intake and food quality.\n\nBoth males and females will be recruited, but its not stated if the aim is to recruit at a gender balanced level. Use of oral contraceptives and menstrual cycle should be recorded (oral contraceptives possibly excluded).\n\nPage 3, sentence: “Short-chain fatty acids, products of microbial fermentation, have also been shown to stimulate the secretion of GLP-1 and PYY.” This sentence requires a reference.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4969", "date": "11 Oct 2019", "name": "Aygoul Dagbasi", "role": "Author Response", "response": "Thank you for your time to review our article and provide valuable comments.We agree with all your comments.We will be performing a direct analysis of the dietary fibre profile of each dietary intervention to be able to better interpret the data as suggested. As you highlighted, high fibre groups (whole and blended) will have the same profiles since these diets consist of the same foods however the low fibre group will have a different profile.The cross-over design of the study allows each participant to act as their own control. This will eliminate confounding factors like habitual dietary intake and gut microbiota profile. Current dietary assessment methods provide an inaccurate measure of dietary intake (Shim et al. 2014). As a result of these; we decided not to collect dietary intake data. We will be excluding participants following extreme diets such as vegan or vegetarian diets.We will aim to recruit at a gender-balanced level. We will note information regarding females’ contraception methods and menstrual cycles.Thank you again for your valuable comments" } ] } ]	1	https://f1000research.com/articles/8-258
https://f1000research.com/articles/8-1783/v1	22 Oct 19	{ "type": "Case Report", "title": "Case Report: Xanthogranulomatous prostatitis, a difficult differential diagnosis of prostate adenocarcinoma", "authors": [ "Reda El Moussaoui", "Ali El Moussaoui", "Mohamed Dakir", "Amal Benkirane", "Ali El Moussaoui", "Mohamed Dakir", "Amal Benkirane" ], "abstract": "Background: Xanthogranulomatous prostatitis is a rare benign inflammatory process of the prostate. Clinical, biochemical and imaging similarities make xanthogranulomatous prostatitis a difficult differential diagnosis of prostatic adenocarcinoma. Case presentation: We report the case of a 62 year-old diabetic patient, with 3 months of lower urinary tract symptoms. On digital rectal examination, the prostate was hard and nodular. Initial prostate-specific antigen (PSA) was 43.97 ng/mL, with urine analysis and transabdominal ultrasonography not showing any alterations. A multiparametric magnetic resonance imaging (mpMRI) of the prostate was performed and showed two foci that were classified as PI-RADS 4 lesions. A transrectal ultrasound guided prostate biopsy was then realized. After an initial suspicion of prostatic adenocarcinoma, on clinical, biochemical and radiological grounds, histopathological examination revealed a suppurated xanthogranulomatous prostatitis, with no evidence of malignancy. Conclusions: Xanthogranulomatous prostatitis is an uncommon benign inflammatory condition of the prostate that can clinically, biochemically and even radiologically simulate prostatic adenocarcinoma. We advise that if an older male with low urinary tract symptoms and a hard nodular prostate on digital rectal examination presents, the first diagnosis that one should think of is prostate adenocarcinoma, especially if PSA is high. However, only histopathological examination can differentiate with certitude between these two pathologies and is therefore essential for the diagnosis of xanthogranulomatous prostatitis.", "keywords": [ "Xanthogranulomatous", "Prostatitis", "Adenocarcinoma" ], "content": "Introduction\n\nA variety of benign conditions of the prostate can clinically mimic prostatic adenocarcinoma, thus raising a diagnostic problem. Xanthogranulomatous prostatitis, a rare form of non-specific granulomatous prostatitis, is considered among those pathologies that can clinically, biochemically and even radiologically behave as a prostate carcinoma1,2. Symmers was the first author to mention this disease in 19503, while Miekoś et al. produced the first case report in Poland in 19864,5. Very few similar cases have since then been described in the literature.\n\nWe report an additional case of a patient with initial clinical suspicion of prostatic carcinoma, but whose histopathological report revealed a xanthogranulomatous prostatitis.\n\n\nCase report\n\nThe patient is a 62 year old diabetic male, with no particular medical history, whose main symptoms started 3 months ago with urinary incontinence, increased urinary frequency and urgency, alongside haematuria. The patient also had micturition burning, which came with a recent onset of fever. Digital rectal examination (DRE) was indolent and revealed an asymmetric and enlarged prostate, with a hard fixed nodule on the right lobe. The patient’s general physical examination was normal, and revealed no alterations of the penis, the testicles or the epididymis. A prostate-specific antigen (PSA) test was performed and showed an increase in serum PSA level that reached 43.97 ng/ml (normal level <4 ng/ml). Urine analysis were within normal limits with no growth in urine culture. A transabdominal ultrasonography showed a normal upper renal tract and a normal bladder wall, with a post-micturition residual volume of 48.21 ml. Transrectal ultrasound (TRUS) was not performed, as it was too painful for the patient to sustain. From the clinical, radiological and biochemical data, a locally advanced prostate carcinoma was suspected. A multiparametric magnetic resonance imaging (mpMRI) of the prostate was then performed. The MRI showed an enlarged prostate, with an estimated weight of 51 grams. Two foci were found (Figure 1), which were classified as PI-RADS 4 lesions, thus requiring histological examination. There was no infiltration of the peri-prostatic fat.\n\n(a) The first focus, of 18 mm, was located in the central zone of the prostate, in the right posterolateral region (blue arrow), with a low signal intensity on T2-weighted images. (b) The other focus was a 22 mm nodule located in the transitional zone of the prostate (blue arrow). Both were classified as PIRADS 4 lesions.\n\nA TRUS guided prostate biopsy was then performed. A systematic 12-core prostate biopsy was realized, alongside three more core samples on zones that were found suspicious on the MRI images. Histological examination of the 15 needle biopsy core samples showed glandular atrophy, fibrosis, and an accumulation of inflammatory cells including polymorphs with eosinophils and neutrophils, with prostatic abscess, as well as the presence of foamy macrophages (lipid-laden histiocytes), also known as xanthoma cells (Figure 2). Tissue cultures and immunohistochemistry tests were not performed. The histopathological examination concluded a suppurated xanthogranulomatous prostatitis, with no evidence of malignancy.\n\n(a) Prostatic parenchyma with abscess and a polymorph inflammatory infiltrate of neutrophils. (b) Xanthomatous infiltrate with foamy histiocytes, with no evidence of malignancy.\n\nConservative treatment was chosen, and the patient was given Ciprofloxacin for 4 weeks (500mg, twice a day). After three months, a PSA test was performed, and showed a significant decrease in PSA that reached 7.27 ng/ml (compared to the initial 43.97 ng/ml). A transabdominal ultrasonography showed a decrease in prostate volume, with an estimated prostate weight of 31 grams (versus 51 grams previously). A close follow-up will be needed for this case, with a clinical and biochemical check-up every trimester, until PSA levels are within normal limits (<4 ng/ml).\n\n\nDiscussion\n\nBenign conditions that mimic prostate carcinoma have been divided into six groups5, among them, inflammatory diseases. Granulomatous prostatitis is an unusual prostatic entity that was first described by Tanner and McDonald in 19436 and classified in 1984 by Epstein and Hutchin7 into five groups, based on aetiology and histopathology: idiopathic (non-specific), infectious (specific), malakoplakia, iatrogenic, and cases associated with systematic diseases and allergy3,7,8. Infectious agents that have been encountered in specific granulomatous prostatitis are Mycobacterium tuberculosis, Treponema pallidum, as well as some fungi and viruses3,8. Specific granulomatous prostatitis may also be due to intravesical Bacillus Calmette-Guerin (BCG) therapy for bladder cancer8. Xanthogranulomatous prostatitis is an uncommon form of non-specific granulomatous prostatitis. The aetiology of xanthogranulomatous prostatitis is still unclear; it has been often associated with hyperlipidemia (in our case, the patient had no history of hyperlipidemia) or recurrent urinary tract infection8, although some authors have considered that it might be caused by an autoimmune disease with a HLA-DR15-linked T-cell response against proteins in prostatic secretions4,9,10. Bostwick and Chang brought the theory of ductal obstruction, speculating that blockage of prostate ducts and stasis of secretions cause cellular debris, bacterial toxins, prostatic secretions, sperm, and semen to escape into the stroma through the destroyed epithelium, eliciting a localized inflammatory response4,9,10.\n\nXanthogranulomatous inflammation occurs very rarely in the prostate. It is vastly known in the kidneys and gallbladder, and has been described in other anatomic sites, such as the mandible, retro peritoneum, third ventricle, choroid plexus, orbit, vagina, lung, stomach, pericardium, and ovary10. Less than 20 cases have been reported in the literature since its first description4,9 with a similar case of suppurated xanthogranulomatous prostatitis recently discovered also in Morocco9.\n\nHistologically, the typical lesion in granulomatous prostatitis is a large inflammatory nodular infiltrate of epithelioid histiocytes, plasma cells, lymphocytes and sometimes polymorphs with eosinophils5,9,11. The specific and distinctive feature of xanthogranulomatous prostatitis is the presence of foamy macrophages (lipid-laden histiocytes), also known as xanthoma cells5,9,11. Immunohistochemistry tests reveal T-lymphocytes in close association with damaged epithelium while B-lymphocytes occur in a more peripheral location or form follicular structures9,11. The presence of xanthoma cells may cause diagnostic confusion with high-grade prostatic carcinoma3,9,12, especially with the hypernephroid pattern of prostate carcinoma (Gleason 4B)3, as well as clear cell carcinoma12. A panel of immunohistochemistry tests, such as cytokeratin, PSA, prostatic acid phosphatase (PAP), leukocyte common antigen (LCA) and CD68 can be useful in differentiating between these two conditions, by showing results more consistent with an inflammatory process3,9,11. Those tests were not performed in our case, as the foamy macrophages were judged sufficient for the histopathological diagnosis of this condition.\n\nIn our case, even though all the signs were pointing towards prostate adenocarcinoma, we decided not to rush the diagnosis. Multi-parametric MRI was very helpful, as it allowed us to locate the suspicious foci. We decided then to perform a 15-core needle biopsy, rather than the systematic 12-core biopsy that is usually performed for prostate carcinoma, to increase our chances of finding pathological cells.\n\nXanthogranulomatous prostatitis causes serious confusion with prostate carcinoma when it comes to diagnosis. It occurs usually in elderly men, usually in the sixth decade3,8. In most cases, xanthogranulomatous prostatitis is diagnosed incidentally on TURP chips or needle biopsy3,8,9. Clinically, the symptoms are either those of urinary obstruction, with low urinary tract symptoms, or severe low urinary tract infection symptoms. Two recent studies have shown that the most encountered symptoms were increased urinary frequency and urgency, sometimes with dysuria, micturition burns, haematuria and urinary incontinence3,8. Few episodes of fever and chills have been reported8. On DRE, it is difficult to distinguish from prostate carcinoma, as the prostate feels hard and nodular3,8. In some cases, DRE finds an asymmetry or an enlargement of the prostate. In addition, xanthogranulomatous prostatitis may cause a transient increase in serum PSA level, which decreases with a resolution of the inflammation4,9,11. In some cases, PSA serum levels reach 150 ng/ml11. In our case, PSA decreased from 43.97 ng/ml to 7.27 ng/ml in the space of 3 months. Furthermore, no imaging (TRUS, MRI) can differentiate between xanthogranulomatous prostatitis and prostate adenocarcinoma, given the radiological similarities, and the absence of a specific feature of xanthogranulomatous prostatitis1,2. In a recent study, it has been shown that 63.6% of patients with PI-RADS (V2) 4 lesions who underwent transrectal biopsy were diagnosed with prostate cancer13. Hence, the only way to differentiate between these two conditions is histopathological examination.\n\nXanthogranulomatous prostatitis can occur in a normal gland, nodular hyperplastic gland or carcinomatous prostate. It is mostly located in the peripheral or transitional zone3,8. In some cases, xanthogranulomatous prostatitis can co-exist with prostate carcinoma3,8,11.\n\nUnlike prostatic adenocarcinoma, conservative management is the rule for xanthogranulomatous prostatitis8,9. Inflammation is often self-limiting and resolves slowly over time. Surgical management may be considered if there is failure of conservative treatment, and might be needed in case of severe low urinary tract symptoms or due to occurrence of complications that may require radical prostatectomy4,9,10. However, surgical treatment of xanthogranulomatous prostatitis can lead to complications like vesical neck contraction, and might require repeated resections12.\n\nLong-term follow-up is needed for patients with xanthogranulomatous prostatitis, as they require a regular clinical and biochemical check-up. MRI in xanthogranulomatous prostatitis follow-up is not required, but it might be necessary if DRE remains suspicious, or if there is no decrease in PSA serum levels. In addition, benign prostatic hyperplasia and prostatic adenocarcinoma can still occur in patients who previously dealt with xanthogranulomatous prostatitis3,8,11, thus making long-term follow-up inevitable.\n\n\nConclusion\n\nXanthogranulomatous prostatitis is an uncommon inflammatory pathology that can mimic prostatic carcinoma both clinically and/or biochemically. Furthermore, no radiological features can help differentiate between these two conditions. Precise histopathological examination is essential for the final diagnosis of xanthogranulomatous prostatitis.\n\nGiven clinical, biochemical and imaging similarities with prostatic carcinoma, as well as the rare nature of this condition, patients with xanthogranulomatous prostatitis raise a major diagnostic issue. Conservative treatment is the rule, with long-term follow-up needed, especially in patients with persisting elevated serum PSA values.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report and accompanying images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nKitzing YX, Prando A, Varol C, et al.: Benign Conditions That Mimic Prostate Carcinoma: MR Imaging Features with Histopathologic Correlation. Radiographics. 2016; 36(1): 162–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee SM, Joshi J, Wolfe K, et al.: Radiologic presentation of chronic granulomatous prostatitis mimicking locally advanced prostate adenocarcinoma. Radiol Case Rep. 2016; 11(2): 78–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShukla P, Gulwani HV, Kaur S: Granulomatous prostatitis: clinical and histomorphologic survey of the disease in a tertiary care hospital. Prostate Int. 2017; 5(1): 29–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiekoś E, Włodarczyk W, Szram S: Xanthogranulomatous prostatitis. Int Urol Nephrol. 1986; 18(4): 433–437. PubMed Abstract \| Publisher Full Text\n\nNoyola A, Gil JF, Lujano H, et al.: Xanthogranulomatous Prostatitis, a Rare Prostatic Entity. Urol Case Rep. 2017; 10: 4–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanner FH, McDonald JR: Granulomatous prostatitis: a histologic study of a group of granulomatous lesions collected from prostate glands. Archives of Pathology and Laboratory Medicine. 1943; 36: 358–370.\n\nEpstein JI, Hutchins GM: Granulomatous prostatitis: distinction among allergic, nonspecific, and post-transurethral resection lesions. Hum Pathol. 1984; 15(9): 818–825. PubMed Abstract \| Publisher Full Text\n\nKumbar R, Dravid N, Nikumbh D, et al.: Clinicopathological Overview of Granulomatous Prostatitis: An Appraisal. J Clin Diagn Res. 2016; 10(1): EC20–EC23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJabbour Y, Lamchahab H, Harrison S, et al.: Prostatic Abscess on Xanthogranulomatous Prostatitis: Uncommon Complication of an Uncommon Disease. Case Rep Urol. 2018; 2018: 5417903. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXing L, Liu Z, Deng G, et al.: Xanthogranulomatous prostatitis with prostato-rectal fistula: a case report and review of the literature. Res Rep Urol. 2016; 8: 165–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRafique M, Yaqoob N: Xanthogranulomatous prostatitis: a mimic of carcinoma of prostate. World J Surg Oncol. 2006; 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPunia R, Amanjit, Mohan H, et al.: Granulomatous Prostatitis – an infrequent diagnosis. Indian J Urol. 2002; 19(1): 16–19. Reference Source\n\nD’Agostino D, Mineo Bianchi F, Romagnoli D, et al.: Comparison between “In-bore” MRI guided prostate biopsy and standard ultrasound guided biopsy in the patient with suspicious prostate cancer: Preliminary results. Arch Ital Urol Androl. 2019; 91(2). PubMed Abstract \| Publisher Full Text" }	[ { "id": "55593", "date": "04 Feb 2020", "name": "Paari Murugan", "expertise": [ "Reviewer Expertise Genitourinary Pathology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA fairly well written report with useful, if not novel information. This case is also interesting because the PSA was quite high at over 40 ng/ml. It is imperative to carefully evaluate these cases for prostatic adenocarcinoma. This not only includes the \"hypernephroid\" pattern (a term that is no longer used) of Gleason grade 4 carcinoma that may mimic xanthomatous inflammation but also typical coexisting adenocarcinoma that may be hard to identify due to large amounts of inflammation and parenchymal distortion. The discussion can also include the fact that multinucleated giant cells are often seen in this condition.\nPlease perform the corrections recommended below:\nThe images should say axial and coronal in Figure 1. Also, please check if the blue arrow in Figure 1b is in the right place.\n\nThe word polymorph can be deleted from Figure 2.\n\nThe pathology image quality should be improved.\n\nIn the discussion, the author name Cheng is spelt wrong and is not referenced.\n\nIt is unclear what the authors refer to as Gleason 4B. This is not used in current practice.\n\nClear cell carcinoma should be changed to clear cell renal carcinoma.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "60973", "date": "14 Apr 2020", "name": "Nausheen Yaqoob", "expertise": [ "Reviewer Expertise Histopathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHigh Power magnification pictures of xanthogranulomatous areas are required.\n\nReferences are very old. References should be refreshed with recent references within last 5 years. (Reference no 4,6,7).\n\nAre you sure only 20 cases are reported to date?\n\nOther antibodies like PSAP, NKX3.1 or AMACR were performed?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1783
https://f1000research.com/articles/8-1782/v1	22 Oct 19	{ "type": "Research Article", "title": "Depression-inducing drugs and the frequency of depression in Alzheimer’s disease and APOE ε4 carriers", "authors": [ "Dorothy Keine" ], "abstract": "Background: Depression is associated with a greater risk of Alzheimer’s disease (AD). Drug-induced depression is a well-known side effect of many medications and is more likely to occur in those who have a higher risk of depressive disorder. Methods: A total of 292 individuals ages 65 and older were included in this dataset. Depressive symptoms were determined through self-reporting, the Short Form Geriatric Depression Scale (SF-GDS), prior diagnosis, or use of antidepressant medication. Depression-inducing drugs (DIDs) were identified using published references. Results: Individuals took 11.51 (SD 8.86) medications and 1.16 (SD 1.27) DIDs per person. Depressed patients were more likely to be taking at least one DID (71.15% vs 28.85%, P value 0.005). Of the total population, 60.56% were taking at least one DID. Those with APOE ε4 had a significantly higher rate of depression than those without (69.12% vs 30.88%, P value 0.03). Conclusions: DIDs are a substantial clinical, medical, and public health problem in older populations. DID consideration is important in populations with an increased risk or diagnosis of AD. Clinical decision support software (CDSS) provides a reliable method to help with DIDs.", "keywords": [ "Depression", "Alzheimer's", "drug-induced depression", "APOE", "clinical decision support software" ], "content": "Introduction\n\nAlzheimer’s disease (AD) is the most common form of dementia1. It is a degenerative brain disease that eventually leads to death. Characteristic symptoms of the disease include difficulties with memory, language problems, agitation, and cognitive deficits that affect a person’s ability to perform everyday tasks. As the disease progresses, the burden increases and the individual will need help with even basic activities of daily living1.\n\nAn estimated 5.7 million people in the United States have AD, and over 35.6 million people are affected worldwide2. This number is projected to grow as the population aged 65 and older in the USA is anticipated to increase from its current level of 53 million to 88 million by 2050. Today, the total US spending on AD is estimated to be $277 billion1.\n\nResearch now suggests that the brain changes associated with AD begin as much as 20 years before initial symptoms3. Individuals often begin to show early signs of cognitive decline when they develop mild cognitive impairment (MCI). A systemic review of 32 studies found that 32% of people with MCI will go on to develop AD within five years1.\n\nLike many chronic diseases, AD is not caused by one factor but instead develops because of multiple factors. Contributing factors include age, family history, the APOE ε4 gene4,5, as well as modifiable lifestyle factors1.\n\nOne factor often associated with AD is depression, both late-life (depression occurring after the age of 60 or 65) and recurring earlier-life cases. One in five individuals experience at least one depressive episode in their lifespan6, and it occurs more often in older populations2,7. In 2009–2012, depression was estimated to affect more than 5% of all US adults8. Depression has also been found to be more common among women9. Many people with depression go undiagnosed and untreated8.\n\nEvidence suggests that depression is preventable and treatable, and may be a modifiable risk factor for preventing or delaying dementia in later life7. Depression can lead to cognitive deficits in some individuals, sometimes referred to as “pseudodementia”2,10. Older adults are at an even greater risk of developing pseudodementia9,11.\n\nIt is currently unknown if depression is a risk factor for developing AD, or a prodromal sign of the disease2,5,9,12–14. The weight of the evidence suggests that depression is likely a risk factor, though2,10,15. This question remains partly due to the timing of studies that have been conducted in late-life, the variability in follow-up time, and also the fact that depression frequency and duration times are often not recorded2.\n\nResearch has established that depression is associated with a greater risk for AD. Many studies have concluded that depression doubles an individual’s risk of developing dementia, particularly AD7,9,10,16,17. Other studies on depression and AD have shown that those who develop depression are 1.5 times more likely to develop AD compared to those who are not depressed9. The evidence is not yet conclusive as to if early or late-onset depression might be a greater risk factor, but the severity of late-life depression has been found to be an important risk factor for dementia2,7. Some studies have shown that depression in earlier life is more closely associated with vascular dementia, and depression in later life is a greater risk for developing AD18.\n\nAPOE ε4 status has been linked to higher rates of depression and is a definitive risk factor for AD5,19. A study completed in India found that those with APOE ε4 had a 4.7 times higher risk of developing depression in old age20. Populations in Sweden with the APOE ε4 gene also had a higher risk of developing depression. Furthermore, APOE ε4 is associated with more severe depressive symptoms4. Post-mortem studies have reported greater hippocampal amyloid plaque and neurofibrillary tangle pathology in AD patients with a history of depression than those without21.\n\nWhile the specific pathway, or pathways, linking depression and AD have not been defined yet, many mechanisms have been hypothesized. Depression may lead to an increase in amyloid plaques through the depression-associated stress response. This response ultimately results in an increase in β amyloid production22. Chronic inflammation also plays a significant role in the pathophysiology of AD23 and is seen in both depression and AD24. It is hypothesized that stress leads to chronic inflammation in depressed persons. Neuroendocrine changes comparable to those found in chronic stress have also been found in depression and AD models10. Additionally, pro-inflammatory cytokines have been shown to interfere with serotonin metabolism, leading to a reduction in synaptic plasticity and hippocampal neurogenesis22,23. Increased cortisol production can also lead to atrophy in the hippocampus and cognitive deficit25. Interleukin-6 (IL-6), C-reactive protein, and tumor necrosis factor are all increased in depression and may be risk factors for dementia26. Brain-derived neurotrophic factor (BDNF), often associated with synaptic plasticity, is decreased in depression and AD27. Furthermore, MRI studies have found that hippocampal atrophy occurs in cases of depression, suggestive of AD pathology28. Lastly, many lifestyle factors associated with depression are also known risk factors for AD, including poor diet, lack of physical activity, and low social engagement1,9.\n\nOf the studies conducted on depression and AD, this author could not find one that examined the link between depression, AD, APOE, and depression-inducing-drugs (DIDs). Drug-induced depression, also referred to as substance-induced mood disorder in the literature, is a well-known side effect of many medications. The first notion of a “depressogenic” drug was documented in 1954, referring to long periods of treatment with the antihypertensive drug reserpine29.\n\nOlder populations have a greater risk of exposure to DIDs, as they are more likely to be on multiple medications8,30–32. It has been found that the use of multiple medications increases the risk of simultaneous depression and that older age is significantly associated with the use of DIDs8.\n\nThe most common DIDs include narcotics, benzodiazepines, anticholinergic medications, antidepressants, sedatives, beta-blockers, calcium channel blockers, and oral contraceptives8,30,33. The mechanisms for a few depressogenic drugs have been studied, and their additive risk factor for AD cannot be ruled out. In one longitudinal study, seven out of 15 individuals who developed AD had been taking antidepressants5.\n\nPossible mechanisms for DIDs have been proposed34. These mechanisms include inhibiting calcium-dependent neurotransmitter release by blocking the inflow of calcium into cells35, decreased serotonin36, reduction in G-protein-coupled catecholaminergic or cholinergic receptors37, decreased levels of dopamine38, and displacing nicotine from acetylcholine receptors38. Because of the wide range of drugs that can cause this side effect, there is “no known reason to presume that all drugs produce depression via a common pathophysiological mechanism”34.\n\nDrug-induced depression is more likely to occur in those who have a higher risk factor for depressive disorder, as may be the case for individuals with the APOE ε4 allele. For those already predisposed to depression, such as carriers of APOE ε4, DIDs might have an amplified effect, leading to more frequent occurrences of depression.\n\nHere, the author examined the prevalence of DIDs in elderly non-symptomatic and symptomatic AD patients to find how many DIDs the population was taking, the prevalence of depression, and whether APOE ε4 status has any effect on depression levels with concurrent DIDs.\n\n\nMethods\n\nThe data presented here is from uMETHOD Health’s (uMH) database of patients. uMH has created a precision-medicine platform to create personalized treatment plans for patients with and at risk of dementia and AD39. All individuals self-selected to follow their treatment plan. The treatment program was recommended to them by their physician, or they found uMH online and chose to purchase the personalized evaluation and treatment.\n\nuMH’s platform identifies and addresses active issues and creates repeatable and practical treatment plans for use in doctors’ practices.\n\nThe bioinformatics platform’s internal software is written in the Python language. It interfaces to an external portal, used by the medical and coaching teams to gather input, and returns reports written in PHP and Java. External medical databases support internal rules-processing algorithms. These are sourced from bodies such as the National Institutes of Health (NIH), Food and Drug Administration (FDA), pharmaceutical trade groups, and consortia focused on topics such as genetics or allergies.\n\nNatural language processing (NLP) techniques are used throughout the input steps, particularly for precise identification of lab tests, medications, drug indications, and comorbidities. A range of NLP techniques are employed to normalize input data. The algorithms that implement this information platform go through a consistent set of steps each time they load a person’s input to generate a new set of reports. These steps are rules-based, so the logic and evidence sources can be tracked (and evaluated).\n\nThe platform was used as a source of data only and was not used for data analysis in this study.\n\nThe data set consists of all the patients in uMH’s database who were 65 or older.\n\nThe inputs to uMH’s data-analytics engine comprise genomics, bio-specimen measurements, medical histories, medications, allergies, comorbidities, lifestyle data, and cognitive evaluations. Data was collected from physicians, patients, and caregivers from annual wellness visits and through uMH’s patient forms. Forms were completed online via uMH’s secure portal or in paper format, depending on the individual’s computer skills and access. Physicians were instructed to follow a set of inclusion and exclusion criteria from Keine et al. 201839 before enrolling patients.\n\nEach patient was evaluated by a physician to determine if they were symptomatic for AD following standard of care guidelines40. This evaluation included administering a baseline cognitive test to determine patients’ cognitive status at the time of enrollment. The physician chose to administer either the Self-Administered Gerocognitive Exam (SAGE) or the Montreal Cognitive Assessment. Cognitive impairment was determined following each tests’ specific guidelines41,42. Tests were scored at the physician’s office by trained personnel, and completed tests were either uploaded to uMH’s secure portal or were sent to uMH by the physician’s office to be entered into the database. The physician then used the findings to qualify the patient as symptomatic or non-symptomatic in uMH’s database.\n\nGenomic data was obtained through consumer-focused companies such as 23andMe or Ancestry.com. Interested patients purchased a genomic kit through the company of their choice and followed the procedures outlined by the genomic testing company. Raw datasets were accessed by the patient directly from the genomic company’s website and uploaded to uMH’s secure portal. As this was an optional step, many people chose to complete their analysis without genomic information. Therefore, APOE ε4 status is only known for a subset of the population presented here.\n\nDepressive symptoms were determined through patients’ self-reporting, the Short Form Geriatric Depression Scale (SF-GDS) at the physician’s office43, prior physician diagnosis as indicated by the patient’s medical history, or a prescription for antidepressant medication.\n\nDIDs were identified from Qato et al. Table 28. The DIDs listed here were coded into uMH’s bioinformatics platform so that each patients’ medication list from uMH’s database was checked against this published list by the platform’s algorithms.\n\nThe data was analyzed using statistical methods to examine the prevalence of DIDs and depression in an elderly population currently at risk of or diagnosed with AD. The frequency of DIDs, antidepressant medications, and depression was analyzed against gender, cognitive impairment levels, depression symptoms, and current medications. This information was then subsequently compared to APOE status to investigate if a possible relationship exists between the APOE ε4 allele and depression levels, with or without DIDs.\n\nAll statistical analyses were performed using SAS University Edition. Power calculations for independent, two-sided t-tests were completed to compare population means. Either pooled or Satterthwaite P values were used based on equality of variance testing. Chi-square tests were completed for categorical data. Means, standard deviations, and frequencies were established using SAS. Significance was determined using a 95% confidence interval.\n\nFollowing the guidelines put forth in the 21st Century Cures Act44, Clinical Decision Support Software (CDSS) is not regulated by the FDA, and uMH’s treatment protocol does not include any investigational drugs. Because of this, the platform was taken straight to market and did not need approval by an ethics committee or board.\n\nuMH’s algorithm platform automatically assigns every participant a random eight-character ID when they are first entered into the system to de-identify their information. All data was collected under their randomly assigned ID, and the investigators here only had access to this de-identified information.\n\nThe data presented here is an analysis of data already gathered from patients; no experiments were performed on humans. Each participant self-selected to participate under the guidance of their individual physician. No patients were recruited by uMETHOD Health. All patients gave consent upon initial data collection and were informed that their de-identified information would be used for future research purposes. For patients who presented with cognitive decline, their caregiver or power of attorney was also informed of all data collection and gave consent to use de-identified data for future research.\n\n\nResults\n\nUtilizing uMH’s data set, 292 individuals were analyzed for this paper (Table 1). A total of 189 people chose to provide genomic data. Of these 189, 111 have at least one copy of the APOE ε4 allele (Table 1). Those with the APOE ε4 allele are referred to as ε4 and those without are referred to as nε4. This population took an average of 11.51 (SD 8.86) medications per person, and 1.16 (SD 1.27) DIDs per person. The maximum number of DIDs seen for one person was five (Table 1).\n\nBMI, body mass index; DIDs, depression-inducing drugs.\n\n36.21% of the population had depressive symptoms (Table 1). Individuals with depression were significantly more likely to be taking at least one DID (71.15% vs 28.85%, P value 0.005) (Table 2 and Figure 1). Those with ε4 had a significantly higher rate of depression than nε4s (69.12% vs 30.88%, P value 0.03) (Table 2). Females were also more likely to be depressed than males (65.71% vs 34.29%, P value 0.005) (Figure 2). The same held true when APOE ε4 status was considered with gender (68.09% vs 31.91%, P value 0.002) (Table 2).\n\nDIDs, depression-inducing drugs.\n\nProportion of depressed patients taking a DID versus depressed patients not on DID. DID, depression-inducing drug,\n\n60.56% of the total population was taking at least one DID (Table 1). Females were more likely to be taking a greater number of concurrent DIDs than males (1.29 [SD 1.32] vs 0.99 [SD 1.19], P value 0.045) (Table 2 and Table 3 and Figure 2). Those taking a DID were more likely to be taking a higher number of antidepressants (0.58 [SD 0.88] vs 0.2 [SD 0.46], P value <0.0001) (Table 2 and Table 3).\n\nAnalysis of the percentage of each gender with depression, depression and APOE ε4, taking antidepressant medication, taking a depression-inducing drug (DID), possessing the APOE ε4 allele, and measurable cognitive impairment.\n\nDIDs, depression-inducing drugs.\n\n29.58% of the overall population was taking at least one antidepressant medication (Table 1). Those taking an antidepressant were more likely to be taking concurrent DIDs (77.38% vs 22.62%, P value 0.0002) (Table 2). Gender was also a significant factor for antidepressants. Females were more likely to be on at least one antidepressant (67.86% vs 32.14%, P value 0.005) (Figure 2), as well as taking a greater number of concurrent antidepressants (0.52 [SD 0.80] vs 0.32 [SD 0.71], P value 0.029) (Table 2 and Table 3).\n\nA higher, though not statistically significant, number of people taking antidepressants were more likely to have ε4 (70% vs 30%, P value 0.084) (Table 2).\n\n75.33% of this population had measurable cognitive impairment (Table 1). Those who were impaired took a higher number of concurrent antidepressant medications (0.46 [SD 0.83] vs 0.25 [SD 0.52], P value 0.036) (Table 2 and Table 3). There was also a higher percentage of cognitively impaired individuals taking antidepressants, but the number did not reach significance (80% vs 20%, P value 0.316). A higher percentage of people with cognitive impairment were also taking DIDs (57.49% vs 42.51%, P value 0.394), but again the number did not reach significance (Table 2).\n\n\nDiscussion\n\nDIDs are a substantial clinical, medical, and public health problem34 that has yet to be addressed in older populations. DID consideration is especially important in populations with an increased risk or a current diagnosis of AD. Prescriptions for elderly patients, especially those with a family history of AD or who currently have symptoms of cognitive decline, should be closely monitored and should avoid taking DIDs whenever possible.\n\nThe results of this work support that ε4 populations are at a greater risk of depression. Whether depression is a prodromal symptom or a risk factor for AD cannot be concluded from this data, but many other studies provide compelling evidence that depression may contribute to a person’s risk of developing AD. If depression is a precursor to AD, then DIDs may accelerate the clinical presentation of AD. It is worth further study to see if those who possess the ε4 allele should avoid drugs likely to cause depression symptoms, as they may be predisposed to develop depression. Further research into the possible mechanisms of DIDs is also warranted and could possibly help further elucidate the correlation between depression and the development of AD.\n\nThis work also showed that DIDs are abundant in elderly individuals’ medication regimes. Re-evaluation of medications that have been documented to cause depression could potentially lead to better cognitive function for many patients. This may be especially beneficial to the ε4 population. Further research may improve guidelines for prescribing DIDs to elderly patients, and additional specifications may be needed for those with the ε4 allele.\n\nFemales appear to be at an even greater risk for DIDs as this study found they may already be pre-disposed to depression, with the risk increasing with the ε4 gene, and are more likely to be taking DIDs and taking a higher number.\n\nFurther research is warranted to see if patients with different levels of cognitive impairment respond differently to DIDs, or if there are other contributors to developing depression as a side effect. Other factors such as age, body mass index, and comorbidities could also be explored to see if they impact patients’ risk of developing DID.\n\nWith the rising numbers of elderly in the US, along with concurrent use of multiple prescription drugs, DID burden on cognitive health continues to grow. 30% of older adults in the United States take five or more medications daily45, known as polypharmacy. Each additional drug increases a patient’s risk of experiencing adverse effects, including depression and cognitive impairment.\n\nCDSS provides a reliable and reproducible method to help with polypharmacy and DIDs. CDSS is software that is created to provide decision support for the diagnosis, treatment, prevention, cure, or mitigation of diseases or other conditions44. The goal of CDSS is to provide clinicians with actionable recommendations to improve clinical decision making46,47. One considerable advantage of CDSS is its inherent ability to process immense amounts of data quickly. This makes CDSS ideal for dealing with complicated medication regimes.\n\nMachine-learning algorithms, one element of CDSS, can be equipped to help physicians and patients better curate their medications to prevent adverse events, unwanted side effects, and even duplicated prescriptions. It’s estimated that 25% of people aged 65–69 take at least five prescriptions, and it is not uncommon for people to take 20 or more drugs48. One individual in this dataset was on a total of 67 medications. The considerable number of patients with polypharmacy and an average of 32 new drugs approved by the FDA each year make this an insurmountable task for a physician to take on by themselves49.\n\nCDSS can quickly review a patient’s medication list, no matter how large, matching it to current guidelines to provide physicians with reproducible guidance. This guidance could include recommendations for deprescribing medications that are therapeutically repetitive, medications that interact with other drugs, and medications that interact or have diminished therapeutic benefit due to an individual’s genetics. CDSS can also alert physicians to medications such as DIDs and anticholinergic drugs that have been shown to cause cognitive burden for elderly patients. CDSS also has the ability to suggest formulary alternatives to reduce the amount of DIDs in a medication list.\n\nCDSS has the potential to support pharmacogenomics. Pharmacogenomics is the study of identifying genomic variants that affect drug efficacy and pharmacokinetics (PK) or pharmacodynamics (PD)50. There are many examples of medications that are effective or ineffective for patients based on their genome, including antidepressants. As of 2015, 20 genetic variations have been found that can affect around 80 medications50. In fact, one gene can affect multiple medications. Many of uMH’s clients choose to get their genome sequenced to find out more about their genetic risk and protection against AD. This same data can also be used to help select the most beneficial medications for each person.\n\nuMH’s bioinformatics platform currently mines publicly available databases to check for drug-gene interactions (DGIs). The platform analyzes more than 2,000 single nucleotide polymorphism (SNPs) per individual, some of which pertain to DGIs, others are used to quantify the genetic risk of AD as well as other genetic diseases.\n\nDGI information is drawn from three sources of pharmacogenomic information: the Clinical Pharmacogenetics Implementation Consortium (CPIC) effort51, DrugBank52,53, and SNPedia’s SNP and genoset compilations54,55. DGIs are looked at in two categories: genetics that influence how enzymes metabolize each drug, and relationships between specific genes and drugs. Each medication a person is currently taking is compared against a table of genes that affect it, and these genes are, in turn, compared to those in the person’s genome.\n\nApproximately half of the people placed on an antidepressant medication do not show signs of improvement and over half will experience an adverse side effect. This is partly due to each person’s genetic makeup and its impact on the PK and PD of the medication56. Many pharmacogenomic studies have been conducted on antidepressant medications and found that their efficacy is highly correlated with SNP variations. Metabolizing enzymes such as P450 and the CYP2D6 polymorphism have both been found to have an effect on the drug plasma levels of antidepressant medications57.\n\nPrecision-medicine platforms, like uMH’s CDSS, offer the opportunity to improve the fitting of a drug to a patient with less trial and error, based on their genetics. This would allow for patients to be placed on the most effective medication as soon as possible, potentially speeding up recovery time from depressive symptoms.\n\nCDSS could also be made more effective if linked to systems pharmacology models. These models have been developed to help predict in vivo drug effects in biological networks58,59. This approach seeks to look at the whole, instead of the more traditional single transduction pathway of drug administration and response. Drugs often have off-target or secondary effects that can be hard to predict as multiple systems can be involved and must be accounted for. Patients’ responses to drugs are also inherently variable due to their unique genome, environment, disease state, and the high prevalence of polypharmacy32,58.\n\nA systems biology analysis could be useful in further defining the mechanisms behind DIDs. This information could then further inform future research into potentially lessening the depressive effects of these drugs, and also inform research into whether depression is a true risk factor for AD or a prodromal symptom.\n\nMany older adults who experience late-life depression never fully recover from their depression-induced cognitive impairment, even after successful treatment of the depression. All these observations magnify the urgent need to provide better vigilance when prescribing medications for the elderly.\n\nA more technology-friendly view is needed when it comes to managing complex medication regimes, especially when medications that can affect patients’ cognitive abilities are involved (as evidenced by the lack of publications on DIDs and the AD population).\n\nThe findings presented here support numerous previous publications indicating a relationship between depression and cognitive decline. While our observational data can only provide conjecture and find correlations, it is clearly worth closely monitoring DIDs in elderly populations. It is of even more importance when a patient may already be at increased risk of AD. There is a better path forward for patients with precision medicine and CDSS.\n\n\nData availability\n\nThe data that support the findings of this study are available from uMETHOD Health but restrictions apply to the availability of these data, which are proprietary company information, and so are not publicly available. Data are, however, available with permission of uMETHOD Health. Requests will be granted on a case-by-case basis to researchers affiliated with an accredited institution following the submission of an ethically approved and relevant research proposal. Readers and reviewers can request access to the dataset used in this manuscript by sending a request to info@umethod.com. The National Alzheimer’s Coordinating Center (NACC) database is a publicly available dataset that is representative of the dataset analyzed in this study and can be used to apply the methodology described in this manuscript.", "appendix": "Acknowledgments\n\nAuthors had full access to all of the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis.\n\nThanks to Dr. John Q Walker and Mark Zelek for their support.\n\nDr. John R. Cirrito confirms that the author has an appropriate level of expertise to conduct this research and confirms that the submission is of an acceptable scientific standard. Dr. John R. Cirrito declares they have no competing interests. Affiliation: Department of Neurology, Washington University, St. Louis, USA.\n\n\nReferences\n\nAlzheimer's Association: 2018 Alzheimer’s disease facts and figures. Alzheimers Dement. 2018; 14(3): 367–429. 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PubMed Abstract \| Publisher Full Text\n\nBroderick PA: Alprazolam, diazepam, yohimbine, clonidine: in vivo CA1 hippocampal norepinephrine and serotonin release profiles under chloral hydrate anesthesia. Prog Neuropsychopharmacol Biol Psychiatry. 1997; 21(7): 1117–1140. PubMed Abstract \| Publisher Full Text\n\nKreider ML, Tate CA, Cousins MM, et al.: Lasting effects of developmental dexamethasone treatment on neural cell number and size, synaptic activity, and cell signaling: critical periods of vulnerability, dose-effect relationships, regional targets, and sex selectivity. Neuropsychopharmacology. 2006; 31(1): 12–35. PubMed Abstract \| Publisher Full Text\n\nPumariega AJ, Nelson R, Rotenberg L: Varenicline-induced mixed mood and psychotic episode in a patient with a past history of depression. CNS Spectr. 2008; 13(6): 511–514. 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PubMed Abstract \| Publisher Full Text\n\nKawamoto K, Houlihan CA, Balas EA, et al.: Improving clinical practice using clinical decision support systems: a systematic review of trials to identify features critical to success. BMJ. 2005; 330(7494): 765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharlesworth CJ, Smit E, Lee DSH, et al.: Polypharmacy Among Adults Aged 65 Years and Older in the United States: 1988-2010. J Gerontol A Biol Sci Med Sci. 2015; 70(8): 989–995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEngagemen I: Advancing Health Through Innovation: 2017 New Drug Therapy Approvals Report (PDF - 2.8MB). 2017.\n\nRelling MV, Evans WE: Pharmacogenomics in the clinic. Nature. 2015; 526(7573): 343–350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClinical Pharmacogenetics Implementation Consortium: Prioritization. Clinical Pharmacogenetics Implementation Consortium. Reference Source\n\nAyvaz S, Horn J, Hassanzadeh O, et al.: Toward a complete dataset of drug-drug interaction information from publicly available sources. J Biomed Inform. 2015; 55: 206–217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrug Bank: Pharmaco-transcriptomics: Up/down regulation of genes due to the metabolism of pharmaceutical compounds. DrugBank. Reference Source\n\nLu M, Lewis CM, Traylor M: Pharmacogenetic testing through the direct-to-consumer genetic testing company 23andMe. BMC Med Genomics. 2017; 10(1): 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSNPedia: Genoset. SNPedia. 2013. Reference Source\n\nBousman CA, Forbes M, Jayaram M, et al.: Antidepressant prescribing in the precision medicine era: a prescriber's primer on pharmacogenetic tools. BMC Psychiatry. 2017; 17(1): 60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinder EB, Holsboer F: Pharmacogenomics and antidepressant drugs. Ann Med. 2006; 38(2): 82–94. PubMed Abstract \| Publisher Full Text\n\nDanhof M: Systems pharmacology - Towards the modeling of network interactions. Eur J Pharm Sci. 2016; 94: 4–14. PubMed Abstract \| Publisher Full Text\n\nvan Hasselt JGC, Iyengar R: Systems Pharmacology: Defining the Interactions of Drug Combinations. Annu Rev Pharmacol Toxicol. 2019; 59: 21–40. PubMed Abstract \| Publisher Full Text" }	[ { "id": "67265", "date": "20 Aug 2020", "name": "Carola G. Schipke", "expertise": [ "Reviewer Expertise biochemistry", "molecular neuroscience", "biomarkers", "Alzheimer´s disease", "diagnosis of Alzheimer´s disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript aims to analyse the frequency of use of depression-inducing drugs in Alzheimer´s disease (AD) and APOE ε4 carriers. The author collected datasets from a commercial platform (uMETHOD health) that advertises that it combines big data, medical research, and advanced algorithms to enable personalized care.\nThe manuscript picks up an important and highly relevant question but needs some major revisions since the way the data is presented, it does not allow for all the conclusions made.\nIn the introduction, the only reference given regarding Alzheimer´s disease in any aspect is a publication on facts by the Alzheimer´s Association, no original research work is cited. In the 2nd paragraph of the introduction, the author writes that 35.6 million people worldwide have AD. This is wrong, the paper that is cited says that \"..Currently, there are 35.6 million individuals with dementia worldwide..\". It is crucial to understand and to state that dementia is not synonymous with AD. Also, no citation is given for the number of AD patients in the US, the number stated in the manuscript is not in reference 2 (that is given for the complete sentence). The 1st sentence in the 3rd paragraph of the introduction that starts with the words \"Research now suggests\" gives a reference that is 20 years old. References need to be updated.\nIn the methods section (and table 1) it is not completely clear, why the overall number of 292 patients is given, but only for 284 information on medication is available. Apparently, percentages in table 1 refer to 284, which is logical. Also, the author states that patients were evaluated to determine if the had AD, but no numbers or results are given, not even the methods used are stated. At least the type of examinations that were used to exclude other forms of dementia should be listed. A major drawback is the lack of information on the severity of cognitive impairment. This can largely influence depressive symptoms - and measures for depressive symptoms, the author needs to state the values (total and z-scores) for the mentioned cognitive test for the cohort. It should be discussed if patients presenting with moderate dementia or worse should be included at all into this study.\nResults: Table 1 states that 84 patients or 29.58% of the population take antidepressants. Figure 2 states that 67.86% of the females and 32.14% of the males take antidepressant. How come the overall percentage is lower than the percentages in males and females? Please clarify. For Figure 2: How many patients do take DIDs because of a diagnosed depression? This number should be looked at and should be taken into consideration for all analyses, since depressive symptoms only improve slowly, and since antidepressant medication is counted as a DID, it is somehow clear - and misleading with regard to the main point of the manuscript - that people diagnosed with depression take antidepressant drugs.\nDiscussion: The discussion picks up important points regarding the interrelations of AD and depression. What is puzzling me is the extreme focus on the software and the platform, paragraphs 7-12 discusses the software/platform. While topics discussed in these paragraphs are generally important, this is like a commercial for me and in my opinion, large parts have nothing to do with the scientific facts reported in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "88465", "date": "26 Jul 2021", "name": "Marina Sagud", "expertise": [ "Reviewer Expertise Psychopharmacology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study has used data from the uMH’s data set, in order to establish the association between the presence of at least one APOE ε4 allele and depression-inducing drugs, and symptoms of depression (widely defined), in individuals aged 65 and older. The results highlight the connection of both APOE ε4 alleles and the intake of depression-inducing drugs, with symptoms of depression in elderly population. The study needs revision, as provided below.\nIntroduction\nInstead of, \"the APOE ε4 gene\", which is mentioned several times in the text, I think it is more accurate to say: \"the APOE ε gene\".\n\nPlease, provide a short description about APOE ε gene, and its three major alleles (epsilon 2, epsilon 3, and epsilon 4), prior to APOE ε gene subsection.\n\nThe notion that antidepressants are among the depression-inducing-drugs appears counter-intuitive. Please, explain further.\nMethods\nDataset-Depressive symptoms were determined through patients’ self-reporting, the Short Form Geriatric Depression Scale (SF-GDS), prior physician diagnosis as indicated by the patient’s medical history, or a prescription for antidepressant medication - antidepressants are also prescribed for other disorders, such as PTSD, anxiety and chronic pain disorders. How can you differentiate those patients?\n\nPlease, provide the short description of the SF-GDS, the Self-Administered Gerocognitive Exam (SAGE) and the Montreal Cognitive Assessment scale. What were the cut-off values for the depression from the SF-GDS, and for the cognitive impairment from the cognitive scales?\nResults\nWas the power analysis conducted to calculate the minimal sample size?\n\nParticipants with at least APOE ε4 allele were referred to as ε4, but the number of APOE ε4 alleles was not analyzed. Because there is difference in the AD risk between the carriers if one or two APOE ε4 alleles, this should also be mentioned in the Limitations.\n\n\"Those with ε4\", or \"ε4 populations\" should be replaced with \"ε4 allele carriers\".\nDiscussion\nI propose to organize the discussion in several subsections.\n\nThe term \"depression\" measured in this study refers to both current (as measured by the SF-GDS scale) or past (from the patient’s medical history). Please, emphasize in the text that this study addressed \"life-time\", rather than current depression.\n\n29.58% of the overall population was taking at least one antidepressant medication-please, compare this data from other authors in the similar samples.\n\nThe findings presented here support numerous previous publications indicating a relationship between depression and cognitive decline-please, provide references.\n\nMany older adults who experience late-life depression never fully recover from their depression-induced cognitive impairment, even after successful treatment of the depression -please, provide references.\n\nPlease, mention study limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1782
https://f1000research.com/articles/8-1563/v1	02 Sep 19	{ "type": "Opinion Article", "title": "NGOs’ experiences of navigating the open access landscape", "authors": [ "Nilam McGrath" ], "abstract": "Grant-led consortia working in the global development sector rely on the input of local and national non-government organisations in low- and middle-income countries. However, the open access mandates and mechanisms embedded within grants and promoted by funders and publishers are designed almost exclusively with large universities and research institutions in mind. Experiences from the consortium of health research non-government organisations comprising the Communicable Diseases Health Service Delivery research programme show that implementing open access mandates is not as simple or frictionless as it initially appears.", "keywords": [ "Open Access", "NGOs", "Global South", "global development", "funders", "publishers", "low middle income country", "Nepal", "Bangladesh", "China", "Pakistan", "Swaziland", "Ghana" ], "content": "Introduction\n\nGrants aimed at tackling issues prevalent within low- and middle-income countries (LMICs) often encourage collaboration or consortium working between Northern universities and organisations in the Global South, thereby bringing together research institutions, non-government organisations (NGOs), the private sector and government agencies.\n\nNational NGOs in the Global South are particularly important to the research process as their responsiveness to national agendas makes them ideal global development partners, making them crucial drivers of research design and implementation. In many research programmes, it is their labour, their insight, their networks and their relationships that influence how an intervention will be received and taken up in their country context. This makes NGOs extremely powerful actors in the research evidence production cycle.\n\nLarge, collaborative grants now come with open access (OA) mandates to aid research uptake. Many funders from the Global North (for example Wellcome Trust, UK Research and Innovation, and the Department for International Development) are clear about the mechanisms by which Northern university-led research programmes can make their work available via open access mechanisms. Similarly, scholarly publishers have mechanisms in place to grant waivers for article processing charges (APCs) to aid funders’ OA mandates.\n\nHowever, existing OA mechanisms are overwhelmingly geared towards supporting universities and large research institutions in the Global North to share research evidence, with the assumption that all collaborating partners, including NGO partners based in LMICs, are able to adequately adapt their working practices to meet open access requirements. In an effort to meet OA requirements, this results in NGO partners funnelling their efforts through Northern universities, thereby creating an over-reliance on their Northern partners to, firstly, access evidence to inform and shape their country-specific research interventions, and secondly, to publish new research they have generated within their country setting. This over-reliance inevitably results in stalled or slower research design processes, information bottlenecks, increased bureaucracy when attempting to publish papers and unanticipated costs.\n\n\nAn over-reliance on Northern universities to access research evidence\n\nWorking at a Northern university undoubtedly has its research access privileges; it gives employees (academic researchers, undergraduates, postgraduates, operational and administration staff) unparalleled access to research evidence through its library. NGO partners and their researchers based in LMICs know this, and many rely on their university counterparts to help them access relevant literature and research findings so that they can then design their country-specific research interventions. This creates a dependency on Northern universities by Southern NGO partners to access information. Furthermore, it generates frequent and one could argue, unnecessary requests for information to Northern universities, creating information bottlenecks and stalling the research process at stages where evidence is needed.\n\nIt also results in Northern universities and their researchers inadvertently becoming gatekeepers of information. Take an example from my own work which involved writing up the results of a media monitoring exercise in Nepal. My colleague in Nepal was relying on my privileged access at a UK university to search for similar and contrasting studies, which I would then send to him in Nepal to review and synthesise. Aside from being time-consuming, this raises questions about my suitability and ability as a non-Nepali, Northern-based researcher to accurately spot and explore the nuances and contextual issues of a particular topic that only my colleague would recognise when searching academic and grey literature.\n\nThis type of back-and-forth between Southern and Northern researchers occurs more than we realise in operational research and in consortia that rely on NGO partnerships. The information dependency that it promotes decreases the exposure that NGOs in LMICs have to larger bodies of research and it has huge implications for building the capacity of individuals and organisations working in LMICs to independently access, interpret, synthesise, generate and share their research evidence. The nature of this dependency is at the core of debates surrounding decolonising development and structural inequalities in knowledge production and sharing (Chan et al., 2011) (see the work of the Open and Collaborative Science in Development Network and The Knowledge Gap). Failure to address access privileges and their associated power further embeds structural inequality within South-North partnerships.\n\n\nBut they’ve got Research4Life, right?\n\nLack of access can be countered somewhat with initiatives such as Research4Life, but this is not the panacea that many claim (Chan et al., 2011; Zachariah et al., 2014). There is a 2-tier access system: free access to papers via Research4Life is restricted to NGOs in Group A countries only, with NGOs based in Group B countries charged $1500 annually to access the same content. However, any blanket exclusion of NGOs from countries deemed to be based in more affluent countries, for example in China, assumes that NGOs are in the same stable economic position as their established, affluent national counterparts. For ‘transitional’ countries, for example, the Philippines or Indonesia, the same assumption applies: NGOs in these countries are financially sustainable and therefore able to pay US$1500 annually for accessing research.\n\nThis assumption does not acknowledge that the non-profit sector works to tight programme budgets and restricted funding. Indeed, restricted or conditional funding is the norm for the global development sector within which NGOs operate. Access to new and existing research via paid subscriptions or annual fees, therefore, remains low on the list of priorities for NGOs, who instead are committed to using grant funding to pay solely for field research, staff time and other implementation costs. So, with limited budgets, and with Research4Life out of reach, where do NGOs go when they need to access research legally? That’s right – their colleagues at Northern universities. And so, the dependency cycle repeats itself, with this South to North pipeline of access becoming the default option for many grant collaborations and consortia. The illegal route involves accessing sites such as Sci-Hub, and judging by the download statistics, it is a popular alternative (Bohannon, 2016).\n\n\nNorthern universities’ role in publishing papers\n\nIn consortia working, the process of publishing papers is also often funnelled through a Northern university partner. On paper, this would appear to be a logical and straight forward process, but experiences from the Communicable Diseases Health Service Delivery (COMDIS-HSD) research programme show that this is not the frictionless process that publishers like to promote. There is almost always an issue with securing waivers, with NGOs often resorting to time-consuming and bureaucratic workarounds to secure waivers for APCs.\n\nWhen publishing research, APCs are the most obvious cost for all grant recipients. Waivers are designed to counter these costs, but the COMDIS-HSD experience was that the process for securing an APC waiver was inconsistent both in terms of how to ask for waivers and also in how ‘corresponding authors’ were unfairly judged on their ability to pay depending on their geographical location. For example, COMDIS-HSD routinely used UK-based corresponding authors to expedite APCs using a university corporate credit card, thereby alleviating the financial burden of high APC payments for their NGO partners. However, publishers incorrectly viewed the UK base of a corresponding author as an indication of the ability to pay the full open access APC, adding a value added tax of 20% to the original cost. The consortium then had to enter into lengthy correspondences with several journals to, firstly, prove that the majority of their authors were from LMICs (and therefore automatically entitled to a waiver), and secondly, to secure the waiver in writing. In short, NGOs continually have to prove how ‘poor’ they are.\n\n\nUnanticipated costs of sharing research\n\nAdditionally, the consortium encountered unanticipated costs and hidden charges whilst navigating the open access process, for example, $50/£50, for using invoicing rather than direct online payment systems. The issue here is not the cost of APCs or invoicing per se, but the lack of transparency on publishers’ websites that these costs exist. Authors often have to navigate away from the journals’ homepage to find APCs, waiver processing information and other caveats, once again adding unnecessary time and bureaucracy to what should be relatively swift submissions, waiver applications, and payment processes.\n\nThere is a fine balance between ensuring research evidence is made available quickly and giving people adequate time to pay APCs. The nature of NGO work means that authors are often conducting field research, meaning that email access is limited and that demands for APC payments can go unnoticed for days or weeks, missing the 7 or 14-day payment deadline of some publishers. Once received, the payment request may be forwarded to a paying institution (the University of Leeds in the case of COMDIS-HSD), adding more time to the payment process. In such cases where short payment deadlines are missed, publishers automatically add additional charges of £50/$75 for not being able to pay APCs within 7–14 days; an unreasonably short time, given that most companies allow 30 days for payment.\n\nIn one instance in July 2018, Springer Nature instructed COMDIS-HSD to pay an APC online within 7 days; after 7 days an invoice would be issued and an administration charge of £50/EUR55/USD$75 added to the APC, with no option to revert to online payment thereafter. This was in spite of a 30-day payment deadline being advertised on their website. My requests for extending the deadline while we waited for the relevant conference discount code (from administrators at BMC Health Services Research, owned by Springer Nature), and so that the university’s finance department could authorise the payment (which can take 1–3 working days) were met with further reminders to pay within 7 days. Pleading phone calls and emails to finance staff and conference organisers meant that we were able to pay the invoice on Day 7, but why would any publisher subject any author to this in the first place?\n\nTo avoid additional ‘late payment’ fees and to meet short payment deadlines such as this, authors will often attempt to pay on personal credit cards; an unreasonable situation for any individual to be placed in. What this ultimately amounts to is NGOs in LMICs being forced to adhere to payment schedules and processes that make them more financially vulnerable than their larger, established, university partners.\n\nThis is just one example of many. Experiences gained over six years with COMDIS-HSD show that the path to publication for NGOs can be a murky and complex process riddled with maddening bureaucracy on one end of the spectrum, to a slightly more transparent and slightly swifter process on the other.\n\n\nSummary\n\nIn consortia that rely on Northern universities partnering with Southern NGOs, NGO efforts to access and publish papers are being funnelled through their Northern counterparts. This creates an unnecessary dependency on the privileges afforded to Northern universities to access relevant research literature, and an over-reliance on Northern university partners to help negotiate and secure APC waivers and pay APCs.\n\nUltimately, NGOs operating in LMICs experience more friction than their Northern university partners in accessing research and in publishing their research findings. What this shows is that NGO partners in LMICs are less supported by publishers and funders to access research and to make their work accessible. This points to a fundamental lack of understanding about NGO organisational structures and their operational practices and protocols, as well as a lack of understanding of their value in the research evidence production cycle.\n\nFurthermore, NGOs in the Global South remain excluded from open access debates and unrepresented in planning and advisory committees, particularly those convened by funders and Northern universities and research institutes, for example, Coalition S Ambassadors, Research4Life partners, and FORCE11 advisors. Their exclusion is an example of how knowledge access inequalities are embedded in the research production cycle. To redress this, a step forward would be to actively include these important research organisations from the Global South in OA discussions and implementation plans.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nBohannon J: Who’s downloading pirated papers? Everyone. Science \| AAAS. Apr. 28 2016, 2:00 PM. (Accessed: 11 August 2019). Publisher Full Text\n\nChan L, Kirsop B, Arunachalam S: Towards Open and Equitable Access to Research and Knowledge for Development. PLoS Med. 2011; 8(3): e1001016. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZachariah R, Kumar AMV, Reid AJ, et al.: Open access for operational research publications from low- and middle-income countries: who pays? Public Health Action. 2014; 4(3): 142–4. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "53316", "date": "16 Sep 2019", "name": "Ross Mounce", "expertise": [ "Reviewer Expertise Open Access" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI thank the author for this insightful opinion article documenting her experience of navigating the open access publishing landscape with NGOs. There are some really novel insights in here which to my knowledge have not yet been communicated in a peer-reviewed forum, so thanks to the author for taking the time to do so. The anecdotes contained within this article are extremely important to be conveyed and to be more widely known amongst the Global North research, publishing and policy communities who may appear otherwise sometimes oblivious to many of the problems discussed here.\nHere are some small constructive comments:\n\"However, publishers incorrectly viewed the UK base of a corresponding author as an indication of the ability to pay the full open access APC, adding a value added tax of 20% to the original cost.\"\nWith respect, I think this conflates two slightly different issues. If an APC is being paid from the UK, then legally-speaking Value Added Tax (which is currently set at 20%) has to be paid, and it not the publisher that is mandating this, but instead the UK government. If the APC could be paid by another author not based in the UK, not from a UK bank account, then that author/institution/NGO might not need to pay UK VAT. The second problem intermingled is the publisher assuming that because a corresponding author affiliation appears to be from the UK, that they somehow automatically have the money to pay a full APC.\nBoth are legitimate problems.\nBut I just think they need to be teased apart slightly to make it clear that there are two problems there not just one. In defence of the publishers, sometimes APCs are known to be invoiced on the basis of who is indicated as the corresponding author of the paper. For some publishers it doesn't matter if the majority of the authors are from LMICs, all that matters is who is the corresponding author. Nilam is right to highlight this as a seemingly unjust problem. To be clear I am not defending publisher policies here (far from it) but if the policy is transparent as least NGOs and authors might know about it before submitting.\nFor example BMC (owned by Springer Nature) write: \"BMC offers APC waivers to papers whose corresponding authors are based in countries classified by the World Bank as low-income economies as of July 2019.\" https://www.biomedcentral.com/getpublished/article-processing-charges/open-access-waiver-fund\n\nPerhaps the author might consider referring to 'research outputs' rather 'research evidence'? From my point of view in the funder landscape I feel that outputs is the more common term but perhaps there is a specific reason for using 'evidence' rather than outputs? Something like the production of scientific software might not be considered 'research evidence' but would clearly be a 'research output'.\n\nIn the discussion of those in the Global South needing to use Sci-Hub, I would perhaps cite voices from the Global South to support this point such as Bendezú-Quispe et al. 2016 \"Sci-Hub and medical practice: an ethical dilemma in Peru\"1.\nThe well-documented above average intensity of usage of Sci-Hub in Iran might also be worth citing on this point with respect to the Global South (Greshake, 2016 \"Correlating the Sci-Hub data with World Bank Indicators and Identifying Academic Use\"2).\n\nIn general, whilst it is important that Nilam's personal experience makes up the majority of this opinion article, it would be good to include many further references on what others have said about similar problems with the open access landscape such as:\nPeterson, A.; Emmett, A.; Greenberg, M.L. Open access and the author-pays problem: Assuring access for readers and authors in a global community of scholars. J. Librariansh. Sch. Commun. 2013 3\nLawson (2015) \"Fee Waivers for Open Access Journals\" https://doi.org/10.3390/publications3030155 4\nBonaccorso, E., Bozhankova, R., Cadena, C., Čapská, V., Czerniewicz, L., Emmett, A., Oludayo, F., Glukhova, N., et al. 2014. Bottlenecks in the Open-Access System: Voices from Around the Globe. 5\nJørgen Burchardt (2014) Researchers Outside APC-Financed Open Access: Implications for Scholars Without a Paying Institution https://doi.org/10.1177/2158244014551714 6\n\nFinally, as Nilam has been working with Health NGOs, I do think a citation to statements in this paper is warranted...\n\"In fact, hybrid OA journals, which have the most expensive APCs, were the most underrepresented in LMICs. It remains unclear why APCs for hybrid journals remain higher than gold APCs given the fact that these journals also ask for subscription fees.\nSince the APCs are mainly paid to the ten same publishers creating an oligopoly, there is little incentive to keep APCs low. This oligopoly may also run much deeper than costs; it creates an important inequity in publication. Although publishers may wish to include researchers from LMICs through waivers, they have not really included LMICs in the publication industry itself. \"\n-- Smith et al (2017) \"Knowledge sharing in global health research – the impact, uptake and cost of open access to scholarly literature\" Health Research Policy and Systems 7\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "4970", "date": "18 Oct 2019", "name": "Nilam McGrath", "role": "Author Response", "response": "Dear Ross,Many thanks for taking the time to provide an extended review - I appreciate it. Below is my response to the points you raise: I agree that I have conflated two ideas and I have now rewritten the section so that the two separate points are clearer. The rewrite has allowed me to elaborate on the waiver inconsistencies as experienced by COMDIS-HSD and to support the experiences by citing recent studies (thank you for signposting to these papers). The distinction between ‘research evidence’ and ‘research outputs’ is a useful one. There are clearly instances in this article where changing the text to ‘research outputs’ or ‘journal articles’ lends clarity to the argument and I have changed the text where relevant. In a small number of instances, I have retained the use of ‘research evidence’ where this refers to the research production cycle. I have expanded the section on Sci-Hub, citing references that highlight its popularity in China, India and Iran, and the ethical dilemma that researchers for researchers. I have cited recent studies that support the COMDIS-HSD experience, specifically: the inequality of knowledge production and sharing; a more detailed explanation of Research4Life’s offer and how this relates to the nature of competing funding priorities for health NGOs (including a pertinent quote from Smith et al 2017); further references to the popularity of Sci-Hub; and differing waiver and payment processes across publishers. Again, thank you for signposting to these studies. I have also made minor edits to the text, e.g consistent use of ‘OA’ throughout. Thanks again for your thoughtful, detailed and constructive review. I hope that the changes I have made fully address your reviewer comments." } ] } ]	1	https://f1000research.com/articles/8-1563
https://f1000research.com/articles/8-464/v1	14 Apr 19	{ "type": "Research Article", "title": "Preliminary assessment of adaptive evolution of mitochondrial protein coding genes in darters (Percidae: Etheostomatinae)", "authors": [ "Leos G. Kral", "Sara Watson", "Sara Watson" ], "abstract": "Background: Mitochondrial DNA of vertebrates contains genes for 13 proteins involved in oxidative phosphorylation. Some of these genes have been shown to undergo adaptive evolution in a variety of species. This study examines all mitochondrial protein coding genes in 11 darter species to determine if any of these genes show evidence of positive selection. Methods: The mitogenome from four darter was sequenced and annotated. Mitogenome sequences for another seven species were obtained from GenBank. Alignments of each of the protein coding genes were subject to codon-based identification of positive selection by Selecton, MEME and FEL. Results: Evidence of positive selection was obtained for six of the genes by at least one of the methods. CYTB was identified as having evolved under positive selection by all three methods at the same codon location. Conclusions: Given the evidence for positive selection of mitochondrial protein coding genes in darters, a more extensive analysis of mitochondrial gene evolution in all the extant darter species is warranted.", "keywords": [ "Mitogenome", "mtDNA", "adaptive evolution", "positive selection", "Etheostoma", "Percina" ], "content": "Introduction\n\nMitochondria provide cells with nearly all energy as a result of oxidative phosphorylation (OXPHOS). Vertebrate mitochondria are primarily maternally inherited and contain mitochondrial DNA (mtDNA), which contains 13 protein coding genes, the products of which contribute, along with nuclear-encoded proteins (Sunnucks et al., 2017), to the formation of the mitochondrial OXPHOS machinery (Ingman & Gyllensten, 2006; Ladoukakis & Zouros, 2017). While purifying selection acts on deleterious mtDNA mutations (Burr et al., 2018), James et al. (2016) utilized a variety of McDonald-Kreitman type analyses of a large number of animal species to show that mtDNA evolution is dominated by slightly deleterious mutations but also undergoes a significant amount of adaptive evolution. Several studies of the evolution of mtDNA-encoded mitochondrial protein coding genes both within fish species (Consuegra et al., 2015; Teacher et al., 2012) and between fish species (D’Anatro et al., 2017; Garvin et al., 2011; Zhang & Broughton, 2015) identified positive selection acting on some of these genes. This study utilized all currently available mitochondrial genome sequences of darter species to determine if positive selection can be detected in any of the mitochondrial protein coding genes. Such positive selection would imply that adaptation of the OXPHOS machinery to differing environmental niches had occurred in at least some lineages during darter speciation.\n\n\nMethods\n\nAll mitochondrial genome sequences utilized in this study were obtained from GenBank except those from Etheostoma chuckwachatte, Etheostoma jessiae, Etheostoma spectabile, Etheostoma tallapoosae and Percina crypta. The E. spectabile sequence was obtained from Rachel Moran, University of Illinois. Whole genome sequencing was performed on E. chuckwachatte, E. jessiae, E. tallapoosae and P. crypta genomic DNA (purified from fin/muscle tissue with the Qiagen DNeasy Blood and Tissue Kit) by the Georgia Genomic Facility at the University of Georgia utilizing the Illumina NextSeq (PE 150) or MiSeq (PE 250) sequencer. A subset of sequence reads from each species, sufficient for at least 30-fold coverage, was aligned to one of the GenBank obtained darter mitochondrial genomes utilizing the Map to Reference function in Geneious 9.1.8 software (Biomatters Ltd., Auckland, New Zealand). Annotated darter mitochondrial genome sequences from GenBank were aligned with the newly assembled darter mitochondrial genome sequences and annotations were transferred and manually adjusted where necessary with Geneious software.\n\nPhylogenetic analysis was carried out on all 13 concatenated and aligned mitochondrial protein coding sequences of the 11 darter species as well as of Perca flavescens, Sander vitreus and Anarhichas minor (outgroup). Bayesian inference (BI) analysis was performed with MrBayes (Ronquist & Huelsenbeck, 2003), as implemented in Geneious software but utilizing optimal partitioning and optimal substitution models determined by PartitionFinder v2.1.1 (AICc, greedy algorithm, MrBayes specific models) (Guindon et al., 2010; Lanfear et al., 2012; Lanfear et al., 2017). The rooted darter sub-tree (Figure 1) was extracted with MEGA 7.0.26 software (Kumar et al., 2016). Aligned darter mitochondrial protein coding genes were subject to site-specific tests for positive selection utilizing the Selecton implementation of an empirical Bayes approach (Doron-Faigenboim et al., 2005; Stern et al., 2007) and also the MEME and FEL methods implemented on the Datamonkey webserver (Delport et al., 2010). The darter tree was used in all three of these analyses.\n\nBranch lengths measured in number of substitutions per site. Posterior probabilities of all branches are greater than 0.91. Pm, Percina macrolepida; Pc, Percina crypta; Ecw, Etheostoma chuckwachatte; Ec, Etheostoma caeruleum; Er, Etheostoma radiosum; Eo, Etheostoma okaloosae; Es, Etheostoma spectabile; Ejs, Etheostoma jessiae; En, Etheostoma nigrum; Ez, Etheostoma zonale; Et, Etheostoma tallapoosae.\n\n\nResults and discussion\n\nCodons under positive selection were identified in seven of the thirteen mitochondrial protein coding genes by at least one of the methods utilized. Specifically, as shown in Table 1, the MEME method identified two codons in COX1, two codons in CYTB, three codons in ND2, and one codon each in ND3 and in ND5. The FEL method identified one codon in COX1, CYTB and ND3. The codons identified as being under positive selection by the FEL method were also identified as being under positive selection by MEME method. Specifically, codon 489 in COX1, codon 96 in CYTB and codon 9 in ND3. As shown in Table 2, the Selecton method identified one codon in ATP6, one codon in COX3, one codon in CYTB and two codons in ND5. While codons under positive selection were identified in ND5 by MEME and Selecton, these methods did not identify the same codons. Specifically, codon 32 was identified in ND5 by MEME and codons 479 and 573 were identified by Selecton. Only codon 96 in CYTB was identified as being under positive selection by all three methods.\n\nDefault significance cutoff value of p<0.1 was used.\n\nKa/Ks ratio indicates the ratio of nonsynonymous/synonymous mutation rates, CI is the confidence interval of the Ka/Ks ratio defined by the 5th and 95th percentiles of the posterior distribution inferred for the position. The p value is the significance of the likelihood ratio test between the M8 model (selection allowed) vs. M8a model (selection not allowed) of the gene.\n\nWhile the Bayesian estimates of the Ka/Ks ratio of the sites identified by Selecton are greater than 1, the lower bounds of the confidence intervals defined by the 5th and 95th percentiles of the posterior distribution inferred for these sites are less than 1. Therefore, the reliability of positive selection of these sites identified by Selecton is not very strong. However, positive selection of the four proteins identified by Selecton has been found to be significant (p = 0.001) by the likelihood ratio test of log-likelihood for model M8 (allowing positive selection) versus model M8a (not allowing positive selection).\n\nThese results indicate that it is likely that the evolution of various darter lineages included positive selection of at least some of the mitochondrially encoded OXPHOS machinery variants. Presumably, this positive selection would have been driven by adaptation to specific environmental factors. As an example of mitochondrial adaptation to environmental factors, Ma et al. (2015) found that adaptation of Chinese glyptosternoid fishes to high-elevation of the Tibetan Plateau is correlated to signals of positive selection of the Cox1 gene. That adaptation to hypoxia is correlated to physiological adaptation of the OXPHOS machinery has been demonstrated by O2 binding kinetics of cytochrome c oxidase (COX) in hypoxia tolerant vs. intolerant sculpin species (Lau et al., 2017). In this study, several nonsynonymous substitutions in COX3 were identified by in silico analysis as candidates for explaining the adaptive variation of COX O2 binding. That mitochondrial haplotype variants are subject selection by environmental factors has been demonstrated experimentally in Drosophila. When a mixed population of “cold adapted” haplogroups and “warm adapted” haplogroups were maintained for a number of generations at differing temperatures, the frequency of the “cold adapted” haplogroup increased at the cooler temperature and decreased at the warmer temperature (Lajbner et al., 2018).\n\nGiven the preliminary results indicating positive mitochondrial gene selection in darters, it would seem that a more expansive analysis of the evolution of mitochondrial protein coding genes in the approximately 200 extant darter species (Near et al., 2011) may be warranted to identify lineage specific adaptations and, potentially, their correlations to relevant life history traits.\n\n\nData availability\n\nAll mitogenome sequences are available in GenBank.\n\nPercina macrolepida: accession number DQ536430, https://identifiers.org/ncbigi/gi:105873653.\n\nPercina crypta: accession number KY965073, https://identifiers.org/ncbigi/gi:1238789905.\n\nEtheostoma chuckwachatte: accession number KY965071, https://identifiers.org/ncbigi/gi:1238789877.\n\nEtheostoma caeruleum: accession number KY660678, https://identifiers.org/ncbigi/gi:1229242602.\n\nEtheostoma radiosum: accession number AY341348, https://identifiers.org/ncbigi/gi:48762649.\n\nEtheostoma okaloosae: accession number KY747492, https://identifiers.org/ncbigi/gi:1215348329.\n\nEtheostoma spectabile: accession number MK243404, https://identifiers.org/ncbigi/gi:1605131112.\n\nEtheostoma jessiae: accession number KY965072, https://identifiers.org/ncbigi/gi:1605131112.\n\nEtheostoma nigrum: accession number KT289926, https://identifiers.org/ncbigi/gi:1035438550.\n\nEtheostoma zonale: accession number AP005994, https://identifiers.org/ncbigi/gi:1173213864.\n\nEtheostoma tallapoosae: accession number KY952221, https://identifiers.org/ncbigi/gi:1236181000.", "appendix": "Grant information\n\nThis study was supported by a Faculty Research Grant funded by the University of West Georgia.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nSpecial thanks to Rachel Moran for access to the E. spectabile mitogenome sequence, to Frank Fontanella for guidance in the use of Mr. Bayes software, and the Warm Springs Fish Technology Center for darter specimens.\n\n\nReferences\n\nBurr SP, Pezet M, Chinnery PF: Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line. Dev Growth Differ. 2018; 60(1): 21–32. PubMed Abstract \| Publisher Full Text\n\nConsuegra S, John E, Verspoor E, et al.: Patterns of natural selection acting on the mitochondrial genome of a locally adapted fish species. Genet Sel Evol. 2015; 47(1): 58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nD’Anatro A, Giorello F, Feijoo M, et al.: Testing for the Occurrence of Selective Episodes During the Divergence of Otophysan Fishes: Insights from Mitogenomics. J Mol Evol. 2017; 84(4): 162–173. PubMed Abstract \| Publisher Full Text\n\nDelport W, Poon AF, Frost SD, et al.: Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics. 2010; 26(19): 2455–2457. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoron-Faigenboim A, Stern A, Mayrose I, et al.: Selecton: a server for detecting evolutionary forces at a single amino-acid site. Bioinformatics. 2005; 21(9): 2101–2103. PubMed Abstract \| Publisher Full Text\n\nGarvin MR, Bielawski JP, Gharrett AJ: Positive Darwinian selection in the piston that powers proton pumps in complex I of the mitochondria of Pacific salmon. PLoS One. 2011; 6(9): e24127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuindon S, Dufayard JF, Lefort V, et al.: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010; 59(3): 307–321. PubMed Abstract \| Publisher Full Text\n\nIngman M, Gyllensten U: Vertebrate Mitochondrial DNA. In: Meyers RA. ed. Encyclopedia of molecular cell biology and molecular medicine. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA. 2006. Publisher Full Text\n\nJames JE, Piganeau G, Eyre-Walker A: The rate of adaptive evolution in animal mitochondria. Mol Ecol. 2016; 25(1): 67–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–1874. PubMed Abstract \| Publisher Full Text\n\nLadoukakis ED, Zouros E: Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. J Biol Res (Thessalon). 2017; 24: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLajbner Z, Pnini R, Camus MF, et al.: Experimental evidence that thermal selection shapes mitochondrial genome evolution. Sci Rep. 2018; 8(1): 9500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanfear R, Calcott B, Ho SYW, et al.: Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol Biol Evol. 2012; 29(6): 1695–1701. PubMed Abstract \| Publisher Full Text\n\nLanfear R, Frandsen PB, Wright AM, et al.: PartitionFinder 2: New Methods for Selecting Partitioned Models of Evolution for Molecular and Morphological Phylogenetic Analyses. Mol Biol Evol. 2017; 34(3): 772–773. PubMed Abstract \| Publisher Full Text\n\nLau GY, Mandic M, Richards JG: Evolution of Cytochrome c Oxidase in Hypoxia Tolerant Sculpins (Cottidae, Actinopterygii). Mol Biol Evol. 2017; 34(9): 2153–2162. PubMed Abstract \| Publisher Full Text\n\nMa X, Kang J, Chen W, et al.: Biogeographic history and high-elevation adaptations inferred from the mitochondrial genome of Glyptosternoid fishes (Sisoridae, Siluriformes) from the southeastern Tibetan Plateau. BMC Evol Biol. 2015; 15: 233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNear TJ, Bossu CM, Bradburd GS, et al.: Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae). Syst Biol. 2011; 60(5): 565–595. PubMed Abstract \| Publisher Full Text\n\nRonquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003; 19(12): 1572–1574. PubMed Abstract \| Publisher Full Text\n\nStern A, Doron-Faigenboim A, Erez E, et al.: Selecton 2007: advanced models for detecting positive and purifying selection using a Bayesian inference approach. Nucleic Acids Res. 2007; 35(Web Server issue): W506–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSunnucks P, Morales HE, Lamb AM, et al.: Integrative Approaches for Studying Mitochondrial and Nuclear Genome Co-evolution in Oxidative Phosphorylation. Front Genet. 2017; 8: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeacher AG, André C, Merilä J, et al.: Whole mitochondrial genome scan for population structure and selection in the Atlantic herring. BMC Evol Biol. 2012; 12: 248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang F, Broughton RE: Heterogeneous natural selection on oxidative phosphorylation genes among fishes with extreme high and low aerobic performance. BMC Evol Biol. 2015; 15: 173. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "47213", "date": "09 May 2019", "name": "Dusan Kordis", "expertise": [ "Reviewer Expertise evolutionary genomics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors tried to find the evidence of positive selection in 13 mitochondrial proteins, involved in oxidative phosphorylation, in 11 species of darters. Darters are the second largest family of North American fishes and only the minnows (Cyprinidae) have more species. Kral and Watson have found some evidence for positive selection acting on 6 mitochondrial genes, by at least one method used (M8 in Selecton, FEL and MEME in Datamonkey). However, they found that only CYTB gene has evolved under positive selection by all three methods used. The authors expect that a more extensive analysis of mitochondrial gene evolution in all extant darter species may identify lineage specific adaptations and their contributions to the relevant life history traits.\n\nI found a number of problems in the present version of the manuscript:\n\nBiology and some special adaptations of darters should be explained in the Introduction. Are there any differences between darter species regarding their life styles, ecology, physiology, metabolism etc.? This information can be used in the interpretation of the observed positive selection in some mitochondrial genes.\n\nThe authors have just listed the codon positions that have been found to be under positive selection. From the codon numbers, it is very difficult to see what the impact of positively selected sites is. It could be actually biologically important or meaningless.\n\nTherefore, the authors must use 3D models of the analysed mitochondrial proteins to show the consequences of the positively selected sites (PSS)/amino acids on the protein secondary and tertiary structure. These amino acids may be functionally important or neutral. The authors should follow some good examples of such analyses (e.g. da Fonseca et al., 20081, Castoe et al., 20082)\n\nThe authors should indicate the chemical nature of amino acids that are encoded by positively selected codons. Some particular polar amino acids are highly mutable and may not be associated with positive selection (e.g. Xia and Kumar, 20063).\n\nThe authors should compare their results (e.g. PSS) with the data from literature (e.g. from fishes and other vertebrates) to demonstrate that some positions are conserved PSS or are unique/different PSS.\n\nDiscussion is very poor and should be strongly improved by obtaining new data about the locations of PSS on the 3D models of the analysed mitochondrial genes. In such a way, their data can be put into biological perspective.\n\nThe title is uncompelling, merely indicating that the authors haven't obtained any serious conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4958", "date": "18 Oct 2019", "name": "Leos Kral", "role": "Author Response", "response": "Thank you for your review and suggestions. I have made revisions to address some of them and I am also providing rationale here for not addressing others. To address point 1, the introduction was expanded to include the most likely difference between darter species that may lead to OXPHOS genes adaptation. To address points 2 and 4, table 3 has been added and the results discussion expanded accordingly. Points 3, 5 and 6 were not incorporated into revision for several reasons. Firstly, I do not have the necessary expertise to confidently relate amino acid changes in protein 3D structure to biochemical function and any such discussion would be only speculative. Furthermore, such an analysis would not necessarily determine if a particular substitution is functionally important or neutral. Work carried out on directed evolution of proteins has demonstrated that mutations that influence the evolution of a protein are not obviously identified as such from a structural analysis since such mutations do not always alter the catalytic sites (see references below). I believe that the Ka/Ks analysis is only indicative of possible adaptive changes and should be first verified by direct assessment of the function of the proteins. I have incorporated this view into the revision. Point 7. The title is a truthful representation of the study. The number of species for which mtDNA genome sequences are available is just large enough to meet the minimum number of species acceptable for Ka/Ks analysis (at least for Selecton). The scope of this study was simply to ascertain if adaptive evolution can be detected to warrant further study. This would include verification of actual changes in OXPHOS function suggested by Ka/Ks analysis. References: Romero, P.A. and Arnold, F.H. 2009. Exploring protein fitness landscapes by directed evolution. Nature Reviews. Molecular Cell Biology10(12), pp. 866–876. Fasan, R., Meharenna, Y.T., Snow, C.D., Poulos, T.L. and Arnold, F.H. 2008. Evolutionary history of a specialized p450 propane monooxygenase. Journal of Molecular Biology383(5), pp. 1069–1080. Shimotohno, A., Oue, S., Yano, T., Kuramitsu, S. and Kagamiyama, royuki 2001. Demonstration of the Importance and Usefulness of Manipulating Non-Active-Site Residues in Protein Design. J. Biochem.129, pp. 943–948. Spiller, B., Gershenson, A., Arnold, F.H. and Stevens, R.C. 1999. A structural view of evolutionary divergence. Proceedings of the National Academy of Sciences of the United States of America96(22), pp. 12305–12310." } ] } ]	1	https://f1000research.com/articles/8-464
https://f1000research.com/articles/8-663/v1	15 May 19	{ "type": "Research Article", "title": "The yield of continuous EEG monitoring in the intensive care unit at a tertiary care hospital in Saudi Arabia: A retrospective study", "authors": [ "Haythum O. Tayeb" ], "abstract": "Background: The practice of continuous EEG monitoring (CEEG) in the intensive care unit (ICU) has been spreading over the past decade. Building an effective ICU CEEG program with sufficient quality demands adequate EEG equipment and significant human resources. While this is available in large tertiary care centers where the practice of CEEG has developed, it may not be available in developing healthcare systems. This study sought to provide data generated from a CEEG program in the adult ICU at a tertiary healthcare center in Saudi Arabia, shedding light on the real-life utility of CEEG in a developing healthcare system. Methods: This is a retrospective review of CEEG findings, along with mortality and duration of hospitalization of patients who had CEEG during a 12-month period at the adult ICU at the King Abdulaziz University Hospital (KAUH) in Jeddah, Saudi Arabia. Results: A total of 202 CEEG records were identified. A total of 52 patients had non-convulsive seizures (NCS); 10 clearly fulfilled criteria for non-convulsive status epilepticus. There were 120 patients that had clinical seizures upon presentation. Among them, 36 (30%) had NCS on EEG. The proportion of patients who were deceased at 60 days was higher in patients who had NCS (42%) than those who didn’t (26%, χ2 (2, n=200)= 4.4, p=0.03). The duration of hospital stay was longer for those who had periodic or rhythmic CEEG patterns (χ2 (2, n=200)= 7.6, p=0.02) but there was no significant relationship with mortality at 60 days. Conclusion: This study demonstrates a real-world experience from a tertiary care center in Saudi Arabia, a developing healthcare system. Findings are consistent with prior experience with ICU CEEG, demonstrating that finding ictal, rhythmic or periodic patterns is associated with morbidity and mortality. Further studies are needed to demonstrate how the practice of CEEG may alter patient outcomes.", "keywords": [ "Neurocritical care", "EEG", "non-convulsive seizures", "status epilepticus", "Saudi Arabia" ], "content": "Introduction\n\nContinuous electroencephalography (CEEG), the practice of continuously recording an electroencephalogram and a time-synchronized video of the patient, is commonly utilized to monitor critically ill patients with acute brain injury or altered mental status1. CEEG is instrumental in the diagnosis and management of nonconvulsive seizures (NCS) and status epilepticus, detection of cerebral ischemia, prognostication of outcomes after cardiorespiratory arrest, and evaluation of abnormal movements and altered mental status1. The practice of CEEG monitoring of critically ill patients in the intensive care unit (ICU) has been spreading over the past decade, particularly in Europe and North America1,2. Building an effective ICU CEEG program with sufficient quality demands not only adequate EEG equipment but also significant human resources2. This includes trained electroencephalographers and technologists who have enough time to devote to reviewing the large amounts of EEG data that are generated through continuous monitoring2. While this is available in large tertiary care centers where the practice of CEEG has developed, it may not be available in developing healthcare systems. Most of the published CEEG data also come from these advanced centers in North America and Europe.\n\nThis study sought to provide data generated from a CEEG program in the adult ICU at a tertiary healthcare center in Saudi Arabia, aiming to shed light on the real-life utility of CEEG in a developing healthcare system outside North America and Europe.\n\n\nMethods\n\nThis is a retrospective review of ICU CEEG findings, as well as mortality status and duration of hospitalization of all patients who underwent CEEG monitoring during a 12-month period from September 2016 to August 2017 at the adult ICU at the King Abdulaziz University Hospital (KAUH) in Jeddah, Saudi Arabia. This is an academic, tertiary-care, 600-bed hospital. Its adult ICU is comprised of 30 beds and is divided into medical and surgical divisions. CEEGs are requested by ICU physician or neurologists according to the clinical needs. An EEG technologist is available during the day time to set up ICU CEEGs. EEG leads are placed using the 10–20 international system of lead placement. CEEGs are digitally recorded, including synchronized video recording of the patient. The duration of CEEG monitoring is decided by the neurology consultation or ICU physicians. Studies whose duration was less than 2 hours were not included in the study as they were considered extended but not long-term studies. An epileptologist with fellowship training in CEEG interpretation reviewed the records on daily basis and reported them using the American Clinical Neurophysiology Society (ACNS) ICU EEG consortium proposed nomenclature for ICU EEG reporting, and the Salzburg criteria for non-convulsive status epilepticus3. Management decisions were made by the physicians in the ICU and neurology services.\n\nReports of CEEGs performed in the adult ICU during the study period were retrieved from the hospital’s electronic medical records (EMR). The author extracted key data from the reports, including background characteristics, the presence of rhythmic and periodic patterns or NCS. The author retrieved relevant demographic and clinical patient data from the hospital’s EMR, including diagnoses, ICU and hospital stay, and mortality status at 60 days. Frequencies, percentages, means, standard deviation, and Chi square were performed using the IBM SPSS Statistics for Windows, version 20.0.\n\nThis study was approved by the Institutional Review Board of KAUH as a retrospective study of anonymized clinical data with waiver of additional patient consent.\n\n\nResults\n\nA total of 202 CEEG records fulfilling the criteria were identified; complete, raw figures are available as Underlying data4. There were 116 female patients. The mean age was 53 (standard deviation=21). The duration of CEEG recording varied, with 48 (24%) recorded for 2–6 hours and 154 (76%) recorded for 6–24 hours. Table 1 shows the frequency of clinical diagnoses of our patients. The most common diagnostic categories were cerebrovascular disease and epilepsy. Table 2 shows the frequency of CEEG findings. Among the 52 patients that had NCS on CEEG, 10 patients clearly fulfilled criteria for non-convulsive status epilepticus. There were 120 patients that had clinical seizures upon presentation prior to CEEG monitoring. Among them, 36 (30%) had NCS on EEG. The proportion of patients who were deceased at 60 days was significantly higher in patients who had NCS (42%) than those who didn’t (27%, χ2 (2, n=200)= 4.4, p=0.03) (Table 3). There was no significant difference in the duration of hospital stay between those who had seizures and those who didn’t (p=0.2) (Table 3). The duration of hospital stay was longer for those who had periodic or rhythmic CEEG patterns (χ2 (2, n=200)= 7.6, p=0.02) but there was no significant relationship with mortality at 60 days (Table 3).\n\n*p<0.05.\n\n\nDiscussion\n\nThe practice of using CEEG in the ICU has developed rapidly over the past decade, particularly in North America and Europe1,5. This study is one of the first to report the experience of using ICU CEEG in Saudi Arabia, a country with a rapidly developing healthcare system that faces economic constraints. The data are consistent with prior knowledge and experience from other countries that CEEG is effective in detecting NCS and other likely harmful subclinical EEG patterns on the ictal-interictal continuum5,6. This study also shows a significant association between NCS and mortality. In addition, having periodic or rhythmic patterns was significantly associated with longer hospital stays.\n\nPrior studies have not definitively proven that utilizing CEEG leads to better outcomes2,5. This, coupled with the significant resources required to effectively run an ICU CEEG program2, may lead decision makers in healthcare systems to hesitate to support the development of CEEG practices. This study presents local data that demonstrate the need for CEEG. The data also raises questions whether CEEG is being utilized optimally. For example, few patients with brain tumors had CEEG, even though this is a patient population at risk of NCS. This suggests a need for a protocol for CEEG in the ICU, with focus on indications, required duration of monitoring, and management of NCS.\n\nThis study is a retrospective analysis with limitations. Data extracted from the EMR did not allow clarity with regards to the mental status of patients, use of sedatives, and other management decisions. Physicians did not follow a clear protocol when deciding the duration of the CEEG study. Longer studies may lead to higher detection rates of relevant CEEG patterns. The number of cases in some diagnostic categories was not high enough to permit subgroup analyses. The clinical setting is that of a developing program with limited resources and must be interpreted in this context. Further studies from developing healthcare systems like Saudi Arabia’s are needed to illuminate how the practice of CEEG monitoring may be integrated in the region.\n\n\nData availability\n\nOpen Science Framework: The yield of continuous EEG monitoring in the ICU at a tertiary care hospital in Saudi Arabia: A retrospective study. https://doi.org/10.17605/OSF.IO/Q56J34.\n\nThis project contains all raw de-identified data associated with this study.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHerman ST, Abend NS, Bleck TP, et al.: Consensus statement on continuous EEG in critically ill adults and children, part I: indications. J Clin Neurophysiol. 2015; 32(2): 87–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerman ST, Abend NS, Bleck TP, et al.: Consensus statement on continuous EEG in critically ill adults and children, part II: personnel, technical specifications, and clinical practice. J Clin Neurophysiol. 2015; 32(2): 96–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeniczky S, Hirsch LJ, Kaplan PW, et al.: Unified EEG terminology and criteria for nonconvulsive status epilepticus. Epilepsia. 2013; 54 Suppl 6: 28–9. PubMed Abstract \| Publisher Full Text\n\nTayeb H: The yield of continuous EEG monitoring in the ICU at a tertiary care hospital in Saudi Arabia: A retrospective study. 2019. http://www.doi.org/10.17605/OSF.IO/Q56J3\n\nSutter R, Semmlack S, Kaplan PW: Nonconvulsive status epilepticus in adults - insights into the invisible. Nat Rev Neurol. 2016; 12(5): 281–93. PubMed Abstract \| Publisher Full Text\n\nRodríguez V, Rodden MF, LaRoche SM: Ictal-interictal continuum: A proposed treatment algorithm. Clin Neurophysiol. 2016; 127(4): 2056–64. PubMed Abstract \| Publisher Full Text" }	[ { "id": "48822", "date": "23 May 2019", "name": "Peter W Kaplan", "expertise": [ "Reviewer Expertise EEG" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHow many of the NCSE were focal versus generalized?\n\nDid not the mortality vary with etiology of NCSE and PDs?\nI would add in the final section on limitations of the study that there is perforce selection bias as the EEG techs were not available during certain periods (if I remember, overnight and other times) and this should be stated.\n\nPossibly an EEG example of NCSE should be provided.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4854", "date": "27 Aug 2019", "name": "Haythum Tayeb", "role": "Author Response", "response": "Dear Dr. Kaplan,Thank you very much for kindly reviewing my manuscript entitled: “The yield of continuous EEG monitoring in the intensive care unit at a tertiary care hospital in Saudi Arabia: A retrospective study”. I value your insights. I have submitted a new version of this manuscript that took in consideration your comments and followed your suggestions as well as the other peer review I received. I briefly respond to your comments below as well:1. How many of the NCSE were focal versus generalized? I included in the new version of the article that \"There were 52 patients with NCS. Among them, 30 were of focal onset (57.7%)\".2. Did not the mortality vary with etiology of NCSE and PDs?The frequency of NCS etiologies in the group that was deceased at 60 days is now shown in a flow chart figure (figure 1). There was no statistically significant difference in mortality risk between the etiologies. Unfortunately, the number of cases in most subgroups was too small to permit subgroup analysis. I addressed this in the new version of the manuscript.3. I would add in the final section on limitations of the study that there is perforce selection bias as the EEG techs were not available during certain periods (if I remember, overnight and other times) and this should be stated. Added. 4. Possibly an EEG example of NCSE should be provided.Added. Once again, thank very much for your time and insights. Sincerely,Haythum Tayeb" } ] }, { "id": "51103", "date": "23 Jul 2019", "name": "Robert C. Tasker", "expertise": [ "Reviewer Expertise Pediatric Neurocritical Care", "Pediatric ICU" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author has described a practice of continuous EEG monitoring in an intensive care unit in a University Hospital in Jeddah, Saudi Arabia.\nI have a number of comments that will help readers better understand what these data mean, and how they might reflect on the implications for ICU practice.\nMethod (page 3)\nIn the first paragraph (line 8) the author describes \"CEEG are requested by ICU physician or neurologists according to the clinical needs\". In order to understand the significance of some of the analyses provided, it would be helpful to know what were the inclusion criteria, or the 'standard operating procedure' for the ICU. For example, were all patients comatose, and the clinician was unable to assess a clinical response?\n\nIn the first paragraph (lines 13-15) the authors describes \"The duration of CEEG monitoring is decided by the neurology consultation or ICU physicians\":\nIn order to understand the significance of some of the analyses later in the manuscript, it would be helpful to know when CEEG was started and finished in relation to admission and discharge/death. It would also be helpful to know the timing of when NCS occurred.\n\nSince this population has been gathered from ICU admissions, it would be helpful to have some description of ICU severity-of-illness, according to the risk-adjustment score used by the unit. The examination of any illness feature that might be associated with death needs to be adjusted for severity of illness. There were 62 observed deaths in 200 cases - what was the expected number of deaths from the admission data?\n\nResults (pages 3 and 4)\nThe presentation of findings in the Results section and the Abstract is a little confusing:\nThe author has a starting population of 202 patients undergoing CEEG monitoring. Then, the denominators being used in the data summaries are n=120, and n=200. I think that a flow chart would help here.\n\nTable 2 can be summarized as text, which will make room for a flow-diagram and better review of the data presented in Table 3.\n\nTable 3 needs some attention to data accuracy. For example, in Row 2 (Hospital stay data) the percentages add up to 101%, which is just a rounding error. In Row 3 (Hospital stay data) the percentages add up to 99%.\n\nYes, the author has found some associations with death, and length of stay, but does the information gained from CEEG have the potential to help with decision making? For example, if we imagined that CEEG was a 'diagnostic test', then:\nThe pre-CEEG probability of death in this population was 31%. The finding of EEG-seizures could change your pre-test to a post-test probability of death of 39%. I don't think that physicians would find this a helpful piece of information. That is, it is not about death, rather it is something we should use to identify a need for treatment, for example. Similarly, the pre-CEEG probability of hospital stay >1 month was 32%. The finding of periodic or rhythmic patterns could change the pre-test to post-test probability of hospital stay >1 month to 34%. Again, I don't think that physicians would find this a helpful piece of information. Obviously, both of these points would need to be tested and validated in a prospective study (as well as knowing details of when these CEEG features were found), but it may lead to a change in emphasis in the first paragraph of the Discussion.\n\nDiscussion (pages 3 and 4)\nBased on the above results, one could conclude that identifying EEG seizures on CEEG does not provide a significant 'quantum' in prognostic evidence. Similarly, identifying periodic or rhythmic patterns on CEEG does not provide a significant 'quantum' in identifying cases that will be likely be in-hospital >1 month.\nI am wondering whether the focus should be on 'what are we doing the CEEG': surely it is to identify something that we can/should treat; and, to identify something that will help us determine that we no longer need to do CEEG.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4856", "date": "27 Aug 2019", "name": "Haythum Tayeb", "role": "Author Response", "response": "Dear Dr. Tasker,Thank you very much for your time and detailed review of my manuscript entitled: “The yield of continuous EEG monitoring in the intensive care unit at a tertiary care hospital in Saudi Arabia: A retrospective study”. I value your insights. I have submitted a new version of this manuscript that took into consideration all of your comments and followed your suggestions as well as the other peer review I received. I respond to your comments below as well:1. it would be helpful to know what were the inclusion criteria, or the 'standard operating procedure' for the ICU.I added this statement in the Data Gathering section of the new version of the article to clarify the standard practice of the physicians when it comes to ordering, starting and stopping CEEG: “ICU physicians or neurologists request CEEG to search for NCS or patterns on the ictal-interictal continuum when critically ill patients have a disturbance in the level of consciousness that is unexplained by apparent underlying neurological or medical conditions. CEEG is initiated and stopped based on the clinical judgment of the treating teams. Generally, physicians aim to continue monitoring for 24 hours in patients with altered mental status but may allow discontinuation of CEEG for clinical or practical constraints (e.g. EEG machine availability or the need to relocate the patients).”In this developing program, there is no hospital protocol with regards to patient selection, CEEG initiation or termination. This is entirely left to the discretion of the treating physicians and is typically reached through discussion and consultation between neurology and ICU. Generally, all patients with unexplained altered mental status were included. It was difficult to ascertain from the EMR documentation how the mental status evolved during the time of the CEEG, unfortunately. This is a limitation I state in the Discussion section.2. In order to understand the significance of some of the analyses later in the manuscript, it would be helpful to know when CEEG was started and finished in relation to admission and discharge/death. It would also be helpful to know the timing of when NCS occurred.It was difficult to determine with consistency retrospectively from EMR how the duration of monitoring was decided and when the seizures occurred relative to the outcomes of interest. I agree that this is a limitation and would be best addressed in a prospective study. I added this to the new version of the article in the discussion section: “Retrospective EMR data did not contain accurate information with regards to the extent and evolution of mental status changes relative to the timing of the CEEG, use and titration of sedatives, and other management decisions. It was difficult to ascertain the timing of EEG initiation in relation to these dynamic variables of interest. Physicians did not follow a clear protocol when deciding the duration of the CEEG study.”3. Since this population has been gathered from ICU admissions, it would be helpful to have some description of ICU severity-of-illness, according to the risk-adjustment score used by the unit. The examination of any illness feature that might be associated with death needs to be adjusted for severity of illness. There were 62 observed deaths in 200 cases - what was the expected number of deaths from the admission data? Thank you for pointing this out. I added this to the methods section: “The average APACHE II (Acute Physiology and Chronic Health Evaluation II) score of medical patients admitted to the ICU ranges between 10-30 on average. The scores are routinely calculated but not recorded in the electronic medical records (EMR).” This was a limitation of the study and we could not remedy this by calculating scores retrospectively. 4. The author has a starting population of 202 patients undergoing CEEG monitoring. Then, the denominators being used in the data summaries are n=120, and n=200. I think that a flow chart would help here.I added a flow chart. Thank you for suggesting it. n=120 is the number of patients who had clinical seizures prior to CEEG initiation. This is a group of interest since data have previously shown a ~30% risk of NCS. We replicated this. I removed n=200 from the prior reporting of the Chi-square to avoid ambiguity. Thank you. 5. Table 2 can be summarized as text, which will make room for a flow-diagram and better review of the data presented in Table 3.Done. I expanded table 1 (formerly table 3) by adding row and column percentages. 6. Table 3 needs some attention to data accuracy. Thank you for pointing this out. I scrutinized the data and ensured accuracy. I fixed several coding errors that altered the proportions slightly without altering the overall outlook of the results as well.7. Does the information gained from CEEG have the potential to help with decision making?Thank you very much for highlighting this issue. To address it, I reported the “pre-test” proportions to show that the “post-test” proportions are not of large magnitudes. I also added row and column percentages in table 1.8. I am wondering whether the focus should be on 'what are we doing the CEEG': surely it is to identify something that we can/should treat; and, to identify something that will help us determine that we no longer need to do CEEG.Thank you for this insight. I altered the discussion to reflect the uncertainty about the clinical meaningfulness of the prognostic information and highlight the need for prospective studies to address the important questions. This is from the new version of the manuscript: “This study reveals statistically significant associations between NCS and mortality and between RPPs and longer hospital stays, supporting the clinical gestalt of identifying and treating potentially harmful CEEG patterns is worthwhile. However, the incurred excess risks of morbidity and mortality in patients with NCS or RPPs was relatively modest. Such modest increases are not likely to be of clinical significance to clinicians evaluating patient prognoses. Nonetheless, this study was a retrospective study with limitations that preclude definitive conclusions about morbidity and mortality risk magnitudes, which are better assessed with prospective studies and in well-developed CEEG programs.Prior studies have not definitively proven that utilizing CEEG leads to better outcomes 2, 5 . This, coupled with the significant resources required to effectively run an ICU CEEG program 2 , may lead decision-makers in developing healthcare systems to hesitate to support the development of CEEG practices and research. This study presents data from a small and developing program to demonstrate the real-world effectiveness of CEEG in detecting potentially harmful electrophysiological patterns. In addition, the study highlights the uncertainties regarding the clinical significance of the prognostic information provided by CEEG. Hopefully, this should lead to further development of ICU CEEG programs with embedded prospective, patient-centered research programs. Such research should focus on how CEEG may be used effectively and optimally and how the generated data may impact clinical decisions and patient outcomes in the ICU.” Once again, thank you very much for your thorough review of this manuscript and your valuable insights. Much appreciated. Sincerely,Haythum Tayeb" } ] }, { "id": "54081", "date": "18 Sep 2019", "name": "Yara Mikhaeil-Demo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a one-year experience using continuous EEG monitoring in a developing healthcare system. The authors experience with a growing cEEG program is a valuable addition to the literature. We have some comments and questions to better understand the results described:\n\nMethods:\n\nWhat were the inclusions and exclusions criteria? Was EEG duration less than two hours the only exclusion criteria?\n\nHow frequently were studies reviewed and results communicated to treating team? It would be helpful to know the timing of when the seizure occurred on EEG and the identification of these findings on EEG and communication with the treating physician.\n\nWhen discussing mortality and morbidity, it would be helpful to adjust for comorbid conditions and severity of underlying illness.\n\nIn the statistical analysis were p values adjusted for multiple comparisons?\n\nResults:\n\nThe author indicates a total of 202 cEEG records were identified. Are these for 202 unique patients? Did any patients have more than one cEEG study during this time? If so, were the subsequent studies excluded if the patients had more than one cEEG study during the one-year period?\n\nHow were the clinical diagnoses defined? For example, was “epilepsy” used only if the patient had a prior diagnosis of epilepsy, or was it also used for new-onset seizures? Subcategorization of “cerebrovascular disease” would also be helpful. (Based on prior studies intracranial hemorrhage is much more likely to be associated with seizures than ischemic stroke.)\n\nFor Table 2, did any patients have more than one of the periodic patterns? Did patients have NCS and periodic patterns? Why is NCSE not listed in the table?\n\nIt would be interesting to look at the diagnoses associated with NCS, NCSE and periodic patterns.\n\nIn addition to describing the primary diagnosis of the patients it would be helpful to know why they were connected to cEEG. What were the indications for EEG monitoring and frequency for each indication? It would be helpful to describe a table of indication for EEG monitoring and percentage for each reason (example, clinical seizure, altered mental status. abnormal movements, etc).\n\nDiscussion:\n\nThe author states: “The data are consistent with prior knowledge and experience from other countries that CEEG is effective in detecting NCS and other likely harmful subclinical EEG patterns on the ictal-interictal continuum.” This paper does not define nor discuss the ictal-interictal continuum.\n\nThe discussion should compare the findings at the current center to prior publications on the yield of CEEG.\n\nWithout adjusting for underlying diagnosis and comorbidities, it is difficult to claim that there is “a significant association between NCS and mortality.”\n\nIt would be helpful to provide a more detailed discussion as to why the following finding was noted “periodic or rhythmic patterns was significantly associated with longer hospital stays.”\n\nIn the method section, the author states: “The duration of CEEG monitoring is decided by the neurology consultation or ICU physicians.” It appears that the longest duration for a study was 24 hours; however, as mentioned 52 patients had nonconvulsive seizures, with 10 patients fulfilling the criteria for non-convulsive status epilepticus. How were these patients monitored for response to treatment? Were NCS and NCSE successfully treated by the time of disconnect?\n\nThroughout the article the authors refer to the resource requirement for CEEG monitoring. It would be interesting to know if and how resource limitations affected monitoring. What was the time from request to connection? Is there any reason to suspect this affected outcomes?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-663
https://f1000research.com/articles/8-726/v1	23 May 19	{ "type": "Software Tool Article", "title": "MetaDEGalaxy: Galaxy workflow for differential abundance analysis of 16s metagenomic data", "authors": [ "Mike W.C. Thang", "Xin-Yi Chua", "Gareth Price", "Dominique Gorse", "Matt A. Field", "Mike W.C. Thang", "Xin-Yi Chua", "Gareth Price", "Dominique Gorse" ], "abstract": "Metagenomic sequencing is an increasingly common tool in environmental and biomedical sciences yet analysis workflows remain immature relative to other field such as DNASeq and RNASeq analysis pipelines. While software for detailing the composition of microbial communities using 16S rRNA marker genes is constantly improving, increasingly researchers are interested in identifying changes exhibited within microbial communities under differing environmental conditions. In order to gain maximum value from metagenomic sequence data we must improve the existing analysis environment by providing accessible and scalable computational workflows able to generate reproducible results.\n\nHere we describe a complete end-to-end open-source metagenomics workflow running within Galaxy for 16S differential abundance analysis. The workflow accepts 454 or Illumina sequence data (either overlapping or non-overlapping paired end reads) and outputs lists of the operational taxonomic unit (OTUs) exhibiting the greatest change under differing conditions. A range of analysis steps and graphing options are available giving users a high-level of control over their data and analyses. Additionally, users are able to input complex sample-specific metadata information which can be incorporated into differential analysis and used for grouping / colouring within graphs. Detailed tutorials containing sample data and existing workflows are available for three different input types: overlapping and non-overlapping read pairs as well as for pre-generated Biological Observation Matrix (BIOM) files.\n\nUsing the Galaxy platform we developed MetaDEGalaxy, a complete metagenomics differential abundance analysis workflow. MetaDEGalaxy is designed for bench scientists working with 16S data who are interested in comparative metagenomics. MetaDEGalaxy builds on momentum within the wider Galaxy metagenomics community with the hope that more tools will be added as existing methods mature.", "keywords": [ "Galaxy", "metagenomics", "differential abundance", "high throughput sequencing", "phyloseq" ], "content": "Introduction\n\nIt is now recognized that there is a strong link between microbial communities in the human body and human health1. While the importance of such communities is understood, the composition and function of the human micro-biome largely remains a mystery. Uncovering how the composition and function of the micro-biome impacts human health represents a significant area of growth. Another important area of research growth is the study of environmental microbial communities in fields such as agriculture, marine science, and ecology. By identifying the composition of microbial communities, researchers are able to link microbes to specific environments and using comparative metagenomics identify how microbial communities’ changes under altered environmental conditions.\n\nCentral to elucidating the link between the metagenomic data and human health or altered environmental conditions is sequencing; however, obtaining useful research outcomes from large volumes of unprocessed sequence data represents a challenge for many bench scientists. The major bottleneck in obtaining value from such data is the huge computational and logistic task required for analysing the large volumes of sequencing data routinely generated in a single sequencing run.\n\nThe sequencing of entire microbial communities requires metagenomic analysis tools. These tools rely on the ability to analyse unbroken sequence reads covering the 16S variable regions. Due to limitations of short read sequencing platforms such as IIlumina, the longest fragment of variable regions of a 16S gene that can be sequenced is shorter than the ideal full 600 bp. Illumina paired-end sequencing of 300 bp on forward read and reverse read produces only 550 bp to allow for stitching the forward end and reverse end together. With 550 bp fragment length, the reads can cover both variable region 3 (V3) and variable region 4 (V4). The length of V3 and V4 are 393bp and 440bp respectively.\n\nA major challenge for bench scientists working with metagenomic data is that many popular software programs requires a 64-bit Linux environment, an environment often unavailable and unfamiliar to researchers. Furthermore, even when such an environment is available, the complexity of the rapidly changing metagenomic algorithms means no gold standard methodologies exist. As such, there are currently over 100 metagenomic analysis tools available, making it challenging to select the appropriate software. For example, the popular metagenomic tool QIIME2 consists of more than 150 python scripts, many of which are wrappers to external programs.\n\nAn increasingly common alternative for the growing number of non-bioinformaticians working with NGS data is the availability of user-friendly interfaces. These interfaces are typically attached to significant compute resources with pre-installed software packages readily available. Interfaces such as Galaxy3 or the Genomics Virtual Lab4 are examples of powerful platforms that grant non-bioinformaticians access to the latest NGS methodologies. The Galaxy platform enables scientists to use bioinformatics tools in an easy to use graphical user interface (GUI) environment, where tool resource management is handled by the administrators of each Galaxy service. The platform’s functionality power comes from the ability to chain tools into workflows, and share the data and workflows. Further, the flexibility of Galaxy platform allows developers to integrate new tools and workflows into the platform. Galaxy maintains a single tool shed repository of pre-wrapped tools that cover an abundance of next generation sequence analyses.\n\nDespite these solutions however, challenges remain in fast moving research areas such as metagenomics with limited metagenomic workflows currently available within the popular Galaxy framework. Currently, there is one end-to-end existing metagenomics workflow offering, the recently published ASaiM5 for both 16S and shotgun metagenomic analysis. While there is overlap between their workflows, MetaDEGalaxy differs in its focus on differential abundance by incorporating the capabilities of phyloseq6 and DESeq27 for complex differential bacterial analysis. DESeq2 contains tests specifically developed to detect significant differences in abundances for counts data. While DESeq2 is most commonly utilised for differential gene expression in RNASeq, using the phyloseq API a Biological Observation Matrix (BIOM) file is formatted for use within DESeq2 and differences in taxa abundances discovered. MetaDEGalaxy also offers extensive graphing capabilities by wrapping the comprehensive metagenomics R-package phyloseq6. Extensive graphing options are available within MetaDEGalaxy wrapping most functions offered within phyloseq which offer the user a high level of control. Additionally, user supplied metadata files can be input to DESeq2 for model generation and to phyloseq for enhanced graphing capabilities allowing for grouping, clustering, and colouring of all graph types based on metadata information. All software wrapped within the workflow is open-source software, a current limitation of existing workflows such as usearch8 within the popular QIIME package 2. Finally, MetaDEGalaxy is designed within the popular Genomic Virtual Lab4 leveraging the functionality of this robust infrastructure.\n\n\nMethods\n\nMetaDEGalaxy accepts either 454 or Illumina paired end sequence FASTQ files that can be overlapping or non-overlapping. Users may alternatively input a pre-computed BIOM file if they do not require BIOM file generation. Additional functionality requires a sample specific tab-delimited metadata file formatted according to QIIME map file standards. This metadata information can be utilised for determining the model to employ within DESeq2 and to generate graphs grouped by various metadata attributes.\n\nIn total, there are four workflows in MetaDEGalaxy (Table 1) which utilise a combination of external software and custom code.\n\nExternal software available include Trimmomatic (v0.32.2)9, FastQC (v0.52), PEAR (v0.9.6)10, SAMTools (v1.1.2)11, BWA (0.7.12.1)12, VSEARCH (v1.9.7)13, the BIOM API, DESeq2 (v2.1.8)7 and phyloseq (Galaxy v1.0)6.\n\nFour comprehensive MetaDEGalaxy tutorial are currently available in github which demonstrate how to work with both overlapping and non-overlapping 16S paired end Illumina reads.\n\nTutorial #1 details the workflow for data QC and the detection of paired end overlap in sequencing data and preparing FastQ files for metagenomic analysis (Figure 1). Tutorial #2 details the entire workflow for overlapping paired end Illumina reads (Figure 2) using the same data set employed by the Mothur_SOP run with the popular Mothur software (v1.35.1)14. This workflow inputs a group of paired-end MiSeq files and a metadata map file and generates overlapping FASTQ files, an annotated BIOM file, a DESeq2 table of differentially expressed microbes, and a variety of phyloseq graphs. Tutorial #3 details the entire workflow for non-overlapping paired end Illumina reads and is similar to tutorial #2 with the exception of pre-processing steps transforming FASTQ files into a Fasta file where PEAR10 software is not run. Finally, tutorial #4 details a workflow for BIOM file processing and analyses detailing how to utilise the platform for analyses starting from an input BIOM file.\n\nBoth workflow 2 and 3 use all the components in the workflow, the only difference is workflow 2 takes in paired-end reads data as input and workflow 3 take single-end reads data as input. The workflow 4 is the subset of the main workflow which starts with blue boxes and ends with all plots generated.\n\nThe Galaxy environment is available for testing purposes at http://203.101.224.124/galaxy and will be available on Galaxy Australia server by the end of 2019 (https://usegalaxy.org.au/). The minimum system requirements for installing the MetaDEGalaxy are a 64-bit unix environment at 4Gb of memory.\n\n\nResults\n\nTo demonstrate some of the advanced functionality of MetaDEGalaxy, we follow tutorial #2 using the Mothur_SOP data to first generate a normalised count table and a table of differentially abundant OTUs (Table 2). The differentially abundant OTU table is formatted in DESeq2 output with additional taxonomic information appended to each row.\n\nWe use this table of differentially abundant OTUs to next generate a symmetric plot. Users are able to select any taxonomic level as well as any metadata variable for comparison and further to pick two values of this variable for direct comparison (Figure 3). In this example, we pick Phylum for our taxonomy level and time as our variable of interest and group the graph according to ‘Early’ or ‘Late’. The resulting symmetric plot shows the differences in OTUs for ‘Early’ and ‘Late’ samples across different phylum (Figure 4). We are also able to generate alpha diversity abundance plots according to various sample attributes grouped here for ‘Replicate Group’ and coloured by ‘Food’ (Figure 5). As a final example, we generate a network plot where we select ‘Replicate group’ for the correlation and select ‘Food’ as the legend (Figure 6).\n\nUsers are able to select the taxonomic rank to examine in addition to two values within any user-defined metadata category.\n\nMetaDEGalaxy is compared to existing software in Table 3. There are comparable web and/or GUI based tools such as QIIME2, Calypso, Explicet, and Megan, however none of these tools are currently available within the popular Galaxy framework. There is currently one actively developed end to end 16S workflows within Galaxy, ASaiM5.\n\nOf the available web-based or GUI-based options, only QIIME2, ASaiM, and MetaDEGalaxy offer end-to-end workflows beginning from input FASTQ files. Only MetaDEGalaxy and Calypso offer extensive differential abundance tools incorporating the algorithms of sophisticated tools for finding differences in count data such as DESeq2. Finally, only ASaiM and MetaDEGalaxy run in the popular Galaxy framework making the set of attributes available within MetaDEGalaxy unique.\n\n\nUse cases\n\nTo demonstrate how to use MetaDEGalaxy we offer four in-depth tutorials describing available workflows. Tutorials 1, 2 and 4 utilise the same input data as the well-documented Mothur_SOP while tutorial 3 utilises custom 300bp paired end, non-overlapping Illumina MiSeq data. In either use case, reads can be accessed and pre-processed via Galaxy Interface with the following steps:\n\n1) click on \"Operations on multiple dataset\" on the top of the history panel\n\n2) check the box for all paired-end files listed on the history panel\n\n3) click on the \"For all selected...\" button the top of the history panel\n\n4) click on \"Build list of Dataset Pairs\" on the drop-down menu\n\n5) Type in a common field of the file name for both forward and reverse paired end data\n\n6) click on the \"Auto-pair\"\n\n7) Enter a name for the collection of paired datasets and click \"Create list\"\n\nApart from the paired-end reads in data collection, users are required to have loaded the metadata table and both 16S reference genome and annotation files. When the paired-end reads from a data collection is imported into a Galaxy history, an important step for the later in the workflow is the renaming of the FASTA sequence header by appending the sample ID to end at the end of each read ID using the reheader tool in Galaxy. This information will be used as the column header for OTU table generated by the workflows.\n\nWorkflow 1 (Figure 1) is designed to detect the status of overlapped paired-end reads data using PEAR. Users should proceed with workflow 2 if the percentage of overlapped paired-end reads data is high. Otherwise, workflow 3 should be used for non-overlapping reads. Both workflow 2 and 3 are fundamentally the same (Figure 2), however, workflow 3 can take single-end reads data as input when the overlapped paired-end reads are not overlapping.\n\nWorkflow 4 is designed to take a precomputed BIOM file as input. BIOM file format is designed to store OTU counts, metadata, and OTU annotation into one file. When users input a BIOM file, workflow 4 can be used to add metadata to an existing BIOM file and create abundance bar plot, network plot and symmetric plots using phyloseq R package.\n\nMore detailed tutorial documentation is available in the github repository.\n\n\nConclusion\n\nMetaDEGalaxy is a complete end-to-end Galaxy workflow for 16S differential abundance analysis. Harnessing the power of open source algorithms such as vsearch, phyloseq, and DESeq2, MetaDEGalaxy offers users high-level of control over their data and analysis options. Focusing on discovering the most differentially abundant OTUs between samples, MetaDEGalaxy allows users to assess the impact of different environmental condition on overall microbial community composition.\n\n\nData availability\n\nData used for the tutorials are available from Zenodo:\n\nZenodo: Mothur MiSeq SOP Galaxy Tutorial Data. https://doi.org/10.5281/zenodo.80065115\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSoftware available from: http://203.101.224.124/galaxy\n\nSource code available from: https://github.com/QFAB-Bioinformatics/jcu.microgvl.ansible.playbook\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.265883516\n\nLicence: GNU General Public License v3.0 for all script/wrappers", "appendix": "Grant information\n\nThis work was funded by an internal capacity building grant within the Australian Institute of Tropical Health and Medicine [15025].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to acknowledge Nectar Australia for compute resources for hosting Galaxy.\n\n\nReferences\n\nClemente JC, Ursell LK, Parfrey LW, et al.: The impact of the gut microbiota on human health: an integrative view. Cell. 2012; 148(6): 1258–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaporaso JG, Kuczynski J, Stombaugh J, et al.: QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010; 7(5): 335–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfgan E, Sloggett C, Goonasekera N, et al.: Genomics Virtual Laboratory: A Practical Bioinformatics Workbench for the Cloud. PLoS One. 2015; 10(10): e0140829. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBatut B, Gravouil K, Defois C, et al.: ASaiM: a Galaxy-based framework to analyze microbiota data. GigaScience. 2018; 7(6): giy057. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcMurdie PJ, Holmes S: Phyloseq: a bioconductor package for handling and analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput. 2012; 235–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010; 26(19): 2460–1. PubMed Abstract \| Publisher Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J, Kobert K, Flouri T, et al.: PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics. 2014; 30(5): 614–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRognes T, Flouri T, Nichols B, et al.: VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016; 4: e2584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchloss PD, Westcott SL, Ryabin T, et al.: Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009; 75(23): 7537–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiltemann S: Mothur MiSeq SOP Galaxy Tutorial Data [Data set]. Zenodo. 2016. http://www.doi.org/10.5281/zenodo.800651\n\nmthang: QFAB-Bioinformatics/jcu.microgvl.ansible.playbook: First release of MetaDEGalaxy (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2658835" }	[ { "id": "50121", "date": "24 Jul 2019", "name": "Saskia Hiltemann", "expertise": [ "Reviewer Expertise Bioinformatics", "Galaxy", "16S metagenomics", "training" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe MetaDEGalaxy, a set of Galaxy tools and workflows for differential abundance analysis of 16S metagenomics data. They have enabled DESeq -a tool designed primarily for RNASeq data- to be used on metagenomics datasets. They provide Galaxy workflows and training materials and a Galaxy server for testing. Furthermore, they have integrated a number of Galaxy tools for visualisation using phyloseq, which is a valuable addition to the existing ecosystem of Galaxy metagenomics tools.\nRemarks:\nIn the abstract: \"Metagenomic sequencing [..] analysis workflows remain immature compared to other fields\"\nThis is a strong claim and requires more justification or be made less broad. Metagenomics (and especially 16S metagenomics) tool suites such as QIIME and Mothur have quite well-established pipelines. And end-to-end online analysis portals such as MG-RAST (https://www.mg-rast.org/), MGnify (https://www.ebi.ac.uk/metagenomics/pipelines/2.0), MOCAT2 (https://mocat.embl.de/), META-pipe1 and others have also been around for some time, and are also user-friendly GUI options.\nIn general, the discussion of existing methods could be expanded. Please describe in more detail how MetaDEGalaxy fits in this ecosystem.\n\nIn the introduction: \"Currently, there is one end-to-end existing metagenomics workflow offering, ASaiM\":\na. Referring to ASaiM as a workflow may be confusing. In Galaxy, the term workflow has a very specific meaning, and ASaiM is a collection of tools, workflows and tutorials with a common topic, and it includes multiple Galaxy workflows within it. Perhaps refer to these solutions as Galaxy environments or similar, and reserve the word workflows for Galaxy workflows?\nb. ASaiM is also by no means the only metagenomics workflow available in Galaxy, for example: - GmT : Mothur SOP 16S end-to-end pipelines have been made available as Galaxy workflows previously.2 - FROGS: Metagenomics pipelines in Galaxy has been previously described.3 - Other Galaxy environments and workflows such as A-Game,4 ANASTASIA,5 or this functional annotation workflow,6 and several others have also been previously described. While these examples have a different focus than MetaDEGalaxy (i.e. functional metagenomics rather than 16S), the authors make the very broad claim that ASaiM is the only other \"existing metagenomics workflow on offering\", which is inaccurate.\nPlease consider expanding the discussion of existing work and Table 3 to include some or all of the above.\n\nIn the results section, please discuss how the differential abundance results obtained with the MetaDEGalaxy pipelines compare to the results described in the Mothur SOP (https://www.mothur.org/wiki/MiSeq_SOP, e.g. under subsection \"population level analysis\" of section \"OTU-based analysis\") Do you determine the same OTUs to be statistically significantly different between the two groups? Explain any differences in results, as well as the added value of your approach over the statistical methods used in the SOP.\n\nThe Phyloseq wrappers the authors have created do not appear to be available from the Galaxy toolshed currently While I appreciate that the authors have developed Ansible playbooks for the installation of the wrappers, such a custom approach is not recommended practice, and adding the tools to the tool shed will greatly increase their accessibility.\nOne option for this would be to submit the wrappers to the IUC tool repository on github (https://github.com/galaxyproject/tools-iuc) where they will be reviewed and automatically uploaded to the toolshed upon acceptance.\n\nMinor Remarks:\nTraining materials for the use of the MetaDEGalaxy workflows are available in the form of PDF files. I would strongly urge the authors to consider contributing these materials to the Galaxy Training Network (https://training.galaxyproject.org) so that they are more readily available for use by the community. I think that MetaDEGalaxy tutorial would be happily accepted there, and the GTN community can provide support to transform the tutorials into the right format.\n\nSince the 4 workflows offered in this manuscript are designed to be run in succession (e.g. workflow 1,2,4 or 1,3,4), have the authors considered creating some full end-to-end workflows, using Galaxy's concept of sub-workflows?\n\nA set of Qiime2 Galaxy tool wrappers have recently been made available in the toolshed (https://toolshed.g2.bx.psu.edu/repository?repository_id=7af460fa907bf4a3), could you update he text in the \"software comparison\" section & table to reflect this?\n\nCompliments:\nI really like the visualisation tools you added, and I would love to add a symmetric plot to the existing 16S Galaxy tutorial on the Galaxy Training Networks site if you put the tools on the tool shed!\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4973", "date": "16 Oct 2019", "name": "Matt Field", "role": "Author Response", "response": "Thank you for taking the time to review MetaDEGalaxy. I have submitted a modified manuscript to address the points you raised.1) Software background / comparison (Major Remark 1 and 2 and minor remark 3)The most significant change to the manuscript is the dramatic expansion of the software discussion and comparison sections to include more web based and Galaxy based metagenomics offerings. Additions include MG-RAST, MetaPipe, MOCAT2, FROGS, GmT, A-Game, and ANASTASIA to name a few.I added a broader discussion about where MetaDEGalaxy fits in relative to the ever expanding metagenomic software environment.2) Expansion of differential abundance comparisonI expanded the manuscript to include more details on tools with differential abundance options including calypso and mothur methods metastats and lefse. I performed a small side-by-side comparison however MetaDEGalaxy had results identical to calypso (which also uses phyloseq_to_deseq) and very different to both mothur techniques so I simply state this fact in the manuscript and cite previous work that has shown this already (Jonsson V, et al. Statistical evaluation of methods for identification of differentially abundant genes in comparative metagenomics. BMC Genomics. 2016).3) Availability of wrapper scripts and tutorials (Major remark 4 and minor remark 1)Thank you for the suggestions regarding better ways to make the software more widely available, I wasn't aware of these resources. The tutorials will indeed be made widely available to the training network once the installation is completed within Galaxy Australia which should be done by the end of 2019. Currently, the code is installed on a demo server however it will be given a permanent home within Galaxy Australia very shortly. We will also make the wrapper scripts available as per your suggestion to the IUC tool repository on github.Also, for testing please note the temporary IP address for the demo server changed to http://203.101.224.202/galaxy/ which is now reflected in the new manuscript." } ] }, { "id": "55128", "date": "15 Oct 2019", "name": "Leo Lahti", "expertise": [ "Reviewer Expertise microbiome research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis submission introduces MetaDEGalaxy, which is a workflow intended for 16S differential abundance analysis in the open source Galaxy platform. The workflow incorporates various currently popular open source algorithms, the proposed workflow support the application of such methods by Galaxy users. In particular, the workflow supports differential OTU abundance testing for common measurement platforms (454 and Illumina). Step-by-step tutorials are provided to support the use.\nThe overall work is sound and clearly written. Appropriate references are provided, and the work is based on commonly used methodologies and open source resources. Data and software are openly available with a unique DOI and permanent archiving through Zenodo.\nThe work does not contribute to methods criticism, validation, or benchmarking. This work is a technical contribution that provide new software plugin for the broader Galaxy platform. This is relevant for the limited community of researchers who use Galaxy for 16S microbiome analysis. The contribution is a contribution to scientific software, rather than scientific discussion. This, in my understanding, fits the F1000Research scope.\nMinor:\nWhy the software has GPL3 license that is more restrictive than e.g. MIT which is often recommended for research software? See DOI: 10.1371/journal.pcbi.10025981\n\nInstead of QIIME, it could be more appropriate to cite QIIME2?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-726
https://f1000research.com/articles/8-1771/v1	17 Oct 19	{ "type": "Case Report", "title": "Case Report: Acute hemorrhagic edema of infancy (Seidlmayer purpura) – a dramatic presentation for a benign disease", "authors": [ "Elena Carboni", "Maria Scavone", "Ettore Stefanelli", "Valentina Talarico", "Stefania Zampogna", "Maria Concetta Galati", "Giuseppe Raiola", "Elena Carboni", "Maria Scavone", "Ettore Stefanelli", "Stefania Zampogna", "Maria Concetta Galati", "Giuseppe Raiola" ], "abstract": "We present a case of an 11-month-old girl who was referred to our unit for an erythematous rash that appeared on the face and extremities. Personal and family history was not relevant. Laboratory tests were normal. During recovery, diameter and colour intensity of the cutaneous lesions increased, but after some weeks, lesions had a self-limited resolution without any treatment. Based on clinical and laboratory findings, a diagnosis of acute hemorrhagic edema of infancy (AHEI) was made. AHEI is a rare cutaneous leukocytoclastic vasculitis that usually affects children aged between 4 and 24 months. Etiology is unknown but almost of 75% of cases are preceded by infectious episodes, vaccinations or use of medications. In contrast to the dramatic cutaneous eruption, clinical conditions are usually optimal. Classically, AHEI is characterized by a triad of symptoms: fever, edema and purpura. Skin lesions are erythematous, annular, medallion-like, purpuric plaques that have a rapid onset and appear on the face and extremities, sparing trunk and mucosal membranes. Initially interpreted as a variant of Henoch-Schönlein purpura, now it is considered a distinct disease. In the majority of cases the disease is benign and self-limited without a visceral involvement, so a conservative approach is most often chosen.", "keywords": [ "edema", "leukocytoclastic vasculitis", "Seidlmayer purpura", "erythematous rash", "children" ], "content": "Introduction\n\nAcute hemorrhagic edema of infancy (AHEI), also known as Seidlmayer purpura, is a rare cutaneous leukocytoclastic vasculitis. It was described for the first time in 1913 and currently there are more than 300 cases in the literature1. Initially it has been interpreted as a Henoch-Schönlein purpura variant, but now it is considered a distinct disease. Although it has a dramatic clinical presentation, it is a benign and self-limited disease2. We report the case of an 11-month-old girl who was referred to our unit for an erythematous rash appeared on the face and extremities, which was indicative of a rare but non negligible diagnosis.\n\n\nCase presentation\n\nAn 11-month-old girl from France presented with fever and oval purpuric lesions on the face and extremities, which had appeared one hour before. In the previous week, she had bilateral conjunctivitis and gastroenteritis, treated with oral rehydration. At admission she was in good clinical condition and her vital signs were normal. Physical examination showed purpuric confluent elements with a cockade pattern on cheeks, left auricle, upper and lower limbs, particularly on distal ends, sparing trunk and back (Figure 1). Hands appeared edematous without joint swelling or tenderness. Bilateral conjunctivitis was still present. No other physical abnormalities were observed. Complete blood count, extended biochemistry, coagulation tests (prothrombin time, partial thromboplastin time, fibrinogen and D-dimer) and urinalysis were normal with C-reactive protein of 18.9 mg/l (normal value <5 mg/l) and procalcitonin of 0.88 ng/ml (normal value <0.05 ng/ml). During the hospitalization the child maintained good clinical condition, with stable vital parameters. Dermatological lesions showed a worsening clinical outcome, with increased diameter and colour intensity during the following three days from admission. Based on clinical and laboratory findings, a diagnosis of AHEI was made. We decided to not perform any therapy and after about two weeks lesions had a self-limited resolution. The child was monitored clinically for about six months and she did not present any relapse of the disease during the follow-up period.\n\n(a) Ecchymotic target-like skin lesions with edema of the hand. (b) Purpuric confluent elements with a cockade pattern on the lower limbs.\n\n\nDiscussion\n\nThe first description of AHEI was made by Snow in 19131. There have been approximately 300 cases reported in the literature2 under a variety of denominations: Seidlmayer purpura, Finkelstein disease, rosette form purpura, medallion-like purpura, infantile post-infectious iris-like purpura and edema3. AHEI is a rare cutaneous leukocytoclastic vasculitis that usually affects children aged between 4 and 24 months and it is more common in males2,4. Most cases of AHEI occur during winter, and almost of 75% of cases are preceded by infectious episodes, such as viral and bacterial infections of the upper respiratory tract, otitis media, conjunctivitis, bronchopneumonia, urinary tract infections and enteritis. Many organisms including adenovirus, varicella zoster virus, cytomegalovirus, rotavirus, herpes simplex virus, tuberculosis, streptococci, and staphylococci are associated with AHEI. Vaccinations or medications could also trigger AHEI5. In this case, the patient had a week-long history of bilateral conjunctivitis and gastroenteritis, compatible with a recent viral infection. A peculiar feature of AHEI is the unusual dramatic cutaneous eruption that contrasts with good general clinical condition (normal vital parameters with normal blood tests) that allowed to exclude more serious diseases. Diagnosis is clinical and it is classically detectable by observance of the clinical triad of symptoms: fever, edema and purpura6. Skin lesions are erythematous, annular, medallion-like, rosette-shaped purpuric plaques that cluster and often coalesce3. These lesions have a rapid onset and appear on face and extremities, sparing trunk and mucosal membranes3,4. The edema typically occurs on feet, hands, face and auricles, and can involve the scrotum in males2,7. In the majority of cases there is no visceral involvement and the disease is benign and self-limited. However, in the literature there are descriptions of cases of renal involvement, arthralgias and two cases of intestinal involvement followed by intussusception2.\n\nKrause et al. suggested the following criteria for diagnosing AHEI8:\n\n- Age <2 years;\n\n- Purpuric or ecchymotic target-like skin lesions with edema on the head and face, with or without mucosal involvement;\n\n- Lack of systemic disease or visceral involvement;\n\n- Spontaneous recovery within few days or weeks.\n\nOur patient presented all of the above conditions. Laboratory data are usually normal, but leukocytosis, thrombocytosis, eosinophilia and high levels of C-reactive protein and erythrocyte sedimentation rate can be observed7. Skin biopsy shows a leukocytoclastic vasculitis of the small dermal vessels characterized by infiltration of perivascular neutrophils, showing fragmentation of nuclei, resulting in fibrinoid necrosis9. Differential diagnoses include urticaria, erythema multiforme, idiopathic thrombocytopenia, meningococcemia, Kawasaki disease, Sweet syndrome, Gianotti-Crosti disease, drug-induced vasculitis, child abuse and trauma-induced purpura, but Henoch-Schönlein purpura is probably the most important9,10. Initially interpreted as a variant of Henoch-Schnölein purpura, now it is considered a distinct disease with different epidemiological, clinical and pathological features4.\n\nTreatment of AHEI remains controversial; conservative management is the most frequently approach, because this disease is a self-limited condition4,7,9. AHEI certainly represents a challenge for the pediatrician at the emergency department and it requires, at least initially, a high level of suspicion for potentially serious pathologies that need adequate, urgent treatment such as for infectious (meningococcemia) or hematological diseases (autoimmune thrombocytopenia, coagulopathies). In fact, in our case some easily laboratory tests, such as complete blood count, prothrombin time, partial thromboplastin time, fibrinogen, D-dimer, C-reactive protein and procalcitonin tests were supportive in the rapid exclusion of these conditions. This approach, together with the typical benign course of the AHEI, quickly guaranteed the exclusion of these conditions, avoiding the execution of unnecessary and/or invasive diagnostic procedures and unrequired therapies.\n\nWe reported this case because it is expression of a rare and often under-recognized disease by pediatricians. A typical feature of AHEI is the discrepancy between dramatic cutaneous involvement and good clinical conditions of the affected children. This characteristic can reassure clinicians about the absence of a serious medical condition, but it is possible only through the knowledge of the disease.\n\n\nConclusion\n\nWe describe a case of AHEI with striking cutaneous involvement that resolved spontaneously. AHEI is an uncommon disease, often under-recognized. For this reason, it is crucial that physicians have the skill to recognize this self-limited disease to avoid parental anxiety and inappropriate procedures or therapies.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWe received written informed consent from the patient’s family for the publication of this manuscript.", "appendix": "References\n\nSnow IM: Purpura, urticaria and angioneurotic edema of the hands and feet in a nursing baby. JAMA. 1913; 61(1): 18–9. Publisher Full Text\n\nFiore E, Rizzi M, Ragazzi M, et al.: Acute hemorrhagic edema of young children (cockade purpura and edema): a case series and systematic review. J Am Acad Dermatol. 2008; 59(4): 684–695. PubMed Abstract \| Publisher Full Text\n\nHomme JL, Block JM: Acute hemorrhagic edema of infancy and common mimics. Am J Emerg Med. 2016; 34(5): 936.e3–6. PubMed Abstract \| Publisher Full Text\n\nCunha DF, Darcie AL, Benevides GN, et al.: Acute Hemorrhagic Edema of Infancy: an unusual diagnosis for the general pediatrician. Autopsy Case Rep. 2015; 5(3): 37–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFotis L, Nikorelou S, Lariou MS, et al.: Acute hemorrhagic edema of infancy: a frightening but benign disease. Clin Pediatr (Phila). 2012; 51(4): 391–3. PubMed Abstract \| Publisher Full Text\n\nMiorin E, Meneghini A, Don B, et al.: Edema emorragico acuto del lattante, descrizione di un caso clinico e revisione della letteratura. Medico e Bambino pagine elettroniche. 2002; 5(3). Reference Source\n\nAlhammadi AH, Adel A, Hendaus MA: Acute hemorrhagic edema of infancy: a worrisome presentation, but benign course. Clin Cosmet Investig Dermatol. 2013; 6: 197–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrause I, Lazarov A, Rachmel A, et al.: Acute haemorrhagic oedema of infancy, a benign variant of leucocytoclastic vasculitis. Acta Paediatr. 1996; 85(1): 114–7. PubMed Abstract \| Publisher Full Text\n\nSavino F, Lupica MM, Tarasco V, et al.: Acute hemorrhagic edema of infancy: a troubling cutaneous presentation with a self-limiting course. Pediatr Dermatol. 2013; 30(6): e149–52. PubMed Abstract \| Publisher Full Text\n\nTagliabue A, Bettinelli A, Cogliati F: Edema acuto emorragico della prima infanzia (porpora di seidlmayer). Medico e Bambino pagine elettroniche. 2009; 12(6). Reference Source" }	[ { "id": "57576", "date": "09 Dec 2019", "name": "Mario Bianchetti", "expertise": [ "Reviewer Expertise Pediatric kidney disease specialist with broad expertise with “childhood vasculitis”" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report entitled “Acute hemorrhagic edema of infancy (Seidlmayer purpura) – a dramatic presentation for a benign disease” is interesting and rather well-presented. The following 12 changes might ameliorate the attractiveness of the report:\nThe term “Seidlmayer purpura” is popular in Germany (and perhaps in Italy). Most authors use the term “Finkelstein-Seidlmayer purpura” (Finkelstein reported his case in 1929, Seidlmayer in 1939). Please modify the manuscript accordingly.\n\nPresent manuscript: “…is a rare cutaneous leukocytoclastic vasculitis.”\nMy suggestion: “…is a rare small vessel cutaneous leukocytoclastic vasculitis affecting infants 4 weeks to 24 months of age.”\n\nStatement: “Initially it has been interpreted as a Henoch-Schönlein purpura variant, but now it is considered a distinct disease.” Remove this statement from the Introduction section (this issue is addressed in the Discussion section).\n\nStatement: “An 11-month-old girl from France presented with fever and oval purpuric lesions on the face and extremities, which had appeared one hour before.” Please provide temperature value or at least “moderate” respectively “high” fever (furthermore: state either “axillary” or “rectal”).\n\nSkin lesions are sometimes painful in Finkelstein-Seidlmayer disease. I wonder if lesions were painful in this case (important information).\n\nWas pruritus present (it is usually absent in this vasculitis)?\n\nStatement: “Many organisms including adenovirus, varicella zoster virus, cytomegalovirus, rotavirus, herpes simplex virus, tuberculosis, streptococci, and staphylococci are associated with AHEI.”\nMy suggestion: remove “tuberculosis” (because tuberculosis has never been associated with this vasculitis after 1969).\n\nStatement: “Vaccinations or medications could also trigger AHEI”. However, no cases of AHEI have been causally associated, to the best of my knowledge, with the medications.\nMy suggestion: modify the statement accordingly.\n\nStatement: “In the majority of cases there is no visceral involvement and the disease is benign and self-limited.”\nMy suggestion: “In the vast majority of cases there is no visceral involvement and the disease is benign and self-limited.\n\nThe statement “Treatment of AHEI remains controversial; conservative management is the most frequently approach, because this disease is a self-limited condition” is poorly presented.\n\nMy suggestion: AHEI is a self-limited condition. The lesions disappear within 3 weeks in ≥80% of the cases. Systemic steroids, non-steroidal anti-inflammatory drugs or antihistamines are prescribed in many cases. It is currently assumed, however, that these drugs do not alter the disease course. Once the diagnosis is established, supportive care and reassurance are advised.\n\nThe total length of the current report is excessive (please reduce the total length by ≥10%).\n\nReferences: - Please remove references number 6 (Miorin) and number 10 (Tagliabue) because they were not published in English language (local journals). - Please add the following new reference: Lava et al. (20171) (recent review, more up-to-date than current reference number 2.)\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "57605", "date": "27 Dec 2019", "name": "Antonio Francesco Urbino", "expertise": [ "Reviewer Expertise Pediatric Emergency specialist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is interesting, well written and provides complete information for paediatricians who have never met the acute hemorrhagic edema of infancy in their practice. Just a few minor observations:\nThe acute hemorrhagic edema of infancy is more known as Finkelstein disease than as Seidlmayer purpura: I suggest to change the eponym both in the introduction and in the keywords.\n\nThe French origin of the girl does not influence the diagnosis and is redundant: please remove it.\n\nIn the discussion section, Finkelstein disease is said to be distinguished from Gianotti-Crosti disease, whose papular and not usually purpuric presentation is quite different: I suggest to remove it.\n\nAuthors state the the Finkelstein disease is more frequent in winter time: when did the girl arrived in the Emergency Department?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1771
https://f1000research.com/articles/8-1768/v1	17 Oct 19	{ "type": "Brief Report", "title": "4T don't stand for tacos: An analysis of food and environmental security considerations in the new Mexican government's agricultural agenda", "authors": [ "Erick de la Barrera", "Ernesto A. Villalvazo-Figueroa", "Edison A. Díaz-Álvarez", "Itzel A. Aguirre-Pérez", "Alexis A. Alcázar-Aragón", "Ángela A. Alvarado-Rodríguez", "Daniella Americano-Guerrero", "Alejandra Andrade-Campos", "Andrea Arias-González", "Rodrigo A. Arriaga-Suárez", "Rodrigo Burciaga", "K. Alejandra Cabrera-Cuamba", "Beatriz A. Cancio-Coyac", "Celeste Contreras-Guízar", "Sofía Cristóbal-Reyes", "T. Alheli Cruz", "J. Pablo del-Río-Gómez", "Carmen Díaz-Trasviña", "Arielle Gaona-Villa", "Jaritzi García-García", "V. Viridiana González-Estrada", "Isis Granados-García", "Bruno A. Ibarra-Otero", "Julio E. Lara-Tello", "Pilar Martínez-Mota-Velasco", "Tziraat Molina-Salgado", "Ananda M. Monteforte-Cariño", "Alan R. Ortega Arroyo", "Amaranta Paz-Navarro", "J. Pamela Pérez-Ríos", "Daniel Piña-Torres", "Cynthia Ramos-Ortíz", "M. Vianey Rangel-César", "Valeria Reyes-Ávila", "Cecilia L. Reyes-Cervantes", "Pamela Saavedra-Tovar", "F. Aldair Valencia-Vázquez", "Alejandra Villaseñor-Villanueva", "Ernesto A. Villalvazo-Figueroa", "Edison A. Díaz-Álvarez", "Itzel A. Aguirre-Pérez", "Alexis A. Alcázar-Aragón", "Ángela A. Alvarado-Rodríguez", "Daniella Americano-Guerrero", "Alejandra Andrade-Campos", "Andrea Arias-González", "Rodrigo A. Arriaga-Suárez", "Rodrigo Burciaga", "K. Alejandra Cabrera-Cuamba", "Beatriz A. Cancio-Coyac", "Celeste Contreras-Guízar", "Sofía Cristóbal-Reyes", "T. Alheli Cruz", "J. Pablo del-Río-Gómez", "Carmen Díaz-Trasviña", "Arielle Gaona-Villa", "Jaritzi García-García", "V. Viridiana González-Estrada", "Isis Granados-García", "Bruno A. Ibarra-Otero", "Julio E. Lara-Tello", "Pilar Martínez-Mota-Velasco", "Tziraat Molina-Salgado", "Ananda M. Monteforte-Cariño", "Alan R. Ortega Arroyo", "Amaranta Paz-Navarro", "J. Pamela Pérez-Ríos", "Daniel Piña-Torres", "Cynthia Ramos-Ortíz", "M. Vianey Rangel-César", "Valeria Reyes-Ávila", "Cecilia L. Reyes-Cervantes", "Pamela Saavedra-Tovar", "F. Aldair Valencia-Vázquez", "Alejandra Villaseñor-Villanueva" ], "abstract": "On his first day in office, on 1 December 2018, freshman President of Mexico, Andrés Manuel López Obrador (AMLO) delivered a speech outlining 100 policy priorities of his administration. The present study analyzed the contributions of this government’s program relating to food security and their environmental implications, and whether they contributed to strengthen the state or improved human security, considering that the poor and marginalized were at the center of AMLO's campaign. In total 45 policy priorities were geared to consolidate the state, while 55 contributed to improving human security. Only six were related to food security, including stipends to food producers and purchasing grains at guaranteed prices, a fertilizer distribution program and subsidies for cattle husbandry and fisheries/aquaculture. These programs contributed to advancing 10 of the 17 Sustainable Development Goals, especially those related to Zero Hunger and Reduced Inequalities. Various policy programs had explicit considerations towards climate change and land degradation, including the exclusion of natural protected areas from agricultural subsidies, and recognized that food production is vulnerable to climate change. The four agricultural programs analyzed may advance AMLO’s goal of avoiding food imports, while curbing rural poverty. However, available evidence is mixed regarding animal acquisition loans, which are likely to have adverse environmental outcomes. Finally, the program for developing agroforestry operations is already contributing to deforestation, and further ecosystem degradation is most likely to occur from the introduction of timber and fruit species to natural forests as this program does not preclude the inclusion of recently cleared plots. If human development goals are to be reached, along with fulfilling the international commitments on sustainable development and environmental conservation, policies need to be implemented that simultaneously tend to a booming transnational industry, while bringing forward the rural poor, who amount to nearly half of the country's population.", "keywords": [ "agricultural policy", "evidence-based policy", "human security", "Mexico", "national security", "socioecological systems", "sustainable development goals" ], "content": "Introduction\n\nOn 1 July 2018, third-time candidate, Andrés Manuel López Obrador (AMLO) was elected president of Mexico in a historical election, obtaining 53% of the vote, a result not seen for various decades. AMLO ran on an anti-neoliberal and anti-establishment campaign, dubbing his regime as the \"4T\" or Fourth Transformation of the Republic, after the wars of Independence (1810), Reform (1857), and Revolution (1910), each one of which redefined Mexico's history. Indeed, AMLO poses that his administration will be equally transformative, but peaceful.\n\nImmediately after being sworn-in in front of Congress, President AMLO headed to the Zócalo, Mexico City's historical central square, where he received a crosier from indigenous leaders and delivered a lengthy speech to a numerous crowd that outlined the 100 priorities to be pursued by his administration. Such a government agenda was ratified in May 2019, when the 2019–2024 National Development Plan was issued1.\n\nRegarding agriculture and rural development, Mexico lives an impossible duplicity. On one hand, Mexico is the 12th largest global food exporter and its agricultural sector contributes ca. 5% of the national gross domestic product, holding the world's 15th largest economy2–4. On the other hand, 61% of its declining and aging rural population lives in poverty, one third of whom are in extreme poverty, while being tasked with producing food for over 120 million Mexicans5. A perception of government abandonment of rural dwellers has become widespread and even commonplace in Mexico, including the notion that peasants are a mere electoral clientele, whose poverty is by design6,7. During his campaign AMLO offered to implement policies to improve rural wellbeing and increase the productivity of small agricultural producers in order to reach food self-sufficiency, invoking food sovereignty as a matter of national security8. This paper considers the food and environmental security implications of the new President's government agenda.\n\n\nMethods\n\nThe authors of this paper participated in an upper division course about \"Sustainability and food security,\" an elective offered at the Escuela Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma de México. In particular, we are college juniors and seniors majoring in environmental sciences, ecology, and agro-genomic sciences and their instructors.\n\nWe utilized the Human Security Framework9 to analyze AMLO's inaugural speech10, which outlined 100 policy priorities for his administration. This framework is appropriate given its explicit focus on human rights, freedoms, and poverty alleviation, which were central in AMLO's discourse during his campaign. In brief, we determined the main area of impact for each enunciated policy, according to the human security components of economic, food, health, environmental, personal, community, and political security9. Those policies that did not contribute directly to human security were classified according to how they strengthen the State, either by supporting the armed forces, reducing government spending, combating corruption, or improving government administration11.\n\nFor the policies related to food security, we investigated whether the corresponding ministry had officially published the program, either as a concept or with an actual budget allocation for the 2019 fiscal year. When rules of operation existed, the policies were analyzed for their potential repercussions on improving food security, their environmental impacts, and their contributions towards reaching the Sustainable Development Goals. For each program the objectives and rules of operation were identified to gathering evidence from similar programs, either in Mexico or elsewhere to assess their coherence. In addition, potential outcomes for the programs that were implemented where constructed based on the literature and evidence gathered for each program that is already being implemented to assess whether its stated objectives can be reached via the published rules of operation12,13.\n\n\nResults\n\nIn total, 45 of the 100 policies outlined by AMLO were geared to strengthen his government (Figure 1). In particular, three of these policies explicitly consider the armed forces, 12 are austerity measures, 11 policies intend to combat corruption, and 19 are designed to improve government administration. The remaining 55 policies are related to human security. In particular, 30 deal with economic or political security, 8 with environmental security, 7 with food security, 5 with personal security, 4 with community security, and one with health security (Figure 1).\n\nFor contributions to national security, the policies were segregated depending on whether they were geared to strengthen the armed forces, to combat corruption, to reduce public spending, or to improve public administration. For the items related to human security, they were assigned to the most relevant component. Numbers in parenthesis indicate the policy count related to each component of national or human security. (See Extended data for details on policies and classification).\n\nSix of the seven food-related policies already had budget and operational rules for the 2019 fiscal year (Table 1). Five of these programs were issued by the Secretary of Agriculture and Rural Development (SADER) and one was issued by the Secretary of Wellbeing. The seventh policy is an intended generalized ban on the use of transgenic seeds. Because this is not an proper program with rules of operation nor a bill under legislative evaluation, and given its contentious nature, its analysis was excluded from the present work and will be discussed elsewhere.\n\nData are from the 2019 fiscal year Federal Budget and the corresponding rules of operation for each program.\n\nThree of the SADER programs (policies 19–21 from Table 1) intend to improve the agricultural productivity of small producers of food (as opposed to producers of cash crops or forage). In particular, Producción para el bienestar (Production for wellbeing) has a budget of MXN$ 9 billion for the 2019 fiscal year to pursue a main objective of fomenting the production of food, i.e. maize, beans, wheat, and rice, by smallholders of up to 20 hectares.\n\nWith a budget of MXN$ 6 billion, the guaranteed prices program will purchase up to 20 tons of white maize and 120 tons of wheat from \"small farmers,\" whose production units do not exceed 5 hectares, and up to 15 liters of milk per cow from small and medium production units throughout the year, and will sell such products to final consumers at a subsidized price. This program is intended to shield basic cereal production from low international commodity prices, as an effort to incentivize their cultivation.\n\nThe third program for improving agricultural productivity is a fertilizer distribution program, which, according to the President, will prevent soil degradation (Table 1). This program has a budget of MXN$ 1.5 billion and will distribute up to 450 tons of fertilizer per hectare, for up to three hectares per participant. The rules of operation consider both the loss of soil fertility from long-term agricultural activity and the noxious effects of excessive or inadequate fertilizer application. For this reason, the program considers the formulation of different fertilizers depending on regional needs, as well as the distribution of biofertilizers where appropriate.\n\nThe two remaining SADER programs are designed to increase the amount of animal protein available for Mexican diets. The first one is a program of unsecured, zero-interest loans for purchasing up to 1 million cows and 50,000 bulls or \"equivalent\" in pigs, goats, sheep, or bees, with an allocation of MXN$ 4 billion in 2019 (Table 1). While the program allows the disbursement of cash to subsidize the acquisition of different types of animals and breeding related services, the wording of the rules of operation suggests that the preferred currency are actual pregnant females and that the eventual payment will be with their offspring.\n\nWith MXN$ 1 billion allotted for the 2019 fiscal year, the last SADER program is designed to improve the productivity of national fisheries and aquaculture (Table 1). This program considers funding various activities, such as boat maintenance or acquisition of fishing nets and equipment. However, its main focus is the acquisition or maintenance of refrigeration equipment for in-boat and at-the-shore conservation of catches and aquacultural production, thus allowing the capture of larger amounts of fish while increasing their shelf life.\n\nSembrando vida (Sowing life) is the flagship program of the Secretary of Wellbeing, which has the main objective of reforesting one million hectares with fruit trees or timber species and was announced by AMLO early during his campaign (Table 1). With a budget of MXN$ 500 million for the 2019 fiscal year, this program will foment the development of agroforestry practices among the poorest communities of 19 states, as long as an adequate environment is available and that each participant allots a 2.5 hectare plot to the program. In return, the participants will be granted a monthly stipend of MXN$ 5,000, 10% of which must be placed in a savings account. In addition to the stipend, participants will be supplied with plants, agricultural materials, and a toolkit to work the land and have access to the community's plant nurseries and fertilizer \"biofactories.\" At the center of this agroforestry program is a community-building intervention called the Comunidades de aprendizaje campesino (Communities for peasant learning; CACs), which are groups of 25 participants from selected localities that co-participate in compulsory training, capacity building, plant propagation, and other communal activities dictated by the program.\n\nThe six agricultural policy priorities of the 4T considered in the present work were aligned with 10 of the 17 SDGs (Table 1). In particular, the goals of Zero hunger (SDG 2) and Reduced inequalities (SDG 10) overlapped with the six programs, followed by Decent work and economic growth (SDG 8), which overlapped with the objectives of five of the policies. In contrast, Responsible consumption and production (SDG 12) was only relevant under Sembrando vida (item 23 from Table 1), while Life below water (SDG 14) only related to the fisheries program (item 22 from Table 1).\n\n\nDiscussion\n\nDuring his electoral campaign, AMLO vowed that Mexico will reach food sufficiency during his administration. This would reverse the pattern that emerged after 20 years of the North American Free Trade Agreement, during which the country has been importing increasing amounts of maize, given the high volume and low price of the production in the USA, while developing a vigorous horticultural industry for exports that often contributes with ca. 5% of the country's annual GDP2,4. In addition, AMLO's discourse has the wellbeing of the poorest at its core, so by improving the livelihoods of rural dwellers, a major contribution towards poverty reduction should be achieved.\n\nThe Producción para el bienestar and Guaranteed prices programs are designed to support small producers (with up to 5 hectares under maize cultivation), who collectively contribute with 25% of the national maize production21. However, given the low international prices for North American corn, 47% lower than the guaranteed price for the 2019 fiscal year, small production units cannot bear the cost of production from a commercial standpoint22. In fact, the harvest from most of these small production units is usually destined to self-consumption, and the tenants usually have communal land rights23. Both programs also intend to decrease the dependency on cereal imports and reduce rural poverty via direct cash transfers, with a special focus on southern Mexico, where the higher rates of poverty are concentrated5. Both programs can be traced back to previous agricultural policies. In particular, Producción para el bienestar is the most recent version of PROCAMPO (created in 1994 under the North American Free Trade Agreement) and PROAGRO Productivo (issued in 2014), whose main objective was to reduce the income disparity between US and Mexican cereal producers24. In turn, the country had implemented guaranteed price policies since the mid 1950's, which were precisely substituted by the direct cash transfers of PROCAMPO24,25. The liberalization of the agricultural sector has had multiple consequences, including reductions in both the net income of small producers and in the price of cereals26,27.\n\nDuring his campaign, AMLO announced a program for distributing fertilizer, indicating a main purpose of avoiding soil \"damage\". This sounds counterintuitive considering that it has been widely documented that an overuse of agrochemicals, particularly nitrogenous fertilizer, can lead to soil degradation and widespread environmental damages28–30. However, the rules of operation for the program are explicit in considering both a loss of soil fertility following intensive agriculture and the opening of nitrogenous fertilizer factories. That the State manufactures fertilizer is not a new idea; the refurbishing of a fertilizer plant was announced early in 2013 by former President Enrique Peña Nieto, during his first year in office. However, construction works did not begin until the end of 2014 and were eventually halted due to very strong suspicion of corruption that keeps emerging well into the current administration31. Considering that nearly half of the soils in Mexico already have some degree of degradation and erosion32, especially those from agricultural regions, land protection practices should be transferred to producers alongside the agrochemicals, including, for instance, conservation agriculture and intercropping of nitrogen-fixing species, or the use of ferti-irrigation, a component of precision agriculture.\n\nSembrando vida, the flagship program of the Secretary of Wellbeing, is modeled after the Tosepan cooperative33, of which the Secretary, Ms. María Luisa Albores, is a member. Indeed, this indigenous organization from Cuetzalan, Puebla has become one of the most emblematic and successful agroforestry operations in the country, including the production of coffee, spices, biofertilizers, ecotourism, and even a credit union that procures and disburses government grants and loans. The latter is precisely the reason why participants of the program have the obligation to save 10% of their stipend, a fraction of such a saving has to be deposited in a trust fund that appears to be what will eventually become the Banco del Bienestar (Bank for Wellbeing), which will disburse all the government direct cash transfers. One of the basic requirements of the program is that participants are owners or have legal holding of 2.5 hectares, which is reasonable considering that agroforestry operations can take up to several years to become productive. However, a justification for this program indicates that the target audience are the poorest among the rural poor, which is not necessarily the case, as the poorest usually lack access to land, communal or otherwise. Unlike the SADER programs, Sembrando Vida does not have safeguards to prevent that people will clear forest and enroll the resulting \"degraded\" plots of land in the program. In fact, some reports indicate that the clearing of forests is already underway to enroll these \"degraded\" plots in the program34. In addition, the fact that groups of 25 participants are required to cooperate through the CACs will require careful implementation. While these community based interventions for rural development have been most successful in places like Cuetzalan with the Tosepan cooperative or the communities in the state of Michoacán of Cherán and Nuevo San Juan Paricutiro35, within the monarch butterfly natural protected area, numerous factors can preclude adequate development. Care must always be taken when replicating and scaling up programs that are successful in certain contexts if they are expected to be implemented successfully elsewhere36. This should be considered by the President especially considering that a multinational version of Sembrando Vida is at the core of his intended intervention in Central America to curb the flux of political and environmental migrants seeking refuge in the USA, whose concentration at the Mexico-US border has led to a humanitarian crisis and has become a source of tension between these countries18,37.\n\nMexico is currently a net importer of meat and animal products38. In this case, the animal loans program aims to increase the domestic production of animal protein, in compliance with the administration's objective of reaching food sovereignty through self-sufficiency. Animal products are indeed a main source of protein in many Mexican diets and, in some cases, the primary source of nutrients, especially in arid and semiarid regions where the environment does not permit agriculture, so animal mediated transformation of inedible plants essentially constitutes the only available food39. However, it is unclear whether this kind of program actually works. On one hand, based solely on anecdotal evidence, a number of journalists and pundits have denounced that the animals are usually eaten within a few months, instead of being used for reproduction. On the other hand, animal-rights organizations have frequently raised concerns against animal gifting, including the cost of rearing animals, which can be exceedingly onerous for poor households, who usually cannot afford feed, water, and veterinary care40. What are undeniable are the environmental consequences of \"loaning\" 1 million cows, whose land, water, and food requirements will add on to the resources already required by the 32 million cows currently raised in Mexico21,41.\n\nDespite having access to two oceans, along 10,000 km of the coasts of one third of the 32 states, fish and seafood consumption is relatively low in Mexico21. This fuels the perception that the country should increase its fishing efforts to improve the availability of marine sources of protein, including the processing of incidental catches for human consumption42. However, fisheries have been depleted worldwide, including in Mexico, which has led to the establishment of aquacultural operations, which potentially have a lower environmental impact than fishing at sea43. By improving the refrigeration capacity of fisheries and aquacultural units, availability of marine protein will increase throughout the year. Care must be taken during implementation of new aquacultural units under this program to avoid environmental damage, which has been amply documented in the existing shrimp farms along the coast of Sinaloa, including from mangrove clearing and by pollution of the surrounding land and sea44.\n\nAn unexpected finding during our review of the rules of operation of the SADER programs for 2019, was that several of such programs include environmental safeguards to prepare agriculture for impending climate change, but that also explicitly acknowledge that a spillover of agriculture can be damaging to the environment. In fact, Fomento a la agricultura45 explicitly excludes plots that are already registered in ecosystem service payment schemes or other biodiversity protection programs in order to prevent land transformation. Moreover, participating plots should already be enrolled in a productive-land plots registry, which attenuates the risk of transforming natural lands for inclusion, a problem that has already been documented for Sembrando vida34.\n\nFomento a la agricultura also considers that Mexican farmers seldom contract agricultural insurance, given high premiums and limited coverage, but also that an impending increase in the frequency and severity of extreme weather events requires better access to insurance that is negotiated in favorable terms for small producers, based on an accurate risk assessment. In this respect, two important lessons emerged from the landing of hurricane Patricia in 2015, the most intense hurricane in recorded history so far. First, that adequate risk assessment and planning are crucial in the event of so called 'acts of god'. Indeed, no casualties were recorded in Mexico as the hurricane hit land, despite severe damage to the forest, crops and plantations, housing, and infrastructure along its path, especially in the central coast of Jalisco, at the point of landing. The second lesson was actually a pleasant surprise, even for most experts. Indeed, after landing as a category 5 hurricane, Patricia reached the city of Guadalajara, the second largest city in Mexico, as a mere tropical storm. The Sierra Madre Occidental was able to prevent a catastrophe similar to those from the Philippines and Haiti, but only because minimum ecosystem integrity was in place. Otherwise, the hurricane could have been lethal for numerous communities, both coastal and inland, as initially forecasted. In stark contrast, reports of landslides that cut off several villages in the highly deforested municipality of Arteaga, Michoacán (as far as 350 km from the landing point!), illustrate the vulnerability of degraded ecosystems46.\n\nClimate change scenarios for Mexico anticipate a reduction in annual precipitation that is likely to be more damaging for agriculture than it would from a mere air temperature increase47,48. Considering that more than half of the country is already arid or semiarid, marginal for agriculture at best, and that up to 80% of agricultural operations are rain-fed, climate change poses a severe risk for those states with agriculture-based economies and for the whole country's food security2,4,23. In contrast, the few agricultural operations that have access to irrigation consume almost 80% of the country's available fresh water, most of which is lost during transport along open-air irrigation canals and delivery with inefficient methods such as flooding and large area aspersion49. The sole access to irrigation improves agricultural productivity; for instance, maize yields increase by a 3.5-fold in contrast with rain-fed agriculture, thus increasing infrastructure coverage and a simultaneous improvement in agricultural water use efficiency would result in higher yields while potentially reducing the actual demand for water4. Water conservation is indeed a national priority and its further privatization should be prevented, as offered by AMLO towards the end of his speech (item 76)10 and implemented within Fomento a la agricultura. This could certainly contribute to a centralized and more efficient water management, which is crucial for a country that, as noted above, has water limitations in ample regions and that is a principal global exporter of embedded water through agricultural commodities50.\n\n\nFinal considerations: agricultural policies for sustainable human development\n\nHalf of the policy priorities posed by President López Obrador for his administration may explicitly consider the various components of human security, mostly focused on aspects of economic and political securities. Four of the six policies related to food security considered here are designed to improve food production by small farmers. The respective rules of operation appear to advance the objectives of each program and could contribute to a principal goal of the president of reaching food self-sufficiency and sovereignty. However, for their implementation, environmental safeguards should be strictly observed, including the prohibition of clearing more forests and a proper and strict use of agrochemicals. For the case of the unsecured loan program for acquiring reproductive cattle, the evidence indicates mixed results and the program should be carefully monitored, both to ensure that the animals actually increase the national production of meat and for mitigating the environmental degradation that is most likely to result. The most objectionable of the policies was Sembrando vida, AMLO's flagship program for rural development that is also being exported to Central America. This program has already induced the clearing of forests in the states where it has been implemented and the replacement of native species with timber and fruit trees will most likely result in further ecosystem degradation.\n\nA constitutionally mandated wellbeing for the rural poor has implications for national security in the form of water and food security and sovereignty, as well as very strong bi-directional interactions with various components of global environmental change. After all, the most intense and long-standing relationship that humans have had with nature has been through food11,51. Policies and programs are needed that simultaneously tend to a booming transnational industry, bring forward nearly half of the population that has been historically disenfranchised, and reduce biodiversity loss and environmental degradation52. In addition, an increasing urban population that is greatly disconnected from production, but which demands larger amounts of higher quality food needs to be taken into consideration. Integrative and evidence-based agricultural policies are indeed necessary for successfully facing these emerging challenges and reaching the goal of food self-sufficiency. Their design and implementation should consider contributions of various scientific disciplines in addition to displaying a profound social sensibility if a substantial improvement of peasant quality of life is sought.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nResearchGate: delaBarreraetal-4THumanSecurity, https://doi.org/10.13140/RG.2.2.23419.0592153.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nDiario Oficial de la Federación: Plan Nacional de Desarrollo. (Accessed on 24 August 2019). Reference Source\n\nInstituto Nacional de Estadística y Geografía: Producto Interno Bruto y Cuentas Nacionales. (Accessed on 24 August 2019). Reference Source\n\nOrganization for Economic Co-operation and Development: OECD Data: Gross Domestic Product. (Accessed on 24 August 2019). 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Intl For Rev. 2015; 17(S1): 65–84. Publisher Full Text\n\nZarazúa-Escobar JA, Almaguer-Vargas G, Ocampo-Ledezma JG: El programa de apoyos directos al campo (PROCAMPO) y su impacto sobre la gestión del conocimiento productivo y comercial del estado de México. Agricultura Sociedad y Desarrollo. 2011; 8(1): 89–105. Reference Source\n\nSantoyo S: La política de precios de garantía: antecedentes, situación actual y perspectivas. Estud Demogr Urban. 1977; 11: 77–98. Publisher Full Text\n\nTrujillo Félix JD, Schwentesius Rindermann Cruz R, Gómez Cruz MA: Las políticas agrícolas de EUA, la Unión Europea y México. Resultados de las reformas. Rev Anal Econ. 2007; 50: 35–56. Reference Source\n\nHernández Ortíz J, Martínez Damián MA: Effects of the change from support price to PROCAMPO on producer prices, excluding imports effects. Rev Fitotec Mex. 2009; 32(2): 155–159. Reference Source\n\nSala OE, Chapin FS 3rd, Armesto JJ, et al.: Global biodiversity scenarios for the year 2100. Science. 2000; 287(5459): 1770–1774. PubMed Abstract \| Publisher Full Text\n\nShaviv A, Mikkelsen RL: Controlled-release fertilizers to increase efficiency of nutrient use and minimize environmental degradation - A review. Fert Res. 1993; 35(1–2): 1–12. Publisher Full Text\n\nPhoenix GK, Hicks WK, Cinderby S, et al.: Atmospheric nitrogen deposition in world biodiversity hotspots: the need for a greater global perspective in assessing N deposition impacts. Glob Change Biol. 2006; 12(3): 470–476. Publisher Full Text\n\nAgro nitrogenados y Fertinal, plantas con pérdidas y en desuso. (Accessed 24 August 2019). Reference Source\n\nBolaños GM, Paz FP, Cruz GC, et al.: Mapa de erosión de los suelos de México y posibles implicaciones en el almacenamiento de carbono orgánico del suelo. Terra Latinoam. 2016; 34(3): 271–288. Reference Source\n\nCooperativa Tosepan Titataniske: Proyectos y estructura de la Cooperativa Tosepan Titataniske. Revista Vinculando. 2004. (Accessed on 11 October 2019). 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Reference Source\n\nWillet W, Rockström J, Loken B, et al.: Food in the Anthropocene: the EAT-Lancet Commission on healthy diets from sustainable food systems. Lancet. 2019; 393(10170): 447–492. PubMed Abstract \| Publisher Full Text\n\nCifuentes L, Frías M, Torres GM, et al.: El Océano y sus recursos: X. Pesquerías. 1st ed Fondo de Cultura Económica: Mexico, 1990. Reference Source\n\nGodfray HCJ, Beddington JR, Crute IR, et al.: Food security: the challenge of feeding 9 billion people. Science. 2012; 327(5967): 812–818. PubMed Abstract \| Publisher Full Text\n\nPaéz-Osuna F, Guerrero-Galván R, Ruiz-Fernández AC: The environmental impact of shrimp aquaculture and the coastal pollution in Mexico. Marine Pollution Bulletin. 1998; 36: 65–75. Publisher Full Text\n\nReglas de Operación del programa Fomento a la Agricultura de la Secretaría de Agricultura y Desarrollo Rural. (Accesed on 29 August 2019). Reference Source\n\nHuracán Patricia: En Arteaga, 400 comunidades aisladas y 100 viviendas dañadas. (Accesed on Ausgust 2019). Reference Source\n\nSáenz-Romero C, Rehfeldt GE, Crookston NL, et al.: Spline models of contemporary, 2030, 2060 and 2090 climates for Mexico and their use in understanding climate-change impacts on the vegetation. Clim Change. 2010; 102(3–4): 595–623. Publisher Full Text\n\nConde C, Ferrer R, Gay C, et al.: Impactos del cambio climático en la agricultura en México. In Cambio Climático: Una visión desde México., Martínez J., Fernández A., Eds.; Instituto Nacional de Ecología: Mexico City, Mexico. 2004. Reference Source\n\nComisión Nacional del Agua: Estadísticas del Agua en México. 1st ed CONAGUA: Mexico, 2014. Reference Source\n\nDalin C, Wada Y, Kastner T, et al.: Groundwater depletion embedded in international food trade. Nature. 2017; 543(7647): 700–704. PubMed Abstract \| Publisher Full Text\n\nde la Barrera E: COP-eration for global food security [version 1; peer review: 2 approved]. F1000 Res. 2016; 5: 2814. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunguía-Rosas MA, Montiel S, Castillo-Burguete T, et al.: Investigación transdisciplinaria para reconciliar seguridad alimentaria, conservación biológica y bienestar. Revista Avance y Perspectiva. 2018; 4. Reference Source\n\nde la Barrera E, Díaz-Álvarez EA, Villalvazo-Figueroa EA: delaBarreraetal-4THumanSecurity. 2019. http://www.doi.org/10.13140/RG.2.2.23419.05921" }	[ { "id": "55339", "date": "04 Dec 2019", "name": "María-José Ibarrola-Rivas", "expertise": [ "Reviewer Expertise Environmental Sciences." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article links the 100 priorities for AMLO’s government to the SDG and the human and governmental security components. The idea is interesting and I think it has potential for a good quality especially one year after AMLO started as president. However, the authors should think carefully what is the aim of the paper: is it really linking to the SDG? Then frame your results in this approach. I give detailed comments below.\n\nTitle: I do not understand what does “don’t stand for tacos” mean in the context of the paper. It should be clear for the reader, so better explain it in the paper or change the title.\nIntroduction: There is not a literature review. You must show what studies have been done about “human and government security” with the human security framework, and with other approaches. Use other studies with similar research aims.\nWhy do you link them with the SDG? (The answer could be obvious, but you need to clarify it in your introduction).\nWhat is the aim and research question of your study? It should not only be about linking the policies with the SDG, what you want to say? What do you want to question?\nMethods: You use the “Human Security Framework”, but you do not describe what it is. Your reference of that framework is a 2-page-link of the “Resolution adopted by the General Assembly on 10 September 2012”. There is no framework explained here. Please give a clear description of what it is and how you decide that each of the 100 policy priorities is linked to each component of the national and human security; as well as for the SDG.\nResults: Figure 1: It is a very nice figure.\nIn the subsidies section: you mention: “450 tons of fertilizer per hectare, for up to three hectares per participant.” Check this value, this is enormous. Please check the units.\nAlso in this section. Could you elaborate more on “the rules of operation for loss of soil fertility and excessive use of fertilizer”? First it was mentioned that use of “subsidies for fertilizer” were to “prevent soil degradation”. How are they specifying that will be done “depending on the region”?\nIn the animal protein for the people:\nWhy bees? There are no proteins in honey.\n\nWho will receive these loans for increasing livestock heads?\nDiscussion: 2nd paragraph you mention that “In fact, the harvest from most of these small production units … have communal rights” but you do not elaborate why having communal land is a reason to not bear the costs.\nAbout the discussion on the fertilizer policies, in the results you mention that it has to be “depending on the region”. The problem with the fertilizer use is the overuse or misuse of them. So the policy should be done with a local-specific recommendation of what is needed. And indeed, as you mention, promote other practices to reduce soil degradation and erosion.\nDescribe what is the Tosepan cooperative.\nAbout the livestock policies, cite the journalist that mentioned that animals have been eaten and not used for reproduction.\nMention that “foment a la agricultura” is a program or a policy (same for the others), and translate it to English. In this respect, maybe you can add them in Table 1. That is easier to refer to.\nThe paragraph of “fomento a la agricultura” is not clear, you describe a lot about the hurricanes, but not very related with agricultural impacts or insurances, or what has been done in risk management. Also you mention a “First” and not a second etc. Check this sentence and make it relevant for the discussion.\nIn the discussion, you do not compare to any existing literature doing similar analysis of human or governmental security, or about policies related with SDG. So, this looks more like an essay than a scientific article. I think your paper has a strong potential and is very interesting but make it stronger. The last suggestion for the discussion: you give clear suggestions about the fertilizer policy, could you give specific suggestion to the other policies that you discuss?\n\nConclusion and discussion:\nYou do not discuss anything about the SDG. It seems like it was added up by force. You only discuss the components of human and government security. So think if the SDG are what you aim to discuss or not. If you do so, then put all your introduction and discussion in this framework.\n\nFinally, AMLO has been president for a year. Which of these policies have happened or started to take place? Is there something different? Or a preference to one of the components of your Figure 1?\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72989", "date": "03 Nov 2020", "name": "Nazirul Islam Sarker", "expertise": [ "Reviewer Expertise Food security", "public policy", "governance", "resilience", "disaster and environmental management." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments The topic focuses on the assessment of the Mexican government’s policy related to food security and environmental sustainability. It has practical implications for practioners, policymakers and researchers. But this manuscript requires substantial improvement in some aspects. The author is advised to consider following suggestions for its improvement.\nAbstract Abstract is not well structured. IMRAD style should be followed for structuring the abstract. It is better to avoid some unnecessary and irrelevant texts from abstract like “On his first day in office, on 1 December 2018, freshman President of Mexico, Andrés Manuel López Obrador (AMLO) delivered a speech outlining 100 policy priorities of his administration”.\nKey practical implication and recommendations should be included in the last part of the abstract.\nIntroduction\nThe importance of the study has been described nicely but introduction of some key issues like major dimensions of food security, environmental security and sustainability are absent. The authors are advised to add an explanation on “how government policy can influence on main components of food security like food availability, access, utilization and nutritional security as well as environmental sustainability?”.\n\nResearch gap is not clear. Can you add at least one paragraph and make clear explanation about research gap?\nMethodology This section is poor. There is no clear indication about the assessment approach. The authors are advised to describe the methodology step-by-step by explain how did you conduct this study. As we know, for policy analysis, main three criteria should be followed like analyzing policy options, implementation and inclusiveness consisting of accountability, transparency and peoples’ participation in the governing process.\nWhat criteria did you follow to assess the policy for food and environmental security?\n\nPlease avoid unnecessary text and include the main steps of your methodology.\nResults\n\nResults seem correct but it will be fine if authors revised the presentation according to the major dimensions of food and environmental security.\n\nFigure 1 is fine and self-explanatory.\nDiscussion Discussion section seems fine. It will be more logical if author focus on the major dimensions of food and environmental security.\nConclusion The author is advised to add conclusion section and add limitation under conclusion section.\nReferences Good.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1768
https://f1000research.com/articles/7-1663/v2	06 Feb 19	{ "type": "Research Article", "title": "Molecular identification and phylogenetic analysis of GABA-producing lactic acid bacteria isolated from indigenous dadih of West Sumatera, Indonesia", "authors": [ "Lili Anggraini", "Yetti Marlida", "Wizna Wizna", "Jamsari Jamsari", "Mirzah Mirzah", "Frederick Adzitey", "Nurul Huda", "Lili Anggraini", "Wizna Wizna", "Jamsari Jamsari", "Mirzah Mirzah", "Frederick Adzitey", "Nurul Huda" ], "abstract": "Background: Dadih (fermented buffalo milk) is a traditional Indonesian food originating from West Sumatra province. The fermentation process is carried out by lactic acid bacteria (LAB), which are naturally present in buffalo milk. Lactic acid bacteria have been reported as one of potential producers of γ-aminobutyric acid (GABA). GABA acts as a neurotransmitter inhibitor of the central nervous system. Methods: In this study, molecular identification and phylogenetic analysis of GABA producing LAB isolated from indigenous dadih of West Sumatera were determined. Identification of the GABA-producing LAB DS15 was based on conventional polymerase chain reaction. 16S rRNA gene sequence analysis was used to identify LAB DS15. Results: PCR of the 16S rRNA gene sequence of LAB DS15 gave an approximately 1400 bp amplicon. Phylogenetic analysis showed that LAB DS15 was Pediococcus acidilactici, with high similarity of 99% at 100% query coverage to Pediococcus acidilactici strain DSM 20284. Conclusions: It can be concluded that GABA producing LAB isolated from indigenous dadih was Pediococcus acidilactici.", "keywords": [ "indigenous dadih", "GABA", "LAB", "16S rRNA gene", "phylogenetic analysis" ], "content": "Introduction\n\nThe non-proteinogenic amino acid γ-aminobutyric acid (GABA) is widely found in bacteria, animals, plants, and fungi (Dhakal et al., 2012; Nonaka et al., 2017). GABA acts as a neurotransmitter inhibitor of the central nervous system (Olsen & Li, 2012). It is formed by decarboxylation of L-glutamate, a reaction catalyzed by an enzyme that depends on the peridoxal phosphate of decarboxylated L-glutamate (Murray et al., 2003). Lactic acid bacteria (LAB) have been reported as a potential producer of GABA (Seo et al., 2013; Wu & Shah, 2017). LAB are generally regarded as safe and non-pathogenic microbes, and has been referred to as ‘generally recognized as safe’. Therefore, GABA-producing LAB can be used directly in functional foods (Zhao et al., 2017). Some LAB can be found in the dairy industry for the production of cheese, yogurt, and other fermented milk products (Yamada et al., 2018).\n\nDadih (fermented buffalo milk) is an Indonesian traditional food originating from West Sumatra Province; it is an extremely popular dairy product in Bukittinggi, Padangpanjang, Solok, Lima Puluh Kota, and Tanah Datar, Indonesia (Surono, 2015). Dadih is made from buffalo milk which is fermented in bamboo for 24–48 hours. The fermentation process is carried out by LAB which are naturally present in buffalo milk (Rizqiati et al., 2015) and the environment (Wirawati et al., 2017). Studies have found that, the LAB strains present in dadih are generally Lactobacillus, Streptococcus, Leuconostoc and Lactococcus (Collado et al., 2007; Surono, 2003).\n\nExtraction of DNA is a basic principle in molecular analysis and it is one of the success factors in DNA amplification that is used in the analysis of genetic characters (Mustafa et al., 2016). Polymerase chain reaction (PCR) and phylogenetic analysis based on 16S rRNA gene sequences have been used for successful identification of isolates from various fermented food products (Malik et al., 2015). These molecular approaches have allowed Lactobacillus species to be reliably identified (Henry et al., 2015). This research was conducted to identify and to characterize GABA producing LAB isolated from indigenous dadih of West Sumatera based on 16 S rRNA gene sequence analysis.\n\n\nMethods\n\nThis study used lactic acid bacteria (LAB) DS15, a GABA-producing LAB isolated from dadih of West Sumatera origin. This bacterium was isolated previously according to the method described by Ali et al. (2009). The experiment was carried out at the Feed Technology Industry Laboratory, Faculty of Animal Science, Andalas University, West Sumatra, Indonesia. LAB DS15 was grown anaerobically in MRS medium (Merck, Darmstadt, Germany) at 30°C and stored for further analysis.\n\nIsolation of the total genome of LAB DS15 was done using Genomic DNA Mini Kit purchased from Invitrogen (PureLinkTM, USA) by following the manufacturer’s instructions. We used Lysozyme (PureLinkTM, USA) at a concentration of 20 mg/ml to break down the bacterial cell wall to improve protein or nucleic acid extraction efficiency.\n\nGenomic DNA of LAB DS15 was used for amplification of 16S rRNA gene. Amplification was done using forward primer 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3') and reverse primer 138R (5'-GGG CGG GGT GTA CAA GGC-3'). The reaction was carried out in a volume of 50 μl. The PCR mixture contained 22 μl of MQ, 25 μl DreamTaq Green DNA Polymerase (Thermo Fisher Scientific, USA), 1 μl of each forward and reverse primer (10 μM each, IDT synthesized) and 1 μl template. Amplification conditions were 5 minutes of preheating at 95°C, 30 seconds denaturation at 95°C, 30 seconds of primer annealing at 58°C, 1 minute extension step at 72°C and post cycling extension of 5 minutes at 72°C for 35 cycles. The reactions were carried out in a thermal cycler (Biometra's T-Personal Thermal Cycler, USA).\n\nPCR products were stored at 4°C for further examination using 1% agarose electrophoresis in 1X, 100 V TAE for 30 minutes. The DNA bands formed from electrophoresis process was visualized using UV transluminator.\n\nSequencing of the 16S rRNA gene was performed at the Laboratory of Medical Molecular Biology and Diagnostic, Indonesian Institute of Sciences, Jakarta. Sequencing results were edited (contig and peak chromatogram verification) using the SeqManTM II program. Analysis of 16S rRNA sequences of LAB DS15 was performed using NCBI BLAST. Multiple alignment was done using the ClustalX 2.1 program. BioEdit version 7.2.5 in edit mode to see the absence of an inverted sequence and align the sequence length. Kinship visualization was done using the combined phylogenetic tree of the MEGA 7.0.20 program with the Neighbor-Joining hood method (Saitou & Nei, 1987).\n\n\nResults and discussion\n\nThe identification of LAB DS15 to determine the strain was done based on 16S rRNA gene. The first step was amplification using PCR method, with the electrophoresis image shown in Supplementary File 1. The amplification process was carried out to obtain more copies of the 16S rRNA gene for the sequencing process. Analysis of sequencing results begun by aligning the base sequence of the 63F forward sequence and reverse 138R using the SegMan program. PCR of the 16S rRNA gene of LAB DS15 gave an approximately 1400 bp amplicon (Figure 1).\n\nM, DNA marker; 1, PCR product of LAB DS18.\n\nSaitou & Nei (1987) indicated that the evolutionary history of organisms can be known using the neighbour-joining method. Organisms within the same taxa are normally clustered together in the phylogenetic tree and have better bootstrap values (Felsenstein, 1985). In this study, we drew a phylogenetic tree to scale and determined the evolutionary distances using the p-distance method. A total of 26 nucleotide sequences and codon positions 1st + 2nd + and 3rd + noncoding were considered, using MEGA 7.0 as reported by Kumar et al. (2016) for evolutionary analyses.\n\nDNA sequencing results were analyzed using NCBI BLAST. According to Willey et al. (2009), 16S rRNA sequencing looks at the similarity of isolates to those already available in GenBank; this is one molecular detection method that is ideal enough to know the kinship relationship between bacteria because the 16S rRNA sequence is a gene found in all microbes and is indispensable in maintain life. The 16S rRNA gene sequencing identified the LAB DS15 to belong to the genus Pediococcus, forming a well-defined cluster with Pediococcus acidilactici. This cluster was recovered in 100% of bootstrap analysis. Pediococcus spp. are widely described as probiotics (Porto et al., 2017). Abbasiliasi et al. (2012) also found Pediococcus acidilactici in fermented milk products. Pediococcus acidilactici are important LAB which have been used as starter cultures in meat, vegetable and dairy fermentation causing characteristic flavor changes, improving hygiene and extending the shelf life of these products (Mora et al., 1997; Porto et al., 2017).\n\nA phylogenetic tree (Figure 2) was constructed to determine the kinship relationship of LAB DS15. The phylogenetic tree is known to show a high consistency of the relationships between organisms. In this study, the isolate showed similarity of 99% at 100% query coverage to Pediococcus acidilactici strain DSM 20284. A value of 99% indicates that the isolate can be considered as the same species with Pediococcus acidilactici strain DSM 20284. The sequence of homology levels was high, as shown by the red color with a score of ≥200 (Figure 3). From the results of this homology it can be concluded that the two sequences are the same and have an evolutionary relationship.\n\nThe next closest species for which a sequence alignment of at least 100% query coverage was observed were Pediococcus pentosaceus strain DSM 20336, Pediococcus acidilactici strain NGRI 0510Q and Pediococcus argentini strain CRL 776 at 98% similarity to the DS15 isolate. Pediococcus stilesi strain FAIR-E 180 showed 98% similarity with 99% query coverage. An alignment query result of 100% indicates a significant alignment, which means the search sequence in this study was identical with the identified genus, even at the species level.\n\n\nConclusion\n\nThe PCR of 16S rRNA gene sequence gave an approximately 1400 bp amplicon for LAB DS15, isolated from indigenous dadih of West Sumatera. Phylogenetic analysis showed that LAB DS15 was Pediococcus acidilactici, with 99% similarity to Pediococcus acidilactici strain DSM 20284.\n\n\nData availability\n\nPediococcus acidilactici strain DS32 16S ribosomal RNA gene, partial sequence, obtained during this study. GenBank accession number MH938236: http://identifiers.org/ncbigi/GI:1481059229.", "appendix": "Grant information\n\nThis research was supported by Ministry of Research, Technology and Higher Education Republic of Indonesia through Master of Education Towards Doctoral Scholarship Program for Excellence Undergraduate and the support through World Class Professor Program Scheme-B No. 123.57/D2.3/KP/2018.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1. Electrophoresis image of the PCR amplification product.\n\nClick here to access the data.\n\n\nReferences\n\nAbbasiliasi S, Tan JS, Ibrahim TA, et al.: Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry. BMC Microbiol. 2012; 12: 260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli FWO, Abdulamir AS, Mohammed AS, et al.: Novel, practical and cheap source for isolating beneficial γ-aminobutyric acid-producing leuconostoc NC5 bacteria. Res J Med Sci. 2009; 3(4): 146–153. Reference Source\n\nCollado CM, Surono IS, Meriluoto J, et al.: Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization. J Food Prot. 2007; 70(3): 700–705. PubMed Abstract \| Publisher Full Text\n\nDhakal R, Baipai VK, Baek KH: Production of gaba (γ - Aminobutyric acid) by microorganisms: a review. Braz J Microbiol. 2012; 43(4): 1230–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFelsenstein J: Confidence Limits On Phylogenies: An Approach Using The Bootstrap. Evolution. 1985; 39(4): 783–791. PubMed Abstract \| Publisher Full Text\n\nHenry DE, Halami PM, Prapulla SG: Lactobacillus plantarum mcc2034, a novel isolate from traditional Indian lactic fermented preparation: molecular identification and evaluation of its in vitro probiotic potential. J Microbiol Biotechnol Food Sci. 2015; 4(4): 328–331. Publisher Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7): 1870–1874. PubMed Abstract \| Publisher Full Text\n\nMalik V, Devi U, Yadav RNS, et al.: 16s rRNA based phylogenetic analysis of lactobacillus plantarum isolated from various fermented food products of Assam. J Microbiol Biotechnol Food Sci. 2015; 5(1): 20–22. Publisher Full Text\n\nMora D, Fortina MG, Parini C, et al.: Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16s rRNA and ldhD gene-targeted multiplex PCR analysis. FEMS Microbiol Lett. 1997; 151(2): 231–236. PubMed Abstract \| Publisher Full Text\n\nMurray RK, Granner DK, Rodwell VW, et al.: Harper’s Illustrated Biochemistry 23th edn. McGraw-Hill Companies, Inc. USA. 2003.\n\nMustafa H, Rachmawati I, Udin Y: Genomic DNA concentration and purity measurement of Anopheles barbirostris. Journal of Disease Vector. 2016; 1: 7–10. Publisher Full Text\n\nNonaka S, Arai C, Takayama M, et al.: Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci Rep. 2017; 7(1): 7057. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlsen RW, Li GD: Gaba. In Brady ST, Siegel GJ, Albers LW, Price DL, (Editors). Basic Neurochemistry (Eight edition): Principles of molecular, cellular, and medical neurobiology. 2012; 367–376. Publisher Full Text\n\nPorto MC, Kuniyoshi TM, Azevedo PO, et al.: Pediococcus spp.: An important genus of lactic acid bacteria and pediocin producers. Biotechnol Adv. 2017; 35(3): 361–374. PubMed Abstract \| Publisher Full Text\n\nRizqiati H, Sumantri C, Noor RR, et al.: Isolation and identification of indigenous lactic acid bacteria from North Sumatra river buffalo milk. Indonesian Journal of Animal and Veterinary Sciences. 2015; 20(2): 87–94. Publisher Full Text\n\nSaitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4): 406–425. PubMed Abstract \| Publisher Full Text\n\nSeo MJ, Lee JY, Nam YD, et al.: Production of γ-Aminobutyric Acid by Lactobacillus brevis 340G Isolated from Kimchi and Its Application to Skim Milk. Food Eng Prog. 2013; 17(4): 418–423. Publisher Full Text\n\nSurono IS: In vitro probiotic properties of indigenous dadih lactic acid bacteria. Asian-Australas J Anim Sci. 2003; 16(5): 726–731. Publisher Full Text\n\nSurono IS: Traditional Indonesian dairy foods. Asia Pac J Clin Nutr. 2015; 24 Suppl 1: S26–S30. PubMed Abstract \| Publisher Full Text\n\nWilley JM, Sherwood LM, Woolverton CJ: Prescott’s Principles of Microbiology. Boston: McGraw-Hill Higher Education. 2009. Reference Source\n\nWirawati CU, Sudarwanto MB, Lukman DW, et al.: Characteristic and development of cow’s milk dadih as an alternate of buffalo’s milk dadih. WARTAZOA Indonesian Bulletin of Animal and Veterinary Sciences. 2017; 27(2): 95–103. Publisher Full Text\n\nWu Q, Shah NP: High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter. Crit Rev Food Sci Nutr. 2017; 57(17): 3661–3672. PubMed Abstract \| Publisher Full Text\n\nYamada Y, Endou M, Morikawa S, et al.: Lactic Acid Bacteria Isolated from Japanese Fermented Fish (Funa-Sushi) Inhibit Mesangial Proliferative Glomerulonephritis by Alcohol Intake with Stress. J Nutr Metab. 2018; 2018: 6491907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao A, Hu X, Wang X: Metabolic engineering of Escherichia coli to produce gamma-aminobutyric acid using xylose. Appl Microbiol Biotechnol. 2017; 101(9): 3587–3603. PubMed Abstract \| Publisher Full Text" }	[ { "id": "46425", "date": "01 Apr 2019", "name": "Qinglong Wu", "expertise": [ "Reviewer Expertise Food microbiology", "microbiome science", "microbial genomics", "functional genomics", "microbial GABA biosynthesis", "biochemistry" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors detailed the 16S rRNA gene-based to be a good lab protocol without demonstrating any scientific value. There is no experimental data to support the GABA production from isolate DS15. They have to present the GABA data in terms of GABA yield under defined fermentation conditions. Meanwhile, they have to demonstrate the pathway in isolate DS15 that is responsible for GABA biosynthesis and GABA export in this strain.\nSecondly, the authors just use one isolate to achieve the claim \"GABA producing LAB isolated from indigenous dadih was Pediococcus acidilactici\". This is not a rigorous way.\nHere are my questions:\nWhat is the level of GABA in dadih? How is GABA production capacity of DS15? There is no massive bacterial isolation from dadih; neither no microbial community profiling for dadih, nor pathway identification of GABA production for microbial community of dadih; so one isolate from dadih does not mean anything.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4559", "date": "10 Apr 2019", "name": "Nurul Huda", "role": "Author Response", "response": "What is the level of GABA in dadih? We don't count the amount of GABA on dadih. We did not count the amount of GABA produced in dadih. GABA is produced by lactic acid bacteria of dadih origin but not the dadih, so we don’t count or determine GABA level of dadih How is GABA production capacity of DS15? GABA production capacity of DS15 was 49.365 mg/L There is no massive bacterial isolation from dadih; neither no microbial community profiling for dadih, nor pathway identification of GABA production for microbial community of dadih; so, one isolate from dadih does not mean anything. In this study, we isolated bacteria from various fermented food products (dadih, ikan budu, asam durian and tape singkong), determined their GABA producing ability and selected the isolate with the highest GABA production for further identification. The results of isolation and characterization are explained in other articles. The distribution of LAB isolates from the indigenous West Sumatera fermented food (dadih only) is; Origin from Aiadingin area. Number of isolate 131; Number of LAB isolate 125; Number of GABA producing LAB isolate 23. Origin from Sijunjung area. Number of isolate 166; Number of LAB isolate 93; Number of GABA producing LAB isolate 19. Origin from Solok area. Number of isolate 100; Number of LAB isolate 96; Number of GABA producing LAB isolate 19. In total, from 3 areas, number of isolate 397; number of LAB isolate 314; number of GABA producing LAB isolate 62." } ] }, { "id": "46293", "date": "08 Apr 2019", "name": "Jagadish Mahanta", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors wanted to identify and characterize GABA producing LAB isolated from “Dadih”.\nHowever, authors have taken a strain already isolated and identified in 2009. Authors have not mentioned anything about the gap in the previous research that necessitated undertaking the present exercise. Authors may clarify the issue. Authors have done elaborate molecular testing and phylogenetic analysis of the bacteria taken from the stock. Authors should elaborate the achievement of this exercise. As emphasized by the authors, they should elaborate, about characterization and GABA production potential of the strain Authors should elaborate on the novelty of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4560", "date": "10 Apr 2019", "name": "Nurul Huda", "role": "Author Response", "response": "Authors wanted to identify and characterize GABA producing LAB isolated from “Dadih”. 1. However, authors have taken a strain already isolated and identified in 2009. Authors have not mentioned anything about the gap in the previous research that necessitated undertaking the present exercise. Authors may clarify the issue. Exploration of isolates from dadih has been carried out, but no studies have used these isolates as GABA producers. In this study we obtained DS15 isolate from the isolation of various fermented foods which had the highest GABA production. We did this isolation based on the method of Ali et al., (2019), not isolates from the author. 2. Authors have done elaborate molecular testing and phylogenetic analysis of the bacteria taken from the stock. Authors should elaborate the achievement of this exercise. The result of BLAST at the NCBI GenBank site from the sequences showed that DS15 isolate were Pediococcus acidilactici. Based on the phylogenetic tree, DS15 has a 99% similarity or homology with P. acidilactici DSM 20284, with the difference of one base pair. The next closest species were P. acidilactici NGRI 0510Q, P. pentosaceus DSM 20336 and P. argentinicus CRL 776 with 98% similarity. P. stilesi FAIR-E 180 shows 98% similarity with 99% query coverage. 3. As emphasized by the authors, they should elaborate, about characterization and GABA production potential of the strain. We have carried out quantitative screening on some of the isolates we obtained from dadih, and we found that DS15 isolates produced the highest amount of GABA. Data and discussion are used in another publications. 4. Authors should elaborate on the novelty of the study. The novelty of this study was the use of bacterial isolates from dadih as a GABA producer." } ] }, { "id": "48482", "date": "11 Jun 2019", "name": "Sahilah Abd Mutalib", "expertise": [ "Reviewer Expertise Food microbiology", "Halal Science", "biomass degradation (EFB and POME)" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Introduction - fairly good and can be improved i. Dadih from Indonesia has- Lactobacillus, Streptococcus, Leuconostoc and Lactococcus - How did they identify the bacteria? Biochemical tests or using molecular approaches? ii. Is there any data on dadih from Malaysia as well for comparison.\n2. Methods - can be improved\n2.1 Sample - Subtopic sample suggested to change - Bacterial strain The month and year of the bacterium should be mentioned for ex: in June 2009. Why too long to continue the partial sequence 16s rRNA analysis?\n2. 2 Isolation of bacterial genomic DNA We used lysozyme-change to \"Twenty(20) mg/ml of lysozyme was used to break down ....\" Please state where did you keep the genomic DNA. Example in -20oC freezer or 4oC refrigerator prior analysis\n2.3 16S rRNA gene amplification Please state the reference after forward and reverse primers is mentioned.\n2.4 Electrophoresis 1% change to 1% (w/v) in 1x change to1x TAE, 100 V The marker should be mentioned in this section, 1 Kb ladder? What kind of dye did you used? Red dye, syber green, ethidium bromide? State their brand as well\nResults and discussion Good - due to a single strain/isolate was studied, thus, the explanation is straight forward.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4840", "date": "17 Oct 2019", "name": "Nurul Huda", "role": "Author Response", "response": "1. Introduction - fairly good and can be improved i. Dadih from Indonesia has- Lactobacillus, Streptococcus, Leuconostoc and Lactococcus - How did they identify the bacteria? Biochemical tests or using molecular approaches? They identify bacteria with a molecular approach using the 16SsRNA technique ii. Is there any data on dadih from Malaysia as well for comparison. No, we don’t have data on dadih from Malaysia. 2. Methods - can be improved 2.1 Sample - Subtopic sample suggested to change - Bacterial strain The month and year of the bacterium should be mentioned for ex: in June 2009. Why too long to continue the partial sequence 16s rRNA analysis? Bacterial strains isolated in July 2017. We did this isolation based on the method of Ali et al., (2009), not isolates from the author. 2. 2 Isolation of bacterial genomic DNA We used lysozyme-change to \"Twenty (20) mg/ml of lysozyme was used to break down ....\" Please state where did you keep the genomic DNA. Example in -20 C freezer or 4 C refrigerator prior Analysis We keep the genomic DNA in 4oC refrigerator. 2.3 16S rRNA gene amplification Please state the reference after forward and reverse primers is mentioned. We got reference for forward primer 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3') and reverse primer 1387R (5'-GGG CGG GGT GTA CAA GGC-3') from Laboratory of Medical Molecular Biology and Diagnostic, Indonesian Institute of Sciences, Jakarta, Indonesia. 2.4 Electrophoresis 1% change to 1% (w/v) Ok we will change in 1x change to1x TAE, 100 V Ok we will change The marker should be mentioned in this section, 1 Kb ladder? What kind of dye did you used? Red dye, syber green, ethidium bromide? State their brand as well We used 1 Kb Plus DNA Ladder (ThermoFisher Scientific) Results and discussion Good - due to a single strain/isolate was studied, thus, the explanation is straight forward. Thank You." } ] } ]	2	https://f1000research.com/articles/7-1663
https://f1000research.com/articles/8-303/v1	18 Mar 19	{ "type": "Research Article", "title": "Burden of health behaviours and socioeconomic position on health care expenditure in Ontario", "authors": [ "Douglas G. Manuel", "Carol Bennett", "Richard Perez", "Andrew S. Wilton", "Adrian Rohit Dass", "Audrey Laporte", "David A. Henry", "Carol Bennett", "Richard Perez", "Andrew S. Wilton", "Adrian Rohit Dass", "Audrey Laporte", "David A. Henry" ], "abstract": "Background: Smoking, unhealthy alcohol consumption, poor diet and physical inactivity are leading risk factors for morbidity and mortality, and contribute substantially to overall healthcare costs. The availability of health surveys linked to health care provides population-based estimates of direct healthcare costs. We estimated health behaviour and socioeconomic-attribute healthcare costs, and how these have changed during a period when government policies have aimed to reduce their burden. Methods: The Ontario samples of the Canadian Community Health Surveys (conducted in 2003, 2005, and 2007-2008) were linked at the individual level to all records of health care use of publicly funded healthcare. Generalized linear models were estimated with a negative binomial distribution to ascertain the relationship of health behaviours and socioeconomic risk factors on health care costs. The multivariable cost model was then applied to unlinked, cross-sectional CCHS samples for each year from 2004 to 2013 to examine the evolution of health behaviour and socioeconomic-attributable direct health care expenditures over a 10-year period. Results: We included 80,749 respondents, aged 25 years and older, and 312,952 person-years of follow-up. The cost model was applied to 200,324 respondents aged 25 years and older (CCHS 2004 to 2013). During the 10-year period from 2004 to 2013, smoking, unhealthy alcohol consumption, poor diet and physical inactivity attributed to 22% of Ontario’s direct health care costs. Ontarians in the most disadvantaged socioeconomic position contributed to 15% of the province’s direct health care costs. Taken together, health behaviours and socioeconomic position were associated with 34% ($134 billion) of direct health care costs (2004 to 2013). Over this time period, we estimated a 1.9% reduction in health care expenditure ($5.0 billion) attributable to improvements in some health behaviours, most importantly reduced rates of smoking. Conclusions: Health behaviours and socioeconomic position cause a large direct health care system cost burden.", "keywords": [ "Burden", "smoking", "alcohol", "diet", "physical activity", "healthcare cost" ], "content": "Introduction\n\nSmoking, unhealthy alcohol consumption, poor diet and physical inactivity are leading risk factors for morbidity and mortality worldwide1. Despite this knowledge, prevalence of these risk factors remains high and reduction efforts may be hindered by failure to understand the full human and cost burdens these risk factors impose on societies2. In an era of increasing health care expenditure most political focus has been on payments for services and the growing impacts of diagnostic and therapeutic technologies. There is also a need to consider costs resulting from upstream health behaviours, and how these have changed over time, to help prioritize public health strategies and support public health decision makers. These relationships are likely to be complex because of some conflicting trends in the prevalence of health behaviours3,4.\n\nWhile health behaviours have a leading role in morbidity and mortality, it is also recognized that there is uneven distribution of health across socioeconomic position (i.e., social and economic factors that influence what position individuals hold within the structure of a society5). Canadian research indicates that individuals with lower socioeconomic position tend to be less healthy than those who enjoy greater educational, income and occupational advantages6–9. As with health behaviour-attributable health care use, evidence of the economic cost of these health disparities helps us understand the health and financial benefits of reducing the gap6.\n\nWe sought to estimate the economic burden attributable to four health behaviour risk factors (smoking, unhealthy alcohol consumption, poor diet and physical inactivity), how these have changed over time, and how they interact with socioeconomic position. The study had three objectives: 1) to examine the direct healthcare costs associated with smoking, unhealthy alcohol consumption, poor diet and physical inactivity; 2) to examine the change in direct health care costs as a consequence of changes, over time, in health behaviours; and, 3) to examine the direct health care costs associated with socioeconomic position (i.e., education, family income, home ownership, and neighbourhood deprivation).\n\nPast studies typically infer health care costs indirectly—where aggregate health care expenditure data is categorized by disease, for example. We used unique Canadian data that individually link respondents from large repeated population health surveys to comprehensive health care utilisation and cost data covering hospital and primary care sectors in Ontario. These data provide, to our knowledge, the largest and most complete population-based examination of the relationship between health behaviours and direct public healthcare costs. We believe this is the first study to measure directly how changes in health behaviours result in changes in health care use. The linked data also provide the means to assess the degree to which health costs are associated with socioeconomic inequalities.\n\n\nMethods\n\nThis study was approved by the Ottawa Health Science Network Research Ethics Board. Datasets were linked using unique encoded identifiers and analysed at ICES. ICES is an independent, non-profit research institute whose legal status under Ontario’s health information privacy law allows it to collect and analyse health care and demographic data, without consent, for health system evaluation and improvement.\n\nWe used the Ontario sample drawn from a national population health survey—the Canadian Community Health Survey (CCHS), conducted in 2003, 2005, 2007-2008, 2009-2010, 2011-2012, and 2013-2014 to develop linked and unlinked study cohorts. The CCHS is a cross-sectional survey conducted by Statistics Canada that collects data related to health determinants, health status and health care use—details of data collected and used in the analyses are provided below. The survey employs a complex multistage sampling strategy to randomly select households in each health region. Details of the survey methodology have previously been published10. A weight, which reflects the number of individuals represented in the target population, is assigned to each respondent; the target population includes individuals aged 12 years and older living in Canada’s ten provinces and three territories. Individuals living on First Nation Reserves, institutionalized residents, full-time members of the Canadian Forces and residents of certain remote areas are excluded from the survey’s sampling frame.\n\nFor the linked study cohort, the Ontario sample of the 2003, 2005, and 2007-2008 CCHS cycles provided 128,501 valid interviews. Of these respondents, a subset agreed to share and link their interview information and 101,506 were successfully linked to their provincial health card number using a deterministic and probabilistic algorithm. We included respondents aged 25 years and older if they were eligible for provincially funded health care and not pregnant at the time of survey administration. For individuals with multiple interviews, only the earliest interview was included. We excluded individuals who were lost to follow-up in the first year following their interview (i.e., they were not available at the beginning of the study). This resulted in a final cohort of 80,749 unique Ontario respondents (see Figure 1).\n\nCCHS, Canadian Community Health Survey; OHIP, Ontario Health Insurance Program.\n\nThe Ontario sample of all six CCHS waves provided 200,324 valid respondents aged 25 years and older for the unlinked study cohort. This cohort was used to estimate direct health care costs by applying the multivariable model that was derived using the linked cohort CCHS data (see Model Development).\n\nThe CCHS waves were used to examine the following risk factors for their association with health care use: age, sex, four health behaviours (smoking, alcohol consumption, diet and physical activity; see Table 1), sociodemographic factors (immigrant status, education level, urban dwelling, neighbourhood deprivation, household income, home ownership, marital status), self-perceived stress, preventive health behaviour (flu vaccination), and health status indicators (body mass index, hypertension, diabetes, heart disease, cancer, history of stroke, dementia, and extent of difficulty in performing basic tasks or participating in activities).\n\nHighest risk levels are in bold and lowest risk levels (reference group) are in italics.\n\n‡Bingeing: five or more drinks on any day in the previous week or weekly bingeing behaviour in the previous month.\n\n†Index score: the healthiness of a diet based on consumption of fruit and vegetables. Individuals start with 2 points and achieve up to 8 additional points for each average daily serving of fruits and vegetables (maximum score = 10). Points are deducted for daily fruit juice servings exceeding 1 (-2 points), no carrot consumption (-2 points), or daily potato consumption exceeding 1 serving for males and 0.7 servings for females (-2 points). Scores that result in negative values after deductions are recoded to zero, resulting in a final range of 0 to 10 for the index.\n\nMET, metabolic equivalent of task (a measure of calories burned by type, duration and frequency of physical activity).\n\nWe examined person-level health care costs across three sectors: 1) hospital care (inpatient hospitalizations, same day surgeries, emergency department visits, rehabilitation hospitals, and complex continuing care centres); 2) drugs (for Ontarians age 65 and older and Ontarians receiving social assistance, Ontario Drug Benefit costs were captured); and 3) community care (primary care billings, specialist billings, lab billings, capitation services, and home care services).\n\nCosts to operate the health care system (e.g., health ministry administrative costs) and capital costs for large scale projects (e.g., building new hospitals) are not reflected in the person-level costs. To account for these exclusions in the health care cost analysis, we obtained annual total health care expenditures from publicly available Ontario Ministry of Finance records (fiscal years 2003 to 2013, where fiscal year is April 1 to March 31), the Canadian Institute for Heath Information’s National Health Expenditure Trends publication, and the MOHLTC’s Report Card for the Ontario Drug Benefit Program publications11–23. The expenditures from each year were categorized into our three health care sectors and expenditures that did not correspond with any of the three sectors were assigned to an ‘other’ category.\n\nAll costs are expressed in 2014 Canadian dollars with past costs inflated using the annual general Consumer Price Index reported by Statistics Canada.\n\nTo develop multivariable cost models, Ontario respondents to the 2003, 2005 and 2007-2008 cycles of CCHS were linked, at the individual level, to all records of health care use that were paid for by the Ontario Ministry of Health and Long-Term Care (MOHLTC). The cost associated with each record was estimated using costing methods developed for Ontario health administrative data24. Briefly, a payer (the MOHLTC) cost perspective was taken, using person-level health care utilization and per-use fee information or budgetary data. Cost information for sectors (i.e., acute hospitalization, same day surgery, emergency department, inpatient rehabilitation, and complex continuing care) that are funded using global budgets (e.g., by institution) are determined using a top-down approach through case-mix methodology. Sectors that have fee payments associated with each use (e.g., prescription drugs, physician services, and home care) have costs estimated directly.\n\nBeginning one year after survey administration, CCHS respondents were followed for a four-year period (between 2004 and 2013) to develop multivariable models estimating the effect of health behaviours on health care costs. We used a generalized linear model with a negative binomial distribution and an offset to account for variation in follow-up times to create separate, sex-specific models for each of the health care sectors: hospital care, drugs, and community care. To assess confounding and mediation, we use a pre-specified, stepwise modelling approach. We started with a health behaviour model followed by a basic sociodemographic model, a primary attribution model that adjusted for additional sociodemographic risk factors, a distal mediator model that included health status indicators, and a proximal mediator model that included a measure of fragility (see Figure 2). This stepwise analysis resulted in 15 models for each sex.\n\nThe model building approach sought to address three issues in assessing the contribution of health behaviours to health care costs. First, we were interested in having appropriate adjustment for other risk factors for increased costs that are correlated with health behaviours (e.g., age and sociodemographic risk factors). Second, we were attentive to risk factors that may mediate the relationship between health behaviours and health care costs (e.g., body mass index or hypertension). Our concern was that their inclusion in the model could inappropriately attenuate the risk from health behaviours. Third, we considered pre-existing illness that may have led to health behaviour change. For example, as people become ill and frail they may become less physically active; in such a situation, physical inactivity may be associated with increased health care cost that is more appropriately identified as illness-associated inactivity.\n\nWe calculated the proportion of the health care costs that can be attributed to health behaviours—the population attributable fraction—for fiscal years 2003 to 2013, for each health care sector. Using the unlinked CCHS cohort, we estimated annual population attributable fractions using each CCHS cycle and for years between CCHS cycles, by averaging the population attributable fractions from the preceding and succeeding year.\n\nWe used a factor-deleted approach to calculate population attributable fractions that involved three steps. In the first step, we estimated expected annual population health care costs for a specific sector by applying the corresponding sector-specific primary attribution models to the weighted CCHS cycle. In the second step, we repeated the calculation after recoding each respondent’s health behaviour to the counterfactual reference or “no exposure” category. For example, taking the weighted cohort, we estimated hospital costs using all smoking exposures (i.e., current, former, and non-smokers) and re-estimated hospital costs assuming all current and former smokers were non-smokers. The difference between the two calculations was an estimate of the annual contribution of smoking to hospital costs. In the final step, we divided this difference by the original population estimate (from the first step) to produce a population attributable fraction. In our example, this would be the population attributable fraction of hospital costs associated with smoking. Health sector specific population attributable fractions were calculated for each health behaviour, and the combination of health behaviours. The same analysis was performed for different socioeconomic groups defined by education, family income, home ownership, and neighbourhood deprivation.\n\nWe calculated annual estimates of costs attributable to health behaviours and socioeconomic position (fiscal years 2004 to 2013) by applying the sector-specific population attributable fractions to the annual public health care expenditures and summing the health care sector results together.\n\nThe change in health care costs attributable to the change in health behaviours was estimated for fiscal years 2004 to 2013. A baseline population attributable fraction for total health care costs associated with all health behaviours was estimated for fiscal year 2003 using the previously described methods for population attributable fractions and attributable costs. The overall health care budget was estimated over the subsequent decade, assuming that health behaviours in 2003 remained constant (e.g., the baseline population attributable fraction did not change over time). The difference between the counterfactual health care budget and the actual health care budget provided an annual estimate of the change in health care costs attributable to changes in health behaviours.\n\nWe performed three sets of sensitivity analyses. First, we created five models representing a stepwise approach to assess confounding and mediation. The five models assessed health care costs and burden estimates with progressive adjustment for risk factors: age, sex and health behaviours (Model 1), basic socio-demographic characteristics (Model 2), additional sociodemographic characteristics (Model 3), health status mediators (Model 4), and fragility measures (Model 5) (Figure 2). The estimate derived from Model 3 was assumed to be our most accurate and appropriate estimate of the attributable burden due to health behaviours. The estimates derived from Model 1 (simply age and health behaviours) and Model 5 (the over-adjusted model) were used as upper and lower bounds of uncertainty.\n\nFor our second sensitivity analysis, we compared age-standardized cost ratios after excluding the top 5% of health care users to assess the possibility of overly influential respondents. The use of health care varies considerably between people, particularly for hospital care and other speciality services. Only a small proportion of people are hospitalized and, of those hospitalized, a small proportion has multiple admissions and complicated long hospital stays. The skewed distribution of health care services has potential to distort the attributable health care expenditure analysis because a small proportion of CCHS respondents may have a strong influence on overall or total population estimates.\n\nThird, we replicated analyses using an inverse propensity-weighted model to assess robustness of the health care cost ratios attributable to smoking. The inverse propensity-weighted model, a complimentary approach to the generalized linear model, is an alternative approach to adjust cost ratios for multivariable risk factors. Our inverse propensity-weighted analyses included several covariates in addition to all those used in the multivariable analyses (i.e., the primary attribution model): household type, highest level of household education, main source of household income, labour force participation, sense of belonging to the community, regional health authority, and survey cycle.\n\n\nResults\n\nThe population attributable fractions for the four behavioural risks were calculated using responses from 80,749 Ontarians surveyed between 2003 and 2008. In total, there were 312,952 person-years of follow-up. Characteristics of the study cohort are presented in Table 2.\n\nData source: Canadian Community Health Survey (CCHS) 2.1, 3.1 and 4.1 (2003, 2005 and 2007/08).\n\n‡Population estimated using the CCHS sampling weights.\n\nFrom fiscal years 2004 to 2013, 22% of Ontario’s health care costs could be attributed to the four health behaviour risk factors (Figure 3). Physical activity had the largest attribution (13%), followed by smoking (10%). However, uncertainty for the burden estimates indicates potential overestimation for physical activity and underestimation for diet. Alcohol-attributable health care costs were also likely underestimated (see limitations section).\n\nError bars represent high and low boundaries on burden estimates.\n\nDuring the 10-year period (2004 to 2013), $89.3 billion in health care costs were attributable to health behaviours. In that same period, the costs attributable to health behaviours improved by 1.9% (23.3% of total healthcare costs in 2004 to 21.4% in 2013). If the proportion of health care expenditure that can be attributed to health behaviours (the population attributable fraction) had remained at 23.3%, use of health care would have been $5.0 billion greater (what we term ‘avoided cost’).\n\nTable 3 presents the burden of health behaviours related to health care costs ($89.3 billion) and the costs avoided by the adoption of healthy behaviours ($5.0 billion). Physical inactivity and smoking contributed the largest proportion of the burden (53% and 41%, respectively). However, a decline in smoking between 2004 and 2013 was responsible for 84% of the avoided costs.\n\nThe scenarios from our sensitivity analysis demonstrate results that were similar to our main analysis. Not unexpectedly, the attribution of health care costs to health behaviours decreased as we increased the number of risk factors adjusted for in the model. Excluding high-cost health care users demonstrated slightly attenuated age-standardised cost ratios for men and women for almost all health behaviour risks. The inverse propensity-weighted model, which adjusted for additional variables, had similar cost ratios to the main analysis from the multivariable model.\n\nBetween 2004 and 2013, $60.7 billion dollars in health care costs (15% of all health care costs for Ontarians aged 25 years and older) were attributable to low socioeconomic position (Figure 4). When health behaviours and socioeconomic position are considered jointly, the health care cost burden was $134 billion (34% of all health care costs for those aged 25 years and older). The break down by health care sector was similar for health behaviour and socioeconomic position. The largest portion of the burden is due to hospital care costs (46% of health behaviour attribution and 54% of socioeconomic position attribution to health care costs); followed by community care costs (22% and 19%), ‘other’ health care costs (21% and 19%), and drug costs (10% and 9%).\n\n\nDiscussion\n\nWe estimated that smoking, unhealthy alcohol consumption, poor diet and physical inactivity attributed to 22% of Ontario’s direct health care costs during the ten-year period from 2004 to 2013. During this same period, improving health behaviours equated to a nearly 2% reduction in direct health care expenditure. Ontarians in the most disadvantaged socioeconomic group contributed to 15% of the province’s direct health care costs. Taken together, health behaviours and socioeconomic position contributed to a burden of $134 billion in direct health care costs (Ontario, 2004 to 2013).\n\nEstimates of the cost of modifiable health behaviours and socioeconomic risk factors provides evidence to allow policy-makers to prioritize interventions aimed at reducing health care costs. Our estimate of a 1.9% reduction in health care expenditure (i.e., $5 billion) through improved health behaviours, suggests that investments in promotion of healthy living have potential for substantial savings in health care costs in Canada. In our study, reduced smoking was the main contributor to avoided health behaviour attributable health care costs (accounting for 84% of the 1.9% cost reduction). This large cost reduction reflects prominent smoking prevention strategies that were introduced during the study period—including 100% smoke-free public places including restaurants25. From 2004 to 2013 there was an 11% reduction in the attributable fraction of smoking, which was solely a consequence of a reduction in the prevalence of current smoking. The large remaining burden from health behaviours and social inequalities suggests that there are significant opportunities to further reduce health care costs through population health strategies.\n\nIt is difficult to compare our findings with previous studies for two main reasons. First, various studies have estimated the economic burden in terms of costs for treatment and management of chronic diseases related to smoking, alcohol, diet or physical activity, but very few have evaluated the simultaneous impact of multiple risk factors in a population. These latter studies have used traditional population aggregated-data attributable fraction methods to estimate economic burden26–31. Second, the significant methodological differences between our study and previous literature limits direct comparison of findings. Briefly, traditional population attributable fraction methods identify diseases where health behaviours are risk factors, estimate the health care costs of these diseases, calculate the proportion of the disease that can be attributed to the risk factors (based on relative risk of disease from an external source and prevalence of exposure in the population of interest), and apply these population attributable fractions to the cost data. There are limitations associated with these methods that stem from combining ecological summary measures of exposure, outcome, and hazards across different sources of data32. Our use of multivariable algorithms and the direct attribution of health behaviours to health care costs offers advantages over analyses that have been performed to date: controlling for confounders, accounting for complexities in the relationship between multiple exposures and covariates, using consistent definitions of exposure, and using specific measures of risk derived internally from the study population.\n\nOur study has several limitations that we believe underestimate the actual burden of health care costs attributable to the four health behaviours. First, we excluded individuals younger than 25 years of age. In general, health care costs for this age group are small; however, alcohol burden for younger people is a notable omission. Alcohol has an important attribution for injury, suicide and other social burdens that occur disproportionately among young people33–35. Second, our reliance on self-reported health risk exposures will generally result in an underestimation of risk burden, especially for food and alcohol36–39. In what is referred to as “social desirability bias”, survey respondents tend to over-report what they perceive as healthy behaviour and underreport unhealthy behaviour40. As an example, self-reported alcohol consumption in surveys accounts about half the volume of alcohol sold41–42. While reporting accuracy affects all risks in this study, burden estimates are mostly affected when people report they are in the healthiest category and they are actually in an unhealthy category. Third, the survey’s brief questions about risks may not capture the full spectrum of behaviour. For example, our measure of physical activity (leisure-time physical activity) did not include active transportation (such as walking and bicycling to work), work activity, or sedentary time and our measure of diet (fruit and vegetable consumption) did not specifically ascertain intake of sodium, trans fats, calories or other aspects of healthy and unhealthy eating. Fourth, we used respondents’ answers to health behaviours and other risks that correspond to their health behaviour at the time of the survey. In general, studies that consider lifetime changes in risks generate higher burden estimates. Our physical activity burden estimates may be an exception, where reverse-causality (i.e., ill health is the cause of reduced physical activity) results in overestimation. Fifth, “other” health care costs—including health care system operating costs and capital costs—are not included our health care cost data and were estimated indirectly. Sixth, we did not include indirect costs (such as lost productivity, wages and income related to illness associated with unhealthy living, or costs borne by individuals to care for their illness), nor did we include costs for health care beyond those paid by the provincial government (e.g., employee health plans).\n\nDespite these limitations, our study demonstrates the importance of integrating public health and prevention within the health care system. A greater investment in disease prevention and population health could help increase the sustainability of publicly funded health care by reducing spending on illness, particularly with respect to hospital care. Combining this effort with strategies that address social determinants of health could secure further benefits. Indeed, our study suggests that interventions outside the health care system, such as improving levels of income and education, and other risk factors that influence socioeconomic position, will reduce health care costs. The health system is one determinant of population health and attention must be paid to the needs of disadvantaged individuals, populations, and communities in order to avoid increasing health disparities43. While health inequities play a significant role in health system costs, estimating the cost of the gap in health care as a result of socioeconomic position can be difficult because of the complexity of the problem. Our study is one of the few that has been able to translate this gap into a dollar figure.\n\n\nConclusions\n\nOur study shows that health behaviours and socioeconomic position contribute to a large health care cost burden. Better health and well-being is the primary goal of improving health behaviours and reducing social inequity. However, it is important to recognize that existing investments in public health also results in a large reduction in expenditure in the acute care sector, particularly hospital care. The health premium of prevention and social equity is an overlooked opportunity for a sustainable health care system.\n\n\nData availability\n\nThe dataset from this study is held securely in coded form at ICES. While data sharing agreements prohibit ICES from making the dataset publicly available, access may be granted to those who meet pre-specified criteria for confidential access, available at www.ices.on.ca/DAS. The full dataset creation plan and underlying analytic code are available from the authors upon request, understanding that the computer programs may rely upon coding templates or macros that are unique to ICES and are therefore either inaccessible or may require modification.\n\nOpen Science Framework: Burden of health behaviours and socioeconomic position on health care expenditure in Ontario. https://doi.org/10.17605/OSF.IO/KW4C544.\n\nThis project contains the sensitivity analysis supplementary file.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was supported by ICES, which is funded by an annual grant from the Ontario Ministry of Health and Long-Term Care (MOHLTC). The opinions, results and conclusions reported in this paper are those of the authors and are independent from the funding sources. No endorsement by ICES or the Ontario MOHLTC is intended or should be inferred.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful for the contributions of Sima Gandhi and Erika Yates as well as our Scientific and Policy Advisory Committees—namely, Peter Austin, Bernard Choi, Prabhat Jha, Isra Levy, Heather Manson, Claudia Sanmartin, Joanne Thanos, and Pegeen Walsh.\n\n\nReferences\n\nLim SS, Vos T, Flaxman AD, et al.: A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2224–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing D, Lawson KD, Kolbe-Alexander TL, et al.: The economic burden of physical inactivity: a global analysis of major non-communicable diseases. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPublic Health Agency of Canada: The Chief Public Health Officer's Report on the State of Public Health in Canada, 2015: Alcohol Consumption in Canada. Ottawa, Canada; 2016. Reference Source\n\nWorld Health Organization: Alcohol and injury in emergency departments: summary of the report from the WHO Collaborative Study on Alcohol and Injuries. 2007. Reference Source\n\nWhitford JL, Widner SC, Mellick D, et al.: Self-report of drinking compared to objective markers of alcohol consumption. Am J Drug Alcohol Abuse. 2009; 35(2): 55–8. PubMed Abstract \| Publisher Full Text\n\nShields M, Connor Gorber S, Tremblay MS: Estimates of obesity based on self-report versus direct measures. Health Rep. 2008; 19(2): 61–76. PubMed Abstract\n\nWong SL, Shields M, Leatherdale S, et al.: Assessment of validity of self-reported smoking status. Health Rep. 2012; 23(1): 47–53. PubMed Abstract\n\nMuggah E, Graves E, Bennett C, et al.: Ascertainment of chronic diseases using population health data: a comparison of health administrative data and patient self-report. BMC Public Health. 2013; 13: 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKreuter F, Presser S, Tourangeau R: Social desirability bias in CATI, IVR, and Web surveys the effects of mode and question sensitivity. Public Opin Q. 2008; 72(5): 847–65. Publisher Full Text\n\nGmel G, Rehm J: Measuring alcohol consumption. Contemp Drug Probl. 2004; 31(3): 467–540. Reference Source\n\nRehm J, Patra J, Popova S: Alcohol-attributable mortality and potential years of life lost in Canada 2001: implications for prevention and policy. Addiction. 2006; 101(3): 373–84. PubMed Abstract \| Publisher Full Text\n\nWodchis WP, Austin PC, Henry DA: A 3-year study of high-cost users of health care. CMAJ. 2016; 188(3): 182–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManuel DG, Bennett C: Burden of health behaviours and socioeconomic position on health care expenditure in Ontario. 2019. http://www.doi.org/10.17605/OSF.IO/KW4C5" }	[ { "id": "45912", "date": "23 Apr 2019", "name": "Suneela Mehta", "expertise": [ "Reviewer Expertise Epidemiology of chronic diseases", "cardiovascular risk prediction modelling", "linked administrative health data" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis elegant analysis aims to estimate direct healthcare costs in Ontario associated with four adverse health behaviours, the change in direct health care costs associated with these health behaviours over time and the direct healthcare costs associated with low socioeconomic position. This is an extremely valuable endeavour; clarifying the economic cost associated with health behaviour risk factors and lower socioeconomic status assists decision makers in prioritising public health strategies focussed on these domains. The introduction provides a thoughtful background to the study, the definition of the behavioural risk factors (Table 1) and representativeness of the cohort (Table 2) are clearly outlined and the discussion of the study limitations is excellent.\nHowever, some additional details and minor amendments would be useful:\nThe analysis relates to publicly-funded health care costs in Ontario but as an international reader, it would be helpful to get a brief background of the structure of health system in Canada, and any aspects that are unique to Ontario. In particular, what proportion of healthcare is delivered in the private health sector? Related to point 1), how generalisable are the findings to Canada as a whole and other countries? The descriptions of the public health care spending data considered (on page 4) and the linkage to person-level health care costs (outlined in the Model development section beginning on page 4) are very clear, but the incorporation of the costs to operate the health system into the modelling process needs some further explanation. Are these non-person level costs considered in the MOHLTC costing perspective developed for Ontario health administrative data that is mentioned at the top of page 5? Also, fifteen models have been developed (five per sex for each of the three identified health care sectors) so how have the expenditures associated with the ‘Other” category mentioned on page 4 been considered in the analysis? In the first paragraph of the section entitled “Sensitivity analysis”, is the explanation of the stepwise model approach describing something different to the description of the model building process detailed on page 5? If not, then I would delete the latter and just leave the last sentence that mentions that models 1 and 5 were used as upper and lower bounds. Furthermore, I would move the information that the primary attribution models were assumed to provide the most accurate estimate of attributable burden of adverse health behaviours to the description on page 5. It would be good to briefly outline the rationale for using a four year period of follow-up on page 5 where this is first mentioned. Also, the titles of tables A-5 to A-8 in the supplementary data state that a five year period between 2004 and 2013 was analysed so these should either be corrected if they are typos or further detail provided in the sensitivity analysis section of the methods. Results are presented in Figure 4 for health care costs attributable to low socioeconomic position but there is no corresponding definition of low socioeconomic position in the methods or results.\n\nMinor amendments:\nIn the methods section of the abstract, it would be helpful to add that the unlinked, cross-sectional CCHS samples for each year from 2004 to 2013 were Ontario samples only. The sentence in the results section of the abstract beginning “Taken together, health behaviours…” would be more accurate if stated as “Taken together, these four adverse health behaviours and low socioeconomic position were associated with 34% ….”. Similarly, the conclusion of the abstract and other sentences similarly worded throughout the paper as well as the labels assigned to Figure 4 would benefit from being reworded to something like “adverse health behaviours” and “low socioeconomic position”. In the results section describing the sensitivity analyses, it would be helpful to the reader to include the corresponding table numbers in the supplementary data. The latter part of the sentence in the discussion on page 12 beginning “Fifth, “other” health care costs…” should read ‘are not included in our health care cost data…’\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4612", "date": "07 May 2019", "name": "Doug Manuel", "role": "Author Response", "response": "We thank you for the constructive review and agree with your suggested edits to improve the comprehensiveness of the paper. Our plan is to submit a revised version, addressing the proportion of health care costs covered by Ontario’s public health care system and generalizability of results; clarifying the methods for modelling person-level health care costs, the stepped approach to model building, and follow-up period; and, defining measurement of SEP. However, given the current version is under review by another reviewer, we will wait to submit a revised version." }, { "c_id": "4951", "date": "16 Oct 2019", "name": "Doug Manuel", "role": "Author Response", "response": "Thank you for your constructive review. We provide a point-by-point summary of response to your comments below. 1. The analysis relates to publicly-funded health care costs in Ontario but as an international reader, it would be helpful to get a brief background of the structure of health system in Canada, and any aspects that are unique to Ontario. In particular, what proportion of healthcare is delivered in the private health sector? We have added a description of Canada’s health care system to the methods section Public health care spending data. 2. Related to point 1), how generalisable are the findings to Canada as a whole and other countries? We have added a new paragraph in the Discussion (paragraph 3) that focuses solely on generalizability. 3. The descriptions of the public health care spending data considered (on page 4) and the linkage to person-level health care costs (outlined in the Model development section beginning on page 4) are very clear, but the incorporation of the costs to operate the health system into the modelling process needs some further explanation. Are these non-person level costs considered in the MOHLTC costing perspective developed for Ontario health administrative data that is mentioned at the top of page 5? Also, fifteen models have been developed (five per sex for each of the three identified health care sectors) so how have the expenditures associated with the ‘Other” category mentioned on page 4 been considered in the analysis? Failure to indicate our method to incorporate ‘other’ health care costs (i.e., total provincial health care expenditures minus expenditures in hospital care, community care and drugs) was an oversight. We applied the population attributable fraction from community care to the indirectly estimated other health care costs. We have added this detail to the methods section Estimating the health care cost burden of health behaviours and socioeconomic position in Ontario. 4. In the first paragraph of the section entitled “Sensitivity analysis”, is the explanation of the stepwise model approach describing something different to the description of the model building process detailed on page 5? If not, then I would delete the latter and just leave the last sentence that mentions that models 1 and 5 were used as upper and lower bounds. Furthermore, I would move the information that the primary attribution models were assumed to provide the most accurate estimate of attributable burden of adverse health behaviours to the description on page 5. We have revised the text as suggested to address the confusion regarding the model building approach. 5. It would be good to briefly outline the rationale for using a four year period of follow-up on page 5 where this is first mentioned. Also, the titles of tables A-5 to A-8 in the supplementary data state that a five year period between 2004 and 2013 was analysed so these should either be corrected if they are typos or further detail provided in the sensitivity analysis section of the methods. We have added details to the Model development section to clarify the choice of a four-year follow-up time frame. The supplementary appendices were erroneously labelled. We have revised accordingly. 6. Results are presented in Figure 4 for health care costs attributable to low socioeconomic position but there is no corresponding definition of low socioeconomic position in the methods or results. We have added details to the methods section Estimating population attributable fractions of health care costs to define the comparator group the the SEP analysis. Minor amendments: 1. In the methods section of the abstract, it would be helpful to add that the unlinked, cross-sectional CCHS samples for each year from 2004 to 2013 were Ontario samples only. Done 2. The sentence in the results section of the abstract beginning “Taken together, health behaviours…” would be more accurate if stated as “Taken together, these four adverse health behaviours and low socioeconomic position were associated with 34% ….”. Similarly, the conclusion of the abstract and other sentences similarly worded throughout the paper as well as the labels assigned to Figure 4 would benefit from being reworded to something like “adverse health behaviours” and “low socioeconomic position”. Done 3. In the results section describing the sensitivity analyses, it would be helpful to the reader to include the corresponding table numbers in the supplementary data. Done 4. The latter part of the sentence in the discussion on page 12 beginning “Fifth, “other” health care costs…” should read ‘are not included in our health care cost data…’ Done" }, { "c_id": "4952", "date": "16 Oct 2019", "name": "Doug Manuel", "role": "Author Response", "response": "Thank you for your helpful review. We have added a number of details to respond to your points. See our point-by-point response below.1. The information regarding the measurement of lifestyle risk factors and SEP is critical and should be presented with details. What measurement instruments were used? What are the reliability and validity of these measures, what is the rationale for the categorisation? Particularly, the small proportion of healthcare cost attributable to poor diet may be a result of the limitations of the measure (I consider the dietary measure to be a fruit and vegetable intake measure only). We have added details to the methods section Behavioural and other risk factors for health care use to more fully describe measurement of health behaviour and sociodemographic risk factors. We have also expanded the discussion Limitations section.2. Although it is interesting and informative to examine healthcare cost associated with low SEP, I think this idea may require a little bit more consideration. SEP is a relative concept, so what is the practical interpretation of PAF associated with SEP? Were the authors considering the healthcare cost that could have been averted if everyone was of high socioeconomic status? Particularly, how do we correctly interpret PAF associated with ethnicity? What are the counterfactuals? We agree, SEP is a relative concept. We have framed the analysis as an equity gap and added details to the Estimating population attributable fractions of health care costs to describe the exposures used for the analysis.3. To me, it may be more meaningful to estimate the PAF of socioeconomic inequality, rather than SEP. See above.4. It seems that the authors have a-priori decided to model women and men separately? What is the rationale? However, later on in the paper, when describing the economic cost, all results were presented for both sexes combined. Is there an explanation for this? Yes, we did a priori model men and women separately. There were several considerations such as differential misclassification bias of exposure (e.g., men and women have different pattern of misclassification error for weight and height). Many/most studies of the underlying leading causes of diseases sex-stratify based, for example, on the observed effect modification for cardiovascular disease and most leading causes of cancer. The results were then combined to clearly present our main focus on health behaviours and SEP.5. It looks like the authors have conducted thorough and comprehensive statistical analysis. However, most results are not presented. For example, results from the regression analysis (at least for Model 3) could be of interest to readers. We agree with your suggestion. As you can appreciate, there was a wide range of components for the study. However, on reflection we agree that these models are of interest, particularly for replication or for use by other jurisdictions who may use the estimated to indirectly model burden. These models will be added to the on-line figure repository. (The analyst who created those models is currently on leave).6. The change analysis between 2004 and 2013: is this based on modelling using all data points or the two time points only? We have revised the methods section Estimating costs attributable to changes in health behaviours to more clearly indicate that this analysis is based on annual estimates.7. Could you please provide more information and a reference for inverse propensity-weighted analyses? We have added additional details and references to the description of the inverse propensity-weighted sensitivity analysis.8. Sorry if I have missed this point from the paper. The modelling of SEP is based on adjusted analysis considering lifestyle risk factors? As we know SEP is a strong predictor of lifestyle behaviour, so parts of the association between SEP and healthcare cost would be explained by lifestyle behaviours. It would be useful if the authors more explicitly define the part of PAF attributable SEP independent of lifestyle risk factors. The findings for both health behaviours and SEP were estimated using Model 3 simultaneously for health behaviours and SEP. We have clarified this in the manuscript.We agree with the reviewer’s perspective that SEP and health behaviours have strong relationships with complex pathways.As such, Model 3 likely represents a somewhat conservative estimate of the independent effects of either health behaviours or SEP. The sensitivity analyses were performed with less and more specified models to gauge the potential effect of adding or removing mediators. Model 1 was a simple model of health behaviours without SEP measures. Of note, we didn’t create to complementary model—i.e., a simple model of SEP without health behaviour measures.Given the complex relationships, we recognize that there are alternative methods approaches that we hope will be further explored in future studies.9. Given that the data spans over 10 years, have the authors considered inflation? Particularly healthcare specific inflation? It may be worth discussing that the change in PAF over time could be a result of a change in population health behaviour of that in the average costs of certain health care procedures/items. For this study, all costs are expressed in 2014 Canadian dollars with past costs inflated using the annual general Consumer Price Index from Statistics Canada. It is unclear what is meant by “…could be a result of a change in population health behaviour of that in the average costs of certain health care procedures/items.”10. Furthermore, the authors may consider the checklist we developed for reporting cost of illness analysis.We’ve completed the checklist and added it to the on-line repository. We had been holding off using a reporting guideline until those for population modelling studies (http://www.mrc-epid.cam.ac.uk/ph-modelling-guidelines/) became available.We have also performed studies that have examined how modelling/burden studies can be reported and found the CHEERS guidelines are relevant.Minor comments/questions: 1. Abstract: Taken together, health behaviours and socioeconomic position were associated with 34% ($134 billion)… suggest add \"these\" before health behaviours, because this is only referring to the 4 selected behaviours, not health behaviour in general.We have modified the language in the abstract to more clearly indicate the attributable costs are related to select health behaviours and socioeconomic measures.2. Methods: could the authors provide some justification regarding why these lifestyle behaviours were selected? In the Introduction we highlight our focus on smoking, unhealthy alcohol consumption, poor diet and physical inactivity as the leading risk factors for morbidity and mortality worldwide.3. Results: “uncertainty for the burden estimates indicates potential overestimation for physical activity and underestimation for diet.” Could the authors provide some justification for this statement?We have added details to the results section Health behaviour attributable healthcare use to indicate we are referring to the sensitivity analyses represented by the error bars in Figure 3.4. The units for the physical activity measure seem incorrect. Could it be MET hour, instead of MET?We have clarified in the table that our measure was MET-hours/day." } ] }, { "id": "49969", "date": "04 Jul 2019", "name": "Melody Ding", "expertise": [ "Reviewer Expertise Lifestyle epidemiology", "physical activity and health", "cost of illness analysis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this paper. In this study, the authors presented some novel and interesting analyses to explore health care cost attributable to unhealthy lifestyle patterns and socioeconomic position (SEP; or, perhaps more meaningful, socioeconomic inequalities?). Compared with existing literature on this topic, this study has a few unique contributions: 1) The authors modelled several lifestyle risk factors simultaneously, both in terms of contribution of individual risk factors and combined lifestyle profiles; 2) the authors used data across multiple years and considered the change in attributable and avoided costs over time; 3) the authors considered both lifestyle behaviours and low SEP as risk factors, both individually and combined. However, I have several recommendations for the authors to consider.\n\nThe information regarding the measurement of lifestyle risk factors and SEP is critical and should be presented with details. What measurement instruments were used? What are the reliability and validity of these measures, what is the rationale for the categorisation? Particularly, the small proportion of healthcare cost attributable to poor diet may be a result of the limitations of the measure (I consider the dietary measure to be a fruit and vegetable intake measure only).\n\nAlthough it is interesting and informative to examine healthcare cost associated with low SEP, I think this idea may require a little bit more consideration. SEP is a relative concept, so what is the practical interpretation of PAF associated with SEP? Were the authors considering the healthcare cost that could have been averted if everyone was of high socioeconomic status? Particularly, how do we correctly interpret PAF associated with ethnicity? What are the counterfactuals?\n\nTo me, it may be more meaningful to estimate the PAF of socioeconomic inequality, rather than SEP.\n\nIt seems that the authors have a-priori decided to model women and men separately? What is the rationale? However, later on in the paper, when describing the economic cost, all results were presented for both sexes combined. Is there an explanation for this?\n\nIt looks like the authors have conducted thorough and comprehensive statistical analysis. However, most results are not presented. For example, results from the regression analysis (at least for Model 3) could be of interest to readers.\n\nThe change analysis between 2004 and 2013: is this based on modelling using all data points or the two time points only?\n\nCould you please provide more information and a reference for inverse propensity-weighted analyses?\n\nSorry if I have missed this point from the paper. The modelling of SEP is based on adjusted analysis considering lifestyle risk factors? As we know SEP is a strong predictor of lifestyle behaviour, so parts of the association between SEP and healthcare cost would be explained by lifestyle behaviours. It would be useful if the authors more explicitly define the part of PAF attributable SEP independent of lifestyle risk factors.\n\nGiven that the data spans over 10 years, have the authors considered inflation? Particularly healthcare specific inflation? It may be worth discussing that the change in PAF over time could be a result of a change in population health behaviour of that in the average costs of certain health care procedures/items.\n\nFurthermore, the authors may consider the checklist we developed for reporting cost of illness analysis.1\n\nMinor comments/questions:\n\nAbstract: Taken together, health behaviours and socioeconomic position were associated with 34% ($134 billion)… suggest add \"these\" before health behaviours, because this is only referring to the 4 selected behaviours, not health behaviour in general.\n\nMethods: could the authors provide some justification regarding why these lifestyle behaviours were selected?\n\nResults: “uncertainty for the burden estimates indicates potential overestimation for physical activity and underestimation for diet.” Could the authors provide some justification for this statement?\n\nThe units for the physical activity measure seem incorrect. Could it be MET hour, instead of MET?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-303
https://f1000research.com/articles/8-1764/v1	16 Oct 19	{ "type": "Research Article", "title": "Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth", "authors": [ "Mohamed Shamel", "Mahmoud M. Al-Ankily", "Mahmoud M. Bakr", "Mahmoud M. Al-Ankily", "Mahmoud M. Bakr" ], "abstract": "Background: Tooth whitening usually includes the direct use of gels containing carbamide or hydrogen peroxide on the tooth enamel surface through a wide variety of products formulas. A generally new advancement in whitening of teeth uses the significant importance of the tooth color shift from yellow to blue in delivering a general enhancement in the observation of tooth whiteness. The aim of the current work was to measure the tooth whitening effects, surface roughness and enamel morphology of six different types of blue covarine-containing and blue covarine-free toothpastes using in vitro models. Methods: A total of 70 sound extracted human premolars were randomly and equally divided into seven groups, and each subjected to tooth brushing using different toothpastes. Tooth color and enamel surface roughness were measured before and after the brushing procedure using a white light interferometer, and scanning electron microscopy (SEM) was used to assess tooth surface after the procedure. Results: Toothpaste containing blue covarine resulted in the greatest improvement in tooth color amongst all groups as well as a statistically significant color difference when compared to blue covarine-free toothpaste. Furthermore, blue covarine-containing toothpaste resulted in fewer morphological changes to the enamel surface. This was confirmed with SEM images that showed smooth enamel surfaces with fine scratches.\n\nConclusions: The results from the present study show that blue covarine containing toothpastes are reliable, effective in tooth whitening and produce less surface abrasion when compared to blue covarine-free toothpastes.", "keywords": [ "Tooth whitening", "Blue covarine", "Enamel", "tooth color", "enamel surface roughness", "SEM" ], "content": "Introduction\n\nPatients are generally dissatisfied with their present teeth color, as demonstrated by many studies in different regions of the world1–6. This is most evident from the expanded interest for orthodontic treatment and for using products, either in office or at home, for whitening of teeth7.\n\nAttempts to improve the shade of teeth is presently possible using multiple methods, including professional scaling and prophylaxis performed in the dental clinic, fabrication of tooth crowns and laminate veneers and the use of tooth-whitening toothpastes8. Tooth-whitening formulations work by one of two different methods, either by the elimination of extrinsic tooth stain or by bleaching of the tooth itself9.\n\nTooth whitening usually includes the direct use of gels containing carbamide or hydrogen peroxide on the tooth enamel surface and are available in a wide variety of products formulas. The peroxide moves into the enamel structure to brighten any internal discoloration and hence making the teeth look whiter. Nonetheless, the efficient delivery of peroxides from the toothpaste to the tooth is multifactorial, including design factors, controlling boundaries and the comparatively limited contact periods during brushing9,10.\n\nHistorically, one of the main components in toothpastes used for whitening has been abrasives, which help to eliminate extrinsic stains as well as prevent their formation. A wide range of other components is usually added to this abrasive system, such as surfactants, calcium chelators, polymers and enzymes; however, evidence indicates that the abrasive is the most crucial component in the toothpastes for stain removal11,12. Yet, there are worldwide governing limitations on the maximum levels of abrasives allowed in any toothpaste and thus there is some restrictions to how much whitening can be effectively and (most importantly) safely obtained through abrasive advancements8.\n\nTo numerically evaluate color, a system was suggested by the International Commission on Illumination (CIE) in 1976 which consisted of three different coordinates designed as L, a and b. Any color is totally identified by three coordinates: L, a* and b. L values range from 0 to 100 and denotes darkness/brightness; a* represents the green–red component, with values ranging from –80 (green) to +80 (red); and b* represents the blue–yellow component, with values ranging from –80 (blue) to +80 (yellow)13.\n\nA generally novel advancement in tooth bleaching utilizes the importance of the tooth color change from yellow to blue (i.e. a decrease of b* values) in obtaining a general enhancement in the observation of whiteness of teeth14. The theory is reinforced by tooth whitening studies that show that the change in color from yellow to blue represents significant evidence of tooth whitening and that a decrease in the value of b* color parameter is a crucial factor for tooth whitening recognition by patients15,16. Using previous optical theories, a toothpaste containing a blue pigment, named blue covarine, was introduced. This paste applies the blue pigment onto the tooth enamel during daily cleaning procedures using toothbrushes, changing tooth color from yellow to blue. Several clinical and in vitro studies confirmed that this increase in bluish color and decrease in yellow color makes the teeth look whiter immediately after tooth brushing17,18.\n\nBlue covarine-containing tooth pastes have been additionally improved by supplementing the concentration levels of blue pigment, so as to furtherly improve the optical tooth whitening advantage14,19. The aim of the current study was to measure the tooth whitening effects, surface roughness and enamel morphology of six different types of blue covarine-containing and blue covarine-free toothpastes using in vitro models.\n\n\nMethods\n\nA total of 70 sound extracted human premolar teeth, obtained for purposes of research with informed consent. The study was approved by the Research Ethics Committee at Suez Canal University (approval number Suez- REC 27/2018), where this research was performed. The enamel surfaces were then cleaned with prophylactic paste to ensure elimination of extrinsic stains. The teeth were kept in sterilized artificial saliva for two hours. Teeth were mounted in gypsum blocks by embedding the roots and the lingual half, where the middle part of the middle one-third of the buccal aspect is the highest area of the specimen.\n\nThe specimens (n= 10 per group) were randomly distributed to the tested toothpastes which were as follows:\n\nGroup I: Close up White now (Unilever, São Paulo, Brazil) (with blue covarine)\n\nGroup II: Sensodyne True White (GSK, UK) (whitens by abrasion/sodium triphosphate)\n\nGroup III: Colgate Optic White (Colgate-Palmolive, USA) (whitens by abrasion/sodium monophosphate with hydrogen peroxide)\n\nGroup IV: Close Up (Unilever, São Paulo, Brazil)\n\nGroup V: Sensodyne (GSK, UK)\n\nGroup VI: Colgate (Colgate-Palmolive, USA)\n\nGroup VII: Control: No tooth paste application\n\nGroups I and IV have the same formulation (sodium fluoride), but the former has an additional whitening ingredient. The same is true for groups II and V (potassium nitrate), and III and VI have the same formulation (sodium fluoride), but the former have an additional whitening component. Thus, groups IV, V and VI serve as non-whitening controls for groups I, II and III, respectively.\n\nThe three parameters of color of each specimen were measured using a VITA Easyshade spectrophotometer Advance 4.01 (VITA Zahnfabric, Bad Sackingen, Germany) according to the CIE Lab* color order system. Mean measurements of the middle part of the middle one-third of the buccal aspect were recorded.\n\nThe Baseline surface roughness was studied using Zygo Maxim GP-200 white light interferometer (Artisan Technology Group, Illinois, USA) with a surface area 2 mm x 2 mm. Surface roughness was quantified as deviation from the ideal surface in µm using white light interferometer. Mean measurements of the middle part of the middle one-third of the buccal aspect were recorded.\n\nFor the purpose of standardization of the brushing procedure, a specially designed brushing apparatus was designed and fabricated (Figure 1). The brushing apparatus was set at 120 strokes/min, and a load of 250 g. For each experimental group, the specimens were fixed in the machine and brushed with a toothpaste corresponding to its group for 3.5 minutes, simulating 4 weeks of brushing three times a day. Every toothpaste was mixed with distilled water with a ratio of toothpaste: water 2:115. After tooth brushing, every specimen was rinsed in water to ensure removing all the toothpaste, and the teeth color was remeasured before they were restored in artificial saliva.\n\nThe Brushing apparatus consists of: (A) a gear box to reduce the speed of the motor to 2 cycle/second with a crankshaft and connecting rod attached to a slider in order to change the rotation movement into linear movement to provide a standardized 5 mm horizontal movement; (B) a brush holder; (C) double-pane balance; (D) samples holding pane; and (E) weight holding pane.\n\nThe change in color (ΔE) was calculated as follows:\n\nΔE=[(ΔL)2+(Δa)2+(Δb)2]1/2\n\nwhere ΔL, Δa, and Δb are the difference between the final and initial L, a, and b* color parameters, respectively.\n\nSurface roughness measures after the brushing procedure were recorded and the difference between the baseline was calculated.\n\nSurface Morphology was studied using images obtained from SEM of one sample from each group after the brushing procedure. The specimens were rinsed ultrasonically with water for ten minutes and prepared for SEM (FEI, QUANTA FEG 250) analysis. After dehydration, enamel surfaces were sputter-coated with gold (approximately 30–35 nm) and photomicrographs of representative areas were taken at 1000x and 2000x magnifications. The classification for the enamel changes were as follows; no alterations, mild or slight alterations, significant alterations and loss of superficial structures.\n\nAll data was analyzed with the statistical program SPSS version 21 (SPSS Armonk, NY, USA). Data from baseline as well as from final measurements were analyzed using one-way analysis of variance (ANOVA) when there is only one factor, which is the type of toothpaste, and Tukey’s test as a post hoc test. A paired Student’s t-test was used to compare the initial and final color parameters for each experimental group.\n\n\nResults\n\nResults for initial and final color parameters for all specimens of all groups are tabulated in Table 1. One-way ANOVA revealed mean color changes which was statistically significant between groups (p=0.01). The mean changes in color parameters which were informed by the International Commission on Illumination (CIELAB) values directly after brushing as well as difference in b value alone showed that toothpaste containing blue covarine (Group I, Close Up White Now) gave the highest decrease in b, revealing a bluish change in the color of the teeth and thus the highest improvement in tooth whiteness correspondingly, while the control group showed the least reduction in b and tooth whiteness (Figure 2).\n\nToothpaste which contained blue covarine (Group I) had a color statistically significant difference compared with the non-whitening control, group IV (Close up regular tooth paste) (p = 0.02, Student’s t-test) and also a statistically significant difference with other types of whitening tooth pastes not containing blue covarine group II (p=0.005) and group III (p=0.04), . Mean color changes of specimens treated with whitening blue covarine-free tooth pastes (groups II and III) showed a positive but statistically non-significant increase in the overall color change and a slight increase in the b* when compared to their non-whitening controls, groups V and VI, respectively (p = 0.08, Student’s t-test). Raw tooth color values for each group are available as Underlying data20.\n\nResults of surface roughness measurement of specimens of all groups are tabulated in Table 2. There were no statistically significant differences in the mean values of surface roughness difference (Ra) of all groups (p= 0.07). The control group showed the least surface roughness change, with a reduction in median Ra of 0.25 μm; the treatment with the toothpaste containing blue covarine (Group I) showed Ra value of 0.02 μm; the Ra value of tooth paste of group II was 0.02 μm while that of group III showed the highest Ra value of 0.53 (Figure 3 and Figure 4). Raw surface roughness values and white light interferometry images are available as Underlying data20,21.\n\n\nSEM examination\n\nGroup I tooth samples revealed relatively smooth enamel surface with few fine scratches. Group II showed similar surface pattern with some fine scratches on the enamel surface when compared to control group, whereas tooth paste of group III caused greater alteration in enamel morphology manifested as surface irregularities, few pores and also the scratches became more obvious. Samples from groups IV, V and VI exhibited no noticeable differences regarding the morphology of the surface of the enamel in comparison to the control samples (Figure 5). Raw SEM images are available as Underlying data21.\n\nGroups I and II show smooth enamel surface with fine scratches (arrows). Group III shows irregular enamel with deeper surface scratches and pores (arrows). Groups IV, V and VI show fine surface scratches and few pores (arrows).\n\n\nDiscussion\n\nA variety of commercially available toothpastes with and without bleaching agents for home use were chosen for this study to evaluate color change, surface roughness and surface structure of tested teeth. The findings of this study revealed toothpastes containing blue covarine preparations had a measurable improvement in tooth whiteness which was significantly increased directly after toothbrushing in comparison to other products not containing blue covarine, including group II and III toothpastes, which showed a statistically significant color difference, (p=0.005 and p=0.04, respectively).\n\nIn the present work, it was revealed that the blue covarine-containing toothpaste was the only toothpaste tested to give a significant decrease in b* value and improvement in tooth whiteness (∆E) in comparison to the controls. This decrease in b* value is mainly due to the deposition of the pigments on the enamel surface, which causes modification to the optical characters of the tooth surface22. The use of the blue pigment in toothpastes changes the observation of the yellow color in teeth by applying a thin, translucent blue layer on the tooth surface, thus creating a optical whitening effect. Yellow is opposite to blue in the spectrum of colors, thus a change or shift to blue creates an appearance which visually appears whiter by changing the net color towards white. This mechanism of whitening is built on many previous studies performed which showed that the blue shift of the yellow–blue axis (b) is of higher significance in the observation of whiter teeth than other axis, (L) or (a)23–25.\n\nThere are contradictory results in the literature regarding the efficiency of blue covarine to provide an improved whitening result in teeth whiteness. Many past and recent studies showed instant and progressive whitening14,19,26–28; however, some other studies reported no whitening effect of the blue covarine formulations29,30. This contradiction in results might be due to using different techniques in recording the color change following brushing procedure which includes the use of either visual or instrumental tools. In the present study, a Vita Easyshade spectrophotometer was used to measure the color change as it proved in several studies to be more accurate than visual tools31,32.\n\nIn contrast, all other tooth pastes tested in this study had a relatively no significant change in b and overall color change values (∆E) when compared to the control groups. In this study, the tested toothpaste with 1% hydrogen peroxide (Colgate White now) presented a whitening effect similar to that of conventional toothpaste because it contains abrasive particles in the composition. During toothbrushing, the abrasive particles are trapped between the tips of the toothbrush bristles and the stained tooth surface. This was confirmed by results from the surface roughness experiment, where group III specimens had the highest increase in mean difference of surface roughness. Since the abrasive particles are physically harder than the superficial staining, this is removed leaving a clean tooth surface. Thus, the abrasive cleaning mechanism mainly influences the extrinsic stains and does not significantly affect any underlying intrinsic staining or the natural color of teeth33,34. This finding is consistent with previous studies that concluded that conventional dentifrices could outperform or have a similar whitening effect to whitening toothpaste30,35,36.\n\nConcerning enamel surface roughness, Atomic force was not used to measure the surface roughness due to its limited measurement of an area of 25 μm on a flat area only, whereas a white light interferometer was used in this study due to its wider area measurement (2 mm x 2 mm) and ability to assess non-flat surfaces, which is superior to the properties of atomic force.\n\nAlthough there were no statistically significant differences between all groups in our study, group III toothpaste created the roughest surface, while group VI toothpaste resulted in the smoothest surface in comparison to control group. This change in physical properties can occur due to effects of demineralization and remineralization of enamel surface37. This demineralization can be caused by diffusion of hydrogen peroxide found in the toothpaste in group III.\n\nThe previous results of color change and surface roughness are consistent with the study performed by Vieira-Junior et al., which showed that the alteration in surface roughness has no significant correlation with b* values or significant alteration in the general color change represented by ∆E values38.\n\nIn the present study, it was aimed to compare the changes caused by different toothpastes with different compositions on human enamel surface with SEM and visualize the structural changes with photomicrographs. Specimens of the control group revealed a normal enamel surface, while that of groups I and II showed minor changes of the enamel surfaces manifested as fine scratches. However, group III specimens showed some alteration in the enamel surface manifested as surface irregularities, few pores and the scratches became more obvious.\n\n\nConclusion\n\nFrom the results of the current study it can be considered that toothpastes with blue covarine are both an effective and a safe method to improve the whiteness of teeth in the routine home tooth brushing.\n\n\nData availability\n\nFigshare: tooth color (Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth). https://doi.org/10.6084/m9.figshare.9923756.v120.\n\nThis project contains the following underlying data:\n\ntooth color (Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth).xlsx\n\nsurface roughness (Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth).xlsx\n\nFigshare: Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth. https://doi.org/10.6084/m9.figshare.9923780.v121.\n\nThis project contains raw scanning electron microscopy and white light interferometry images for each group.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nVan der Geld P, Oosterveld P, Van Heck G, et al.: Smile attractiveness. 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Gen Dent. 2015; 63(6): e24–28. PubMed Abstract\n\nCasado BGS, Moraes SLD, Souza GFM, et al.: Efficacy of Dental Bleaching with Whitening Dentifrices: A Systematic Review. Int J Dent. 2018; 2018: 7868531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlman DH: Colour Physics for Industry, edited by Roderick McDonald, Society of Dyers and Colourists, Bradford, England, 1987, 301 pp., paperbound. Price £ 20.00. Color Research & Application. 1988; 13(4): 264–265. Publisher Full Text\n\nTao D, Smith RN, Zhang Q, et al.: Tooth whitening evaluation of blue covarine containing toothpastes. J Dent. 2017; 67S: S20–S24. PubMed Abstract \| Publisher Full Text\n\nBergesch V, Baggio Aguiar FH, Turssi CP, et al.: Shade changing effectiveness of plasdone and blue covarine-based whitening toothpaste on teeth stained with chlorhexidine and black tea. Eur J Dent. 2017; 11(4): 432–437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang N, Zhang C, Agingu C, et al.: Comparison of Whitening Dentifrices on the Effectiveness of In-office Tooth Bleaching: A Double-blind Randomized Controlled Clinical Trial. Oper Dent. 2019; 44(2): 138–145. PubMed Abstract \| Publisher Full Text\n\nWestland S, Luo W, Li Y, et al.: Investigation of the perceptual thresholds of tooth whiteness. J Dent. 2017; 67S: S11–S14. PubMed Abstract \| Publisher Full Text\n\nPhilpotts CJ, Cariddi E, Spradbery PS, et al.: In vitro evaluation of a silica whitening toothpaste containing blue covarine on the colour of teeth containing anterior restoration materials. J Dent. 2017; 67S: S29–S33. PubMed Abstract \| Publisher Full Text\n\nTao D, Sun JN, Wang X, et al.: In vitro and clinical evaluation of optical tooth whitening toothpastes. J Dent. 2017; 67s: S25–s28. PubMed Abstract \| Publisher Full Text\n\nAl-Ankily M, shamel m, Bakr M: tooth color (Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth).xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9923756.v1\n\nshamel m, Al-Ankily M, Bakr M: Influence of different types of whitening tooth pastes on the tooth color, enamel surface roughness and enamel morphology of human teeth. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.9923780.v1\n\nOliveira M, Fernandez E, Bortolatto J, et al.: Optical Dental Whitening Efficacy of Blue Covarine Toothpaste in Teeth Stained by Different Colors. J Esthet Restor Dent. 2016; 28 Suppl 1: S68–77. PubMed Abstract \| Publisher Full Text\n\nKleber CJ, Putt MS, Nelson BJ: In vitro tooth whitening by a sodium bicarbonate/peroxide dentifrice. J Clin Dent. 1998; 9(1): 16–21. PubMed Abstract\n\nGerlach RW, Gibb RD, Sagel PA: A randomized clinical trial comparing a novel 5.3% hydrogen peroxide whitening strip to 10%, 15%, and 20% carbamide peroxide tray-based bleaching systems. Compend Contin Educ Dent Suppl. 2000; (29): S22–28; quiz S42-23. PubMed Abstract\n\nGerlach RW, Barker ML, Sagel PA: Objective and subjective whitening response of two self-directed bleaching systems. Am J Dent. 2002; 15(Spec No): 7A–12A. PubMed Abstract\n\nCollins LZ, Naeeni M, Platten SM: Instant tooth whitening from a silica toothpaste containing blue covarine. J Dent. 2008; 36 Suppl 1: S21–25. PubMed Abstract \| Publisher Full Text\n\nJoiner A, Philpotts CJ, Alonso C, et al.: A novel optical approach to achieving tooth whitening. J Dent. 2008; 36 Suppl 1: S8–14. PubMed Abstract \| Publisher Full Text\n\nVaz VTP, Jubilato DP, Oliveira MRM, et al.: Whitening toothpaste containing activated charcoal, blue covarine, hydrogen peroxide or microbeads: which one is the most effective? J Appl Oral Sci. 2019; 27: e20180051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorres CR, Perote LC, Gutierrez NC, et al.: Efficacy of mouth rinses and toothpaste on tooth whitening. Oper Dent. 2013; 38(1): 57–62. PubMed Abstract \| Publisher Full Text\n\nHorn BA, Bittencourt BF, Gomes OM, et al.: Clinical evaluation of the whitening effect of over-the-counter dentifrices on vital teeth. Braz Dent J. 2014; 25(3): 203–6. PubMed Abstract \| Publisher Full Text\n\nLiberato WF, Barreto IC, Costa PP, et al.: A comparison between visual, intraoral scanner, and spectrophotometer shade matching: A clinical study. J Prosthet Dent. 2019; 121(2): 271–275. PubMed Abstract \| Publisher Full Text\n\nChitrarsu VK, Chidambaranathan AS, Balasubramaniam M: Analysis of Shade Matching in Natural Dentitions Using Intraoral Digital Spectrophotometer in LED and Filtered LED Light Sources. J Prosthodont. 2019; 28(1): e68–e73. PubMed Abstract \| Publisher Full Text\n\nGanss C, Marten J, Hara AT, et al.: Toothpastes and enamel erosion/abrasion - Impact of active ingredients and the particulate fraction. J Dent. 2016; 54: 62–67. PubMed Abstract \| Publisher Full Text\n\nPatil PA, Ankola AV, Hebbal MI, et al.: Comparison of effectiveness of abrasive and enzymatic action of whitening toothpastes in removal of extrinsic stains - a clinical trial. Int J Dent Hyg. 2015; 13(1): 25–29. PubMed Abstract \| Publisher Full Text\n\nJurema AL, Claudino ES, Torres CR, et al.: Effect of Over-the-counter Whitening Products associated or Not with 10% Carbamide Peroxide on Color Change and Microhardness: in vitro Study. J Contemp Dent Pract. 2018; 19(4): 359–366. PubMed Abstract \| Publisher Full Text\n\nPintado-Palomino K, Vasconcelos CV, Silva RJ, et al.: Effect of whitening dentifrices: a double-blind randomized controlled trial. Braz Oral Res. 2016; 30(1): e82. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55473", "date": "22 Oct 2019", "name": "Rania Mossad Hassan", "expertise": [ "Reviewer Expertise Oral Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study investigated the effect of different whitening toothpastes on the teeth enamel color surface roughness and morphology.\nThe topic is interesting and the manuscript is well written and well constructed. The introduction was informative and covered the main ideas investigated in the study.\nThe apparatus used to simulate the brushing provided a standardized procedure thus aiding in having accurate results.\nAll data of teeth color and surface roughness are available.The discussion was well written and covered and explained the results thoroughly.\nHowever, some minor comments are suggested for the authors to consider:\nMore than one type of tooth pastes with blue covarine could have been tested to compare their efficacy, or different concentrations of blue covarine.\n\nThe authors didn`t mention if a new toothbrush was used in the experiment for each tooth or not.\n\nIn figure 2 the word \"colour\" is different than the spelling in the rest of the research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "57522", "date": "06 Dec 2019", "name": "Mahmoud Serag", "expertise": [ "Reviewer Expertise Prosthodontic Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study investigated the effect of different types of tooth pastes on the enamel surface properties.\nThe introduction section was informative, brief and well written. The authors highlighted the numerical evaluation of color as well as the different methods and materials for tooth whitening using tooth pastes at home.\nThe white light inteferometer used to measure surface roughness is an accurate method which ensured reliable results. The same applies to the brushing apparatus used although it was not clear the type of tooth brush used nor the frequency of changing the tooth brush.\nDifferent formulations of the investigated tooth pastes were not fully written.\nThe results were clear and the SEM micrographs were explained thoroughly. However, more statistical analysis could have been added to compare between more experimental groups, not only with the control.\nThe discussion was written well and explained the outcomes of the research.\nOverall the present research was written and executed well with clear results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "55277", "date": "06 Dec 2019", "name": "Rizwan Ullah", "expertise": [ "Reviewer Expertise Oral Biology and Dental materials." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the study is quite lengthy. It is suggested to the authors to reduce the length of article title.\n\nDoes the blue covarine toothpaste have any additional whitening agent in its composition?\n\nIn methods, instead of sterilized artificial saliva write \"sterile saliva\".\n\nIn Figure 1, please recheck the labelling, especially B and D.\n\nIn the discussion, give a brief account of the limitations of your study.\nOverall the study was well performed and showed some interesting results. The standardized brushing method was a very good way to ensure the accuracy of the results of the experiment. Statistical analysis of data is available and appropriately interpreted. However some minor suggestions could be taken into consideration: Further analysis between the results of different groups, e.g group II, III and IV could have added to the study. Overall, the study was clear and well written.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1764
https://f1000research.com/articles/8-1039/v1	10 Jul 19	{ "type": "Research Article", "title": "Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence", "authors": [ "Laura McCulloch", "Stuart M. Allan", "Hedley C. Emsley", "Craig J. Smith", "Barry W. McColl", "Stuart M. Allan", "Hedley C. Emsley", "Craig J. Smith", "Barry W. McColl" ], "abstract": "Background: Blockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. Methods: We assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the sympathetic nervous system (SNS) was determined by measuring noradrenaline, a major SNS mediator. Results: There were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. Conclusion: These data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations and is therefore unlikely to further aggravate stroke-associated infection susceptibility through reduced availability of these key anti-microbial mediators.", "keywords": [ "stroke", "IL-1Ra", "immunoglobulins", "complement", "infection" ], "content": "Introduction\n\nBlocking the actions of the inflammatory cytokine interleukin-1 (IL-1) using a highly selective IL-1 receptor antagonist (IL-1Ra) reduced injury and improved outcome in multiple experimental animal models of cerebral ischemia and is in ongoing clinical stroke trials1–4. The inflammatory-modifying properties of IL-1Ra may confer protective effects to the brain after stroke; however, due to its potential for immunosuppression, it may also compromise systemic immune responses important for defence against infection. Systemic immune dysregulation is particularly important to consider in the context of stroke as patients are highly susceptible to infection, which likely involves roles for stroke-induced impairments in some immune functions5.\n\nWe have previously shown deficits in early antibody responses, particularly IgM, associated with innate-like B cells in both experimental animals and stroke patients, which may contribute to post-stroke infection susceptibility6. IL-1β is reported to induce IgM production in innate-like B cells7; therefore, treatment with IL-1Ra may inhibit these important anti-microbial effects. We assessed whether markers associated with antibody-mediated antibacterial defences were compromised in patients treated with IL-1Ra after stroke. In summary, our data suggests treatment with IL-1Ra is unlikely to aggravate antibody-associated immune function deficits induced by stroke.\n\n\nMethods\n\nThis study involved tertiary analysis of plasma samples taken from a randomised, placebo-controlled phase II trial originally designed to determine the safety and biological activity of intravenous (IV) IL-1Ra4. The online clinical trials registries ClinicalTrials.gov and ISRCTN went live online during the year 2000, at which time online trial registration was a relatively new recommendation. The original IV IL-1Ra trial was set-up in 2000 and commenced February 2001 and therefore this trial was not officially registered. Ethical approval for reanalysis of the samples was obtained through the Health Research Authority National Research and Ethics Service Committee (16/NW/0853).\n\nParticipants and study procedures. In brief, patients ≥ 18 years of age with a clinical diagnosis of stroke within six hours of stroke onset were eligible. Exclusion criteria included National Institutes of Health Stroke Scale (NIHSS) score of ≤ 4, pre-stroke modified Rankin Scale (mRS) score of ≥ 4 or rapidly improving neurological deficit. Patients were randomly assigned to treatment with recombinant methionylated human IL-1Ra (n=17) or placebo (n=17) stratified by age (< 70 and ≥ 70 years), baseline stroke severity (NIHSS score 4–9, 10–20, ≥ 21) and time since stroke onset (< 4 or ≥ 4 h), but not by sex. IL-1Ra was initially administered as an IV loading dose of 100 mg over 60 seconds, followed by 72 hours of consecutive infusions at 2 mg/kg/h. Full patient baseline characteristics and stratification of groups are provided as Extended data8.\n\nNon-stroke control patients (n=13) of a similar age range with no previous history of stroke or transient ischemic attack were also recruited. Control patients were living independently at home, free of infection and able to provide written, informed consent. Controls were matched to stroke patients (six to patients receiving IL-1Ra and seven to patients receiving placebo) on a basis of age (±5 years), sex and degree of atherosclerosis, which was determined using a non-invasive assessment of either ankle-brachial pressure or carotid atherosclerosis using Doppler, as previously described9.\n\nBlood sampling. Venous blood samples were collected prior to the initiation of treatment (admission), at the next 9 am time point (if admission was before 7 am or after 11 am), and then at 9 am at 24 hours, 2 days, 3 days, 4 days and at 5–7 days after stroke, into tubes containing a final concentration of 10 μg/ml pyrogen-free heparin and wrapped in cool packs. Control patients were sampled at 9 am and also at matched patient admission time (two hours) if this was not between 7 am and 11 am. Samples were centrifuged one hour after collection at 2000 xg for 30 min at 4°C. Plasma was separated and frozen in aliquots at -70°C until further analysis.\n\nImmunoglobulins and complement components were measured in plasma samples using MILLIPLEX® multiplex assays. Patient details were blinded from samples and coded samples were randomised across plates for analysis. The MILLIPLEX®MAP Human Isotyping Magnetic Bead Panel- Isotyping Multiplex Assay (HGAMMAG-301K-06, Merck Millipore Corporation, Billerica, MA, USA) was used to measure IgG1, IgG2, IgG3, IgG4, IgA and IgM. MILLIPLEX®MAP Human Complement Panel 1 (HCMP1MAG-19K, Merck Millipore Corporation) was used to measure C2, C4b, C5, C9, Mannose-binding lectin (MBL), Factor D (Adipsin) and Factor I. Many samples had concentrations of Factor D and Factor I below the detection range of the standard curve and so results for these analytes are not reported. MILLIPLEX®MAP Human Complement Panel 2 (HCMP2MAG-19K, Merck Millipore Corporation) was used to measure C1q, C3, C3b/ iC3b, C4, Factor B, Properdin and Factor H. Samples were assayed as singlets and all samples, standards and quality controls were prepared in accordance with the manufacturer’s instructions. Samples were incubated with beads on a plate for one hour (isotyping assay) or overnight (complement assays) at 4°C and washes carried out using a magnetic plate washer. Plates were analysed using a Magpix™ Luminex® machine and Luminex xPonent® software version 4.2, with a sample volume of 50 ml per well and a minimum of 50 events counted per sample.\n\nNoradrenaline was measured in plasma samples using a Noradrenaline ELISA kit (BA E-5200; LDN®, Nordhorn, Germany). Patient details were blinded from samples and coded samples were randomised across plates for analysis. Samples were assayed as singlets and all samples, standards and quality controls were prepared in accordance with the manufacturer’s instructions, where noradrenaline is extracted from plasma using a cis-diol-specific affinity gel, acylated, enzymatically converted and then measured by ELISA. Optical density at 450 nm was measured using an MRX microplate Reader (Dynatech Labs, Chantilly, VA).\n\nAll immunoglobulin and complement components were measured in μg/ml and the D’Agostino and Pearson omnibus test was used to determine Gaussian distribution of sample data. As data were non-normally distributed, sample values were log10-transformed. As the precise kinetics of individual patient responses may vary, the maximal and minimal concentrations of each mediator in the first seven days after stroke were compared to non-stroke controls. Maximal and minimal concentrations from IL-1Ra-treated and placebo-treated stroke patients and non-stroke controls were compared by one-way ANOVA with Bonferonni correction. Noradrenaline concentrations were measured in ng/ml and the D’Agostino and Pearson omnibus test was used to confirm Gaussian distribution of sample data. Maximal and minimal noradrenaline concentration from IL-1Ra-treated and placebo-treated stroke patients and non-stroke controls were compared by one-way ANOVA with Bonferonni correction. Data analysis was performed using GraphPad Prism 6.0 statistical analysis software and for all experiments, values of P ≤ 0.05 were accepted as statistically significant.\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/587881).\n\n\nResults\n\nImmunoglobulin M (IgM) is the predominant immunoglobulin isotype associated with early B cell antibody responses to infection by innate-like B cells, which we have previously shown to be depleted after experimental stroke in mice6,10,11. Lower minimum concentrations of IgM were measured after stroke in comparison to non-stroke controls, and no difference was found between placebo and IL-1Ra treated patients. (Figure 1A)12. Maximum IgM concentrations in the first seven days after stroke were also assessed and did not significantly differ in IL-1Ra or placebo treated patients in comparison to non-stroke controls (Supplementary Figure 1A, Extended data)8. This indicates that the reduced minimum IgM concentration measured over the first seven days reflects an actual reduction in circulating IgM in stroke patients and is not an artefact of increased variance in IgM concentration after stroke.\n\n(A) Minimum IgM concentration measured in the first seven days after stroke was lower in both placebo and IL-1Ra treated patients in comparison to healthy controls. Data show mean ±SD, * P<0.05, ** P<0.01, one-way ANOVA with Bonferonni correction. (B) Minimum concentration of IgG1 and measured in the first seven days after stroke was reduced in both placebo and IL-1Ra treated patients in comparison to healthy controls. Minimum IgG4 (C) and IgA (D) concentrations were reduced in placebo-treated stroke patients in comparison to healthy controls. There was no significant difference between placebo-treated and IL-1Ra-treated stroke patients. No significant difference in IgG2 (E) and IgG3 (F) concentration was detected between placebo-treated and IL-1Ra-treated stroke patients in comparison to healthy controls. Data show mean ±SD, * P<0.05; ** P<0.01; one-way ANOVA with Bonferonni correction.\n\nMinimum IgG1 concentration was significantly reduced in both placebo-treated and IL-1Ra-treated stroke patients in comparison to non-stroke controls (Figure 1B)12. Minimum IgG4 (Figure 1C) and IgA (Figure 1D) concentrations were significantly reduced in placebo-treated stroke patients only. However, there was no significant difference in these immunoglobulins between placebo-treated and IL-1Ra-treated patients. Minimum concentrations of IgG2 (Figure 1E) and IgG3 (Figure 1F) were not significantly altered in IL-1Ra or placebo treated patients in comparison to non-stroke controls. Maximal circulating concentrations of all immunoglobulin isotypes measured in the first seven days after stroke were also compared to non-stroke controls and no significant differences were measured in any immunoglobulin isotypes (Supplementary Figure 1B-F, Extended data)8.\n\nAs complement components are directly associated with the antibacterial functions of immunoglobulins, we investigated stroke-induced changes in circulating complement components and if any changes observed were further influenced by treatment with IL-1Ra. Stroke induced a significant reduction in the minimum concentrations of C3b/ iC3b (Figure 2A), C3 (Figure 2B), C4 (Figure 2C), Factor H (Figure 2D) and Properdin (Figure 2E) measured in the first seven days after stroke in both placebo and IL-1Ra treated patients in comparison to non-stroke controls12. Maximum circulating concentrations of these complement components measured in the first seven days after stroke were also compared to non-stroke controls and no significant differences were seen (Supplementary Figure 2A–E, Extended data)8.\n\nMinimum concentrations of (A) C3b/ iC3b, (B) C3, (C) C4, (D) Factor H and (E) Properdin were measured in the first seven days after stroke were reduced in both placebo and IL-1Ra treated patients in comparison to healthy controls. Data show mean ±SD, * P<0.05; ** P<0.01; one-way ANOVA with Bonferonni correction.\n\nIn contrast, stroke induced a significant increase in maximal circulating concentrations of C1q (Figure 3A), C5 (Figure 3D) and C9 (Figure 3E) in both IL-1Ra and placebo treated patients measured in the first seven days after stroke in comparison to non-stroke controls. Maximum concentrations of C2 (Figure 3B) and C4b (Figure 3C) were increased in placebo-treated patients only12. However, no significant difference was apparent between placebo treated and IL-1Ra treated patients for these factors, suggesting IL-1Ra treatment exerts no effects additional to stroke. Minimum concentrations of these complement components measured in the first week after stroke were also compared to non-stroke controls and no significant differences were seen (Supplementary Figure 3A–E, Extended data)8.\n\nMaximum concentrations of (A) C1q, (B) C2, (C) C4b, (D) C5 and (E) C9 were measured in the first seven days after stroke were increased in both placebo and IL-1Ra treated patients in comparison to healthy controls. Data show mean ±SD, * P<0.05; ** P<0.01; one-way ANOVA with Bonferonni correction.\n\nMinimal and maximal levels of Factor B, MBL and C5a measured in the first week after stroke were also compared to non-stroke controls. Concentrations of Factor B (Figure 4A, B) and MBL (Figure 4C, D) were not significantly altered by stroke or by treatment with IL-1-Ra12.\n\nMinimal and maximum concentrations of (A, B) Factor B and (C, D) mannose-binding lectin (MBL) were measured in the first seven days after stroke were unchanged in both placebo and IL-1Ra treated patients in comparison to healthy controls. Data show mean ±SD, one-way ANOVA with Bonferonni correction.\n\nSplenic noradrenaline levels are increased after experimental stroke and may be toxic to IgM producing B cells6. Maximum noradrenaline concentration measured in the first seven days after stroke was increased in both placebo and IL-1Ra treated patients in comparison to non-stroke controls (Figure 5A)12. Treatment with IL-1Ra had no additional effect on noradrenaline concentration when compared to placebo. Minimum noradrenaline concentration in the first seven days after stroke was also measured and was not significantly different to non-stroke controls (Supplementary Figure 4, Extended data) or affected by IL-1Ra treatment8.\n\nMaximal noradrenaline concentration measured in the first seven days after stroke was significantly higher in both placebo and IL-1Ra treated patients in comparison to controls. Data show mean ± SD P<0.05; *P<0.01; one-way ANOVA.\n\n\nDiscussion\n\nIn this study, we assessed if markers associated with antibody-mediated antibacterial defences were compromised in patients treated with IL-1Ra after stroke. Plasma IgM, IgG1, IgG4 and IgA immunoglobulin concentrations were reduced after stroke and this was not further altered by treatment with IL-1Ra. Assessment of complement components indicated induction of the classical pathway of complement activation after stroke but inhibition of the alternative pathway without modulation by IL-1Ra. Plasma noradrenaline was increased after stroke and also not influenced by treatment with IL-1Ra. These data suggest that treatment with IL-1Ra is unlikely to aggravate antibody-associated immune function deficits induced by stroke.\n\nThe IL-1 family of cytokines play a critical role in host defence to pathogens by signalling to a variety of host cells to induce downstream effects including, but not limited to, pro-inflammatory cytokine and chemokine production, immune cell recruitment and upregulation of vascular adhesion molecules13,14. However, in conditions of sterile inflammation and tissue injury, such as stroke, these effects can aggravate primary tissue damage and impair injury repair mechanisms. Blocking IL-1 signalling has shown improved outcome in both experimental animal and patient stroke studies1,4,15. However, the immunosuppressive effects of blocking IL-1 signalling after stroke may additionally inhibit systemic responses to infection, further increasing the risk of infection in patients who are already immune compromised16,17. Indeed, meta-analysis studies have shown an increased risk of serious infection in rheumatoid arthritis patients treated for prolonged periods with the IL-1 blocking drug anakinra16. However, as of yet this has not been observed in stroke patients, potentially reflecting differences in the duration of treatment. No statistically significant differences in infection incidence were seen between IL-1Ra and placebo treated patients in this study, with 5/17 IL-1Ra treated patients experiencing infection between admission and day seven and 4/17 infections in placebo treated patients. Consistent with this pattern, we have shown here that relatively short duration of treatment with IL-1Ra after acute stroke did not further affect stroke-induced changes to circulating immunoglobulin, complement or noradrenaline concentrations and is therefore unlikely to further compromise immune defence against infection through reducing the availability of these antibacterial mediators.\n\nIL-1 cytokine family members are reported to have variable effects on B cell antibody production. IL-1β was reported to be important for the rapid production of anti-bacterial IgM by innate-like B cells important for early containment of infection prior to the induction of adaptive immune responses7,18. This would suggest treatment with IL-1Ra after stroke could further compromise the early production of IgM in innate-like B cells which are already known to be reduced in number after stroke6. However, this effect of IL-1Ra on IgM concentrations was not seen. We know that experimental stroke results in a significant loss of many populations of B cells and associated IgM6; therefore, it is possible that the effects of the stroke itself on B cells overwhelm any additional effects of cytokines that could moderately enhance or inhibit immunoglobulin production. Furthermore, we do not know if remaining B cells are functionally impaired and therefore able to respond to IL-1β signalling as they would under normal homeostatic conditions. We have previously reported that stroke is associated with reduced circulating IgM concentrations in comparison to non-stroke controls6, an effect reproduced here. Further studies will be required to determine if IgM, or any of the mediators assessed in this study, would be useful as biomarkers to determine which patients are likely to develop infection after stroke.\n\nWe have shown for the first time that circulating IgG1, IgG4 and IgA concentrations were reduced in the first seven days after stroke in comparison to non-stroke controls. This is in agreement with previous data showing that pan-IgG concentrations were reduced in patients after stroke, although subclasses of IgG were not assessed in that study and no reduction in IgA was found at the seven day time point assessed19. IgA is the most predominant immunoglobulin isotype at mucosal surfaces including the respiratory tract and is crucial for antibacterial protection at these sites20. Given the early reduction of IgA in placebo-treated stroke patients, determining the effect of stroke on IgA-producing B cells at infection susceptible sites, such as the lung mucosa, could further elucidate if this has an important role in post-stroke infection susceptibility.\n\nIn contrast to the short half-life of IgA and IgM20–22, the half-lives of IgG1 and IgG4 are reported to be 21 days and therefore, an early reduction in IgG concentration is not compatible with a lack of de novo production after stroke due to loss of B cells23. Previous studies have suggested that reduced total-IgG after stroke may be associated with increased loss or catabolism of IgG, which could account for reductions in concentration occurring more rapidly than its natural half-life24. An alternative explanation could be that reduced IgG concentration is indicative of vascular risk factors and inflammatory changes preceding stroke that are associated with stroke risk. However, control patients in this study were matched for risk factors including their degree of atherosclerosis and would be expected to show similar changes to stroke patients if these were associated with risk factors. Understanding that the kinetics of an individual immunoglobulin subset changes both preceding and as a result of stroke, and their associations with post-stroke infections, could be invaluable in providing new therapeutic targets to reduce incidence of infection and improve outcome in patients.\n\nThe complement system has a crucial role in enhancing humoral immune defence and protecting from bacterial infection via interactions with both the innate and adaptive immune systems25. As activation of complement is closely associated with efficient immunoglobulin-mediated clearance of pathogens, we determined whether these pathways were compromised by stroke. We have assessed, for the first time, individual concentrations of multiple complement components covering all pathways of complement activation after stroke. These exploratory data suggest there are no overall deficits in complement activation after stroke. Complement activation pathways converge at multiple points; however, their initial activation mechanisms are distinct. The classical complement pathway is activated when IgM or IgG immune complexes bind to C1 (composed of C1q, C1r and C1s)25,26. Maximum circulating concentration of complement components associated with the classical and lectin pathways of activation, C1q, C2 and C4b, and end stage mediators common to all pathways, C5 and C9, were increased in the first seven days after stroke in comparison to non-stroke controls. As concentrations of MBL itself was not significantly altered by stroke, this suggests the classical complement pathway is specifically activated after stroke.\n\nIn contrast, the alternative pathway of complement activation is initiated by microbial cell surfaces and polysachharide antigen and results in a cascade that generates C325,26. Complement components that were significantly downregulated after stroke, C3b/ iC3b, C3, Factor H (fH) and Properdin, are more associated with the alternative pathway of complement activation, suggesting that the alternative pathway is suppressed. These data are in agreement with previous studies investigating systemic C-reactive protein, C3c and C4 complement concentrations in the serum of patients 24 h after ischemic stroke, which concluded that the classical pathway of complement activation was activated in the first 24 h after ischemic stroke, whereas C3c, associated with the alternative pathway, was reduced27,28. The roles of individual pathways of complement activation in infection susceptibility after stroke remains to be determined, but these data suggest overall deficits in complement concentration are unlikely to contribute to reduced antibody-mediated clearance of pathogens that may occur after stroke, further supporting reduced circulating immunoglobulins as an important influence on infection susceptibility.\n\nIn this study, circulating noradrenaline concentrations measured in the first week after stroke were increased in comparison to non-stroke controls but were not influenced by treatment with IL-1Ra. This is in agreement with previous studies showing activation of the sympathetic nervous system in both stroke and subarachnoid haemorrhage patients that resulted in increased plasma noradrenaline concentrations that persisted up to 10 days29–31. Our previous studies have shown that after experimental stroke, activation of the sympathetic nervous system and release of noradrenaline within the spleen is toxic to resident B cells and preventing noradrenaline signalling using the β-blocker propranolol prevented B cell and IgM loss and resulted in reduced infectious burden6. The cytokine IL-1β is also increased in the spleen after stroke and is reported to activate peripheral nerves, including the splenic nerve, and increase production of splenic noradrenaline32,33. However, blockade of IL-1β signalling did not alter circulating concentrations of noradrenaline after stroke.\n\nIn summary, we have shown that treatment with IL-1Ra after stroke does not affect circulating concentrations of immunoglobulins, complement components or noradrenaline and is therefore unlikely to further increase patient susceptibility to infection via pathways in which these mediators are key participants. This is in agreement with data from IL-1Ra Phase II trials, in which treatment of stroke patients with IL-1Ra did not aggravate incidence of infection4,12. These data suggest that blocking IL-1 in a stroke context may not be concerning from the perspective of increasing infection risk in patients. Additionally, the reductions in circulating immunoglobulin concentrations detected after stroke in this study further support that antibody mediated immune defence may be an important therapeutic target to reduce the burden of infection after stroke.\n\n\nData availability\n\nEdinburgh Data Share: Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence. https://doi.org/10.7488/ds/257012\n\nThis project contains the following underlying data:\n\n- Full noradrenaline data by patient and timepoint.xlsx (noradrenaline concentrations in ng/ml)\n\n- Full complement data by patient and timepoint.xlsx (complement concentrations in μg/ml)\n\n- Full immunoglobulin data by patient and timepoint.xlsx (immunoglobulin concentrations in μg/ml)\n\n- Figure 1A dataset.pzf – Figure 5A dataset.pzf (raw data underlying Figure 1–Figure 5 in GraphPad Prism format)\n\n- Figure 1 full data excel format.xlsx (minimum concentrations of immunoglobulins in μg/ml)\n\n- Figure 2 full data excel format.xlsx (minimum concentrations of complement components C3b/iC3b, C3, C4, Factor H and Properdin in μg/ml)\n\n- Figure 3 full data excel format.xlsx (maximum concentrations of complement components C1q, C2, C4b, C5 and C9 in μg/ml)\n\n- Figure 4 full data excel format.xlsx (concentrations of complement components Factor B and MBL in μg/ml)\n\n- Figure 5 full data excel format.xlsx (maximum concentrations of noradrenaline in ng/ml)\n\nEdinburgh Data Share: Supplementary Figures: Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence. https://doi.org/10.7488/ds/25458\n\nThis project contains the following extended data:\n\n- Supplementary Figure 1.tif (figure showing maximum concentrations of immunoglobulin)\n\n- Supplementary Figure 2.tif (figure showing maximum concentrations of complement components C3b/iC3b, C3, C4, Factor H and Properdin)\n\n- Supplementary Figure 3.tif (figure showing minimum concentrations of complement components C1q, C5, C9, C2 and C4b)\n\n- Supplementary Figure 4.tif (figure showing minimum concentrations of noradrenaline)\n\n- Supplementary Table 1.tif (table of baseline characteristics of stroke patients and stratification of groups)\n\n- Data Supplementary Fig 1.xlsx - Data Supplementary Fig 4.xlsx (data underlying Supplementary Figures 1-4)\n\n- Data Supplementary Fig 5.xlsx (Supplementary Table 1 in spreadsheet format)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis work was funded by grants from BBSRC [BB/J004332/1] and MRC [MR/L003384/1, MR/R001316/1] assigned to Dr Barry W. McColl and the University of Edinburgh Moray Endowment Fund assigned to Dr Laura McCulloch.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank all the participating patients and controls for their participation and consent. We also thank Sharon Hulme for assistance with ethical applications and sample transfer. We thank Merck Millipore Corporation, Billerica, MA, USA for kind provision of the Milliplex® MAP immunoglobulin isotyping and complement panel kits used in this study.\n\n\nReferences\n\nSobowale OA, Parry-Jones AR, Smith CJ, et al.: Interleukin-1 in Stroke: From Bench to Bedside. Stroke. 2016; 47(8): 2160–2167. PubMed Abstract \| Publisher Full Text\n\nTouzani O, Boutin H, Chuquet J, et al.: Potential mechanisms of interleukin-1 involvement in cerebral ischaemia. J Neuroimmunol. 1999; 100(1–2): 203–215. 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J Clin Immunol. 1994; 14(2): 134–140. PubMed Abstract \| Publisher Full Text\n\nMorell A, Terry WD, Waldmann TA: Metabolic properties of IgG subclasses in man. J Clin Invest. 1970; 49(4): 673–680. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiesz A, Dalpke A, Mracsko E, et al.: DAMP signaling is a key pathway inducing immune modulation after brain injury. J Neurosci. 2015; 35(2): 583–598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKemper C, Atkinson JP: T-cell regulation: with complements from innate immunity. Nat Rev Immunol. 2007; 7(1): 9–18. PubMed Abstract \| Publisher Full Text\n\nHolers MV: Complement and its receptors: new insights into human disease. Annu Rev Immunol. 2014; 32: 433–459. PubMed Abstract \| Publisher Full Text\n\nPedersen ED, Waje-Andreassen U, Vedeler CA, et al.: Systemic complement activation following human acute ischaemic stroke. Clin Exp Immunol. 2004; 137(1): 117–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Napoli M: Systemic complement activation in ischemic stroke. Stroke. 2001; 32(6): 1443–1448. PubMed Abstract \| Publisher Full Text\n\nNaredi S, Lambert G, Edén E, et al.: Increased sympathetic nervous activity in patients with nontraumatic subarachnoid hemorrhage. Stroke. 2000; 31(4): 901–906. PubMed Abstract \| Publisher Full Text\n\nUrra X, Cervera A, Obach V, et al.: Monocytes are major players in the prognosis and risk of infection after acute stroke. Stroke. 2009; 40(4): 1262–1268. PubMed Abstract \| Publisher Full Text\n\nChamorro A, Amaro S, Vargas M, et al.: Catecholamines, infection, and death in acute ischemic stroke. J Neurol Sci. 2007; 252(1): 29–35. PubMed Abstract \| Publisher Full Text\n\nSchwarting S, Litwak S, Hao W, et al.: Hematopoietic stem cells reduce postischemic inflammation and ameliorate ischemic brain injury. Stroke. 2008; 39(10): 2867–2875. PubMed Abstract \| Publisher Full Text\n\nNiijima A, Hori T, Aou S, et al.: The effects of interleukin-1 beta on the activity of adrenal, splenic and renal sympathetic nerves in the rat. J Auton Nerv Syst. 1991; 36(3): 183–192. PubMed Abstract \| Publisher Full Text" }	[ { "id": "51097", "date": "22 Jul 2019", "name": "María Ángeles Moro", "expertise": [ "Reviewer Expertise stroke and vascular cognitive impairment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe beneficial properties of IL-1ra have been widely demonstrated in numerous experimental models of cerebral ischemic injury. For this reason, the molecule is currently under evaluation in ongoing clinical trials. However, given the mechanism of action of IL-1ra, possible adverse effects of this drug and derived from its immunosuppressive action, such as the potentiation of stroke-related infections, might hamper its clinical use. In this context, the aim of the work by McCulloch et al. is highly relevant by trying to ascertain whether factors associated with enhanced susceptibility to bacterial infections are increased in IL-1ra-treated patients after stroke. The authors explored a wide array of very well selected parameters, ranging from Ig isotypes, components of the complement system, up to noradrenaline, as a parameter of the stress response. The results show that whereas these factors, as expected, are affected by stroke, none of them was affected by the treatment, clearly supporting that, at least with the protocol of administration used for this study, IL-1ra is safe as regards to immunosuppression-derived complications.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "51311", "date": "26 Jul 2019", "name": "Stefano Fumagalli", "expertise": [ "Reviewer Expertise Stroke neuroimmunology", "complement system" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMcCulloch et al. addressed whether stroke patients treated with IL-1 receptor antagonist (IL-1Ra) had factors associated with antibody-mediated antibacterial defences compromised. Based on their results, the authors conclude that IL-1Ra does not alter circulating immunoglobulin and complement factors in patients’ blood, thus the treatment is not expected to further aggravate stroke-associated infection.\n\nThe addressed issue is of pivotal importance for stroke patient treatment, especially for treatment protocols using anti-inflammatory agents. Post-treatment complications have contributed to the unsuccessful clinical translation of promising anti-inflammatory compounds that proved effective in experimental models.\n\nIn its present form, the work presents some weakness that the authors should tackle.\n\nData are presented as the minimum or maximum levels of immunoglobulins and complement factors observed in a 7 day-period after stroke. Blood was sampled at admission (prior to treatment) and at 1, 2, 3, 4 and 5-7 days after stroke. I think that, being so many sampling times available, and post-stroke inflammation quickly changing over time, a picture of the temporal changes in treated vs. non-treated patients would be very informative. As such, I suggest: - to add the information on when min (or max) concentrations were reached in each condition, comparing them; - to add a statistical analysis for repeated measures comparing IL-1Ra patients’ sampling at each time after treatment vs. admission (prior to treatment).\n\nHad patients received recanalization therapies? Since the temporal window of IL-1Ra for some patients is still within that of thrombolytic therapies (<4.5h), the authors should explain why they did not receive them if so. From what I can understand, the specimens used come from a trial registered in 2000, thus likely explaining the use of the placebo in patients eligible for recanalization therapies according to present guidelines. I think that this needs clarification in the text.\n\nThere is a big difference in placebo vs. IL-1Ra treated patients in the frequency of haemorrhagic stroke (0% vs. 29.4%). Did the authors consider to correct for this potentially impacting difference?\n\nThe measure of complement components may be biased. Indeed blood specimens were collected in the presence of heparin, which does not represent the standard for running unbiased complement protein analysis. Complement proteins and activation products should be measured in blood sampled in the presence of EDTA (Bergseth et al., 20131).\n\nIn complement system biology, the decrease of circulating levels of recognition molecules in acute phases is generally regarded as an index of complement activation (through consumption of the circulating proteins binding their targets). On later times, circulating levels also mirror increased synthesis and they may increase. This should be considered when discussing the observations relative to complement measures. Moreover, complement system activation in stroke patients is reported in the first 6h (Zangari et al., 20162), therefore a comparison of admission vs. later sampling in IL-1Ra treated patients would be informative (do they recover to control physiological complement protein levels as expected?).\n\nThere is now a general consensus that the lectin pathway of complement activation is that mainly involved after ischemic stroke. This has been reviewed in Orsini et al. (20143) and Fumagalli and De Simoni (20164). Besides MBL, the lectin pathway comprises other recognition molecules such as ficolins and collectin-11, that play a major role in ischemic patients (especially ficolin-1 and ficolin-3; refer to the above mentioned reviews). In this regard the conclusions drawn by the authors that: ‘As concentration of MBL itself was not significantly altered by stroke, this suggests the classical complement activation is specifically activated after stroke’ sounds incorrect. Of note, MBL concentrations are genetically determined and about 20% of the general population bears variants causing MBL deficiency (Garred et al., 20065). Indeed in figure 4C the authors show MBL levels below the detection limit. It is acknowledged that a circulating level of MBL < 500 ng/mL is associated with MBL genetic variants (Eisen et al., 20086). Thus it is not surprising that no MBL changes were observed. A more clear analysis could be done excluding the MBL-deficient patients. Interestingly, these latter patients develop less ischemic lesion volume and deficits than those with normal MBL circulating levels (Osthoff et al., 20117), thus further supporting lectin pathway critical contribution to stroke pathophysiology.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4957", "date": "16 Oct 2019", "name": "Laura McCulloch", "role": "Author Response", "response": "We are pleased the reviewer recognises the relevance of this study to the future treatment of stroke patients and thank them for their insightful and constructive comments which we have addressed as detailed below. We hope that these changes meet with the reviewer’s approval.1. Data are presented as the minimum or maximum levels of immunoglobulins and complement factors observed in a 7 day-period after stroke. Blood was sampled at admission (prior to treatment) and at 1, 2, 3, 4 and 5-7 days after stroke. I think that, being so many sampling times available, and post-stroke inflammation quickly changing over time, a picture of the temporal changes in treated vs. non-treated patients would be very informative. As such, I suggest:- to add the information on when min (or max) concentrations were reached in each condition, comparing them;- to add a statistical analysis for repeated measures comparing IL-1Ra patients’ sampling at each time after treatment vs. admission (prior to treatment).We appreciate that there are limitations in analysing the data as the minimum or maximum concentration reached over a sampling time course. However variation in the kinetics of individual patient immune responses to stroke can be affected by many factors including stroke severity, presence of infection and timing of infection onset. In a small patient cohort as reported here (n=17), this variation can have a large impact at individual sampling time points. Assessing minimal or maximal concentrations over a relatively short period of time (e.g. 1 week) within which there are multiple sampling time points can enable discovery of temporal responses to stroke that that are biologically meaningful but where the peaks/troughs may be occurring at slightly different time points in individual patients. We do agree, however, that it is important to ensure more granular temporal patterns are not missed. To ensure this, we have analysed the time points at which minimal or maximal concentrations of mediators significantly changed by stroke were reached, as suggested by the reviewer. The percentage of patients reaching their minimum circulating concentration of immunoglobulins (Supplementary Figure 5, Extended Data) and minimum (Supplementary Figure 6, Extended Data) or maximum circulating concentration of complement components (Supplementary Figure 7, Extended Data) at each time point was compared in placebo and IL-1ra treated groups. These analyses have been added to the relevant results sections (Results, lines 187-190, 203-206, and 217-220). No differences in kinetics were apparent in IL-1Ra versus placebo treated groups further confirming the key findings that IL-1Ra has no significant additional effect on these mediators over and above the effects of stroke. This analysis has been described in the manuscript in Methods section under Statistical analyses section, lines 145-148.We thank the reviewer for the suggestion to include some statistical analyses to account for the repeated sampling that occurred in individual patients. As there were missing samples in a few patients, a mixed effects model with Dunnett’s post-test was used to analyse each sampling time point compared to admission in both IL-1Ra treated patients, as suggested by the reviewer, and placebo treated patients. As expected, due to the variance when minimum and maximum concentrations were reached in individual patients, there were few significant changes at individual time points in comparison to admission (Supplementary Figure 9-12, Extended data). However, many immunoglobulin subsets were significantly reduced at d4 post stroke, and at some other sampling time points, in both IL-1Ra and placebo treated patients showing that the loss of immunoglobulins after stroke can still be detected in a small cohort with high variance in the kinetics of individual responses (Supplementary Figure 8, Extended data). This reanalysis has been acknowledged in the manuscript in Methods section, under statistical analyses. Again, there were no major differences in responses between IL-1ra and placebo treated patients, with the exception of IgM which showed significantly reduced concentrations at d4 In the IL-1Ra treated group but not in the placebo treated group. Overall, these analyses further confirm the overall conclusion that IL-1Ra does not exacerbate changes to circulating immune mediators induced by stroke. 2. Had patients received recanalization therapies? Since the temporal window of IL-1Ra for some patients is still within that of thrombolytic therapies (<4.5h), the authors should explain why they did not receive them if so. From what I can understand, the specimens used come from a trial registered in 2000, thus likely explaining the use of the placebo in patients eligible for recanalization therapies according to present guidelines. I think that this needs clarification in the text.The samples used in this study were tertiary analysis of plasma samples taken from a randomised, placebo-controlled phase II trial originally designed to determine the safety and biological activity of intravenous (IV) IL-1Ra and recruitment for this study began in 2001, completing in 2003. tPA was not approved in the UK for treatment for patients <3 h from stroke onset until 2003 and for patients 3- 4.5 h from onset until 2012, therefore recanalization therapies were not an option for patients in this original study. We thank the reviewer for raising this important point and have provided details in the manuscript (Methods section Standard protocol approvals, registrations and patient consents lines 76-80).3. There is a big difference in placebo vs. IL-1Ra treated patients in the frequency of haemorrhagic stroke (0% vs. 29.4%). Did the authors consider to correct for this potentially impacting difference?Due to the blinding and randomisation of patient recruitment in the original study all haemorrhagic stroke patients were by chance allocated to the IL-1Ra treatment group. We agree with the reviewer that as little is known about the differential effects of haemorrhagic versus ischemic stroke on the immune system, the frequency of haemorrhagic strokes in the IL-1Ra treatment group may have the potential to bias the data. To address this, we identified and highlighted the data points that represent haemorrhagic stroke patients in each mediator analysed in the main figures. The data points representing the minimum circulating concentrations of immunoglobulins measured in the first week after stroke in haemorrhagic stroke patients were distributed throughout the range of concentrations measured in the IL-1Ra treated group suggesting that in this data set, the high proportion of haemorrhagic stroke patients in one treatment group did not skew the interpretation of these results (Figure 1). A similar distribution of haemorrhagic stroke patients was apparent in the majority of complement components significantly reduced (Figure 2), increased (Figure 3) or unchanged (Figure 4) and in increased noradrenaline levels (Figure 5) measured in the first week after stroke. The exception to this was in the minimum concentrations of C3b/ iC3b and C3 (Figure 2A and 2B) where haemorrhagic stroke patient data points localised to the higher end in the range of measured minimum circulating concentrations within the IL-1Ra treated group. However, when data points from haemorrhagic stroke patients were excluded from the IL-1Ra treated data set and statistical analysis was performed, there were no significant differences in minimal circulating concentrations of C3b/ iC3b or C3 between the IL-1Ra and placebo treated groups. Indeed, reanalysis of all mediators excluding the haemorrhagic stroke patient data points did not alter the statistical results or conclusions drawn in the results section.We have amended our discussion to acknowledge the greater frequency of haemorrhagic stroke patients within the IL-1ra treated patient group, highlight the reanalysis we have performed and confirm that future experiments designed to determine if differences exist between immune responses to ischemic versus haemorrhagic stroke are required to further our understanding of post stroke immune suppression (Discussion).4. The measure of complement components may be biased. Indeed blood specimens were collected in the presence of heparin, which does not represent the standard for running unbiased complement protein analysis. Complement proteins and activation products should be measured in blood sampled in the presence of EDTA (Bergseth et al., 20131).The blood samples were collected in heparin containing tubes for the original study, whereas collection in EDTA would have provided optimal conditions for subsequent measurements of complement components1, 2. Heparin is known to have an effect on baseline levels of complement components in serum as complement can spontaneously activate during the clotting process. However, plasma from blood samples collected in EDTA or heparin were reported to have similar baseline levels of complement components1. There may be a minimal increase in absolute measures of complement concentrations in the samples used in this study due to original sample collection methods. However as all plasma samples were collected by this method, any artefact of the heparin collection should be comparable across all samples and therefore not alter our conclusions on the effects of stroke on complement components in the manuscript. We have acknowledged this caveat of the study in our discussion and thank the reviewer for this comment (Discussion).5. In complement system biology, the decrease of circulating levels of recognition molecules in acute phases is generally regarded as an index of complement activation (through consumption of the circulating proteins binding their targets). On later times, circulating levels also mirror increased synthesis and they may increase. This should be considered when discussing the observations relative to complement measures. Moreover, complement system activation in stroke patients is reported in the first 6h (Zangari et al., 20162), therefore a comparison of admission vs. later sampling in IL-1Ra treated patients would be informative (do they recover to control physiological complement protein levels as expected?).As reported in response to point 1 from the reviewer, individual patient trajectories were highly varied with patients reaching their maximal or minimal concentrations at various time points from admission to 5-7 d (Extended data, Supplementary Figures 7 and 8). Due to this variance, no individual time points after stroke showed significantly different concentrations of any complement component in comparison to control samples, including the later 5-7 d sampling time point. Our analysis has detected common changes to complement components after stroke across this time window. We agree with the reviewer our method limits the capacity to discuss these findings in terms of activation of complement pathways as we cannot associate these changes to a specific time post stroke or post infection. We have altered our discussion of the data to remove any speculation about activation or suppression of complement pathways and instead have reported the overall changes in concentration and their relationship to the different arms of the complement cascade only and have added information that reduced concentration of some complement mediators may reflect pathway activation through sequestration (Discussion). We have acknowledged that to determine if the changes to complement components were associated with increased early activation or increased synthesis of components, further studies would be required in a larger patient cohort with more stringent inclusion and exclusion criteria to account for the variance in the kinetics of individual patient responses (Discussion).6. There is now a general consensus that the lectin pathway of complement activation is that mainly involved after ischemic stroke. This has been reviewed in Orsini et al. (20143) and Fumagalli and De Simoni (20164). Besides MBL, the lectin pathway comprises other recognition molecules such as ficolins and collectin-11, that play a major role in ischemic patients (especially ficolin-1 and ficolin-3; refer to the above mentioned reviews). In this regard the conclusions drawn by the authors that: ‘As concentration of MBL itself was not significantly altered by stroke, this suggests the classical complement activation is specifically activated after stroke’ sounds incorrect. Of note, MBL concentrations are genetically determined and about 20% of the general population bears variants causing MBL deficiency (Garred et al., 20065). Indeed in figure 4C the authors show MBL levels below the detection limit. It is acknowledged that a circulating level of MBL < 500 ng/mL is associated with MBL genetic variants (Eisen et al., 20086). Thus it is not surprising that no MBL changes were observed. A more clear analysis could be done excluding the MBL-deficient patients. Interestingly, these latter patients develop less ischemic lesion volume and deficits than those with normal MBL circulating levels (Osthoff et al., 20117), thus further supporting lectin pathway critical contribution to stroke pathophysiology. We thank the reviewer for this insightful comment. In response to this we have reanalysed our data to determine which patients in the study did not reach a circulating level of >500 ng/ml at any sampling time point and excluded these patients from our analysis of MBL concentration. We identified two patients in each of the control, IL-1Ra and placebo groups that met this criteria and reanalysed our minimum and maximum circulating concentration of MBL with these patients excluded (Figure 4). Remaining data points on this graph that are <500 ng/ ml were from patients whose minimum circulating MBL level fell to this value, but had read at >500 ng/ ml at other sampling time points, preventing the possibility that they had a genetic MBL deficiency. With the exclusion of these patients there were still no significant differences in maximum or minimum MBL concentration measured in the first week after stroke in placebo or IL-1Ra treated patients. The potential for these genetic variants to confound our analysis and the results of our reanalysis excluding these patients was added to the discussion. However as we have not measured other components specific to the lectin pathway of complement activation we have amended the manuscript to show that we cannot draw any conclusions on the effect of stroke on the lectin pathway from the data within this manuscript (Discussion). 1. Mollnes TE, Garred P, Bergseth G. Effect of time, temperature and anticoagulants on in vitro complement activation: consequences for collection and preservation of samples to be examined for complement activation. Clinical and experimental immunology 1988;73:484-488.2. Bergseth G, Ludviksen JK, Kirschfink M, Giclas PC, Nilsson B, Mollnes TE. An international serum standard for application in assays to detect human complement activation products. Molecular Immunology 2013;56:232-239." } ] } ]	1	https://f1000research.com/articles/8-1039
https://f1000research.com/articles/8-1762/v1	16 Oct 19	{ "type": "Research Article", "title": "Evaluation of post-bleaching hypersensitivity using desensitizing agent before and /or after in-office bleaching: A randomized clinical trial.", "authors": [ "Silvia Sabry Tawfik", "Mohamed Adel Ezzat Khairy", "Maha Abd Elsalam ElBaz", "Maha Ebrahim Mohamed El korashy" ], "abstract": "Background: Dental bleaching has become one of the most demanded aesthetic procedures as it is very simple and gives fast results that satisfy the patient’s wishes. However, unfortunately, it has the annoying side effect of tooth sensitivity. This clinical trial was designed to evaluate the efficacy of using a desensitizing agent, which was applied during in-office bleaching with 30% HP (hydrogen peroxide). Methods: 36 participants were divided into four groups (N = 9). According to the intended intervention as follows: Group I:—Application of Relief ®Amorphous Calcium Phosphate (ACP) before in-office bleaching; Group II—Application of Relief ®ACP before and after in-office bleaching; Group III—Application of Relief ®ACP after in-office bleaching; and Group IV: placebo was applied before and after in-office bleaching. Then, three hydrogen peroxide bleaching applications for a total of 45 minutes were performed. The primary outcome variable was hypersensitivity, assessed with (VAS) scale immediately and 24h, 1 week and 1 month after the procedure; while the secondary outcome was teeth shade assessed using the VITAPAN ® classical shade guide. Results: Statistical analysis was done using Kolmogorov-Smirnov, Shapiro-Wilk tests, Kruskal-Wallis test and Dunn’s test. Immediately after bleaching, there was statistically significant distinction in the median pain score between the groups. After 1 day, the ACP gel before and after bleaching group showed the lowest median score, while, the control group confirmed the highest median score. After 2 days, no statistically sizable difference was observed between all groups. The group receiving ACP gel before and after showed the highest change in median of classical shade guide scores while, (ACP gel after) and control group; showed the lowest change. Conclusion: The application of the desensitizing agent prior to and after in–office bleaching was successful in lowering post-bleaching hypersensitivity. Trail Registration: Clinical trial.gov NCT02942082 21/10/2016", "keywords": [ "Tooth bleaching", "Hydrogen peroxide", "hyper-sensitivity", "Randomized clinical trial", "in-office bleaching", "VAS", "desensitizing agent", "ACP" ], "content": "Introduction\n\nIn-office bleaching permits close professional control, prevents material ingestion, reduces the total treatment time, and has the potential for quick results, improving patient satisfaction1. Be that as it may, tooth sensitivity (TS) is the most common side-effect of dental bleaching. It has been hypothesized that tooth sensitivity resulting from tooth bleaching occurs because peroxide penetrates the tooth structure and directly activates a neuronal receptor and not because of hydrodynamic effects. In addition, alteration caused by bleaching agents on the morphological enamel surface as increased surface porosity, depressions and superficial irregularities2,3.\n\nFor this reason, a desensitizing agent is used to decrease the tooth sensitivity that patients experience. The mechanism of action of potassium nitrate remains unknown. This acts by diminishing the excitability of the intra-dental nerve endings and blocking the dentinal tubules4. Potassium nitrate and sodium fluoride are broadly used as desensitizing agents to treat tooth sensitivity. Potassium ions cause the depolarization of the sensory nerve5, and fluoride treats tooth sensitivity, probably by blocking uncovered dentinal tubules or decreasing the liquid stream into the mash and blocking the transmission of improvements6. Of late, bioactive materials such as ACP (amorphous calcium phosphate) and CCP-ACP (casein phosphopeptite and amorphous calcium phosphate) as desensitizers and re-mineralizing specialists have been utilized for this purpose; however, there is only limited research in this area7. The objective of this randomized clinical trial is to assess whether the use of a desensitizing agent will diminish the tooth sensitivity induced by dental bleaching.\n\n\nMethods\n\nThe protocol and the informed consent template were reviewed and accepted by the Ethics committee of Scientific Research at the Faculty of Dentistry, Cairo University on October 2016 (Approval number 161038 on 24 October 2016). All participants provided written informed consent regarding all of the trial’s procedural steps and the publication of the trial results and photos. The trial has been registered on clinical-trials.gov (NCT02942082) on 21 October 2016.\n\nThis was a randomized clinical trial with an equal allocation ratio (1:1:1:1). This article was prepared following the protocol established by the consolidation standards of reporting trials, statement 2010. The trial was started in November 2017 and it was completed in November 2018.\n\nAll participants were recruited from the outpatient clinic of the Conservative Department at Cairo University who attended seeking tooth whitening. In total, 36 (6 male and 30 female) adult patients took part in this trial subsequent to satisfying all inclusion criteria. Telephone numbers and addresses of all subjects in the study were recorded as part of the signed consent.\n\nAll subjects received a phone call at the time of the pre-determined follow-up dates. Patients were given a brief explanation about the investigations those who agreed to participate signed an informed consent. The in-office bleaching was performed in a single visit.\n\n\nEligibility criteria\n\nTo be included in this trial, participants needed to be aged between 18 and 40 years old, have good oral health with no periodontal disease and have six upper anterior teeth that are free from either caries or restoration with no sign of spontaneous pain.\n\nParticipants with anterior restorations and carious lesions in their upper labial surfaces were not included in this trial. Additionally, participants suffering from recession and cracks, those taking analgesics, and those with bruxism habits were also not enrolled in this trial. Finally, pregnant women and lactating mothers were also excluded.\n\nSample size calculation was performed using the GPower program version 3.1.9.2 (University of Düsseldorf, Düsseldorf, Germany). Based on post-bleaching hypersensitivity results from previous studies8,9, a sample size of 36 patients was calculated. A sample of 24 patients was enough for the detection of an effect size 0.4, with a power of 85%, and a 5% significance level. The number was increased to 29 to address non-parametric variables in order to increase the reliability of the results, and again increased to 36 for loss to follow up.\n\nUsing a free online website (http://www.random.org/), a sequence generation for patient numbers was created. Each participant chose a number from successively numbered obscure envelopes. They were then allocated into one of the set-ups using a randomization table formed of four columns, and each column contained numbers from one to nine. The table was kept with the senior supervisor (MA) who generated the allocation sequence and he was responsible for ensuring proper randomization and allocation concealment.\n\nOne operator performed the bleaching procedures for all participants (SS).\n\nAfter taking a medical and dental history using medical and dental health charts see (extended data10), patients were given a concise clarification regarding the examinations. A prophylaxis paste and a low-speed brush for teeth cleaning were used a day prior to bleaching. Utilizing a visual analogue scale (VAS, see extended data10), every member set a mark that represents their level of pain on a scale from 0–10. VAS scores were assessed for each patient by measuring the distance in cm from the anchor word (0 cm) to the mark.\n\nIn-office bleaching was done using Dash™ whitening gel syringe 30% hydrogen peroxide (Discus dental, Culver City, 91761 USA) batch number DSE 1001, for three sessions each 15 minutes.\n\nIn-office bleaching procedures. After patient lip lubrication, the operator placed a cheek retractor and a bit block into the patients mouth (Figure 1). Isolation using cotton rolls was performed, and a liquid barrier material, Liquidam™ barrier syringe catalogue number 99-905-26, was applied to cover the tissues. The applied Liquidam was cured for 10 seconds. Then, according to the group specification, the following were performed: Group I: a desensitizing agent Relief® ACP oral care gel syringe (Discus Dental, Culver City, 91761 USA) batch number DSE 1001 which according to the manufacture, contains 5% potassium nitrate; 0.22% sodium fluoride and 0.75% amorphous calcium phosphate (ACP) was applied on the teeth before dental bleaching; Group II: the Relief® ACP was applied before and after bleaching procedures; Group III: Relief ACP was applied after bleaching; and Group IV: glycerin liquid (Human Care International, Egypt, Batch No. SP8P) was applied before and after bleaching procedures. Dash™ whitening gel syringe 30% hydrogen peroxide (Discus dental, Culver City, 91761 USA) batch number DSE 1001 was applied to the facial side of teeth (1–2 mm thick), and the gel was left on the teeth for 15 minutes for a total of three cycles (Figure 2). After each cycle, the gel was removed with surgical suction tip or wiped with gauze. Again, the desensitizing agent Relief® ACP Oral Care Gel or glycerin was applied on the labial surfaces of teeth according to the group specification. For Group II, Group III, and Group IV, glycerin was applied before and after in-office bleaching. Post-operative instructions were given to the participants; for the next two days, they were asked to avoid pigmented consumption, such as tea and cola, and to avoid using toothpaste with desensitizing agents.\n\n\nOutcomes evaluation\n\nPost-bleaching hypersensitivity. Post-bleaching hypersensitivity was assessed using a VAS scale, immediately and 24 hours, one week and one month after the bleaching procedure. VAS was used by the patients from the second day after bleaching on a daily basis to record tooth sensitivity between the schedule of the subsequent evaluations, and patients were asked to bring this pain diary at every point of assessment. The assistant supervisor (ME) kept in contact with the participants by phone to remind them and to ensure accurate adherence to instructions. VAS scores were assessed for each patient by measuring the distance in cm from the anchor word (0 cm) to the mark. The blinded assessor collected the data of evaluation in a printed assessor chart (extended data10).\n\nColor evaluation. The blinded assessor recorded the shade of each participant’s teeth at baseline and at the recall appointments (after 24 hours, 1 week and 6 months). The measurement area of interest for shade matching was the middle third of the facial surface of maxillary anterior teeth. 16 tabs of the shade guide (The VITAPAN ® classical shade guide (Vita Zahnfabrik, Badsä ckingen, Germany) were arranged from highest (B1) to lowest (C4) value. These values were converted into numerical codes using a conversion table (Table 1) (Borges et al., 2012).\n\nThis was a double-blind clinical trial in which the participants and assessors were unaware of the type of the desensitizer used (relief® ACP oral care gel or a placebo) and were not aware of which group they were in. However, for clinical purposes, the operator was not blind, as both materials used had the same color but different consistencies; thus, it was impossible to blind the operator. The assessor collected the data from evaluations in a printed assessor chart (see extended data10).\n\nIn this study, data analysis was performed using IBM SPSS statistical for Windows, Version 23.0 Armonk, NY:IBM Corp. Non-normal (non-parametric) data were represented as the median and Inter-quartile Ranges (IQR). For comparison between the four groups, a Kruskal–Wallis test was used. Friedman’s test was used to study changes over time within each group. For pairwise comparison, Dunn’s test and Bonferroni’s test were used. Quantitative data were represented in terms of frequencies and percentages, and significance was set at P ≤ 0.05.\n\n\nResults\n\nIn this study, 36 (6 male and 30 female) adult patients took part in this trial (see underlying data). A patient flow chart is available as part of the Reporting guidelines10.\n\nPatient ages ranged between 18–40 years old; their mean ages were 32.4± 4.3, 32.1 ± 4.4, 33.1 ± 4.8 and 32.1± 5.3 for the ACP gel before bleaching group, ACP gel before and after bleaching group, ACP gel after bleaching and the control group, respectively, with no statistically significant difference regarding mean age values (P-value = 0.965) between all groups.\n\nRegarding patient gender, the ACP gel before bleaching group included two males (22.2%) and seven females (77.8%), while the ACP gel before and after bleaching group included one male (11.1%) and eight females (88.9%), the ACP gel after bleaching group included on male (11.1%) and eight females (88.9%) and the control group, glycerin before and after bleaching, included two males (22.2%) and seven females (77.8%). Regarding gender distribution, no significant difference (p = 1.000) was found. (Table 2)\n\n: Significant at P ≤ 0.05\n\nACP - Amorphous calcium phosphate\n\nA comparison of the VAS scores is shown in Table 3 and Figure 3. Before bleaching, all cases in the study groups showed no pain (P-value = 1.000, Effect size = 0.000). A significant difference was shown in all groups immediately after bleaching (P-value <0.001, Effect size = 3.149). Regarding pairwise comparison between the groups, the ACP gel after bleaching and glycerin before and after bleaching groups showed the highest median pain score of 5 (4–6.5) and 5 (5–7), respectively, with a non-statistically significant difference between both groups and that of the ACP gel before bleaching group, which was 4 (3.5–4.5). The ACP gel before and after bleaching group showed the lowest median pain score of 3 (0–4), with a non-statistically significant difference from the ACP gel before bleaching group, which was 5 (5–7), and a statistically significantly lower median pain score than the ACP gel after bleaching and glycerin applied before and after bleaching groups, at 5 (4–6.5) and 5 (5–7), respectively. After 1 day, a significant difference was revealed between the groups (P-value < 0.001, Effect size = 3.367). Pairwise comparison between the groups revealed that the glycerin before and after bleaching group showed the highest median pain score of 3 (2–3.5), with a non-statistically significant difference from the ACP gel after bleaching group, which was 2 (1–2.5). The ACP gel before and after bleaching group showed the lowest median pain score 0 (0–1), with a non-statistically significant difference from the ACP gel before bleaching group, at 1 (0.5–1.5), and a statistically significantly lower median pain score than the ACP gel after bleaching and glycerin applied before and after groups, at 2 (1–2.5) and 3 (2–3.5), respectively. On day 2 after bleaching, a statistically significant difference was shown (P-value = 0.099, Effect size = 1.046). For days 3, 4, 5, 6 and 7, and even after 1 month, all cases of the four groups showed no pain.\n\n:P ≤ 0.05 significant, Different superscripts in the same row represent a statistically significant difference between values.\n\nACP - Amorphous calcium phosphate\n\n\nChanges in hypersensitivity scores after bleaching within each group\n\nResults for changes in hypersensitivity are presented in Table 4 and Figure 4. In the ACP gel before bleaching group, the median pain score values changed statistically significantly over time (P-value < 0.001, Effect size = 0.437). The pair-wise comparison showed a statistically significant rise in the 0 median pain results instantly after bleaching followed by a statistically significant reduction in pain results after 1 day. Days 1 to 2 did not alter pain statistically significantly. No statistically significant change was observed in the pain scores after bleaching from day 2 to days 3, 4, 5, 6, 7 and 1 month.\n\nSimilarly, there was no statistically significant variation in pain score in the ACP gel before and after bleaching group after bleaching over time (P-value of < 0.001, Effect size of 0. 484). A comparison of the time points shows that the mean pain scores instantly after bleaching increased statistically significantly followed by a statistically significant reduction in pain results after the first day. Days 1 to 2 did not alter the pain results statistically significantly. No statistically significant changes to pain values after bleaching occurred from day 2 to days 3, 4, 5, 6, 7 and 1 month, as all the cases did not have any pain.\n\n: Significant at P ≤ 0.05, Different superscripts in the same column indicate statistical significant differences between values\n\nACP - Amorphous calcium phosphate\n\nWith regard to the ACP gel after bleaching group, the median pain scores over time (P-value < 0.001, Effect size= 0.298) changed significantly. The comparison between time intervals indicates that median pain scores were statistically significantly increased instantly after bleaching, and that the pain scores decreased statistically significantly after 1 day and from 1 to 2 days. Between day 1 and day 2, there was no statistically significant shift in pain rates.\n\nNo statistically significant changes in pain scores after bleaching occurred between day 2 and days 3, 4, 5, 6, 7 and 1 month, as there was no pain in all cases.\n\nIn the same way, the median pain results changed over time (P-value < 0.001, Effect size = 0.466) in the glycerin before and after bleaching group. A pairwise comparison between times showed that the median pain scores results increased statistically significantly immediately after the bleaching and then decreased after 1 day and from 1 day to 2 days. No significant changes in pain scores were observed from 2 days to 3 days.\n\n\nTooth color related data\n\nComparison between interventions. Results of changes in tooth shade across groups are presented in Table 5 and Figure 5. There was no statistically significant difference between the groups immediately post bleaching, after 1 day and 1 week (P-value = 0.828, Effect size = 0.066), 1 month (P-value = 0.426, Effect size = 0.007), or 6 months (P-value = 0.024, Effect size = 0.202). Pair-wise comparison between the groups revealed that the ACP gel before & after bleaching group showed the statistically significantly highest median change in classical shade guide score. ACP gel after bleaching group showed a lower median change in shade, followed by the ACP gel before bleaching group which where statistical significant. Glycerin before & after bleaching group showed the lowest median change in shade guide score.\n\n*: Significant at P ≤ 0.05, Different superscripts in the same row are statistically significantly different\n\nACP - Amorphous calcium phosphate\n\n\nDiscussion\n\nThe in-office bleaching method offers great efficiency, using hydrogen peroxide with elevated concentrations of 25 to 40%3. High-concentration bleaching agents, however, cause tooth sensitivity, which is the primary adverse impact associated with in-office bleaching, during and up to 24 hours following the bleaching operation. This sensitivity is associated with the pulp tissue inflammation phase11,12.\n\nIn each clinical session, 30% hydrogen peroxide in-office bleaching was performed according to the guidelines of the manufacturer for three 15-minute sessions in this research. Several studies have implemented hydrogen peroxide in three 15-minute sessions13–16. H2O2 produces free radicals, which interact with pigment molecules to have a whitening impact. These free radicals in the bleaching gel break down the double bonds between pigment molecules and change the arrangement and/or size of the pigment molecules, creating a more white tooth appearance17.\n\nThe release of cell-derived factors, such as ATP and prostaglandins, can be caused by tooth bleaching. This interaction can cause pulp nociceptors to be excited or sensitized, and harm the pulp tissue18. Another hypothesis is presently not well recognized; i.e., that the hydrodynamics activate the intradental nerve and release neuropeptide in reaction to this operation3.\n\nPotassium nitrate and sodium fluoride are often used to treat tooth sensitivity as desensitizing agents. These agents can be contained in a bleaching gel and supplied during therapy with a custom tray, or applied separately by being placed in the mouth of the subject for a brief period of time prior to bleaching19,20. In contrast, fluoride used as a desensitizing agents treats tooth sensitivity by blocking exposed dentinal tubules or decreasing liquid pulp flow and blocking stimulus transmission6.\n\nThe mechanism by which potassium nitrate operates is still not known. Various randomized clinical studies to assess the effectiveness of tooth bleaching have been released, reporting that the use of potassium nitrate and sodium fluoride eliminate post bleaching hypersensitivity14,16,18,20–23. A systemic review and meta-analysis assessed the effectiveness of potassium nitrate and sodium fluoride as desensitizing agents in tooth bleaching therapy and concluded that potassium nitrate and +/-sodium fluoride decrease tooth sensitivity24.\n\nThe supply of Ca and PO4 ions, by means of ACP technology, together with whitening processes can be useful to minimize the loss of minerals as well as the occurrence of roughness and erosion in the dental structure due to whitening25. There is limited research on the application of ACP, bioactive materials, CCP-ACP, bio-glass and bio-glass ceramics as desensitizing agents and re-mineralizing agents7.\n\nThe primary aim of these products is to encourage dental structural remineralization through the creation of an amorphous calcium phosphate layer on the surface of the product, initiating apathetic crystallization. This method of remineralization seems far more efficient than sodium nitrate or fluoride alone26, as it not only helps to alleviate the pain, but also stops it from beginning. (Zhao et al. 2011)27 They reviewed the application of amorphous calcium phosphate in dentistry and found that, as a result of increasing microcrystalline, ACP is converted readily into a crystalline phase, such as octacalcium phosphate and apatite.\n\nA study by Kwon et al., 201628 evaluated the time taken by potassium nitrate to reach the pulp cavity and the impact of penetration concentrations on tooth whitening. Relief ACP was used for 0, 5, 15, 30 and 60 minutes before bleaching. The study concluded that, as early as 5 minutes after application, potassium nitrate penetrates into the pulp cavity, and potassium nitrate pretreatment with desensitizers had no adverse effect on the efficacy of tooth whitening. Similar penetration times for hydrogen peroxide and the penetration of potassium nitrate can be described by the small molecular weights of both 34.40 grams per mol and 101,10 grams per mol, respectively; and also, as water soluble agents, their transport into the tooth can be facilitated28. The present clinical trial used oral Relief ACP® gel as a desensitizing agent that, according to the manufacturer, includes 5% potassium nitrate, 0.22% sodium fluoride and 0.75% amorphous calcium phosphate (ACP)4.\n\nIn the present trial, a VAS scale was used, as a great deal of proof is available to support the pain intensity validity of VAS. Such scales show benefits over other pain intensity self-reporting measures and pain-controlled behavior29,30. Many studies have used the VAS scale to assess dental sensitivity intensity after office bleaching8,9,14,31–34. Others have used both the visual analog scale (VAS) and numerical scale (NRS)16,23.\n\nIn this study, participant ages in all groups ranged from 18–40 years old. The mean of ages of patients were 32.4± 4.3, 32.1 ± 4.4, 33.1 ± 4.8 and 32.1± 5.3 for the ACP gel before bleaching group, ACP gel before and after bleaching group, ACP gel after bleaching group and control group, respectively, with no statistically significant difference regarding mean age values (P-value = 0.965) between all groups. A recent review was conducted in 35 (Rezende et al., 2016). Regarding the participants’ ages, the authors did not find any link between age and the risk or level of tooth sensitivity. Regarding gender, 36 patients (30 women and six men) took part in this research. There was no statistical significance in gender distribution among all groups.\n\nHowever, with the incidence of female patients in this research distinct outcomes for other population profiles can be noted. Furthermore, socio-cultural beliefs about femininity and men also seem to be an important factor in pain response between the sexes, as the social acceptance of pain expression among females is usually more acceptable—an influence that could lead to biased pain reporting36. The highest average pain score was found in the ACP gel after bleaching and Glycerin before and after bleaching groups, showing the highest median pain score. The lowest average pain score was shown in the ACP gel applied before and after bleaching group. As mentioned above, potassium nitrate has advantages as a product in that it reduces tooth sensitivity after dental bleaching; several clinical studies have reported this14,16,18,20–23. Moreover, the efficacy of potassium nitrate and sodium fluoride as desensitizing agents was measured in a systematic review meta-analysis24. These studies cannot be directly compared with this clinical trial, as the Relief ACP ® oral care used as a desensitizing agent includes in its ingredients 5% potassium nitrate, 0.22% sodium fluoride and 0.75% amorphous calcium phosphate (ACP).\n\nIn this research, hypersensitivity for all participants tested was not reported by the end of the seven days of the trial. This conclusion was consistent with that in Martin et al., and Farag et al.8,37.\n\nIn a control group in which glycerin was used as a placebo before and after in-office bleaching, a hypersensitivity enhancement was noted. This may be the result of the placebo or Hawthorne effect. The Hawthorne effect can be defined as an unconscious shift in the participant because of the simple understanding of being observed during a clinical trial38.\n\n\nStrength and limitations\n\nThis trial was a randomized clinical trial with a comparatively large sample of patients, performed in a clinical setting. This was the first clinical study to use a desensitizing agent before and after in-office bleaching to reduce hypersensitivity after bleaching. The median age for patients in this study was 32, and this affects the generalizability of the results to the general population and can be regarded as a study limitation. The following limitation should be considered: the generalizability of results may be limited given the number of female participants was much higher than male participants. We assessed one bleaching product, Dash™ whitening gel; different bleaching gels and concentrations are likely to affect sensitivity. A randomized clinical trial should therefore be performed with various bleaching products and demographic profiles using other equivalent measures.\n\n\nConclusion\n\nThe application of the desensitizing agent before and after bleaching was found within the limitations of this research, to be effective in reducing post-bleaching hypersensitivity. Hypersensitivity in all groups gradually decrease two days after in-office bleaching.\n\n\nConsent\n\nWritten approval was acquired from patients to publish their clinical information and clinical images.\n\n\nData availability\n\nOpen Science Framework: Evaluation of post-bleaching hypersensitivity using desensitizing agent before and /or after in-office bleaching: A randomized clinical trial. https://doi.org/10.17605/OSF.IO/XUB2510\n\nThe project contains the following underlying data:\n\nResults data\n\nResults raw.xlsx (Spreadsheet of tooth Sensitivity scores using VAS scale)\n\nAge and gender.xlsx (Spreadsheet of age and gender data for study participants)\n\nColor measurments.xlsx (Spreadsheet of color changes for study participants)\n\nOpen Science Framework: Evaluation of post-bleaching hypersensitivity using desensitizing agent before and /or after in-office bleaching: A randomized clinical trial. https://doi.org/10.17605/OSF.IO/XUB2510\n\nThis project contains the following extended data:\n\nSupplementary files\n\nAssessor chart (Word document containing chart used by assessor to record patient data)\n\nPain diary (Word document containing pain diary with VAS scales used by participants)\n\nMedical and dental health charts (Word document containing chart used to record medical and dental data for participants on requirement)\n\nOpen Science Framework: CONCORT checklist and flow diagram for Evaluation of post-bleaching hypersensitivity using desensitizing agent before and /or after in-office bleaching: A randomized clinical trial. https://doi.org/10.17605/OSF.IO/XUB2510\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nYan M, Yu Y, Zhang G, et al.: A journey from dental pulp stem cells to a bio-tooth. Stem Cell Rev Rep. 2011; 7(1): 161–71. PubMed Abstract \| Publisher Full Text\n\nBrännström M: The hydrodynamic theory of dentinal pain: sensation in preparations, caries, and the dentinal crack syndrome. J Endod. 1986; 12(10): 453–7. PubMed Abstract \| Publisher Full Text\n\nMarkowitz K: Pretty painful: why does tooth bleaching hurt? Med Hypotheses. 2010; 74(5): 835–40. PubMed Abstract \| Publisher Full Text\n\nPinheiro HB, Lopes B, Klautau EB, et al.: Influence of bioactive materials used on the dentin surface whitened with carbamide peroxide 16%. Mater Res. 2010; 13(2): 273–278. Publisher Full Text\n\nHaywood VB, Caughman WF, Frazier KB, et al.: Tray delivery of potassium nitrate-fluoride to reduce bleaching sensitivity. Quintessence Int. 2001; 32(2): 105–9. PubMed Abstract\n\nSaylor CD, Overman PR: Dentinal Hypersensitivity: A Review. RDH. 2011; 31(3): 1–11. Reference Source\n\nRahiotis C, Vougiouklakis G: Effect of a CPP-ACP agent on the demineralization and remineralization of dentine in vitro. J Dent. 2007; 35(8): 695–8. PubMed Abstract \| Publisher Full Text\n\nMartin J, Fernandez E, Bahamondes V, et al.: Dentin hypersensitivity after teeth bleaching with in-office systems. Randomized clinical trial. Am J Dent. 2013; 26(1): 10–4. PubMed Abstract\n\nPintado-Palomino K, P Filho O, Zanotto ED, et al.: A clinical, randomized, controlled study on the use of desensitizing agents during tooth bleaching. J Dent. 2015; 43(9): 1099–1105. PubMed Abstract \| Publisher Full Text\n\nTadros S: Evaluation of post-bleaching hypersensitivity using desensitizing agent before and / or after in-office bleaching: A randomized clinical trial. 2019. http://www.doi.org/10.17605/OSF.IO/XUB25\n\nLoguercio AD, Martins LM, Silva LM, et al.: In-Office Whitening. Tooth Whitening. 2016; 145–167. Publisher Full Text\n\nBlind S, Trial RC, Calixto AL, et al.: Comparison of the Effects of In-office Bleaching Times on Whitening and Tooth Sensitivity : A Comparison of the Effects of In- office Bleaching Times on Whitening and Tooth Sensitivity : A Single Blind , Randomized Clinical. Oper Dent. 2016; 41(2): 138–145. Publisher Full Text\n\nMarson FC, Sensi LG, Vieira LCC, et al.: Clinical evaluation of in-office dental bleaching treatments with and without the use of light-activation sources. Oper Dent. 2008; 33(1): 15–22. PubMed Abstract \| Publisher Full Text\n\nTay LY, Kose C, Loguercio AD, et al.: Assessing the effect of a desensitizing agent used before in-office tooth bleaching. J Am Dent Assoc. 2009; 140(10): 1245–51. PubMed Abstract \| Publisher Full Text\n\nOntiveros JC: In-office vital bleaching with adjunct light. Dent Clin North Am. 2011; 55(2): 241–53. PubMed Abstract \| Publisher Full Text\n\nParreiras SO, Szesz AL, Farago PV, et al.: Effect of an experimental desensitizing agent on reduction of bleaching-induced tooth sensitivity: A triple-blind randomized clinical trial. J Am Dent Assoc. 2018; 149(4): 281–290. PubMed Abstract \| Publisher Full Text\n\nLi Y, Greenwall L: Safety issues of tooth whitening using peroxide-based materials. Br Dent J. 2013; 215(1): 29–34. PubMed Abstract \| Publisher Full Text\n\nReis A, Dalanhol AP, Kossatz S, et al.: Assessment of tooth sensitivity using a desensitizer before light-activated bleaching. Oper Dent. 2011; 36(1): 12–7. PubMed Abstract \| Publisher Full Text\n\nBonafé E, Loguercio AD, Reis A, et al.: Effectiveness of a desensitizing agent before in-office tooth bleaching in restored teeth. Clin Oral Investig. 2014; 18(3): 839–45. PubMed Abstract \| Publisher Full Text\n\nPalé M, Mayoral JR, Llopis J, et al.: Evaluation of the effectiveness of an in-office bleaching system and the effect of potassium nitrate as a desensitizing agent. Odontology. 2014; 102(2): 203–10. PubMed Abstract \| Publisher Full Text\n\nMondelli RF, Azevedo JF, Francisconi AC: Comparative clinical study of the effectiveness of different dental bleaching methods - two year follow-up. J Appl Oral Sci. 2012; 20(4): 435–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrignardello-Petersen R: Desensitizing gel reduces the risk of experiencing dentin hypersensitivity and results in a small decrease in hypersensitivity levels after tooth bleaching. J Am Dent Assoc. 2018; 149(10): e136. PubMed Abstract \| Publisher Full Text\n\nda Costa Poubel LA, de Gouvea CVD, Calazans FS, et al.: Pre-operative use of dexamethasone does not reduce incidence or intensity of bleaching-induced tooth sensitivity. A triple-blind, parallel-design, randomized clinical trial. Clin Oral Investig. 2019; 23(1): 435–444.PubMed Abstract \| Publisher Full Text\n\nWang Y, Gao J, Jiang T, et al.: Evaluation of the efficacy of potassium nitrate and sodium fluoride as desensitizing agents during tooth bleaching treatment — A systematic review and meta-analysis. J Dent. 2015; 43(8): 913–23. PubMed Abstract \| Publisher Full Text\n\nGiniger M, Macdonald J, Ziemba S, et al.: The clinical performance of professionally dispensed bleaching gel with added amorphous calcium phosphate. J Am Dent Assoc. 2005; 136(3): 383–92. PubMed Abstract \| Publisher Full Text\n\nTschoppe P, Neumann K, Mueller J, et al.: Effect of fluoridated bleaching gels on the remineralization of predemineralized bovine enamel in vitro. J Dent. 2009; 37(2): 156–62. PubMed Abstract \| Publisher Full Text\n\nZhao J, Liu Y, Sun WB, et al.: Amorphous calcium phosphate and its application in dentistry. Chem Cent J. 2011; 5: 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwon SR, Dawson DV, Wertz PW: Time Course of Potassium Nitrate Penetration into the Pulp Cavity and the Effect of Penetration Levels on Tooth Whitening Efficacy. J Esthet Restor Dent. 2016; 28 Suppl 1: S14–22. PubMed Abstract \| Publisher Full Text\n\nJensen MP, Karoly P, Braver S: The measurement of clinical pain intensity: a comparison of six methods. Pain. 1986; 27(1): 117–126. PubMed Abstract \| Publisher Full Text\n\nHjermstad MJ, Fayers PM, Haugen DF, et al.: Studies comparing Numerical Rating Scales, Verbal Rating Scales, and Visual Analogue Scales for assessment of pain intensity in adults: a systematic literature review. J Pain Symptom Manage. 2011; 41(6): 1073–1093. PubMed Abstract \| Publisher Full Text\n\nThiesen CH, Rodrigues Filho R, Prates LH, et al.: The influence of desensitizing dentifrices on pain induced by in-office bleaching. Braz Oral Res. 2013; 27(6): 517–523. PubMed Abstract \| Publisher Full Text\n\nMaghaireh GA, Alzraikat H, Guidoum A: Assessment of the effect of casein phosphopeptide-amorphous calcium phosphate on postoperative sensitivity associated with in-office vital tooth whitening. Oper Dent. 2014; 39(3): 239–47. PubMed Abstract \| Publisher Full Text\n\nHegde V, Murkey L: Evaluation of residual root canal filling material after retreatment of canals filled with hydrophilic and hydrophobic obturating system: An in vitro scanning electron microscopy study. Endodontology. 2017; 29(1): 47. Reference Source\n\nLima SNL, Ribeiro IS, Grisotto MA, et al.: Evaluation of several clinical parameters after bleaching with hydrogen peroxide at different concentrations: A randomized clinical trial. J Dent. 2018; 68: 91–97. PubMed Abstract \| Publisher Full Text\n\nRezende M, Loguercio AD, Kossatz S, et al.: Predictive factors on the efficacy and risk/intensity of tooth sensitivity of dental bleaching: A multi regression and logistic analysis. J Dent. 2016; 45: 1–6. PubMed Abstract \| Publisher Full Text\n\nBartley EJ, Fillingim RB: Sex differences in pain: a brief review of clinical and experimental findings. Br J Anaesth. 2013; 111(1): 52–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarag I, Abouelfotouh I, Mohamed O, et al.: A comparative study of different bleaching techniques, regarding the color change, stability and postoperative hypersensitivity: a randomized controlled clinical trial. Stomatological Dis Sci. 2018; 2: 5. Publisher Full Text\n\nTalioti E, Hill R, Gillam DG, et al.: The Efficacy of Selected Desensitizing OTC Products: A Systematic Review. ISRN Dent. 2014; 2014: 865761. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "64696", "date": "02 Jul 2020", "name": "Cristian Bersezio", "expertise": [ "Reviewer Expertise Clinical studies", "Dental materials", "Tooth whitening" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirstly, I congratulate the team of researchers, since all clinical studies present a certain degree of difficulty. There is very interesting data in your research, which I think can be much better used.\n1) I suggest that it be reviewed by experts in the translation of scientific manuscripts since there are certain phrases or very colloquial words.\n2)In the summary:\nI would delete the word \"unfortunately\", simply mention that its main adverse effect is tooth sensitivity.\n\n\"N\" (uppercase) is used when speaking to the total study population, when determining the number of a group, \"n\" (lowercase) is used.\n\nThe results should include the most important data and show their numerical values, not just words.\n\n3) In the introduction:\nThe definition of dental sensitivity is missing, it talks about the cause but not what it is.\n\nThe objective of the study is clear, I would recommend strengthening the introduction with antecedents on ACP, and formulating a hypothesis based on them.\n\n4) In the methodology: The methodology is very well detailed.\nIn the part of the second visit, do not use the term sessions, use the term applications. There are three applications of 15 minutes each, in a single session.\n\nI recommend using better photographs, they are not very aesthetic. It would have been good without the cotton, the bite rest on the molars performs that function.\n\nThe sample size per group seems to be a little low, I recommend justifying it with other publications.\n\nThe history of dental sensitivity was an Exclusion criterion?\n\nFor the evaluation of the color, the ideal would have been to use the Vita Bleachguide 3D Master guides, it is more precise, and the change value between one unit and the other is the same. On the other hand, as the order by value of the vita classical is suggested, the change in color between one tablet (unit) and another is not the same among all.\n\n5) In the results:\nThe results must be summarized, there is an excess of tables and graphs that express practically the same thing. You should choose to use tables or graphs to express the same results. In the written part, the most important thing should be highlighted, not to report all the results that can be seen when reading the tables or graphs.\n\nDemographic data is best used as a table to express it and should include baseline sensitivity and color data, and analyze whether the groups are the same.\n\nTo express the color change, I recommend using the ΔSGU (difference from the scale guide unit), please review the papers:\n\nOper Dent. 2019 Nov / Dec; 44 (6): 581-588.1 Oper Dent. Jan / Feb 2017; 42 (1): 41-52.2\n\n6) In discussion:\nThe first part (second to fifth paragraph) are more a theoretical framework than a discussion. It is more appropriate to use it in the introduction to justify a hypothesis or the use of that desensitizer. It should be summarized and used in the introduction.\n\nTheoretical content can be used to justify the results, but it must be related. For this, the first part of the discussion always remembers the research question and then answers it, and based on that the results can be explained.\n\nAlthough effectiveness is a secondary result, this must be discussed. Especially since the placebo group has a greater recurrence of color over time. It is interesting to analyze and provides valuable information.\n\nMajor changes are required in the preparation of the manuscript for publication, which can be modified by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5691", "date": "09 Jul 2020", "name": "Silvia Sabry Tawfik", "role": "Author Response", "response": "Thanks a lot, dear professor for your meticulous & great review. I'll do my best to rephrase & correct all the points you have mentioned." } ] }, { "id": "66353", "date": "07 Jul 2020", "name": "Eduardo Fernández Godoy", "expertise": [ "Reviewer Expertise Restorative Dentistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI consider a correct study, justification of the proposal and research question.\n\nThe scales used are adequate and the results expected.\n\nI am concerned about the low sample size and the statistical power of the conclusions. I think you should add this to the limitations of the study.\n\nAdequate references, nice work!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5690", "date": "09 Jul 2020", "name": "Silvia Sabry Tawfik", "role": "Author Response", "response": "Thanks a lot dear professor. Regarding the sample size calculation, it was done by the biostatistics department at the faculty of dentistry, Cairo University. Based on the primary outcome post- bleaching hypersensitivity, based on previous studies 8 9. Pintado-Palomino, K., Peitl Filho, O., Zanotto, E. D., & Tirapelli, C. (2015). A clinical, randomized, controlled study on the use of desensitizing agents during tooth bleaching. Journal of dentistry, 43(9), 1099-1105. Martin, J., Fernandez, E., Bahamondes, V., Werner, A., Elphick, K., Oliveira Jr, O. B., & Moncada, G. (2013). Dentin hypersensitivity after teeth bleaching with in-office systems. Randomized clinical trial. Am J Dent, 26(1), 10-4." } ] } ]	1	https://f1000research.com/articles/8-1762
https://f1000research.com/articles/8-1761/v1	16 Oct 19	{ "type": "Case Report", "title": "Case Report: Importance of dietary modification in successful management of eosinophilic gastroenteritis", "authors": [ "Pujitha Kudaravalli", "Sheikh A. Saleem", "Sana Riaz", "Bishnu Sapkota", "Sheikh A. Saleem", "Sana Riaz", "Bishnu Sapkota" ], "abstract": "Introduction: Eosinophilic gastroenteritis (EGE) is an extremely rare inflammatory disorder with an estimated prevalence of 22-28/100 000. We herein, present a case of EGE in an elderly patient which was successfully managed with dietary restriction. Case report: A 70-year-old male with a history of gastroesophageal reflux disorder (GERD), atopic dermatitis and asthma presented with 2 weeks history of foul-smelling non-bloody diarrhea associated with nausea, vomiting and weight loss. Physical examination was significant for dry oral mucosa and loss of skin turgor. Lab findings were significant for a hemoglobin of 13.2 g/dl, hematocrit of 38.5%, mean corpuscular volume of 86.3%, white blood cell count of 24,200/mm3, albumin of 2.2 g/L, stool fat of 70g, stool osmolar gap of 115, C-reactive protein 1.47. Erythrocyte sedimentation rate, HIV test were unremarkable. Infectious stool work-up was negative. Computed tomography of the abdomen was unremarkable. The mucosa appeared mildly inflamed on upper endoscopy and colonoscopy, and biopsies showed eosinophilic infiltration of the mucosal and muscular layers. A diagnosis of eosinophilic gastroenteritis was made after other causes such as parasitic infection, drug use and malignancy were ruled out. The patient was counseled on a six-food elimination diet which successfully resolved his diarrhea. The patient did not have any relapses with dietary modification on follow-up. Discussion: The recurrence rate of EGE is 50%. Steroids improve symptoms in 90% of cases but the recurrence rates are high. The type, dose and duration of steroid therapy is unclear. Sodium cromoglicate, ketotifen, and Montelukast are other proposed treatments, the results being inconclusive. Bowel resection is performed in intestinal obstruction, but medical therapy is needed as recurrence in other segments in common. Dietary modification, a therapy with no side-effects should be the first line of treatment as it can result in resolution sparing the patient of steroid induced side effects.", "keywords": [ "Eosinophilic gastroenteritis", "high recurrence", "dietary modification", "steroids", "side effects" ], "content": "Introduction\n\nEosinophilic gastroenteritis (EGE) is an extremely rare inflammatory disorder with an estimated prevalence of 22–28/100 000. We herein, present a case of EGE in an elderly patient which was successfully managed with dietary restriction. We would like to stress that food allergy is the main culprit in certain cases of EGE and dietary modification should always be the first step in the management of these cases1.\n\n\nCase presentation\n\nA 70-year-old male with a history of gastroesophageal reflux disorder (GERD), atopic dermatitis and asthma presented in August 2018 with 2 weeks history of foul-smelling non-bloody diarrhea associated with nausea, vomiting and weight loss. He did not have a family history of colonic disorders. He denied alcohol use, smoking or use of any other illicit drugs. Physical examination was significant for dry oral mucosa and loss of skin turgor.\n\nLab findings were significant for a hemoglobin of 13.2 g/dl, hematocrit of 38.5%, mean corpuscular volume of 86.3%, white blood cell count of 24,200/mm3, albumin of 2.2 g/L, stool fat of 70g, stool osmolar gap of 115, C-reactive protein 1.47. Erythrocyte sedimentation rate, HIV test were unremarkable. Infectious stool work-up was negative. Computed tomography of the abdomen was unremarkable. The mucosa appeared mildly inflamed on upper endoscopy and colonoscopy, and biopsies showed eosinophilic infiltration of the mucosal and muscular layers. A diagnosis of eosinophilic gastroenteritis was made after other causes such as parasitic infection, drug use and malignancy were ruled out.\n\nThe patient was suggested a six-food elimination diet which included the elimination of soy, wheat, egg, milk, peanuts, and fish/shellfish and it successfully resolved his diarrhea.\n\nThe patient was followed up in the outpatient GI clinic annually and did not have any relapses after dietary modification.\n\nSimple interventions like dietary changes can prevent recurrences and resolve symptoms in eosinophilic gastroenteritis. Although compliance to dietary changes can be challenging it is nonetheless an intervention without any side effects. The evidence for using steroids, type and duration of steroid therapy is not substantial to indicate its benefits. Use of biological agents in the treatment of EGE is still underway and hence the first line of intervention for the treatment of EGE should start with dietary modification.\n\n\nDiscussion\n\nEGE most commonly affects males in the third decade of life and those with a history of eczema, asthma, allergic rhinitis. The prevalence of EGE is underestimated as it is under diagnosed given the rarity of the condition especially when it presents outside of the age spectrum like in our patient. The pathogenesis of EGE remains unclear and it is hypothesized to be a hypersensitive response to certain allergens. One proposed theory is that infectious and geographical factors interact with certain genetic variants causing esophagic dysbiosis which leads to Th2 cell immune response. Dysregulation of several genes termed EOE transcriptome is also shown to play a role2,3.\n\nLaboratory findings seen in EGE include peripheral eosinophilia, elevated serum IgE levels, increased stool fat excretion, prolonged prothrombin time, hypoalbuminemia, anemia from intestinal malabsorption. Imaging can show thickening or nodularity of the antrum and thickened or saw tooth mucosa in the small bowel and when the muscular layer is involved irregular narrowing especially in the distal antrum and proximal small bowel can also be seen. EGE is a diagnosis of exclusion and other causes of hyperreactivity and eosinophilia should be considered such as parasitic infections, vasculitis disorders, reaction to drugs like enalapril, interferons, non-steroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori infection4. The diagnosis of EGE is established when the biopsy shows more than expected numbers of eosinophils and all the other causes are ruled out.\n\nKlein et al., classified EGE based on the depth of eosinophilic infiltration. Mucosal disease presents with abdominal pain, nausea/vomiting, diarrhea and the patients develop protein losing enteropathy, malabsorption and failure to thrive. Muscular involvement causes wall thickening and impaired motility which may lead to intestinal obstruction. Serosal disease presents as either isolated ascites or ascites in addition to the symptoms of serosal and muscular disease5.\n\nThe recurrence rate of EGE is 50% and a dietary trial could be a long-term solution like in our patient. A six-food elimination diet (soy, wheat, egg, milk, peanut/tree nuts, and fish/shellfish) or an elemental diet is recommended. Steroids improve symptoms in 90% of cases but the recurrence rates are high. The type, dose and duration of steroid therapy is unclear. The most commonly used regimen is prednisolone at 20 to 40 mg/day, for 6 to 8 weeks including the tapering although most patients need a longer course due to relapses. Sodium cromoglicate, ketotifen, and Montelukast are other proposed treatments, the results being inconclusive. Bowel resection is performed in intestinal obstruction, but medical therapy is needed as recurrence in other segments is common6.\n\nThe use of biologics in the treatment of refractory EGE in being studied and could lead to new longer lasting results. Interleukin 5, an eosinophilic growth factor, is shown to play an important role in the pathogenesis of EGE. A few clinical trials assessing the use of anti- interleukin 5 in the form of humanized antibodies have shown improvement in endoscopic appearance, reduced clinical symptoms and improved quality of life while a few clinical trials showed no statistical difference in disease progression with this treatment. Trials to understand if eosinophils are key to the development and progression of eosinophilic esophagitis are underway8\n\n\nConclusion\n\nThe clinical guidelines for the diagnosis of EGE is sparse and the treatment offered is low-evidence based. Dietary modification, a therapy with no side-effects should be the first line of treatment and can result in resolution sparing the patient of steroid induced side effects.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.", "appendix": "Acknowledgements\n\nThe case report has been accepted for a poster presentation at American College of Gastroenterology Conference in October 2019 at San Antonio, Texas.\n\n\nReferences\n\nOkimoto E, Ishimura N, Okada M, et al.: Successful Food-Elimination Diet in an Adult with Eosinophilic Gastroenteritis. ACG case Rep J. 2018; 5(1): e38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLyles J, Rothenberg M: Role of genetics, environment, and their interactions in the pathogenesis of eosinophilic esophagitis. Curr Opin Immunol. 2019; 60: 46–53. PubMed Abstract \| Publisher Full Text\n\nCianferoni A, Spergel JM: Eosinophilic Esophagitis and Gastroenteritis. Curr Allergy Asthma Rep. 2015; 15(9): 58. PubMed Abstract \| Publisher Full Text\n\nLópez-Medina G, Gallo M, Prado A, et al.: Eosinophilic Gastroenteritis: Case Report and Review in Search for Diagnostic Key Points. Case Rep Gastrointest Med. 2015; 2015: 239506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenatti C, Sacchetti C, Pedrazzi A, et al.: Eosinophilic gastroenteritis: a case report and a review of eosinophilic gastrointestinal disorders. Ital J Med. 2009; 3(3): 166–171. Publisher Full Text\n\nSadeghi A, Abdi E, Jamshidfar N, et al.: Eosinophilic gastroenteritis; a report of two cases with different presentations. Gastoenterol Hepatol Bed Bench. 2017; 10(Supp1): S161–S164. PubMed Abstract \| Free Full Text\n\nTemiz T, Yaylaci S, Demir MV, et al.: Eosinophilic gastroenteritis: a rare case report. N Am J Med Sci. 2012; 4(8): 367–368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoufosse F: Targeting the Interleukin-5 Pathway for Treatment of Eosinophilic Conditions Other than Asthma. Front Med (Lausanne). 2018; 5: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "57549", "date": "10 Dec 2019", "name": "Ravi Ranjan Pradhan", "expertise": [ "Reviewer Expertise Internal medicine (cardio", "gastro", "neuro)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for providing me an opportunity for peer review. I have the following comments and I think major revision is required for the article.\n\nAbstract:\nThe authors should not mention all details like history, lab findings and discussion in the abstract. I think the abstract should be concise like this:\n\"Eosinophilic gastroenteritis (EGE) is an extremely rare inflammatory disorder with an estimated prevalence of 22-28/100 000. Steroids improve symptoms in 90% of cases but the recurrence rates are high, and patients are exposed to steroid-related side effects. We herein, present a case of EGE in an elderly patient who was successfully managed with dietary restriction. Dietary modification in patients with EGE is free of steroid-related side effects, and may be considered as initial therapy for EGE.\"\n\nIntroduction:\nAdd more in the introduction about EGE, discuss global scenarios and particular nation scenarios.\n\nThe line “Eosinophilic gastroenteritis (EGE) is an extremely rare inflammatory disorder with an estimated prevalence of 22–28/100 000.\" has no citation. Citation is advised.\n\nCase presentation:\n\nDon’t mention the date (August 2018).\n\nDescribe more about diarrhea: the number of episodes per day, and color. Was it associated with pain in the abdomen? Was there nocturnal diarrhea?\n\nDescribe about vomiting.\n\n\"family history of colonic disorders\" - what does it mean? Colorectal ca or polyp? The authors are advised to mention it.\n\n\"Physical examination was significant for dry oral mucosa and loss of skin turgor.\" Write like: \"Physical examination was significant except for dry oral mucosa and loss of skin turgor.\n\nInvestigations:\nRemove this line: \"Lab findings were significant for a\". Rewrite like: “on investigation hemoglobin was…”.\n\nWrite down important findings in sentence. Make a table for the investigations that shows detailed parameters:\n- Parameters - Reference range, adults - On admission - After dietary restriction\n\nInfectious stool work-up was negative. Mention in detail what infectious work up was done.\n\nKeep the endoscopy and colonoscopy photo.\n\nMention which site the biopsy was taken from - colon or esophagus?\n\nFigure of histopathology is a must and will give the most convincing evidence.\n\nWas endoscopy/biopsy done after dietary restriction? How can you presume that there was no inflammation going on after dietary restriction based on only improvement of clinical history?\n\nFollow up:\nHow long was the patient followed up and after what time of dietary restriction did the patient show clinical improvement?\n\nStrengths, Limitations and Take Away Lessons:\nThe authors are advised to remove this portion.\n\nDiscussion:\nThe discussion seems to be like the introduction and the authors are advised to include these paragraphs in the introduction section.\n\nThe discussion should include how this case report is similar to or different from other reports.\n\nWhat is unique to this case report?\n\nWhat is unique in management? Discuss in detail.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "96347", "date": "29 Oct 2021", "name": "Kalyan Saginala", "expertise": [ "Reviewer Expertise epidemiology of cancers - bladder cancer", "melanoma" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRecommend to add pictures of upper EGD and colonoscopy with histo-pathological pictures as well if possible.\n\nFamily history not significant for gastrointestinal disorders would be better than stating as he did not have a family history of colonic disorders.\n\nWould recommend to document Lab findings in tabular form with reference ranges as well.\n\nBetter to document what is the approximate duration time period when the symptoms diarrhea resolved .\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1761
https://f1000research.com/articles/8-608/v1	01 May 19	{ "type": "Research Article", "title": "Knowledge management awareness assessment in Nigerian tertiary institutions", "authors": [ "Afolakemi Simbo Ogunbanwo", "Julius Olatunji Okesola", "Sheryl Buckley" ], "abstract": "Background: Knowledge management (KM) is a recipe for increasing performance and promoting innovation in tertiary institutions. However, some scholars argue that the Nigerian educational sector is yet to fully appreciate the importance of KM as their KM awareness level is still low. Since measurement is the basic foundation to accomplish success, this paper assesses the KM awareness level in tertiary institutions of south-west Nigeria. Methods: We applied a survey method using a closed ended questionnaire administered to 50 participants from each of the 10 institutions measured by Likert scaling. Employing SPSS for data analysis, frequency count and percentage score were adopted to analyse the demographic data, and the research hypotheses were analysed with chi square test, Pearson chi square and bivariate correlation (Pearson) analysis. Results: A positive relationship between awareness, current status and level of familiarity was noted. KM awareness level in the institutions is high even though there is a significant difference between the public and private universities, as well as between the students and academic staff. Conclusions: Since an increase in the awareness level increases both current status and level of familiarity which often account for KM success, it is recommend that KM awareness level should continuously be improved upon in Nigerian tertiary institutions.", "keywords": [ "Awareness level", "Knowledge", "knowledge management", "tertiary institutions", "performance" ], "content": "Introduction\n\nKM is a process of coordinating, organising and making institutional or organisational knowledge available for knowledge creation, sharing, storage and reuse to achieve institutional aims and objectives. Managing the existing knowledge flow in tertiary institutions is essential. According to Kayıkçı and Ozan1, knowledge is a powerful tool for organisational competition and therefore becomes significant to every industry including banking, education and governmental sectors2–5. Knowledge generated should be properly managed to ensure its future availability. Therefore, tertiary institutions have moved beyond being merely a knowledge provider to students, to also curating current knowledge for future use6. A number of these institutions now operate like business organisations and compete among themselves, with knowledge as their commodity. Tertiary institutions are centres for knowledge creation and sharing7, and are regarded as knowledge business organizations that should devise means of gathering and disseminating knowledge for effective decision making8,9. Therefore, institutions desiring higher performance must identify, capture and circulate valuable institutional knowledge for re-use9,10.\n\nMany studies including Demchig7 and Kidwell et al.11 have worked on the application of KM in tertiary institutions, claiming that it improves institutional capabilities in decision making and reduces the product development cycle time, as well as improving academic and administrative services. They argue that KM adoption and implementation by the institutions could result to exponential improvements in knowledge sharing, as it has a positive impact on academic research, curriculum development, student and alumni services, administrative services and strategic planning. Al-sulami, Rashid and Ali12, claimed that the performance level of an institution can shoot up through the effective and efficient implementation of knowledge management. Similarly, it increases innovation giving institutions a competitive advantage over others13.\n\nKM is an emerging concept in developing countries with varying awareness and maturity levels. Charles & Nawe14 discovered that staff of Mbeya University of Science and Technology (MUST) in Tanzania were not fully aware of KM practices. Demchig7 conducted an assessment on level of KM maturity in Mongolian higher institutions using the Knowledge Management Capability Assessment (KMCA) model; it was revealed that maturity level of KM was in level one, indicating knowledge sharing was not discouraged in Mongolian higher institutions7. Yaakub, Othman & Yousif15 discovered that KM practices in Malaysian higher learning institutions is still very low, while Anvari et al.16 found the level of KM in Firoozabad Islamic Azad University to be below average. Although KM awareness and maturity level is yet to be fully investigated amongst Nigerian tertiary institutions in the southwest geo-political zone, several Nigerian authors17–19 have found that KM is has yet to be fully implemented in Nigerian tertiary institutions.\n\nWhile the literature indicates KM awareness in most developing country institutions is low7,14–16, a number of studies do not agree20,21. Since the position is difficult to generalise due to socio-cultural differences, KM awareness in Nigerian institutions needs to be further investigated. Hence, we formulated the following four hypotheses to test awareness levels, as well as ascertaining the difference between KM awareness levels of the public and private institutions, as well as that of the academic staff and students.\n\nHypothesis1: KM awareness level in universities in the southwest of Nigeria is high.\n\nHypothesis2: There is significant difference in the KM awareness level of the academic staff and student.\n\nHypothesis3: There is significant difference in the KM awareness level between public and private institutions in southwest Nigeria.\n\nHypothesis4: There is a relationship between awareness, current status and KM familiarity in the tertiary institution in in the southwest of Nigeria.\n\n\nMethods\n\nThis section discusses appropriate sampling methods employed as well as the instrumentations adopted, and reported following the STROBE reporting guidelines22.\n\nThis study adopted both probability and non-probability sampling to examine the awareness level of KM in Nigerian tertiary institution. The research population frame is the 46 accredited universities in south west Nigeria while the total population is 550 comprising both academic staff and students of selected tertiary institutions in South West Nigeria. Stratified random sampling was adopted to select 11 universities out of 46 accredited universities only. The stratified random sampling used is as follows:\n\nFirstly the population was grouped into three stratums - federal, state and private containing 7, 11, and 28 universities respectively.\n\nSecondly, systematic random sampling was used to select item from each stratum.\n\nLastly, the size of each stratum was kept proportional to the sizes of the strata thereby resulting in picking two federal, three state and six private universities.\n\nPurposive sampling was used to select the names of the 11 universities involved in the research from each of the stratum, as well as the participants consisting of academic staff and students from the selected universities. The student population outnumbers staff in every university therefore, the authors decided to gather a sample of students to staff at a ratio of 3:2. To avoid data overload and have a manageable sample size, a total number of 50 respondents (30 students and 20 members of academic staff) were selected from each university to arrive at 550 (50x11) sample size.\n\nThe questionnaire (see Extended data23) was personally administered to the 10 universities involved as one university backed out from the research. The 10 universities involved in the research were five public (two federal, three state) and five private. A total number of 50 respondents were selected from each university and 500 questionnaires were administered out of which only 456 were returned and used for the analysis. The Ethical Committee of the University of South Africa issued an authorization memo to approve the questionnaire.\n\nLikert scaling was adopted to measure awareness levels in each institution. Questions on level of KM awareness were assigned a score 1 to 4 for ‘none’, ‘low’, ‘high’ and ‘very high’ respectively while questions on knowledge recognition were respectively tagged with score 1 to 4 for ‘strongly disagree’, ‘disagree’, ‘agree’ and ‘strongly agree’. Similarly, questions on current status were assigned score tags of 1 to 4 for ‘not in existence’, ‘on pipeline’, developing’ and ‘matured’ respectively, while the level of familiarity were assigned a score ranging from 1 to 4 for ’unaware’, ‘introductory’, ‘intermediate’ and ‘advance’ respectively.\n\nTo test the reliability of the research instrument, Cronbach’s alpha reliability test was conducted generating a result of 0.845, thereby confirming the consistency, reliability and acceptability of the factors used. Similarly, the questionnaire was pre-tested using two institutions different from those involved in the study. Administering 60 questionnaires on 30 participants from each institutions, responses and comments obtained helped to identify and address potential hitches prior to performing the actual research.\n\nIn line with UNISA research ethics policy, all participants had the study explained to them before their recruitment. All participants provided written informed consent to participate.\n\nIBM Statistical Programme for Social Sciences version 21 was adopted for this data analysis. Descriptive statistics of frequency counts and percentage scores was employed to analyse the demographic data, while the participants’ responses were analysed using percentage count. Hypothesis 1 was analysed with one sample chi square test, hypotheses 2 and 3 were by Pearson chi square, and hypothesis 4 was by Spearman’s rho – a non-parametric correlations.\n\nFor both the chi square and Pearson correlation coefficient, a p value <0.05 (5% significant) as ruled below.\n\nRule 1 If the p value is greater than 0.05 (p<0.05) accept the null hypothesis\n\nRule 2 If the p value is less than 0.05 (p>0.05) accept the alternate hypothesis\n\nRule 3 0.00<R<0.33 indicates weak relationship\n\nRule 4 0.34<R<0.66 indicates moderate relationship\n\nRule 5 0.67<R<1.0 indicates strong relationship\n\n\nResult\n\nTable 1 shows the demographic characteristics of the respondents. The total number of returned questionnaires was 456, 82% of the 500 questionnaires administered. Of these, 55% were male while 45% were female. Regarding the academic qualification of the respondent, the majority respondents were undergraduates (63%), follow by those with a master’s degree (19%), PhDs (9%) and Bachelor’s degree (9%). In terms of respondents’ status, the majority were students (67%), with academic staff making up 33% of the sample. Public universities constituted 55% while private universities made up 45% (Table 1 and Underlying data24).\n\nTo test the KM awareness level in the sampled institutions, a one-sample chi square test was applied to hypothesis 1. KM awareness levels were defined as ‘none’, ‘low’, ‘high’ and ‘very high’ (Table 2). The expected N for all the variables was 114. The results show that the hypothesis was accepted with test statistics value 295.930a, expected count of 114 and p value of 0.001.\n\nTo test for possible differences in KM awareness level between academic staff and students, a one-sample chi square test was also used to test the hypothesis 2. The hypothesis was accepted as the test statistics value obtained was 24.794, the expected count was 0.64 and p value of 0.001.\n\nIn terms of differences in KM awareness levels between public and private institutions, the outcome of the one sample chi square test on hypothesis 3 confirms the acceptance of the alternate hypothesis with chi square test value of 10.301, expected count 0.90 and p value 0.016.\n\nPearson correlation was applied on hypothesis 4 to determine the correlation between the KM awareness level, KM current status and KM familiarity. The hypothesis was accepted as the p value was 0.001. The result as depicted on Table 3 shows that there is a moderate relationship between the variables (KM awareness level, KM current status and KM familiarity) with a correlation coefficient range (r) of 0.35 < \|r\| < 0.45.\n\nKM – knowledge management\n\n**. Correlation is significant at the 0.01 level (2-tailed).\n\nb. Listwise N=456\n\n\nDiscussion and conclusion\n\nAlthough KM implementation is important to tertiary institutions7,11–13, assessment must come before implementation18,25. This study was therefore conducted in the context of the current literature, the majority of which suggests that KM is still emerging in developing countries7,15–18,26, and yet to be fully implemented in Nigeria17–19.\n\nThe study investigated knowledge management awareness in Nigerian South-west tertiary institutions and addressed the relationship between awareness, familiarity and current status of KM level. It was discovered that there is significant difference in KM awareness level amongst the public and private universities. Awareness levels between academic staff and students is also significantly different, conforming with the findings of Krubu and Krub27 and Akuegwu and Nwiue28 where heads of department were more involved in KM practice. This study also empirically provides evidence for correlation between the awareness, familiarity and current status of KM level. We found a positive relationship between awareness, current status and level of familiarity. This suggests that if awareness levels increases, more people/institutions will practice KM and its current status will improve thereby shifting the state from developing to maturing. Similarly, KM awareness levels in south west tertiary institution was found to be high, confirming the previous studies of Ohiorenoya and Eboreime29 and Oke, Ogunsemi and Adeeko30. However, since KM awareness level in both the public and private institutions in the South West region in Nigeria is also high, this study concludes that Nigerian institutions recognise the importance of KM towards achieving institutional innovations and higher performance.\n\nFurther research may be needed to investigate the level of KM maturity and the relationship between KM and academic performance in Nigerian institutions.\n\n\nData availability\n\nFigshare: Knowledge Management Awareness. https://doi.org/10.6084/m9.figshare.7730480.v124\n\nThis project contains the following underlying data:\n\nknowledge_management_F1000.sav (Study participants knowledge management awareness data)\n\nFigshare: Knowledge management awareness questionnaire. https://doi.org/10.6084/m9.figshare.7764644.v123\n\nThis project contains the following extended data:\n\nF1000_Questionnaire_KM_awareness.docx (Study questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis study was sponsored by the University of South Africa, South Africa.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgment\n\nThe researchers would like to express their gratitude to the management of University of South Africa for making all the resources needed for the research available.\n\n\nReferences\n\nKayikçi K, Ozan Y: Effects of Knowledge Management Competencies of School Principals’ to Quality Studies in School. Int J Bus Soc Sci. 2014; 5(5): 188–198. Reference Source\n\nLee CS, Wong KY: Development and validation of knowledge management performance measurement constructs for small and medium enterprises. J Knowl Manag. 2015; 19(4): 711–734. Publisher Full Text\n\nHaque MM, Ahlan AR, Jalaldeen M, et al.: Factors Affecting Knowledge Sharing on Innovation in the Higher Education Institutions (HEIs). ARPN J Eng Appl Sci. 2015; 10(23): 18200–18210. Reference Source\n\nSharma M, Kaur M: Knowledge Management in Higher Education Institutions. IRA - Int J Manag Soc Sci. 2016; 4(3): 548–555. Publisher Full Text\n\nOkesola OJ, John SN, Okokpujie KO, et al.: An improved Bank Credit Scoring Model: A Naïve Bayesian Approach. 2017 International Conference on Computational Science and Computational Intelligence (CSCI). 2017; 228–233. Publisher Full Text\n\nLaal M: Knowledge Management in Higher Education. Procedia Comput Sci. 2011; 3: 544–549. Publisher Full Text\n\nDemchig B: Knowledge management capability level assessment of the higher education institutions: Case study from Mongolia. Procedia Soc Behav Sci. 2015; 174: 3633–3640. Publisher Full Text\n\nToro U, Joshi MJ: A Review of Literature on Knowledge Management using ICT in Higher Education. Int J Comput Technol Appl. 2013; 4(1): 62–67. Reference Source\n\nGyaase PO, Anane ET, Armah INA: The Use of Information and Communication Technology (ICT) for Knowledge Management in the Second Cycle Educational Institutions in Ghana. Int J Comput Appl. 2015; 128(7): 7–13. Publisher Full Text\n\nFolorunso O, Ogunseye OS, Okesola JO, et al.: Visualizing E-Voting Results. J Theor Appl Inf Technol. 2010; 10(1): 57–69. Reference Source\n\nKidwell JJ, Vander Linde KM, Johnson SL: Applying Corporate Knowledge Management Practices in Higher Education. Educause Quarterly. 2000; 28–33. Reference Source\n\nAl-sulami ZA, Rashid AM, Ali N: The Role of Information Technology to Support Knowledge Management Processes in Higher Education of Malaysian Private Universities. Int J Sci Res Publ. 2014; 4(10): 1–12. Reference Source\n\nNorth K, Kumia G: Knowledge Management. Switzerland: Springer International Publishing, 2014. Publisher Full Text\n\nCharles W, Nawe J: Knowledge Management (KM) Practices in Institutions of Higher Learning in Tanzania with Reference to Mbeya University of Science and Technology. Univ Dar es Salaam Libr Journal. 2017; 12(1): 48–65. Reference Source\n\nYaakub MB, Othman K, Yousif AF: Knowledge Management Practices in Malaysian Higher Learning Institutions: A Review on Selected Cases. Int J Educ Res. 2014; 2(1): 1–10. Reference Source\n\nAnvari A, Alipourian GA, Moghimi R, et al.: An assessment of Knowledge Management (KM): A consideration of information, culture, skills and technology. African J Bus Manag. 2011; 5(28): 11283–11294. Reference Source\n\nOjo A: Knowledge Management in Nigerian Universities: A Conceptual Model. Interdiscip J Information Knowledge Manag. 2016; 11: 331–345. Publisher Full Text\n\nAgarwal NK, Marouf LN: Initiating Knowledge Management in Colleges and Universities: A template. Int J Knowl Content Dev Technol. 2014; 4(2): 67–95. Publisher Full Text\n\nOkesola OJ, Okokpujie K, Goddy-worlu R, et al.: Qualitative Comparisons of Elicitation Techniques in Requirement Engineering. ARPN J Eng Appl Sci. 2019; 14(2): 565–570. Reference Source\n\nAbu Naser SS, Al Shobaki MJ, Abu Amuna YM: Promoting Knowledge Management Components in the Palestinian Higher Education Institutions - A Comparative Study. Int Lett Soc Humanist Sci. 2016; 73: 42–53. Publisher Full Text\n\nAbu Naser SS, Al Shobaki MJ, Abu Amuna YM: Measuring knowledge management maturity at HEI to enhance performance-an empirical study at Al-Azhar University in Palestine. Int J Commer Manag Res. 2016; 2(5): 55–62. Publisher Full Text\n\nvon Elm VJE, Altman DG, Egger M, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies.\n\nOgunbanwo A, Buckley S, Okesola J: Knowledge management awareness questionnaire. figshare. Paper. 2019. http://www.doi.org/10.6084/m9.figshare.7764644.v1\n\nOgunbanwo A, Buckley S, Okesola J: Knowledge Management Awareness. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7730480.v1\n\nKulkarni U, Louis RS: Organizational Self Assessment of Knowledge Management Maturity. In Ninth Americas Conference on Information Systems. 2003; 2542–2551. Reference Source\n\nCharles W, Nawe J: No Title. 48–65.\n\nKrubu DE, Krub SG: Towards Sustainable Development: An Assessment of Knowledge Management Initiatives in Nigerian Universities. J Sustain Dev Africa. 2011; 13(3): 165–177. Reference Source\n\nAkuegwu BA, Nwi-ue FD: Application of Knowledge Management Skills in University Administration in Nigeria: Evidence from Heads of Departments. Br J Educ Soc Behav Sci. 2013; 3(4): 574–588. Reference Source\n\nOhiorenoya JO, Eboreime OF: Knowledge Management Practices and Performance in Nigerian Universities. Eur Sci J. 2014; 10(16): 400–416. Reference Source\n\nOke AE, Ogunsemi DR, Adeeko OC: Assessment of Knowledge Management Practices among Construction Professionals in Nigeria. Int J Constr Eng Manag. 2013; 2(3): 85–92. Reference Source" }	[ { "id": "47958", "date": "30 May 2019", "name": "Aderonke Oni", "expertise": [ "Reviewer Expertise information system and data mining" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe object of the study is clear and the research method was clearly outlined as well. However, the following should be attended to:\nThe authors should work of the grammatical structure. Personal pronoun e.g. \"we\" should be avoided. The authors should add more significant recommendations. Full meaning of \"STROBE\" should be written before abbreviation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "53536", "date": "24 Sep 2019", "name": "Magiswary Dorasamy", "expertise": [ "Reviewer Expertise Knowledge Management Systems", "Cyber Security", "Action Research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper investigated KM awareness among tertiary level institutions. Respondents were academics. Data were collected appropriately. However, the paper seems to be missing theoretical framework. A strong theory will be good to lead the direction of the hypotheses. Any of the KM models would be applied for this studies to enhance its contribution in the context of Nigerian tertiary education. Some unique constructs may play important role in the awareness level of KM in Nigeria. A detail lit review may help to determine this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-608
https://f1000research.com/articles/8-1753/v1	15 Oct 19	{ "type": "Opinion Article", "title": "An intrinsically disordered proteins community for ELIXIR", "authors": [ "Norman E. Davey", "M. Madan Babu", "Martin Blackledge", "Alan Bridge", "Salvador Capella-Gutierrez", "Zsuzsanna Dosztanyi", "Rachel Drysdale", "Richard J. Edwards", "Arne Elofsson", "Isabella C. Felli", "Toby J. Gibson", "Aleksandras Gutmanas", "John M. Hancock", "Jen Harrow", "Desmond Higgins", "Cy M. Jeffries", "Philippe Le Mercier", "Balint Mészáros", "Marco Necci", "Cedric Notredame", "Sandra Orchard", "Christos A. Ouzounis", "Rita Pancsa", "Elena Papaleo", "Roberta Pierattelli", "Damiano Piovesan", "Vasilis J. Promponas", "Patrick Ruch", "Gabriella Rustici", "Pedro Romero", "Sirarat Sarntivijai", "Gary Saunders", "Benjamin Schuler", "Malvika Sharan", "Denis C. Shields", "Joel L. Sussman", "Jonathan A. Tedds", "Peter Tompa", "Michael Turewicz", "Jiri Vondrasek", "Wim F. Vranken", "Bonnie Ann Wallace", "Kanin Wichapong", "Silvio C. E. Tosatto", "M. Madan Babu", "Martin Blackledge", "Alan Bridge", "Salvador Capella-Gutierrez", "Zsuzsanna Dosztanyi", "Rachel Drysdale", "Richard J. Edwards", "Arne Elofsson", "Isabella C. Felli", "Toby J. Gibson", "Aleksandras Gutmanas", "John M. Hancock", "Jen Harrow", "Desmond Higgins", "Cy M. Jeffries", "Philippe Le Mercier", "Balint Mészáros", "Marco Necci", "Cedric Notredame", "Sandra Orchard", "Christos A. Ouzounis", "Rita Pancsa", "Elena Papaleo", "Roberta Pierattelli", "Damiano Piovesan", "Vasilis J. Promponas", "Patrick Ruch", "Gabriella Rustici", "Pedro Romero", "Sirarat Sarntivijai", "Gary Saunders", "Benjamin Schuler", "Malvika Sharan", "Denis C. Shields", "Joel L. Sussman", "Jonathan A. Tedds", "Peter Tompa", "Michael Turewicz", "Jiri Vondrasek", "Wim F. Vranken", "Bonnie Ann Wallace", "Kanin Wichapong" ], "abstract": "Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are now recognised as major determinants in cellular regulation. This white paper presents a roadmap for future e-infrastructure developments in the field of IDP research within the ELIXIR framework. The goal of these developments is to drive the creation of high-quality tools and resources to support the identification, analysis and functional characterisation of IDPs. The roadmap is the result of a workshop titled “An intrinsically disordered protein user community proposal for ELIXIR” held at the University of Padua. The workshop, and further consultation with the members of the wider IDP community, identified the key priority areas for the roadmap including the development of standards for data annotation, storage and dissemination; integration of IDP data into the ELIXIR Core Data Resources; and the creation of benchmarking criteria for IDP-related software. Here, we discuss these areas of priority, how they can be implemented in cooperation with the ELIXIR platforms, and their connections to existing ELIXIR Communities and international consortia. The article provides a preliminary blueprint for an IDP Community in ELIXIR and is an appeal to identify and involve new stakeholders.", "keywords": [ "ELIXIR", "intrinsically disordered proteins", "protein-protein interactions", "protein function", "databases", "community standards", "protein dynamics", "cellular regulation" ], "content": "Introduction\n\nIntrinsically disordered regions (IDRs), protein segments that lack persistent secondary or tertiary structure (Chouard, 2011; Dyson & Wright, 2005; Tompa, 2011), are predicted to cover almost a third of the residues in eukaryotic proteomes (Pancsa & Tompa, 2012; Xue et al., 2012). IDRs play a central role in cell regulation and contribute significantly to the cellular complexity of higher eukaryotes (Dunker et al., 2008; Dyson & Wright, 2005; Forman-Kay & Mittag, 2013; Gouw et al., 2018; Mitrea & Kriwacki, 2016; Schad et al., 2018; Tompa, 2005; Van Roey et al., 2014). They represent a major source of protein diversity and versatility on the level of organisms and during evolution (Babu et al., 2012; Buljan et al., 2012; Davey et al., 2015; Light et al., 2013; Weatheritt & Gibson, 2012). In the human proteome, IDRs are expected to contain up to one hundred thousand interaction interfaces and a million sites of post-translational modification (Tompa et al., 2014). However, to date, only a small fraction of these functional modules have been characterised (Gouw et al., 2018; Schad et al., 2018).\n\nIDR-mediated interactions are commonly found in dynamic and transient complexes that underlie enzyme inhibition, signal transduction and liquid-liquid phase transition (Borgia et al., 2018; Ivarsson & Jemth, 2019; Kriwacki et al., 1996; Martin & Mittag, 2018; Mitrea & Kriwacki, 2016; Olsen et al., 2017; Scott & Pawson, 2009; Wright & Dyson, 2015). IDR-mediated interactions are also major determinants of protein regulation, and are so far known to affect: (i) the post-translational modification state of a protein by acting as docking sites to recruit modifying enzymes; (ii) the cellular half-life of a protein by recruiting E3 ubiquitin ligases resulting in ubiquitin-dependent proteasomal degradation of the protein; and (iii) the localisation of a protein by acting as signals that target proteins to specific subcellular locations (Beltrao et al., 2012; Davey & Morgan, 2016; Gouw et al., 2018; Guharoy et al., 2016; Iakoucheva et al., 2004; Mészáros et al., 2017; Van Roey et al., 2014). Many functions of IDRs are directly associated with their structural attributes, without directly contributing to binding events that result in complex formation; for example, IDRs can act as entropic springs, flexible linkers or spacers (Tompa, 2005; van der Lee et al., 2014), or fly-casting regions to capture binding partners (Shoemaker et al., 2000). Furthermore, IDRs are subject to extensive pre- and post-translational regulation to modulate protein function in response to cellular stimuli (Bah & Forman-Kay, 2016; Csizmok & Forman-Kay, 2018; Van Roey et al., 2013; Van Roey et al., 2012; Weatheritt et al., 2012).\n\nAs a result of the fundamental regulatory functions performed by IDRs, and the cell-state conditionality of these regulatory processes, IDRs encode many of critical steps of a protein life-cycle from the ribosome to the proteasome. Consequently, IDRs play a key role in many human diseases, including cancer (p53), Alzheimer’s disease (Aβ, Tau) or Parkinson’s disease (α-synuclein) (Shigemitsu & Hiroaki, 2018; Uversky et al., 2008) and human IDR interfaces are often mimicked by pathogens to hijack host pathways and deregulate the cell (Davey et al., 2011; Dyson & Wright, 2018; Kruse et al., 2019; Via et al., 2015; Xue et al., 2012). Given their therapeutic relevance, IDP-mediated interactions are now seen as potential drug targets (Corbi-Verge & Kim, 2016). Therefore, a better understanding of their structure and function will help to develop new strategies to fight human diseases.\n\nIDP research spans several experimental fields studying protein structure and function including structural biology, biophysics, biochemistry, cell biology, proteomics, comparative genomics, systems biology, synthetic biology and pharmacology (Figure 1A) (Blikstad & Ivarsson, 2015; Corbi-Verge & Kim, 2016; Felli & Pierattelli, 2015; Forman-Kay & Mittag, 2013; Plitzko et al., 2017). On a structural level, IDRs do not adopt a single stable highly populated structure, instead, they are structurally heterogeneous, continuously sample a wide ensemble of conformations, with preferentially sampled intramolecular contacts driving local transient secondary structure and compaction of conformations (Davey, 2019; Dyson & Wright, 2005; Forman-Kay & Mittag, 2013; Holehouse & Pappu, 2018). Therefore, IDRs are more easily described by probabilistic models than the intuitive visual representations of structures of folded protein regions. Classical methods for structural characterisation of a protein, such as X-Ray crystallography, are unable to capture the dynamic structures of IDRs. Instead, the structural aspects of the IDRs are studied by a range of biophysical methods including Nuclear Magnetic Resonance (NMR), Small-angle X-ray scattering (SAXS), circular dichroism (CD) or Förster resonance energy transfer (FRET) (Felli & Pierattelli, 2015; Fuertes et al., 2017; Holmstrom et al., 2018; Plitzko et al., 2017; Tompa, 2011; Tolchard et al., 2018). The results of these complementary methods can be integrated to build a model of IDR structure and dynamics.\n\n(A) Key experimental methods used across the three major research focuses of the IDP field: structure, interactome and function. (B) The growth in structural IDP data curated by the DisProt database (Piovesan et al., 2017). DisProt was established over a decade ago in the USA, and recently brought to Europe after years of inactivity and completely re-annotated, this explains the lag in the curation. A huge amount of IDP/IDR data in the IDP literature remain uncurated and the vast majority of IDP regions remain to be characterised (Pancsa & Tompa, 2012; Xue et al., 2012). (C) The growth in functional IDP data curated by the Eukaryotic Linear Motif (ELM) resource (Gouw et al., 2018). Similar to the structural IDR data, a huge amount of functional IDP/IDR data in the IDP literature remains uncurated and the vast majority of functional modules in IDPs remain to be characterised (Tompa et al., 2014).\n\nThe functional aspects of IDPs are studied by a battery of well-established cell biology and biophysical approaches (Gibson et al., 2015) (Figure 1). Many of the approaches were developed for protein interaction elucidation, for example, by coupling mutagenesis to affinity-purification. However, interactions mediated by IDRs are often defined by their low affinity and cell-state dependent conditionality, two properties that are not always amenable to the available experimental protein interaction detection methods (Gibson et al., 2015; Van Roey et al., 2014). Therefore, there are a growing number of approaches that have been specifically designed to characterise low affinity IDR-mediated interactions such as peptide arrays, proteomic phage display (ProP-PD) and peptides attached to Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLE-pep) (Blikstad & Ivarsson, 2015; Davey et al., 2017; Nguyen et al., 2017; Volkmer, 2009). As IDRs often adopt a dominant conformation in their bound state, many IDR-containing interfaces can be studied in complex with the structured binding partners using structural approaches including NMR spectroscopy and X-Ray Crystallography (Bonetti et al., 2018; Fuxreiter, 2018; Iešmantavicius et al., 2014; Schad et al., 2018). Distinct IDR functionalities are also studied using isolation, deletion or mutagenesis of a functional module combined with bespoke assays to characterise phase separation, protein localisation, stability and post-translational modification (Gibson et al., 2015; Gouw et al., 2018; Martin & Mittag, 2018).\n\nThe computational IDP field tackles several distinct research tasks including: (i) the prediction of IDRs (Cilia et al., 2014; Dosztányi et al., 2005), (ii) the identification of functional modules in IDRs (Dosztányi et al., 2009; Edwards & Palopoli, 2015; Krystkowiak & Davey, 2017; Neduva & Russell, 2006), (iii) docking of peptides that are intrinsically disordered in their unbound state (Raveh et al., 2011; Trabuco et al., 2012), (iv) force field development and molecular simulations for IDR structure (Best, 2017; Chong et al., 2017; Huang & MacKerell, 2018; Stanley et al., 2015), (v) in silico inhibitor design and development for IDRs (Baggett & Nath, 2018; Santofimia-Castaño et al., 2019; Yu et al., 2016) and (vi) the processing of the data produced by experimental structural and functional analyses of IDRs (Bernadó et al., 2007; Franke et al., 2017; Nodet et al., 2009; Ozenne et al., 2012) (Table 1). The members of the European IDP community are major contributors across these computational IDP fields (Figure 2).\n\nEurope hosts many of the key IDP resources (Table 2). DisProt is the largest database of manually curated experimental data on IDP structure (Piovesan et al., 2017; Sickmeier et al., 2007). MobiDB and D2P2 are central resources of pre-computed structural prediction of IDPs (Piovesan et al., 2018; Oates et al., 2013). ELM is a manually curated database of binding regions residing in IDPs (Gouw et al., 2018). SASBDB and PCDDB are central resources for Small-angle X-ray Scattering (SAXS) and Circular Dichroism (CD) data (Valentini et al., 2015; Whitmore et al., 2017). PED3 is a database of conformational ensembles from Nuclear Magnetic Resonance (NMR) and SAXS data, and Molecular Dynamics (MD) simulations (Varadi et al., 2014). It is important to note that the vast majority of the computational frameworks developed by the IDP field had to be created from scratch given the unique structural and functional aspects of IDRs. For example, the analysis of IDP structure drew predominantly from polymer biophysics rather than the existing structural biology of folded proteins (Holehouse & Pappu, 2018; Milles et al., 2018; Schuler et al., 2016).\n\nThe ELIXIR IDP Community has grown out of the NGP-NET COST Action (Non-globular proteins: From sequence to structure, function and application in molecular physiopathology), a scientific cooperation funded in 2015 under a Horizon 2020 EU Framework Programme. The NGP-NET community spanned 30 different countries, plus EMBL-EBI and EMBL Heidelberg. NGP-NET held a series of thematic workshops on IDPs to drive the development of computational resources and community standards. A strategic workshop titled “Intrinsically Disordered Proteins in Core Data Resources” was organised by NGP-NET at the EBI campus, Hinxton, the UK on June 1–2, 2017 to discuss the integration of IDP-related data and computational resources into the ELIXIR framework. A major outcome of this workshop was the recognition that IDP annotations are significantly underrepresented in the ELIXIR Core Data Resources (CDRs) (Durinx et al., 2016). These initial insights evolved into a comprehensive plan to develop the IDP field and integrate key IDP resources and tools into the CDRs. That plan formed the basis for the ELIXIR IDP Community proposal that was submitted and presented at the ELIXIR Head of Nodes meeting in Basel, Switzerland on September 13th, 2017. After completing the ELIXIR Community application process, including the creation of this white paper, the IDP Community proposal was accepted by the ELIXIR Head of Nodes on May 14th, 2019.\n\nIn parallel with these developments, the IDP research community has developed closer integration with ELIXIR activities. A follow up strategic meeting, “The 2nd Workshop on Intrinsically Disordered Proteins in Core Data Resources”, was held in Prague on March 13–14, 2019 and brought together data producers, database developers and a representative of the ELIXIR Data Platform to discuss the integration of IDP data into the CDRs; and a member of the IDP research community attended the ELIXIR Interoperability Platform Face To Face Meeting on 1–2 April 2019. Furthermore, several actions have already taken place within the ELIXIR framework to tackle time-sensitive priorities including two implementation studies, “Implementation study for the integration of ELIXIR-IIB in ELIXIR Data Curation activities” and “Integration and standardisation of intrinsically disordered protein data”, focussing on interoperability. An additional, fundamental development associated with these implementation studies was the initialisation of the HUPO (Human Proteome Organisation) Proteomics Standards Initiative (PSI) Intrinsic Disorder workgroup.\n\nThe goal of the IDP Community in ELIXIR is to support the development of standards, tools and resources to accelerate the identification, analysis and functional characterisation of intrinsically disordered regions. Here, we introduce the major areas of priority for the development of an e-infrastructure that will allow the community to realise these goals while supporting the needs of the generators, users and consumers of IDP data.\n\n\nIdentification of community challenges\n\nA strategic workshop titled “An intrinsically disordered protein user community proposal for ELIXIR” was held on October 31st, 2018 at the University of Padua, Italy. The meeting was attended by participants from 13 ELIXIR nodes: Belgium, Cyprus, Czech Republic, EBI, Germany, Hungary, Ireland, Israel, Italy, Netherlands, Spain, Switzerland, and the United Kingdom. ELIXIR members in both Greece and Sweden were also interested, but could not attend. The ELIXIR Hub team was represented by John Hancock, the ELIXIR Communities and Services Coordinator and representatives from the Interoperability, Tools and Training ELIXIR platforms. Members of the 3DBioInfo and Proteomics ELIXIR Communities, the Instruct-ERIC structural biology research infrastructure, and the ELIXIR CDRs of high relevance to the IDP research, namely PDBe (Mir et al., 2018), IMEx/IntAct (Orchard et al., 2014) and UniProt (UniProt Consortium, 2019), were also present. During the meeting, the following topics were identified as key areas of priority for the ELIXIR IDP Community.\n\nThe IDP field has not yet established official standards to allow consistent storage and dissemination of data. Hence, a key responsibility of the IDP Community is the definition of guidelines and standards to improve the reproducibility, interpretation, and dissemination of experimental data. The development of novel standards would result in a shift from the current “organic” interoperability of the community, where each group defines their own formats and creates a range of parsers to read the formats developed by other groups, to standardised data dissemination accessible to the larger biological community. For functional data describing protein interactions, molecular interaction interchange standards based on the guidelines of the HUPO-PSI-MI molecular standards (Sivade Dumousseau et al., 2018) can be applied. However, for structural data, no pre-existing solution exists. In the simplest cases, experimental evidence for IDRs can be mapped to protein sequences as sequence features. Structural descriptions such as IDR conformational ensembles (e.g. based on experimental NMR, SAXS or MD simulations data) or secondary structural propensities (e.g. based on NMR data) will require more descriptive standards, especially if they are probabilistic. Only a subset of the required experimental and functional ontologies are already available and most IDP data cannot be described by pre-existing terminology. IDPs will require novel entries to controlled vocabularies describing specific experimental methods and protein structural concepts.\n\nThe first step of this process was undertaken at a workshop for the ELIXIR implementation study: “Integration and standardisation of intrinsically disordered protein data” held at the University of Padua, October 29–30th, 2018. The meeting proposed a first draft Minimum Information About Disorder Experiments (MIADE) standard defining the data required for reporting an IDP experiment. A further key outcome of the meeting was the establishment of the HUPO-PSI intrinsic disorder (HUPO-PSI ID) workgroup to drive the development of the required standards, storage and dissemination formats and controlled vocabularies for the community. Upon completion, the HUPO-PSI ID recommendations will be adopted by the key community resources promoting effortless integration of IDP data into the ELIXIR CDRs. An important goal of the roadmap will be the initiation of collaborations with the experimental communities for each of the distinct structural and functional methods used in IDP research to develop: (i) experimental method specific standards and (ii) workflows with minimal required experimental detail for the characterisation of IDPs. These developments would require extensive collaboration with the data generators and the experimental method specific data repositories including SASBDB (Valentini et al., 2015), PCDDB (Whitmore et al., 2017) and the BMRB (Ulrich et al., 2008) to allow data produced by IDP researchers to be stored and disseminated in the most descriptive and efficient way possible. This will simplify the integration of the results of these analyses into the community resources. The recent development of the PDB-Dev repository of non-atomistic or part-atomistic structural data can provide a prototype for the deposition of experimental IDP data (Vallat et al., 2018; Peng et al., 2019).\n\nThe vast majority of the experimental data describing IDPs, the functional modules encoding their function, the regulatory mechanisms conditionally controlling that function, and their dysregulation in disease, is isolated in the text of research and review articles and in poorly formatted supplementary tables. This hampers the integration of IDP information with data such as protein function, modification, splice variants and disease-causing single-nucleotide polymorphisms (SNPs). Consequently, data created at great expense is significantly underutilised. Annotation of bona fide IDPs, especially for their function, is currently a labour-intensive process. The IDP community has already been experimenting with crowdsourced curation, a topic which is of interest to the ELIXIR Data Platform, and included within Task 3 of the ELIXIR Data Platform 2019–2023 ELIXIR Programme.\n\nDisProt is a successful example, leveraging the collective expertise of around 40 researchers from a dozen different IDP labs in as many countries. The rate and accuracy of curation can be improved by integrating the available ELIXIR e-infrastructure into the curation process through partial annotation of articles and through automatic selection, classification and prioritisation of the relevant articles for curation. For example, the annotation of IDP-mediated interactions directly into the IMEx consortium annotation portal would provide a pre-built environment for such an endeavour. Automatic triage and pre-compiling of data based on Europe PMC data would also greatly boost productivity, allowing the community to cope with the increasing amount and complexity of data being published (Britan et al., 2018). The large number of articles describing the structure and function of IDPs has highlighted the need to exploit text-mining approaches in order to eventually leverage automatic annotation from the literature. ELIXIR would facilitate integration with existing text-mining frameworks. Finally, a future goal of the community is the early capture of IDP data pre-publication directly from the data producers to reduce the need for manual curation. The coordinated experimental and computational fields within the ELIXIR IDP Community can provide a single contact point to lobby journals to require data deposition prior to publication.\n\nIDP annotations are significantly underrepresented in the ELIXIR CDRs and a key goal of the ELIXIR IDP community is to facilitate the integration of IDP data and services into these resources. The CDRs do not currently annotate or import experimental data describing IDR structure and function despite their high abundance and functional importance. Recently, InterPro, PDBe, and UniProt adopted IDP predictions from sequence retrieved from MobiDB, as part of an ELIXIR Implementation Study “Integration and standardisation of intrinsically disordered protein data”. This work can be used as a blueprint for the successful integration of additional IDP data into further CDRs. It would be advantageous both for the IDP Community and the CDRs to comprehensively integrate the available IDP data into these resources. Interoperability can be guaranteed by the implementation of IDP specific ontologies to describe IDP specific features and the availability of standards-compliant RESTful APIs, enabling cross-linking and programmatic access to IDP resources. Many of the key IDP resources already host RESTful APIs and utilise persistent identifiers from CDRs. However, these APIs are currently not fully interoperable with each other without bespoke adaptors, though this will be tackled by Priority Area 1 “Standards and exchange formats for IDP data”. The development of curation guidelines and standards in line with the requirements of the CDRs, or the adoption of CDR guidelines, will streamline the integration process. The IDP resources that annotate functional modules and their interactions are currently not members of the International Molecular Exchange (IMEx) consortium (Orchard et al., 2012). A key step on the roadmap is joint curation efforts between the ELIXIR IDP Community and the IMEx consortium of IDP-mediated interactions, thereby reducing duplication of effort.\n\nThe IDP research field is currently developing best practices for scientific (i/o) file formats, data analysis pipelines and benchmarking of scientific tools. These steps are being taken to raise the quality and accessibility of software developed by the community to produce more accurate, faster, more stable and user-friendly software implementation. The development and adoption of the storage and dissemination formats in Priority Area 1 will help standardise the (i/o) file formats of IDP tools. However, several use cases will not be covered by these formats (e.g. standardised i/o formats for residue-specific IDR scoring) and additional effort will be required to formalise the output formats in these areas. Containerised software, such as those available via BioContainers, or package managers, such as BioConda, are rarely used in IDP research. However, such advances would make IDP tools more accessible to the wider community and would simplify their benchmarking.\n\nThe field of IDP research could also benefit from the development of reusable experimental workflows such as those implemented with the Common Workflow Language for commonly used IDP analysis pipelines built on top of ELIXIR CDRs and Deposition Databases. These workflows could then be managed using the Galaxy workflow manager platform. Benchmarking remains an issue for the community as the lack of common benchmarking datasets has hampered the systematic assessment of IDP tools. As a result, many publications have claimed superior performance for biased datasets, furthering the need for standardisation. Due to the availability of high-quality manually curated IDP data from DisProt, the community can provide gold standard blind datasets to run periodic benchmarking assessments of IDR prediction tools. This approach has recently been successfully applied by the Critical Assessment of Intrinsic Disorder (CAID) initiative. A similar platform for the comparison of methods predicting functional modules within IDPs or aligning homologous IDRs would also benefit the community and drive advances in these developing fields.\n\nThe development of Open Source software to benchmark IDP analysis methods across a wide range of performance metrics will simplify and standardise the assessment of IDP tools. OpenEbench can be used as an information hub to distribute reference datasets, to run comparative assessments and to publish benchmarking results. From a technical point of view, the adoption of BioContainers and/or Galaxy, and the standardisation of input and output formats would streamline the assessment process. Benchmarking results covering both scientific and technical aspects of the available IDP analysis tools can be hosted at OpenEBench to simplify method selection by users. Finally, the addition of these computational tools for the analysis of IDPs to the bio.tools registry would improve the visibility of these tools.\n\nComputational IDP researchers based in Europe develop many of the tools and resources that underlie the global IDP e-infrastructure. However, these assets are currently spread over numerous institutes and universities across Europe. The development of an umbrella resource, DisProtCentral (founded June 2017), consolidating the European IDP resources and tools through a single portal will improve the accessibility of these resources for the wider biological community. The DisProtCentral consortium will provide a central hub to access high-quality curation, annotation and predictions of structural and functional information on IDPs, in addition to providing stable identifiers/URIs to describe regions of disorder within specific proteins, enabling cross-referencing and linking between resources. The consortium will include all the stakeholders in the IDP field and provide a centralised repository for the protein disorder-related databases and tools. This will future-proof these key IDP resources against issues arising from the loss of funding or change of group focus.\n\nThis initiative to produce such a centralised resource draws attention to the need for a fit-for-purpose interoperability tooling adaptor that translates across the metadata annotations of individual resources. This will allow pre-existing data from distinct resources to be integrated via normalisation functionalities such as ontology cross-referencing for data previously mapped to different ontologies/vocabularies, or ontology term mapping for those that have not been mapped to any standard terminologies. Ideally, newly generated data should be produced FAIR-at-source (Findable, Accessible, Interoperable, and Reusable) (Wilkinson et al., 2016). DisProtCentral will play a vital role in encouraging IDP data generators to follow the standardised set of best practices: identifying recommended ontologies that are universally adopted by those data generators, defining a common schema markup strategy, and validating persistent identifiers for data to be deposited into the resource. A wider goal of the DisProtCentral resource is to provide a single point of contact to promote discourse and collaboration to ensure that the needs of the IDP data generators, users and consumers are all being met by the computational IDP researchers. An important aspect of this effort will be the development of training material for the wider biological communities describing the best practices for IDP analyses.\n\n\nAlignment with ELIXIR Activities\n\nThe community challenges identified are already well-aligned with on-going ELIXIR platform goals and activities. In particular:\n\nData Platform: The task proposed in Priority Area 3 “Integration with ELIXIR Core Data Resources” aligns with the goals of the Data Platform. The community is already in the process of integrating IDP annotation into the ELIXIR CDRs as part of two ELIXIR implementation studies, “Implementation study for the integration of ELIXIR-IIB in ELIXIR Data Curation activities” and “Integration and standardisation of intrinsically disordered protein data”. The recent integration of MobiDB into InterPro, PDBe and UniProt can be taken as a blueprint for the further integration of IDP information into ELIXIR CDRs. Furthermore, initial discussions have taken place to plan the integration of additional sources of IDP data. A separate task, which can benefit from ongoing Data Platform activities, is the development of an IDP curation framework as described in Priority Area 2 “Automated and community-driven curation”. This aligns with ELIXIR Data Platform Task 3 “Scalable curation” of the 2019–2023 ELIXIR Programme.\n\nThe distributed community curation for IDP resources will benefit from stronger interaction with Europe PMC, with the SciLite framework providing literature mining. This is especially important where data is scattered throughout the extensive corpus of biological literature and not properly indexed. Together with an automated curation triage step selecting relevant papers, this can boost the productivity of community curation while ensuring high-quality annotation of the IDP literature. Initial efforts have started with the design of a dedicated curation-support prototype, which has been used by DisProt curators since 2018. The service capitalises on the neXtA5 platform (Mottin et al., 2017), designed by the Text Mining group of the Swiss Institute of Bioinformatics. The demonstrator (http://candy.hesge.ch/disprotGUI/) is able to rank articles based on a scoring function, which prioritises articles with a high density of IDP-related concepts. Thanks to the curation-support tool, it will be possible to obtain a high quality curated benchmark, with the aim to evolve the current system from a triage system to a more accurate binary classifier, being able to select not only relevant papers but also to highlight short passages of text in full-text papers likely to support the expert curation.\n\nTools Platform: The tasks proposed in Priority Area 4 “Standardisation, benchmarking and indexing of computational tools” fall under the objectives of the Tools Platform. A major goal of Priority Area 4 is to increase productivity by developing reusable experimental workflows for commonly used IDP analysis pipelines built on top of ELIXIR CDRs. The Tools Platform can advise the ELIXIR IDP Community on the development of such workflows using the Common Workflow Language in collaboration with the ELIXIR Interoperability platform. The IDP analysis tool benchmarking by the CAID initiative is complementary to the OpenEBench benchmarking platform. OpenEBench represents an information hub for the distribution of reference datasets, the application of metrics and comparative assessments and the distribution of benchmarking results. CAID can also drive prototype development for software containers and reusable experimental workflows for the community. The outcomes could then be generalised for other applications. The bio.tools service registry can be a comprehensive census of the available software for IDP analysis and the BioContainers and Bioconda platforms can assist in the generation of software containers and their registry across different technologies to facilitate access and use of software by the IDP communities and beyond.\n\nInteroperability Platform: Several of the identified priorities will benefit from the input of the Interoperability Platform. The main interaction with the platform will be to develop FAIR-compliant standards and guidelines for reporting, data exchange and retrieval of data on IDP structure and function (Wilkinson et al., 2016). The recent development of a HUPO PSI-ID workgroup and a draft MIADE standard are key steps towards this goal. IDP resources themselves will benefit from becoming FAIR and adhering and contributing to the further development of standards, controlled vocabularies and ontologies. These advances are of paramount importance to improve the dissemination of IDP data. FAIR-compliant data will also aid in the FAIRification of analysis tools where reusable computational workflows can make programmatic calls to retrieve reusable data from an integrated centralised source. The Interoperability Platform service framework has already defined the various key activities to support the “FAIRification” of data which can be applied to IDP data. The effort to make IDP data FAIR can exploit the various key services that are offered by the Interoperability Platform such as those identified as recommended practices through the ELIXIR Recommended Interoperability Resources (RIRs), and the platform mission-critical initiatives (i.e., Bioschemas, and Common Workflow Language). As this is an emerging community with new data resources in development, deploying these recommended practices in new IDP databases will highlight the IDP Community as a use case example in the mission to make data FAIR-at-source. This work can be supported by the Interoperability Platform under their remit to provide support for interoperability for ELIXIR Communities.\n\nCompute Platform: Work being carried out in the Compute Platform can be leveraged in two main ways. Community curation requires authentication for a wide range of participants, which can benefit from the federated ELIXIR AAI approach for curator login. This could also facilitate the attribution of credit to curators. A second element would be to use the ELIXIR distributed computing infrastructure including identity and access management, data integration and container deployment across a range of appropriate compute endpoints as required, both for updating large-scale databases and to run the CAID experiment. A range of new Compute Platform activities are underway to enable an integrated approach to the deployment of relevant workflows and containers. The IDP Community can take advantage of these activities to deploy IDP-related workflows and containers.\n\nTraining Platform: The IDP Community is focussed on the development of the next generation of IDP researchers. Extensive training has been funded and carried out by the COST Action NGP-NET and additional training schools are planned as part of the MSCA-RISE-funded IDPfun project. The Italian ELIXIR node, which has been actively providing training, will organise a yearly training course on IDP resources. The input of the ELIXIR Training Platform will be indispensable to facilitate the development of a core set of training materials, its dissemination and inclusion in ELIXIR training courses. These materials will comprise introductory and advanced lessons on databases access, in silico analysis of IDPs (e.g. IDP prediction, IDP docking, MD simulations and IDP-specific primary sequence analysis techniques), and computational processing of experimental data (e.g. NMR spectra, phage display). There will also be a requirement for the development of training material related to IDP interoperability to train experimentalists, software developers, database developers and curators in topics such as IDP standards, IDP controlled vocabularies and Common Workflow Language (CWL) for IDPs. This aligns with the Interoperability Platform’s training and outreach task in the 2019–2023 ELIXIR Programme to collaborate with Training Platform members to provide Interoperability training. In collaboration with the Training Platform, the IDP Community will be able to adopt and implement a wide range of best practices and guidelines available through the ELIXIR Training Toolkit, which aims to provide a comprehensive reference resource for developing training capacity, rolling out new training programs, as well as expanding existing ones. All materials and activities developed for IDP training will be shared through TeSS for further dissemination. Additionally, train-the-trainer activities will be planned to increase the size of the IDP trainers pool and build additional training capacity.\n\n\nAlignment with other communities\n\nStructural Bioinformatics (3DBioInfo): ELIXIR 3DBioInfo is a community of structural bioinformaticians with the remit of continuing the development of the e-infrastructure for the storage, visualisation, analysis, annotation, and prediction of the structure of biological macromolecules and complexes. The probabilistic approach to protein structure employed by the IDP researchers is highly complementary to the work of the ELIXIR 3DBioInfo community and together, a complete structural description of proteins can be achieved. With the two communities addressing different challenges, there are numerous opportunities for synergistic connections between them, particularly in the development of structural ontologies and standards, structure boundary definition for structural studies, methods to predict IDR-globular domain interactions and whole protein structure prediction tools.\n\nProteomics. The ELIXIR Proteomics Community aspires to improve the research on proteoforms including protein forms caused by post-translational modifications (PTMs) and sequence variants. To this end, proteoform-centric annotations of proteomes are needed. This includes information on the co-occurrences of IDRs with PTMs and non-constitutive exons, and if possible, the functional outcomes of these conditional changes to the protein. Close collaboration is desirable between the communities regarding data interoperability to promote the use of IDP and proteomics data in data analysis workflows. IDP research also applies proteomics-related technologies such as ion-mobility mass spectrometry and cross-linking mass spectrometry. In these cases, close collaboration with the proteomics community within the HUPO-PSI Mass Spectrometry workgroup will support the standardisation efforts of the IDP community.\n\nRare Diseases: The overarching goal of the ELIXIR Rare Diseases Community is to create a sustainable, reusable, and interoperable infrastructure that will enable researchers to discover, access, and analyse rare disease data. A key aspect of the analysis of rare diseases data is the elucidation of the mechanism(s) by which genetic change(s) result in a diseased state. While the interpretation of the effect of disease mutations altering globular domains is well established, IDPs represent challenges in this context. Interaction and collaboration with the ELIXIR IDP Community will provide invaluable information to aid in the understanding of disease-causing variations, truncations, and/or chimeric oncogene(s) related to IDRs. The development, and dissemination of standards for IDR structural and functional data as proposed in priority area 1 are important aspects for the collaboration between the ELIXIR IDP and Rare Diseases Communities.\n\nGalaxy: Galaxy is a web-based e-infrastructure for computational biomedical research. It allows users with minimal computational proficiency to run and share data analysis workflows. This promotes reproducibility and simplifies sharing of data and results. Currently, there is limited use of Galaxy by the ELIXIR IDP community. However, one of the priority areas of the roadmap, “Standardisation, benchmarking and indexing of computational tools”, is the development of reusable experimental workflows for commonly used IDP analysis pipelines built on top of ELIXIR CDRs. A collaboration with the ELIXIR Galaxy Community would be highly beneficial for such an endeavour, particularly in the development of IDP pipelines using the ELIXIR scientific workflow platform.\n\nInstruct-ERIC: Instruct-ERIC is a European research infrastructure for structural biology that makes high-end technologies and methods available to European researchers. Similarly to the proposed ELIXIR 3DBioInfo Community, the interdependency of the research performed by Instruct-ERIC and the IDP researchers, covering the structured and unstructured parts of the proteome, allows for extensive synergies. Instruct-ERIC coordinates access to facilities which permit the structural analysis of IDPs including several centres specialised in NMR and biophysical techniques. Furthermore, Instruct-ERIC has developed a set of high-quality training courses in structural biology. Given the complementary teaching focus of the ELIXIR IDP Community and the Instruct-ERIC initiative, future training schools with contributions from both sources would be of great benefit to any participant.\n\nThe Dark Proteome Initiative: The Dark Proteome Initiative is a US consortium of experimentalists with the goal of fostering collaborations amongst IDP researchers and lobbying for funding to address open and biologically important questions in the IDP field. The Dark Proteome Initiative is a readymade community of world-class generators of IDP data utilising a wide range of experimental approaches allowing the ELIXIR IDP Community to access a single entity for guidance on the needs of experimental data generators. The Dark Proteome Initiative will also be able to provide invaluable advice on the detailed definitions and requirements for the development of ontologies and standards.\n\nPLUMED consortium: The PLUMED consortium has been recently established as an open community including PLUMED developers, contributors and users to help to establish reproducibility, open access data, and harmonisation of the protocols for molecular dynamics simulations, free energy calculations and other simulations that can be run with the PLUMED software (PLUMED consortium, 2019).\n\n\nConclusions\n\nRecent years have seen a rapid growth of interest in IDPs. This has coincided with significant advances in the in vivo, in vitro and in silico methods to study the structure and function of IDPs. Unfortunately, the basic requirements for the organisation and dissemination of the tools and data produced by the field have not advanced in step with these developments. The field is on the cusp of an era where the high-throughput characterisation of the extensive intrinsically disordered regions (IDRs) of proteins is possible. This roadmap will provide the foundation that supports this data explosion and provide a solid platform for the future biological research of IDPs.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nBabu MM, Kriwacki RW, Pappu RV: Structural biology. Versatility from protein disorder. Science. 2012; 337(6101): 1460–1461. PubMed Abstract \| Publisher Full Text\n\nBaggett DW, Nath A: The Rational Discovery of a Tau Aggregation Inhibitor. Biochemistry. 2018; 57(42): 6099–6107. PubMed Abstract \| Publisher Full Text\n\nBah A, Forman-Kay JD: Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications. J Biol Chem. 2016; 291(13): 6696–6705. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55886", "date": "21 Nov 2019", "name": "Iva Pritisanac", "expertise": [ "Reviewer Expertise intrinsically disordered protein experimental biophysics", "biochemistry and computational development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere has been a significant contribution from Europe to the field of intrinsically disordered protein (IDP) research, including development of computational tools. This white paper recognizes that science and methodological development, and reports on a meeting focused on organizing the software and the software developers supporting the IDP community in Europe. While it is extremely important to provide infrastructure support for the IDP community and efforts to increase communication and usage of tools are essential, it is not clear that the significant effort to put these resources under a single umbrella and unify data formats is of value beyond that of encouraging independent development with opportunities to interact. This white paper provides a useful report on the meeting that will be of value to the IDP computational community. However, it could be improved to provide greater clarity on the proposal and some of the suggested priorities could be re-evaluated.\n\nSpecific comments:\nThe white paper itself is challenging to read. It is unclear who the target audience of the article is? Is it the IDP/IDR community or the ELIXIR community, or both? In the present form, the article might be confusing to both communities, as it provides a lot (at times too much) detail for some aspects, resources, and individual tools, while omitting crucial information for others. For instance, it is unclear what from the current resources would be relocated into a new ELIXIR infrastructure and what would continue to exist independently and simply being \"pointed at\" from a central resource? It is also unclear exactly where central data repositories and central software platforms would reside. Many presently available resources are outlined and suggested at different points, making it unclear how everything would fit together. In addition, it is unclear if there will be a central resource(s), such as DisProtCentral, and if so would this resource(s) ultimately have independent developers, next to the participating research groups.\n\nSome of the software and the databases that will be the key components are listed in Figure 2. However, not all of the outlined resources are presently functional or consistently maintained. It is therefore unclear if the initiative aims at reviving some of the resources in the future, and if so, whether this be done by the individual research groups. For the present time, it would be important to include a table with links to the websites/repositories of the operational resources, and a note on the current status of all other, e.g. \"not actively developed\", \"under development\", \"under restructuring\", etc.\n\nIt would be very helpful to include schematics to help understanding of the proposed initiative and improve the flow of the article. For instance, it would be good to use flow charts to outline the hierarchy of priorities to help understand how presently available resources fit into different priority areas and what will be the first tasks undertaken by different work groups. The priorities relating to the Area 1 (“Standards and exchange formats for IDP data\") would be especially important to clarify in that sense, so that the community can be informed where to look for updates on the matter and thus help aid these efforts. Finally, the history of the interactions between the IDP community and ELIXIR and the subsequent integration into the ELIXIR core could be represented with a timeline and be in a separate subsection of the article, as this presently interrupts the flow of the article.\n\nIt would be important to define the strategy that the community wants to undertake towards integration of resources. For instance this could be a) integrated, dedicated databases with API access that are continuously maintained for data storage; and b) open source software with modular architecture that will enable integration into, e.g., BioConda for software development. At present, the general strategy is not clear or explicit, making the article quite confusing to follow.\n\nIt would be highly valuable to use this article and this platform to promote open source software development and established platforms for this purpose, such as GitHub. GitHub has proven effective in countless large-scale projects and it connects thousands of developers. The comparatively very small community of IDP computational researchers would benefit by developing, integrating, and sharing their resources through GitHub. In addition, such a platform would ensure inclusiveness. Related to this, it is not clear how the proposed centralization will solve the major issue of long term support for software, including funding for people to continue to maintain and develop the tools. A good way to assure a project will continue is for the project source/algorithm be fully open, in a repository such as GitHub, to enable use of modular code within other projects.\n\nIt is stated that DisProtCentral would be used for consolidating the European IDP resources and tools. However, it has not been mentioned what is the plan (if any) for integration of non-European resources and tools? Would there be some way for developers from outside of Europe to qualify to submit or include their software and resources in DisProtCentral?\n\nThere is a lot of emphasis on 'interoperability', and adaptors that could be used to this end. However, apart from the simplest workflows, e.g. annotation of predicted disordered regions on a protein sequence, it is likely that the demands for the workflows will be rapidly changing in the future. Spending effort on defining formats for data is not likely to be productive since by the time these standards can be applied they are could already be outdated. Technically speaking, it is not a problem if data are used in different formats, as long as those formats are easy to parse, since writing a parser is trivial. Therefore, the emphasis should be shifted to the modular software development that would enable developers to 'mix and match' various routines and dynamically create their own workflows adapt to the task at hand. The proposal for integration into bio.tools or BioConda in this context is a very good one.\n\nThere are two tools that are actively maintained to current standards, GROMACS and PLUMED2, and they are not IDP specific and they run well regardless of ELIXIR. These points should be noted. Capabilities to download the software, see the development flow and interact with the project directly are available for these tools, but it is not clear whether these principles are a general priority.\nMinor points:\nA full name and abbreviation for ELIXIR should be given in the abstract.\n\nInternational Molecular Exchange (IMEx) is mentioned in the article before the abbreviation is introduced (the abbrev. is introduced later).\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "55395", "date": "25 Nov 2019", "name": "Osman Ugur Sezerman", "expertise": [ "Reviewer Expertise bioinformatics", "sysytem biology", "structural bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes importance of intrainsically disordered proteins with thorough set of examples with good references. It also explains computational and experimental challenges involved in IDP research.\nA good summary of available tools and platforms are also provided. Key challenges in IDP research are identified and key activities to be carried out to tackle these challenges are provided. ELIXIR community has many of the researchers working in the field within the consortium. All the needed activities align well with ELIXIR activities as well. So it is natural that ELIXIR plays a centralized role in these activities. The paper also identified key players around the world that they can collaborate with establishing a task force to tackle major challenges IDP research needs to tackle.\n\nOnly issue I have with the paper is the lack of focus on reproducibility issues. That is a major problem life science research is facing today. most of the experimental results are not reproducible even computational models are not reproducible resulting in waste of billions of euros spent in funding such research activities.\nThey should have a special focus group within their consortium to ensure reproducibility of experimental results within IDP community (define key information needed to reproduce results, endorse use of e notebooks and use of protocols established withing the network, etc.). They should also do the same for computational models that are developed to tackle several problems related to IDP. A section added on these issues would strengthen the paper. Also if they include other initiatives focusing on reproducbility that would strengthen their task force.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1753
https://f1000research.com/articles/8-965/v1	25 Jun 19	{ "type": "Study Protocol", "title": "Pragmatic Home-Based Exercise after Total Hip Arthroplasty - Silkeborg: Protocol for a prospective cohort study (PHETHAS-1)", "authors": [ "Lone Ramer Mikkelsen", "Merete Nørgaard Madsen", "Michael Skovdal Rathleff", "Kristian Thorborg", "Camilla Blach Rossen", "Thomas Kallemose", "Thomas Bandholm", "Merete Nørgaard Madsen", "Michael Skovdal Rathleff", "Kristian Thorborg", "Camilla Blach Rossen", "Thomas Kallemose", "Thomas Bandholm" ], "abstract": "Introduction: Rehabilitation exercises are offered to patients after total hip arthroplasty (THA); however, the effectiveness and optimal type and dose of exercise remains unknown. The primary objective of this trial is to indicate the preliminary efficacy of home-based rehabilitation using elastic band exercise on performance-based function after THA, based on the relationship between the performed exercise dose and the change in performance-based function (gait speed) from 3 (start of intervention) to 10 weeks (end of intervention) after surgery. The secondary objective is to investigate if a dose-response relationship exists between the performed exercise dose and changes in: hip-related disability, lower-extremity functional performance, and hip muscle strength Methods: In this prospective cohort study, patients scheduled for THA will be consecutively included until 88 have completed the intervention period from 3 to 10 weeks postoperatively. Participants perform the standard rehabilitation program with elastic band exercises. Exercise dose (exposure) will be objectively quantified using a sensor attached to the elastic band. The primary outcome is gait speed measured by the 40-m fast-paced walk test. Secondary outcomes include: patient reported hip disability (Hip disability and Osteoarthritis Outcome Score (HOOS)), hip muscle strength (hand-held dynamometry) and lower extremity function (30-s chair stand test). Discussion: This trial will add knowledge concerning the relationship between performed exercise dose and post-operative outcomes after THA. The protocol paper describes the study design and methods in detail, including the statistical analysis plan. Trial registration: Pre-registered on March 27, 2017 at ClinicalTrails.gov (ID: NCT03109821).", "keywords": [ "Total Hip Arthroplasty", "Rehabilitation", "Exercise therapy", "Dose-response", "Strength training" ], "content": "Introduction\n\nTotal hip arthroplasty (THA)1 is offered to patients with end-stage hip osteoarthritis to reduce pain and improve function1. Muscle strength and functional performance, such as walking ability, are substantially reduced early after THA2–5; this is why postoperative rehabilitation is offered throughout the municipalities in Denmark. In some municipalities, this is organized as outpatient supervised rehabilitation, whereas in other municipalities, patients receive an initial instruction and perform rehabilitation exercise in their own homes without supervision. In Central Denmark Region (place of this trial), the current predominant clinical practice is home-based rehabilitation for most patients.\n\nSystematic reviews with meta-analyses show that supervised, outpatient rehabilitation exercise is not superior to home-based exercise for performance-based or self-reported function outcomes6,7. It has also been difficult to demonstrate clear superiority with relevant effect size of one type of rehabilitation exercise over another for performance-based or self-reported function outcomes8,9. There is, however, some evidence to indicate that rehabilitation exercise may be superior to no or very little rehabilitation exercise for selected muscle-strength, gait, and function outcomes after THA6,9,10. It suggests that a dose-response relationship exists for post-operative rehabilitation exercise and recovery after THA.\n\nTo be able to investigate a dose-response relationship for post-operative rehabilitation exercise and recovery after THA, objective measures that capture compliance to home-based exercise are needed11. In recent work12–14, we have validated a measure to monitor compliance to home-based exercise in healthy subjects (an in-built sensor attached to an elastic exercise band), and started using it in clinical populations for intervention research15–18. With the PHETHAS-1 trial, we want to use this sensor technology to investigate if a dose-response relationship exists for home-based rehabilitation exercise and recovery after THA, using a prospective cohort study design. By using this technology, we will be able to not only investigate a dose-response relationship on the recovery associated with exercise, but also investigate the preliminary efficacy of home based, rehabilitation exercise after THA. This can be achieved by comparing participants with the least exercise compliance to those with the most. This will indicate whether home-based, rehabilitation exercise “works” better than no or very little rehabilitation exercise, although not a randomized comparison. It will help inform a subsequent large-scale, confirmatory, randomized trial investigating the efficacy of rehabilitation exercise after THA when compared to no or very minimal rehabilitation exercise.\n\nThe primary objective is to indicate the preliminary efficacy of home-based rehabilitation using elastic band exercise on performance-based function after THA, based on the relationship between the performed exercise dose and the change in performance-based function (gait speed measured by 40-m fast-paced walk test) from 3 (start of intervention) to 10 weeks (end of intervention) after surgery.\n\nThe secondary objective is to investigate if a dose-response relationship exists between the performed exercise dose and changes in: hip-related disability, lower-extremity functional performance, and hip muscle strength.\n\n\nMethods\n\nThe study is a pragmatic, single-center, prospective cohort study (single cohort) conducted in Silkeborg, Denmark. Outcome assessments will be performed at 3 (start of home-based strengthening exercise) and 10 weeks (after 7 weeks of home-based strengthening exercise) after surgery. Furthermore, patient-reported outcome measures will be collected pre-surgery (see the participant timeline in Table 1). It is the aim that all outcome assessments will be performed by three physiotherapists who have been thoroughly trained in performing the outcome assessments. The data collection methods, trial logistics and the intervention have been tested in a pilot study including 10 patients and adjustments have been made accordingly. The study will adhere methodologically to the STROBE guideline for prospective cohort studies and the CONSORT statement.\n\n* HOOS: Hip disability and Osteoarthritis Outcome Score\n\n** VAS: Visual Analogue Scale\n\nAll participants will be included from the Elective Surgery Centre at the public hospital, Silkeborg Regional Hospital. Exercise instruction as well as blinded outcome assessments will be performed by physiotherapists from Elective Surgery Centre. The physiotherapists are members of the staff of physiotherapists at Elective Surgery Centre and all have at least 6 months of experience working with THA.\n\nParticipants will be included by consecutive sampling. The inclusion criteria are: age above 18 years, scheduled for a primary THA at the Elective Surgery Centre due to osteoarthritis and able to understand written and spoken Danish. The exclusion criterion is: referral to supervised rehabilitation in the municipality (instead of the home-based rehabilitation exercise-program in the present study).\n\nThe exercise intervention reflects the standard rehabilitation exercise practice at Elective Surgery Centre; hence, a pragmatic approach is used. During a short hospital stay (typically discharge on the day after surgery), all patients are instructed in an exercise program of unloaded exercises (not part of the intervention studied) to be performed at home during the initial 3 postoperative weeks until their scheduled follow up visit at the hospital. At this visit (3 weeks after surgery), and after the outcome assessment, the participants will receive a thorough instruction in the strengthening exercises that they are instructed to perform without supervision in their own homes the following 7 weeks. The instruction is conducted one-to-one by physiotherapists using approximately 20 minutes per participant and supported by an instruction booklet with written and illustrated exercise descriptions. The strengthening exercises included are: hip abduction, flexion and extension with elastic band resistance and sit-to-stand. The prescribed training load will be two sets with repetitions to contraction failure (neuromuscular fatigue) and a relative load of 10 to 20 repetition maximum (RM), performed every second day (3–4 times a week). The strengthening exercises are supplemented with daily stretching of hip flexor muscles and balance exercise (one-legged stance). Exercise compliance for the strengthening exercises will be monitored objectively (see Outcomes section). No efforts will be made to increase compliance beyond normal practice (e.g. SMS encouragements, or likewise), because we intent to measure actual, uninfluenced compliance as close to daily practice as possible. The participants will be advised to gradually increase their activity level after the operation to comply with the recommendations on physical activity from the Danish Health and Medicines Authority. Furthermore, they will be given instructions on how to handle pain during exercises and recreational activities (the pain management guide is available as Extended data)19. To reinforce similar treatment administration, face-to-face meetings among the participating physiotherapists will be held per need to discuss issues experienced in the clinic. The exercise intervention is described in detail according to the exercise-specific Consensus on Exercise Reporting Template (CERT)20 (A completed CERT checklist is available as Extended data)19, supplemented with the full set of strength training descriptors as suggested by Toigo and Boutellier (Table 2)21. Finally, the exercise intervention is described according to the Template for Intervention Description and Replication (TIDieR) checklist, which is a generic intervention-description template (a completed TIDieR checklist is available as Extended data)22,19.\n\n* RM: Repetition Maximum\n\nThe participants will be advised to gradually increase their activity level after the operation. Likewise, they will be instructed to gradually progress their exercises during the 7 weeks of training at home according to the described progression model, where the strengthening exercises are performed to failure in each set; when the possible repetitions exceed 20 in two of the three elastic band exercises they should change the elastic band so that a higher loading is possible. The participants are instructed that pain in relation to exercise is normal, and that up to 5 on a numeric rating scale (NRS) during exercise is considered acceptable based on the suggested pain monitoring system by Thomée et al.23. However, the pain should decrease within 30 minutes after the exercise session. The participants are advised to contact the hospital if they experience increasing pain or other complications such as swelling or wound problems (the pain management guide is available as Extended data)19.\n\nExposure. Performed exercise dose will be quantified as the total physiological exercise stimulus (Time under tension summary dose per week) recorded by a sensor (Bandcizer: commercially available from www.bandcizer.com) attached to the elastic exercise band. The sensor automatically switches on and stores data when the elastic exercise band is used13,14. Furthermore, performed exercise dose will be quantified as the number of days with strengthening exercises being performed.\n\nChange in gait speed is chosen to be primary outcome, as walking ability is considered the most important function to improve by patients undergoing THA surgery24. Furthermore, the 40-m fast-paced walk test is part of the core set of functional tests to include in clinical trials in patients with osteoarthritis in hip or knee recommended by OARSI25,26.\n\nChange in gait speed\n\nMeasured by the 40-m fast-paced walk test25,26. Change from 3 to 10 weeks after surgery.\n\nGait speed\n\nMeasured by the 40-m fast-paced walk test25,26. At 10 weeks after surgery.\n\nChange in patient-reported function\n\nMeasured by the Activities of Daily Living (ADL) subscale of HOOS27. HOOS is a disease-specific patient-reported outcome measure. Change from 3 to 10 weeks after surgery.\n\nChange in patient-reported symptoms\n\nMeasured by the symptoms subscale of HOOS27. Change from 3 to 10 weeks after surgery.\n\nChange in patient-reported pain\n\nMeasured by the pain subscale of HOOS27. Change from 3 to 10 weeks after surgery.\n\nChange in patient-reported hip related quality of life\n\nMeasured by the quality of life subscale of HOOS27. Change from 3 to 10 weeks after surgery.\n\nChange in lower extremity function.\n\nMeasured by the 30-s chair stand test25,26 (The maximal number of rises from a chair within 30 seconds). Change from 3 to 10 weeks after surgery.\n\nChange in hip abductor muscle strength.\n\nTest of isometric muscle strength in hip abduction in the operated leg. The hand-held dynamometer Power Track II Commander will be used to assess this using standardized test procedure28. Change from 3 to 10 weeks after surgery.\n\nChange in hip flexor muscle strength.\n\nTest of isometric muscle strength in hip flexion in the operated leg. The hand-held dynamometer Power Track II Commander will be used to assess this using standardized test procedure28. Change from 3 to 10 weeks after surgery.\n\nSelf-efficacy.\n\nThe general self-efficacy scale29 will be used to measure self-efficacy, defined as an individual's belief in his or her capacity to execute behaviors necessary to produce specific performance attainments. At 3 weeks after surgery.\n\n24-hour physical activity (mean upright time/day and mean number of steps/day).\n\nAn ActivPAL movement-sensor will be used to measure mean time per day in upright position (standing and walking) based on 7 days of data collection. The sensor will be applied 3 weeks after surgery and used the following week. At 4 weeks after surgery.\n\nNumber of participants with adverse events.\n\nNumber and type of adverse events will be registered by the physiotherapist 3 and 10 weeks after surgery in the following pre-defined categories: Hip dislocation, infection, fracture, wound seepage, acute myocardial infarction, deep venous thrombosis, readmission and other.\n\nMean change in pain after each exercise session.\n\nThe visual analogue scale (VAS) will be used to assess pain before and after each exercise session. Data will be summarized as a mean change in pain per exercise session for the entire intervention period. At 10 weeks after surgery.\n\nNumber of pain flares after exercise sessions.\n\nVAS will be used to assess pain before and after each exercise session. Pain flare is defined as an increase in pain of ≥20 mm30. Data will be summarized, both for the first 14 days of the intervention and for the entire intervention period. At 5 and 10 weeks after surgery.\n\nMotivation to perform the prescribed exercises.\n\nThe participants will be asked about their motivation to perform the prescribed exercises. A short questionnaire comprising three questions developed for this purpose will be used (the questionnaire is available as Extended data)19. The possible responses are ordered in 4 levels of motivation on an ordinal scale. At 3 weeks after surgery.\n\nEvaluation of the prescribed exercises\n\nThe participants will be asked to evaluate the exercises. A short questionnaire comprising three questions developed for this purpose will be used (the questionnaire is available as Extended data)19. The possible responses are ordered in 4 levels on an ordinal scale. At 10 weeks after surgery.\n\nAt June 28, 2017, two outcome measures were added to the study. At 10 weeks after surgery, participants will be asked both to describe their perception of the result after surgery and the change in hip problems (from preoperatively to 10 weeks after surgery). The questions will be phrased as \"How would you describe the result of your operation?\" with response categories \"Excellent\", \"Very good\", \"Good\", \"Fair\", \"Poor\". The second question will be asked as \"Overall, how are the problems now in the hip on which you had surgery, compared to before your operation?\" with the response categories \"Much better\", \"A little better\", \"About the same\", \"A little worse\", \"Much worse\". These two questions have been used as anchor questions to establish patient acceptable symptom state (PASS) and minimal clinically important improvement (MCII) cut-points for patient-reported outcomes – including some subscales of HOOS – 1 year after THA31. We will use these questions to group patients according to their perception of result of the operation and changes in hip problems, as well as for exploratory analysis of PASS and MCII cut-points for HOOS, 10 weeks after surgery.\n\nIn April, 2019, pain flare was added as an outcome measure.\n\nCategories of adverse events were defined prior to study start, but they were not specifically described in the trial registration. Motivation to perform prescribed exercises was registered as outcome, but although predefined, the three items in the short questionnaire were not specifically described. Evaluation of prescribed exercises was added as outcome prior to study start.\n\nIn April 2019, the secondary objective was added to the primary and pre-specified objective because the primary objective did not clearly outline the secondary analyses of secondary outcomes for the hypothesized dose-response relationship.\n\nAll the changes outlined above occurred before the last participant was included and the study was unblinded (please see “Blinding” below).\n\nIn addition to collecting quantitative data, we will also conduct an embedded qualitative study concerning the participants' experience with performing home-based exercise and resuming general physical activities. The aim will be to understand the patients' motivation and barriers related to home-based exercise and general physical activity after THA. The participants will be selected through theoretical sampling32, expectedly a maximum of 20. Participants will be recruited partly from the PHETHAS-1 trial, and partly from the population of standard THA patients not involved in an exercise trial. This is done to elucidate the influence of participating in a trial with extra interventions such as exercise diary, outcome assessments, etc. Semi-structured interviews will be conducted 10 weeks postoperatively using an interview guide (available as Extended data)19. This qualitative study is undertaken to refine the home-based intervention for future trials and clinical implementation. The embedded qualitative study will be reported in a separate paper with a clear reference to the PHETHAS-1 trial.\n\nThe sample size estimation is based on a minimal clinical important difference of 0.2 m/sec33 between changes in gait speed among participants with highest performed exercise dose compared to participants with smallest performed exercise dose. Based on results from a pilot study leading up to this trial, we expect a maximal difference of 4 hours in performed exercise dose (total Time under tension summary dose) during the 7-week intervention period between participants with highest and lowest exercise compliance. Also based on the pilot study, a SD of 1.06 hours for exercise dose and 0.16 m/sec for change in gait speed were used. The power is set at 0.90 to increase the power for secondary analyses, and with a 0.05 level of significance. Based on the above, the required sample size is estimated to be 88 participants.\n\nThe basis for recruitment makes the trial highly feasible due to the approximately 800 elective THA procedures performed annually at the Elective Surgery Centre. As there may be more eligible participants per day than for whom there is available equipment (BandCizers and ActivPAL sensors), we restrict inclusion by including consecutive participants from random sections of the department. That is, patients examined and booked for surgery in pre-specified clinics in the outpatient department. Patients are allocated to the specific clinics in the department by a secretary at random and with no influence from any personnel involved in the study. The estimated inclusion rate is approximately one to two participants per week; please see estimated participant flow and current recruitment status in Figure 1.\n\nThe outcome assessors will be blinded to exercise compliance-data. Moreover, we will inform the participants that we measure how they perform their exercises and not how much they exercise or what the study hypothesis is. This is done with the purpose of minimizing sensor-induced influence on compliance and to reduce expectation bias.\n\nThe elastic band sensor (BandCizer) automatically records and stores exercise data during elastic band exercises. It is a valid measure of date, time of day, number of repetitions and sets, total time-under-tension (TUT), and total single repetition TUT during commonly used home-based strength training exercises for the lower extremities14. The 40-m fast-paced walk test measures performance-based function and is part of the recommended core set of tests to assess physical function in people diagnosed with hip or knee osteoarthritis by the Osteoarthritis Research Society International (OARSI)25,26. A high inter-tester reliability is shown (intraclass correlation coefficient (ICC) 0.95) in a population with hip osteoarthritis33. The HOOS questionnaire measures patient-reported outcome in the subscales: symptoms, pain, ADL, function in sport and recreation and hip-related quality of life27. HOOS is shown valid, responsible, and reliable (ICC >0.78) when evaluating patients undergoing THA34. Hip muscle strength28 and 30-s chair stand test will be conducted in accordance with previous published methods26,28 showing acceptable relative and absolute inter-rater reliability when used after THA (ICC 0.83-0.93 and SEM 7–10%)35. General Self-Efficacy Scale is a 10-item validated questionnaire holding a scale assessing optimistic self-beliefs to cope with a variety of difficult demands in life, scored between 1–4 points without any defined cut-off point29. ActivPal movement-sensors measures physical activity as time spent in the sit/lie position (X-axis), standing (Y-axis) and walking (Z-axis). It has been validated in several studies in healthy adults36 and in older adults with a hip fracture37,38.\n\nData collection will continue for participants who discontinue their training. Data collection will only be discontinued if participants explicitly withdraw from the study or any major events or diseases prevent the outcome assessments. If participants do not attend their scheduled follow ups, they will be contacted and offered a new time.\n\nRaw data from the Bandcizer will be uploaded to a secure online database using a tablet or smartphone. Here, the investigator will be able to access and analyze data and extract the following variables; date and number of training sessions, number of repetitions, time under tension for each repetition and total time under tension for each training session. Data from the outcome measurement will be double entered in EpiData 3.1 using anonymous coding with ID numbers and relevant range checks for data values to minimize typing errors. Completed data collection forms will be stored in a locked cabinet at Silkeborg Regional Hospital. Electronic data files will be stored on a secured hospital server with access requiring personal login. The linkage between ID numbers and personal identification data (e.g. civil registration number, name, address) will be stored as an electronic file as described above.\n\nAll the planned analyses are listed in Table 3.\n\nDescriptive analyses will be performed for demographic variables, supplementary descriptive variables, adverse events, motivation to perform prescribed exercises, evaluation of prescribed exercises and pain after exercise sessions (change in pain and pain flares). Data will be presented as means with 95% confidence intervals (CI) or medians with inter quartile ranges (IQR) for continues variables and as frequencies with percentages for categorical variables.\n\nInitially, scatterplots of outcome variables and exercise dose variables will be used to suggest starting model structures and possible more complex alternatives. The structures of the models used for the dose-response analysis will depend on the specific relationship between change in gait speed and the exercise dose variable. Because of this, and not having any prior knowledge of the structure of the relationship, multiple models will be fitted and evaluated by R-squared values to identify the models that fit data the best. As a starting point, the first model will be fitted as a fixed increase in outcome, based on exercise dose-change done by linear regression modelling. If necessary, more complex regression such as polynomial relationship and other nonlinear structures will also be evaluated.\n\nIn the case that none of the models seem to fit the data, a linear regression model with a categorical variable based on intervals of the exercise dose variable will be fitted. This model does not provide a direct dose response relationship but provides an estimate of the association between the outcome variable and the exercise dose variable within the specific intervals.\n\n“Regression to the mean” may be present and will be evaluated by the correlation between the change and the measure at baseline. If regression to the mean is believed to be present for an outcome, the models of the outcome will additional include the baseline measure to adjust for regression to the mean.\n\nPossible confounding variables (self-efficacy (baseline), physical activity (during intervention), and gait speed (baseline)) will also be included in the models. The confounding effect of each variable will be examined by comparison of dose response estimates in models with and without the confounder. If there is no relevant change between the estimates of the models, the confounder will be excluded from the model. Normality assumptions in the models are evaluated by QQ-plots\n\nFor the dose response relationship between change in HOOS ADL, the analysis will be similar to the analysis for change in gait speed outlined above.\n\nThe relationship between exercise compliance and HOOS subscales (symptoms, pain, quality of life), 30-s chair stand test and hip muscle strength will be presented as means with CIs or medians with IQRs within each of the compliance quartiles, as well as graphical representation of these values.\n\nTo better understand what may relate to how patients comply with prescribed rehabilitation exercise after THA, we will investigate how different variables relate to exercise compliance (dependent variables: time under tension summary dose and total number of exercise sessions), using uni-variable modelling. Independent variables will be: pain flares (first two weeks of intervention), pain flares (entire intervention period), HOOS pain (baseline), motivation to perform exercises, belief in effect of exercises, self-belief in compliance to exercising, satisfaction with rehabilitation exercise, physical activity (mean upright time/day and mean number of steps/day) and self-efficacy (baseline).\n\nTo better understand what may relate to how physically active patients are after a THA, we will investigate how different variables relate to physical activity (dependent variables: mean upright time/day and mean number of steps/day), using univariate modelling. Independent variables will be: pain flares (first two weeks of intervention), HOOS pain (baseline), motivation to perform exercises, self-belief in compliance to exercising, and self-efficacy (baseline).\n\nIn the analysis of \"result of the operation\", the change in score from baseline to follow-up will be presented for each HOOS subscale (pain, symptoms, ADL, QOL) and gait speed. Data will be presented both for each response category of the anchor question, and for the subgroup of patients, who answered \"excellent\", \"very good\" or \"good\" data. This subgroup is considered to be reporting a hip-specific acceptable symptom state. Data will be presented by means with 95% CI or medians and interquartile ranges (IQR). In each response category of the question for \"change in hip problems\", the change in score from baseline to follow-up will be presented for each HOOS subscale (pain, symptoms, ADL, QOL) and gait speed. Data will be presented by mean scores with 95% CI or median and inter quartile range (IQR).\n\nFurthermore, for each exercise dose quartile, the percentage of patients in each response category of the questions for \"result of the operation\" and \"change in hip problems\", will be presented graphically. Finally, HOOS cut points for PASS and MCII will be estimated by the mean score or mean change approach39.\n\nMissing items within the HOOS and General Self-efficacy scale will be handled as recommended in the guidelines (HOOS: <50% missing items in each subscale is accepted, self-efficacy: ≤3 missing items is accepted). Concerning ActivPal data, a minimum of four days of data collection will be accepted as sufficient to calculate min/day upright time and steps/day40. In situations where participants have to stop the physical tests due to pain, the data from the best performance are used no matter if the pre-defined number of repetitions is reached. It is noted if tests are interrupted due to pain to be able to perform sensitivity analysis if appropriate. If participants are lost to follow up (despite the before-mentioned efforts to keep every participant in the trial) they will be excluded from the analyses that include change scores. We will not use last-observation-carried-forward or other imputation procedures, as we aim to investigate relationships between performed exercise dose and observed changes in post-operative outcomes to help qualify subsequent research work.\n\nSince the study involves no major changes to current practice it is not deemed necessary to establish a data monitoring committee or perform any interim analyses. Likewise, no provisions for post-trail care will be made.\n\n\nDiscussion\n\nThis trial will add knowledge concerning the preliminary efficacy of home-based rehabilitation using elastic band exercises based on the relationship between performed exercise dose and outcomes after THA. We believe this is the first trial to do so, since earlier attempts have not used objective measurement of exercise dose as in the present trial. In an observational cohort study, Zech et al found no significant associations between the exercise therapy intensity or duration and improvements in patient reported function, pain, and stiffness41. However, the exercise dose was dependent on the participants´ health insurance as well as individual conditions and the physiotherapist's decision, which likely induces a risk of bias by indication.\n\nThe essential need from a clinical perspective is to be able to prescribe evidence-based exercise programs after THA. Despite the growing number of studies, a recent systematic review that included 20 studies concludes that insufficient therapeutic validity and potentially high risk of bias in the included studies limit the ability to assess the effectiveness of exercise after THA42.\n\nThe new knowledge from the present study can potentially identify whether the dose of performed home-based exercise is related to changes in post-operative outcomes after THA. It will give insight concerning the potential influence from other factors than exercise, such as general physical activity and self-efficacy. Furthermore, the embedded qualitative study will give insight to perceived motivation and barriers to perform the prescribed exercise as well as to resuming general physical activities. The results from both the quantitative and qualitative study are expected to be useful in optimizing current practice; however, the results will also be used to plan, power and execute a randomized controlled trial that compares the effectiveness of rehabilitation exercises to no rehabilitation exercises (just resuming general physical activities).\n\nThe strengths of this study include the objectively measured exercise dose, the standardized and thoroughly described intervention and the inclusion of outcome variables at all levels in the International Classification of Function, Disability and Health (ICF). We chose gait speed measured by the 40-m fast-paced walk test as the primary outcome. Walking ability is considered the most important function to improve by patients undergoing THA surgery24, and the 40-m fast-paced walk test is part of the core set of functional tests to include in clinical trials in patients with osteoarthritis in hip or knee recommended by OARSI25. An important candidate for the choice of primary outcome for clinical research has been suggested to be a patient-reported one43. Nevertheless, we chose a performance-based measure as the primary, as we were concerned about ceiling effects on patient reported outcomes that measures function and pain, such as the HOOS questionnaire after THA44.\n\nMultiple factors can potentially affect exercise compliance; therefore, we include measurements of physical activity and self-efficacy. Also, it is not known which outcomes that is most susceptible to exercise dose which is why we include a broad range of different outcome types to be able to explore potential dose-response relationships.\n\nBlinding of participants in randomized exercise trials are often impossible, in the present study we seek to blind the participants to the specific focus om exercise dose, they are just told that we measure \"the way they exercise\". Hypothesis blinding is considered a design strength when blinding of participants regarding treatment is not possible45. Furthermore, we blind the outcome assessor in the sense that they are not allowed to see the exercise diary or BandCizer data prior to the outcome assessment.\n\n\nTrial status\n\nThe trial began recruiting participants in April 2017. After a period with slow inclusion, the inclusion rate is back at 1–2 participants per week, thus, inclusion is expected to be completed in July 2019. See current status on participant flow in Figure 1.\n\nThis paper is based on protocol version 5, March 8, 2019.\n\n\nDeclarations\n\nThe Ethics Committee of Central Denmark Region accepted initiation of the study and reviewed the study as non-notifiable (Inquiry 270/2017). The study was approved by the Danish Data Protection Agency (ref. no: 1-16-02-589-15).\n\nTrained Research staff (nurse or physiotherapist) will provide presentation of comprehensible information about the research to potential participants, confirmation that they understand the research, and assurance that their agreement to participate is voluntary. Potential participants will also receive information sheets. They will be offered deliberation time and, subsequently, written consent will be obtained from those who choose to participate. The informed consent document is available as Extended data19.\n\nAll records that contain names or other personal identifiers, such as informed consent forms, will be stored separately from study records identified by code number to protect confidentiality before, during, and after the trial.\n\nThe principal investigator, as well as all co-authors, will have access to the full dataset as needed. A fully anonymized dataset and statistical analysis code will be made available for the scientific journal reviewing the manuscript within six months in line with the recent proposal from the International Committee of Medical Journal Editors (ICMJE)46.\n\nResults from the trial will be published in international, scientific peer-reviewed journals, no matter the trial outcome. The results will also be presented at relevant scientific conferences and symposiums. Authorships will be allocated according to the ICMJE recommendations. The following papers are planned:\n\n1. Pragmatic Home-Based Exercise after Total Hip Arthroplasty – Silkeborg (PHETHAS-1): Results from a prospective cohort study.\n\n2. Motivation and barriers to perform home-based exercise after Total Hip Arthroplasty – a qualitative embedded study within the PHETHAS-1 trial.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: PHETHAS-1 protocol. https://doi.org/10.6084/m9.figshare.825601419.\n\nThis project contains the following extended data:\n\nWHO Trial Registration Data Set_PHETHAS.docx\n\nConsent document.pdf\n\nManaging pains associated with exercise.docx (pain management guide)\n\nPHETHAS Interview guide_english (PHETHAS-2 interview guide, translated into English)\n\nQuestionnaire_ Motivation to perform exercises.docx\n\nQuestionnaire_Evaluation of prescribed exercises.docx\n\nCERT Checklist.docx\n\nTIDieR Checklist.docx\n\nExytended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: SPIRIT checklist for “Pragmatic Home-Based Exercise after Total Hip Arthroplasty - Silkeborg: Protocol for a prospective cohort study (PHETHAS-1)”. https://doi.org/10.6084/m9.figshare.825601419.", "appendix": "Grant information\n\nThe study has received the following external funding: Regional Hospital Central Jutland Research Foundation (75,000 DKkr), The Danish Rheumatism Association (75,000 DKkr, grant R139-A3844), The Association of Danish Physiotherapists (37,500 DKkr), The Aase and Ejnar Danielsen Foundation (100,000 DKkr, grant: 10-002170) and The family Kjærsgaard foundation (26,928 DKkr).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nFootnotes\n\n1 Abbreviations: ADL, Activities of Daily Living; CI, Confidence Interval; HOOS, Hip disability and Osteoarthritis Outcome Score, ICMJE, International Committee of Medical Journal Editors; IQR, Inter Quartile Range; NRS, Numeric Rating Scale; THA, Total Hip Arthroplasty; PHETHAS, Pragmatic Home-Based Exercise after Total Hip Arthroplasty â€“ Silkeborg; RM, Repetition Maximum; STROBE, STrengthening the Reporting of OBservational studies in Epidemiology; TIDieR, Template for Intervention Description and Replication; TUT, time-under-tension; VAS, Visual Analogue Scale; WHO, World Health Organisation\n\n\nReferences\n\nGossec L, Paternotte S, Maillefert JF, et al.: The role of pain and functional impairment in the decision to recommend total joint replacement in hip and knee osteoarthritis: an international cross-sectional study of 1909 patients. Report of the OARSI-OMERACT Task Force on total joint replacement. Osteoarthritis Cartilage. 2011; 19(2): 147–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJudd DL, Dennis DA, Thomas AC, et al.: Muscle strength and functional recovery during the first year after THA. Clin Orthop Relat Res. 2014; 472(2): 654–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKolk S, Minten MJ, van Bon GE, et al.: Gait and gait-related activities of daily living after total hip arthroplasty: a systematic review. Clin Biomech (Bristol, Avon). 2014; 29(6): 705–18. PubMed Abstract \| Publisher Full Text\n\nHolm B, Thorborg K, Husted H, et al.: Surgery-induced changes and early recovery of hip-muscle strength, leg-press power, and functional performance after fast-track total hip arthroplasty: a prospective cohort study. PLoS One. 2013; 8(4): e62109. 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BMJ Open. 2014; 4(6): e005044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkovdal Rathleff M, Thorborg K, Bandholm T: Concentric and eccentric time-under-tension during strengthening exercises: validity and reliability of stretch-sensor recordings from an elastic exercise-band. PLOS One. 2013; 8(6): e68172. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRathleff MS, Bandholm T, Ahrendt P, et al.: Novel stretch-sensor technology allows quantification of adherence and quality of home-exercises: a validation study. Br J Sports Med. 2014; 48(8): 724–8. PubMed Abstract \| Publisher Full Text\n\nRathleff MS, Thorborg K, Rode LA, et al.: Adherence to commonly prescribed, home-based strength training exercises for the lower extremity can be objectively monitored using the bandcizer. J Strength Cond Res. 2015; 29(3): 627–36. PubMed Abstract \| Publisher Full Text\n\nRathleff MS, Bandholm T, McGirr KA, et al.: New exercise-integrated technology can monitor the dosage and quality of exercise performed against an elastic resistance band by adolescents with patellofemoral pain: an observational study. J Physiother. 2016; 62(3): 159–63. PubMed Abstract \| Publisher Full Text\n\nRiel H, Matthews M, Vicenzino B, et al.: Feedback Leads to Better Exercise Quality in Adolescents with Patellofemoral Pain. Med Sci Sports Exerc. 2018; 50(1): 28–35. PubMed Abstract \| Publisher Full Text\n\nClausen MB, Bandholm T, Rathleff MS, et al.: The Strengthening Exercises in Shoulder Impingement trial (The SExSI-trial) investigating the effectiveness of a simple add-on shoulder strengthening exercise programme in patients with long-lasting subacromial impingement syndrome: Study protocol for a pragmatic, assessor blinded, parallel-group, randomised, controlled trial. Trials. 2018; 19(1): 154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHusted RS, Troelsen A, Thorborg K, et al.: Efficacy of pre-operative quadriceps strength training on knee-extensor strength before and shortly following total knee arthroplasty: protocol for a randomized, dose-response trial (The QUADX-1 trial). Trials. 2018; 19(1): 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikkelsen LR: PHETHAS-1 protocol. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.8256014.v1\n\nSlade SC, Dionne CE, Underwood M, et al.: Consensus on Exercise Reporting Template (CERT): Explanation and Elaboration Statement. Br J Sports Med. 2016; 50(23): 1428–1437. PubMed Abstract \| Publisher Full Text\n\nToigo M, Boutellier U: New fundamental resistance exercise determinants of molecular and cellular muscle adaptations. Eur J Appl Physiol. 2006; 97(6): 643–63. PubMed Abstract \| Publisher Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014; 348: g1687. PubMed Abstract \| Publisher Full Text\n\nThomee R: A comprehensive treatment approach for patellofemoral pain syndrome in young women. Phys Ther. 1997; 77(12): 1690–703. PubMed Abstract \| Publisher Full Text\n\nHeiberg KE, Ekeland A, Mengshoel AM: Functional improvements desired by patients before and in the first year after total hip arthroplasty. BMC Musculoskelet Disord. 2013; 14: 243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDobson F, Hinman RS, Roos EM, et al.: OARSI recommended performance-based tests to assess physical function in people diagnosed with hip or knee osteoarthritis. Osteoarthritis Cartilage. 2013; 21(8): 1042–52. PubMed Abstract \| Publisher Full Text\n\nDobson F, Bennell KL, Hinman RS, et al.: Recommended performance-based tests to assess physical function in people diagnosed with hip or knee osteoarthritis. 2013. Reference Source\n\nNilsdotter AK, Lohmander LS, Klassbo M, et al.: Hip disability and osteoarthritis outcome score (HOOS)--validity and responsiveness in total hip replacement. BMC Musculoskelet Disord. 2003; 4: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorborg K, Petersen J, Magnusson SP, et al.: Clinical assessment of hip strength using a hand-held dynamometer is reliable. Scand J Med Sci Sports. 2010; 20(3): 493–501. PubMed Abstract \| Publisher Full Text\n\nSchwarzer R, Jerusalem M: Measures in Health Psychology: A User's Portfolio. Causal and Control Beliefs. 1995. Reference Source\n\nDworkin RH, Turk DC, Wyrwich KW, et al.: Interpreting the clinical importance of treatment outcomes in chronic pain clinical trials: IMMPACT recommendations. J Pain. 2008; 9(2): 105–21. PubMed Abstract \| Publisher Full Text\n\nPaulsen A, Roos EM, Pedersen AB, et al.: Minimal clinically important improvement (MCII) and patient-acceptable symptom state (PASS) in total hip arthroplasty (THA) patients 1 year postoperatively. Acta Orthop. 2014; 85(1): 39–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharmaz K: Constructing Grounded Theory. 2nd ed. SAGE Publications Ltd; 2014. Reference Source\n\nWright AA, Cook CE, Baxter GD, et al.: A comparison of 3 methodological approaches to defining major clinically important improvement of 4 performance measures in patients with hip osteoarthritis. J Orthop Sports Phys Ther. 2011; 41(5): 319–27. PubMed Abstract \| Publisher Full Text\n\nThorborg K, Roos EM, Bartels EM, et al.: Validity, reliability and responsiveness of patient-reported outcome questionnaires when assessing hip and groin disability: a systematic review. Br J Sports Med. 2010; 44(16): 1186–96. PubMed Abstract \| Publisher Full Text\n\nMikkelsen LR, Mikkelsen S, Soballe K, et al.: A study of the inter-rater reliability of a test battery for use in patients after total hip replacement. Clin Rehabil. 2015; 29(2): 165–74. PubMed Abstract \| Publisher Full Text\n\nGodfrey A, Culhane KM, Lyons GM: Comparison of the performance of the activPAL Professional physical activity logger to a discrete accelerometer-based activity monitor. Med Eng Phys. 2007; 29(8): 930–4. PubMed Abstract \| Publisher Full Text\n\nTaraldsen K, Askim T, Sletvold O, et al.: Evaluation of a body-worn sensor system to measure physical activity in older people with impaired function. Phys Ther. 2011; 91(2): 277–85. PubMed Abstract \| Publisher Full Text\n\nTaraldsen K, Vereijken B, Thingstad P, et al.: Multiple days of monitoring are needed to obtain a reliable estimate of physical activity in hip-fracture patients. J Aging Phys Act. 2014; 22(2): 173–7. PubMed Abstract \| Publisher Full Text\n\nde Vet HC, Terwee CB, Mokkink L, et al.: Interpretability. Measurements in medicine- Practical guides to biostatistics and epidemiology: Cambridge: Cambridge University Press; 2011; 227–68. Publisher Full Text\n\nMigueles JH, Cadenas-Sanchez C, Ekelund U, et al.: Accelerometer Data Collection and Processing Criteria to Assess Physical Activity and Other Outcomes: A Systematic Review and Practical Considerations. Sports Med. 2017; 47(9): 1821–1845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZech A, Hendrich S, Pfeifer K: Association Between Exercise Therapy Dose and Functional Improvements in the Early Postoperative Phase After Hip and Knee Arthroplasty: An Observational Study. PM R. 2015; 7(10): 1064–1072. PubMed Abstract \| Publisher Full Text\n\nWijnen A, Bouma SE, Seeber GH, et al.: The therapeutic validity and effectiveness of physiotherapeutic exercise following total hip arthroplasty for osteoarthritis: A systematic review. PLoS One. 2018; 13(3): e0194517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatrick DL, Burke LB, Powers JH, et al.: Patient-reported outcomes to support medical product labeling claims: FDA perspective. Value Health. 2007; 10 Suppl 2: S125–37. PubMed Abstract \| Publisher Full Text\n\nPaulsen A, Pedersen AB, Overgaard S, et al.: Feasibility of 4 patient-reported outcome measures in a registry setting. Acta Orthop. 2012; 83(4): 321–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoutron I, Guittet L, Estellat C, et al.: Reporting methods of blinding in randomized trials assessing nonpharmacological treatments. PLoS Med. 2007; 4(2): e61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaichman DB, Backus J, Baethge C, et al.: Sharing Clinical Trial Data--A Proposal from the International Committee of Medical Journal Editors. N Engl J Med. 2016; 374(4): 384–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "50853", "date": "02 Aug 2019", "name": "Dana L. Judd", "expertise": [ "Reviewer Expertise Total Hip Arthroplasty and Rehabilitation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol paper details a prospective cohort study which will investigate the initial efficacy of home-based exercise program following total hip arthroplasty (THA). The authors present strong rationale for performing the study, in particular citing evidence regarding the deficits in strength and function following THA as well as the lack of consensus regarding rehabilitation following THA surgery.\nThe study design is described in great detail, and the proposed methods will adequately answer the research question. The strengths of the study design include objective monitoring of exercise adherence, the standardization of the exercise protocol, and the combination of validated strength measures and functional performance outcome measures. The choice of exercises for the intervention target common deficits following THA, and the choice of a gait outcome as a primary outcome measure will represent an important functional outcome for patients. A unique aspect of this proposed trial is the proposed qualitative investigation into patients' perceived barriers to and motivations for participating in the proposed exercise program. Not only will this have potential to inform future trials, but may provide important information from the patients' point of view to better inform clinical practice and home exercise prescription after THA.\n\nThere are also items the authors may wish to consider. First, although there are proposed training and meetings to ensure correct instruction by therapists to patients, the authors may consider including a way to objectively report fidelity data of the therapists to describe how well they adhered to the protocol. Second, due to the importance of collecting the exercise adherence data, is there a plan to handle missing data from the Bandcizer sensors? Or perhaps a way to ensure data is being collected over the weeks of intervention as expected and a plan for troubleshooting. Finally, although the choice of exercises for the protocol aligns well with common impairments following THA, it is possible the few specific exercises and proposed dosing may not yield the expected changes in gait speed without the inclusion of gait-specific training.\n\nOverall, this is a well written manuscript describing a well-designed study and the results should be valuable to the literature regarding rehabilitation after THA.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4965", "date": "14 Oct 2019", "name": "Lone Ramer Mikkelsen", "role": "Author Response", "response": "Response to Reviewer 1 Thank you for your positive evaluation. Below we address your items for consideration one by one. Reviewer 1, question 1: First, although there are proposed training and meetings to ensure correct instruction by therapists to patients, the authors may consider including a way to objectively report fidelity data of the therapists to describe how well they adhered to the protocol.Response: We agree that it would be relevant to include an objective measure of fidelity in the exercise instructions. We did not consider this when we started the trial, and at the present time, inclusion is almost completed. What we did consider, however, was to continuously standardize the instructions through regular meetings and discussions with the therapists administering the intervention. We feel this reflects clinical practice and, hence, the pragmatic approach that we wanted for the trial. The main discussion points at these meetings, until now, have primarily concerned adaptations to the exercise program when the patients experience pain during or after exercise. That is why we have developed the pain management guide which is published as extended data. Furthermore, we use ongoing reminders of the importance of thorough instructions in performing exercise to failure.No changes made in manuscriptReviewer 1, question 2: Second, due to the importance of collecting the exercise adherence data, is there a plan to handle missing data from the Bandcizer sensors? Or perhaps a way to ensure data is being collected over the weeks of intervention as expected and a plan for troubleshootingResponse: This is a good point and based on previous experiences with this technology, which is still under development, we expect that such a plan will be important. We have added plans for handling missing data and troubleshooting. To prevent discontinued use of the Bandcizer sensor during exercise, we inform the patients of the importance of using the Bandcizer sensors in every single exercise session. To estimate if this is accomplished, we ask the patients at the 10 week follow up visit: \"How often was the BandCizer sensor attached to your elastic exercise band when exercising?\"\" (options: Every time, most of the time about half the time, a few times, never). This is described in \"Extended data\" Changes made: Text added in the section \"Handling of missing data\": Based on the importance of the Bandcizer data, we have made a plan for how to use the data if we are not able to extract the data as planned. Thus, we prioritize to use the Bandcizer data the following way depending on what is possible:1) As planned with a valid total time-under-tension (TUT) estimate.2) If 1) is not possible, we will use the total number of repetitions as an estimate of the total exercise dose.3) If 1) and 2) is not possible, we will use the total number of days with performed exercise as an estimate of the total exercise dose. We will report the proportion of missing data, e.g. self-reported non-compliance in Bandcizer use or invalid Bandcizer sensor-data. Furthermore, we will perform a sensitivity analysis using data on total exercise dose (days with exercise) from the exercise diary. This will inform if there is a comparable dose-response relationship when analyzing self-reported compliance data compared to objectively measured data (Bandcizer). The two types of data yield a risk of different types of bias. Self-reported exercise dose is often over-estimated and the Bandcizer data may induce problems with missing data as described above which could led to an underestimation of exercise-dose. We will not use last-observation-carried-forward or other imputation procedures on exposure (exercise dose), as we aim to investigate relationships between actually performed exercise dose and changes in post-operative outcomes. However, on possible confounding variables that are included in the model, we will use multiple imputation if needed to retain the sample size of n=88 in the analysis. The models used in the imputation will include the remaining confounders with measures as predictors. Reviewer 1, question 3: Finally, although the choice of exercises for the protocol aligns well with common impairments following THA, it is possible the few specific exercises and proposed dosing may not yield the expected changes in gait speed without the inclusion of gait-specific training. Response: The type of exercise and the dose reflects our current practice which we want to evaluate. To our knowledge this is not a smaller exercise dose compared to clinical practice many places. It is still unclear if the prescribed exercises, and exercise dose, is sufficient to improve gait speed. However, it is more comprehensive than the \"control group\" intervention in our previous study where supervised resistance training was compared to home-based exercise and found comparable in effect (reference 1). If the instructions are followed correctly, the total time-under-tension is 10 min per session, 3-4 times per week equaling 30-40 min/week during 7 weeks. We could have included some gait-specific exercises, but instead we chose, in a pragmatic approach, to comply with the current clinical practice at our institution. All patients were encouraged to be physically active (e.g. by walking) as per the recommendations from the Danish Health and Medicines Authority. They were recommended to perform daily walking with increasing distance during their rehabilitation. Changes made: Text added in the section \"Intervention\": The exercises in the present trial is comparable to the control intervention in a previous study from our department where we compared usual care (home-based exercise using elastic band resistance) to supervised progressive resistance training in machines and found comparable effects (Mikkelsen et al, 2014, ref 1 in response to reviewer). The patients are recommended to perform daily walking with increasing distance during their rehabilitation. They are advised to gradually increase their general activity level after the operation to comply with the recommendations on physical activity from the Danish Health and Medicines Authority (≥30 minutes/day of physical activity with moderate intensity + 20 minutes twice a week of physical activity with high intensity). Furthermore Table 2 has been corrected since we discovered a mistake concerning time-under-tension for each exercise. The corrected values are 2 seconds concentric (instead of 1 in the first version) + 1 second isometric and 2 seconds eccentric, equaling 150 sec/exercise/set. Reference in response to Reviewer 1 Mikkelsen LR, Mechlenburg I, Søballe K, et al.: Effect of early supervised progressive resistance training compared to unsupervised home-based exercise after fast-track total hip replacement applied to patients with preoperative functional limitations. A single-blinded randomised controlled trial. Osteoarthritis Cartilage. 2014 Dec;22(12):2051-8" } ] }, { "id": "50851", "date": "14 Aug 2019", "name": "Kristi Elisabeth Heiberg", "expertise": [ "Reviewer Expertise Physiotherapy and rehabilitation after THA", "TKA", "OA", "Hip fracture", "elderly. Quantitative and qualitative methodology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a generally well-written, interesting, and well-designed study, on an important topic. Both from a therapeutic and health-economic perspective it is important to make the patients with THA self-reliant in their exercise management. We also consider the qualitative part of the proposed trial as an important contribution to understand the patients’ barriers and motivation for performing home-based exercises after THA.\nHowever, we have some comments to the authors:\nIt is pointed out that there is some evidence to indicate that rehabilitation exercise may be superior to no or very little rehabilitation exercise after THA (Introduction section). Can you clarify the construct ‘rehabilitation exercise’?\n\nThe rationale for using elastic band seems a bit unclear in the section of introduction. How can the authors argue for open kinetic chain strength training with elastic band compared to functional task-oriented training? Is there any prior research indicating the effectiveness of strength training in an open kinetic chain?\n\nThe term ‘pragmatic’ should be clarified (page 3).\n\nParticipants: We consider the inclusion criteria very wide and unspecific (18 years and OA?). There seems to be few exclusion criteria. Why were patients with stroke, other neurological diseases, drug abuse etc., not excluded from the study?\n\nAs walking ability is considered the most important function to improve by patients undergoing THA, why were activities of walking not included in the training? What is the rationale and link between elastic band training and improvements in walking?\n\nRegarding dose-response; how do you deal with the eventual problem of registration of little activity with the use of the elastic band and a high general weight-bearing activity, such as walking, without registration? In total, this can be considered as a large dose of activity that is not registered. The ActivePAL is only applied for one week.\n\nThe planned statistical analyses are well described and seem to be adequate.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4966", "date": "14 Oct 2019", "name": "Lone Ramer Mikkelsen", "role": "Author Response", "response": "Response to Reviewer 2 Thank you for your positive evaluation. Below we address your comments one by one. Reviewer 2, question 1: It is pointed out that there is some evidence to indicate that rehabilitation exercise may be superior to no or very little rehabilitation exercise after THA (Introduction section). Can you clarify the construct ‘rehabilitation exercise’? Response: We define rehabilitation exercise as: \"A regimen or plan of physical activities designed and prescribed for specific therapeutic goals. Its purpose is to restore normal musculoskeletal function or to reduce pain caused by diseases or injuries.\"This definition is synonymous to the MESH term \"Exercise therapy\" as defined in the PubMed MeSH database.Changes made: Definition added in the section \"Intervention\". Reviewer 2, question 2: The rationale for using elastic band seems a bit unclear in the section of introduction. How can the authors argue for open kinetic chain strength training with elastic band compared to functional task-oriented training? Is there any prior research indicating the effectiveness of strength training in an open kinetic chain?Response: The construct functional task-oriented training is difficult to define. To our knowledge, there is currently not evidence to support a specific type of exercise after THA as more efficient than others, as we mention in the introduction: \"It has also been difficult to demonstrate clear superiority with relevant effect size of one type of rehabilitation exercise over another for performance-based or self-reported function outcomes 8, 9\". In a previous study, we have shown that an intervention with open kinetic chain exercises using elastic band (more or less similar elastic band exercises as in the present study) was comparable to closed kinetic chain exercise in strength training machines (reference 1). In that study both groups was also advised to be physically active (e.g. by walking). In the current study we also include the task-oriented, closed kinetic chain exercise \"sit to stand\" as well as advise to perform daily gait training.Changes made: Text added in the section \"Intervention\": The exercises in the present trial is comparable to the control intervention in a previous study from our department where we compared usual care (home-based exercise using elastic band resistance) to supervised progressive resistance training in machines and found comparable effects (Mikkelsen et al, 2014, ref 1 in response to reviewer). The patients are recommended to perform daily walking with increasing distance during their rehabilitation. Reviewer 2, question 3: The term ‘pragmatic’ should be clarified (page 3).Response: By pragmatic study we mean that the study reflects real life for the involved trial stakeholders. In this study this is for instance reflected by the type and dose of exercise which reflects our current practice.Changes made: Definition added in the section \"Study design\". Reviewer 2, question 4: Participants: We consider the inclusion criteria very wide and unspecific (18 years and OA?). There seems to be few exclusion criteria. Why were patients with stroke, other neurological diseases, drug abuse etc., not excluded from the study?Response: As this study is designed to reflect current practice (pragmatic approach) we used a minimum of exclusion criteria. There is a continuum between explanatory trials (many exclusion criteria, undertaken in an idealized setting, to give the intervention under evaluation its best chance to demonstrate a beneficial effect) and pragmatic trials (undertaken in the “real world” and with usual care and is intended to help support a decision on whether to deliver an intervention). For elaboration see PRagmatic Explanatory Continuum Indicator Summary (PRECIS-2) (https://www.precis-2.org/) and reference 2.No changes madeReviewer 2, question 5: As walking ability is considered the most important function to improve by patients undergoing THA, why were activities of walking not included in the training? What is the rationale and link between elastic band training and improvements in walking?Response: Daily walking and increasing general physical activity is recommended to the participants. The focus on elastic band exercise is based on the documented deficits in muscle strength after THA (references 2 and 4 in the manuscript). The rationale for this focus is that adequate muscle strength is a requisite to re-establish a normal, symmetric gait pattern. For many of the patients this is after years of limping due to pain. In the current study we combine specific strengthening exercise (elastic band) with functional task exercise (sit-to-stand) and functional training through physical activities – primarily walking, but also resumption of activities preferred by the patients which is encouraged. Changes made: Text added in the section \"Intervention\": The patients are recommended to perform daily walking with increasing distance during their rehabilitation. They are advised to gradually increase their general activity level after the operation to comply with the recommendations on physical activity from the Danish Health and Medicines Authority (≥30 minutes/day of physical activity with moderate intensity + 20 minutes twice a week of physical activity with high intensity). Reviewer 2, question 6: Regarding dose-response; how do you deal with the eventual problem of registration of little activity with the use of the elastic band and a high general weight-bearing activity, such as walking, without registration? In total, this can be considered as a large dose of activity that is not registered. The ActivePAL is only applied for one week. Response: We agree that it is crucial to measure not only the patients' effort during the specific exercises, but also their other physical activities. This is why we include the ActivPal measurement as well as the exercise diary where the patients also register their physical activities. It would be ideal to have objectively measured activity data from the entire intervention period; however that would be very time consuming for both patients and researchers. We consider the physical activity as a potential confounder on the association between exercise dose and gait speed. We can not guarantee that the one week with activPal measurements is representative for the entire intervention period. However, 7 days is a typical time frame when measuring activity level and (ref 40 in manuscript). We could have repeated the measurement on ActivPal in the end or middle of the intervention. However, it would require an extra effort by both patients and staff because the patients are not at the hospital during that period. We already ask the patients to collect a lot of data using the Bandcizer at every exercise session as well as the ActivPal the first week. All things considered, we decided that one week of objectively measured activity as a proxy for the general activity level during the intervention period should be sufficient. No changes made. References in response to Reviewer 2 Mikkelsen LR, Mechlenburg I, Søballe K, et al.: Effect of early supervised progressive resistance training compared to unsupervised home-based exercise after fast-track total hip replacement applied to patients with preoperative functional limitations. A single-blinded randomised controlled trial. Osteoarthritis Cartilage. 2014 Dec;22(12):2051-8 Loudon K, Treweek S, Sullivan F, et al.: The PRECIS-2 tool: designing trials that are fit for purpose. BMJ. 2015 May 8;350:h2147." } ] }, { "id": "50849", "date": "30 Aug 2019", "name": "Sjoukje Bouma", "expertise": [ "Reviewer Expertise Orthopedics", "physical activity." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well-written and interesting study protocol focusing on the effectiveness of home-based rehabilitation following total hip arthroplasty. Specific strengths of the protocol are the very detailed description of the study design and the content of the home-based exercise intervention in particular, the objectively quantified exercise dose and the combination of quantitative and qualitative methods which will be used to evaluate the home-based exercise intervention. However, a few items were encountered while reading the manuscript which the authors might reflect upon:\nThe different aspects of the home-based exercise intervention are described in great detail (Table 2), which enhances the understanding and replicability of the intervention. However, it is not entirely clear why these specific four strengthening exercises (hip abduction, flexion, extension with elastic band resistance and sit-to-stand) and the intervention duration of 7 weeks were chosen. Please elaborate on the rationale for these choices regarding the design on the home-based exercise intervention (e.g. based on guidelines or recommendations?).\n\nOn page 4, it is stated that participants will be advised to comply with the recommendations on physical activity from the Danish Health and Medicines Authority. Could you give a (short) description of these recommendations?\n\nOn page 4, it is described that all participants will receive a thorough instruction in the strengthening exercises at the baseline measurement and that face-to-face meetings among physiotherapists will be held to reinforce similar treatment administration. Will patients also be monitored during the intervention period to check whether they are still performing the strengthening exercises according to the received instructions?\n\nOutcome measures (1): 24-hour physical activity level will only be determined at the start of the intervention. Why will this outcome measure not be repeated at the end of the intervention as well?\n\nOutcome measures (2): in contrast to hip abductor and hip flexor muscle strength, hip extensor muscle strength is not specified as secondary outcome measure. However, hip extensor muscle strength is one of the four strengthening exercises of the interventions. Could you clarify why this will not be evaluated as secondary outcome?\n\nSelf-efficacy (1): to measure self-efficacy, the general self-efficacy scale will be used. However, it has been stated that the concept of self-efficacy is most useful when it is measured for a particular behavior in a specific context (e.g. postoperative rehabilitation following total hip arthroplasty) (Bandura, 1997)1. Could you elaborate on the decision to include a general self-efficacy scale instead of a more context-specific self-efficacy scale (e.g. The Self-Efficacy For Rehabilitation Outcome Scale, Waldrop et al. 2001)2?\n\nSelf-efficacy (2): Table 1 shows that self-efficacy will be measured both prior to surgery (admission) and 3 weeks after surgery (baseline measurement). Literature indicates that short-term postoperative self-efficacy seems to be a better predictor of long-term outcome following total joint arthroplasty than preoperative self-efficacy (Scheek et al. 2007)3. You seem to incorporate this finding by only including the postoperative self-efficacy as possible confounding variable in the statistical analyses. What will be the additional value of including the preoperative self-efficacy measurement in your study design?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "4967", "date": "14 Oct 2019", "name": "Lone Ramer Mikkelsen", "role": "Author Response", "response": "Response to Reviewer 3 Thank you for your positive evaluation. Below we address your comments one by one. Reviewer 3, question 1: The different aspects of the home-based exercise intervention are described in great detail (Table 2), which enhances the understanding and replicability of the intervention. However, it is not entirely clear why these specific four strengthening exercises (hip abduction, flexion, extension with elastic band resistance and sit-to-stand) and the intervention duration of 7 weeks were chosen. Please elaborate on the rationale for these choices regarding the design on the home-based exercise intervention (e.g. based on guidelines or recommendations?). Response: The type of exercise and the dose reflects our current practice which we want to evaluate (pragmatic approach). To our knowledge, there is currently not evidence to support a specific type of exercise after THA as more efficient than others, as we mention in the introduction: \"It has also been difficult to demonstrate clear superiority with relevant effect size of one type of rehabilitation exercise over another for performance-based or self-reported function outcomes 8, 9\". In a previous study, we have shown that an intervention with strengthening exercises using elastic band (more or less similar exercises to the present study) was comparable to supervised exercise in strength training machines (reference 1). The focus on strengthening exercise is based on the documented deficits in muscle strength after THA (references 2 and 4 in the manuscript). The rationale for this focus is also that adequate muscle strength is a requisite to re-establish a normal, symmetric gait pattern. Changes made. Text added in the section \"Intervention\": The exercises in the present trial are comparable to the control intervention in a previous study from our department where we compared usual care (home-based exercise using elastic band resistance) to supervised progressive resistance training in machines and found comparable effects (Mikkelsen et al, 2014, ref 1 in response to reviewer). Reviewer 3, question 2: On page 4, it is stated that participants will be advised to comply with the recommendations on physical activity from the Danish Health and Medicines Authority. Could you give a (short) description of these recommendations? Response: We agree and have added a description. Changes made: Text added in the section \"Intervention\": (≥30 minutes/day of physical activity with moderate intensity + 20 minutes twice a week of physical activity with high intensity) Reviewer 3, question 3: On page 4, it is described that all participants will receive a thorough instruction in the strengthening exercises at the baseline measurement and that face-to-face meetings among physiotherapists will be held to reinforce similar treatment administration. Will patients also be monitored during the intervention period to check whether they are still performing the strengthening exercises according to the received instructions? Response: We only monitor on compliance concerning exercise dose (the Bandcizer data and the exercise diary). We have no monitoring of movement quality in the exercise. This could be relevant from a clinical point of view, however it would be difficult to standardize for use in research. No changes made Reviewer 3, question 4: Outcome measures (1): 24-hour physical activity level will only be determined at the start of the intervention. Why will this outcome measure not be repeated at the end of the intervention as well? Response: It would be ideal to have objectively measured activity data from the entire intervention period; however that would be very time consuming for both patients and researchers. We consider physical activity as a potential confounder on the association between exercise dose and gait speed. We can not guarantee that the one week with activPal measurements is representative for the entire intervention period. However, 7 days is a typical time frame when measuring activity level and (reference 2). We could have repeated the measurement on ActivPal in the end or middle of the intervention. However, it would require an extra effort by both patients and staff because the patients are not at the hospital during that period. We already ask the patients to collect a lot of data when using the Bandcizer at every exercise session as well as the ActivPal the first week. All things considered, we decided that one week of objectively measured activity, as a proxy for the general activity level during the intervention period, should be sufficient. No changes made Reviewer 3, question 5: Outcome measures (2): in contrast to hip abductor and hip flexor muscle strength, hip extensor muscle strength is not specified as secondary outcome measure. However, hip extensor muscle strength is one of the four strengthening exercises of the interventions. Could you clarify why this will not be evaluated as secondary outcome? Response: Patients after total hip replacement are often limited in their range of motion in hip extension impeding the performance of hip extensions strength test in prone position. We have not been able to find a valid way to measure hip extension strength with handheld dynamometer in these patients which is why we chose to focus solely on hip abduction and flexion. No changes made Reviewer 3, question 6: Self-efficacy (1): to measure self-efficacy, the general self-efficacy scale will be used. However, it has been stated that the concept of self-efficacy is most useful when it is measured for a particular behavior in a specific context (e.g. postoperative rehabilitation following total hip arthroplasty) (Bandura, 1997)1. Could you elaborate on the decision to include a general self-efficacy scale instead of a more context-specific self-efficacy scale (e.g. The Self-Efficacy For Rehabilitation Outcome Scale, Waldrop et al. 2001)2? Response: We agree that a context-specific self-efficacy scale would have been preferable. However, we have not been able to identify a Danish version of the The Self-Efficacy For Rehabilitation Outcome Scale, thus it would require a translation process prior to initiating the study. An advantage of using the general self-efficacy scale is to be able to compare our findings to previous studies on THA patients where that scale has often been used (e.g. reference 3-5). No changes made Reviewer 3, question 7: Self-efficacy (2): Table 1 shows that self-efficacy will be measured both prior to surgery (admission) and 3 weeks after surgery (baseline measurement). Literature indicates that short-term postoperative self-efficacy seems to be a better predictor of long-term outcome following total joint arthroplasty than preoperative self-efficacy (Scheek et al. 2007)3. You seem to incorporate this finding by only including the postoperative self-efficacy as possible confounding variable in the statistical analyses. What will be the additional value of including the preoperative self-efficacy measurement in your study design? Response: Preoperative self-efficacy is only included as a descriptive variable and not included in the analysis. The argument for this is, exactly as stated in your comment, that post-operative self-efficacy seems to be a better predictor than preoperative measurement.The preoperative measurement will increase the possibility to compare the study population to other studies. Also it will give a descriptive indication to whether self-efficacy is a constant variable or if it changes from pre- to postoperative in our sample of patients. No changes made References in response to Reviewer 3 Mikkelsen LR, Mechlenburg I, Søballe K, et al.: Effect of early supervised progressive resistance training compared to unsupervised home-based exercise after fast-track total hip replacement applied to patients with preoperative functional limitations. A single-blinded randomised controlled trial. Osteoarthritis Cartilage. 2014 Dec;22(12):2051-8 Migueles JH, Cadenas-Sanchez C, Ekelund U, et al.: Accelerometer Data Collection and Processing Criteria to Assess Physical Activity and Other Outcomes: A Systematic Review and Practical Considerations. Sports Med. 2017;47(9):1821–1845. Brembo E, Kapstad H, Van Dulmen S, et al.: Role of self-efficacy and social support in short-term recovery after total hip replacement: a prospective cohort study. Health Qual Life Outcomes. 2017 Apr 11;15(1):68. Olsson LE, Hansson E, Ekman I. Evaluation of person-centred care after hip replacement-a controlled before and after study on the effects of fear of movement and self-efficacy compared to standard care. BMC Nurs. 2016 Sep 9;15(1):53. Jørgensen LB, Mikkelsen LR, Noe BB, et al.: The psychosocial effect of web-based information in fast-track surgery. Health Informatics J. 2017 Dec;23(4):304-318." } ] } ]	1	https://f1000research.com/articles/8-965
https://f1000research.com/articles/8-1749/v1	14 Oct 19	{ "type": "Software Tool Article", "title": "Creating and sharing reproducible research code the workflowr way", "authors": [ "John D. Blischak", "Peter Carbonetto", "Matthew Stephens", "Peter Carbonetto", "Matthew Stephens" ], "abstract": "Making scientific analyses reproducible, well documented, and easily shareable is crucial to maximizing their impact and ensuring that others can build on them. However, accomplishing these goals is not easy, requiring careful attention to organization, workflow, and familiarity with tools that are not a regular part of every scientist's toolbox. We have developed an R package, workflowr, to help all scientists, regardless of background, overcome these challenges. Workflowr aims to instill a particular \"workflow\" — a sequence of steps to be repeated and integrated into research practice — that helps make projects more reproducible and accessible.This workflow integrates four key elements: (1) version control (via Git); (2) literate programming (via R Markdown); (3) automatic checks and safeguards that improve code reproducibility; and (4) sharing code and results via a browsable website. These features exploit powerful existing tools, whose mastery would take considerable study. However, the workflowr interface is simple enough that novice users can quickly enjoy its many benefits. By simply following the workflowr \"workflow\", R users can create projects whose results, figures, and development history are easily accessible on a static website — thereby conveniently shareable with collaborators by sending them a URL — and accompanied by source code and reproducibility safeguards. The workflowr R package is open source and available on CRAN, with full documentation and source code available at https://github.com/jdblischak/workflowr.", "keywords": [ "reproducibility", "open science", "workflow", "R", "interactive programming", "literate programming", "version control" ], "content": "Introduction\n\nA central tenet of the scientific method is that results should be independently verifiable — and, ideally, extendable — by other researchers. As computational methods play an increasing role in many disciplines, key scientific results are often produced by computer code. Verifying and extending such results requires that the code be “reproducible”; that is, it can be accessed and run, with outputs that can be corroborated against published results1–9. Unfortunately, this ideal is not usually achieved in practice; most scientific articles do not come with code that can reproduce their results10–13.\n\nThere are many barriers to sharing reproducible code and corresponding computational results14. One barrier is simply that keeping code and results sufficiently organized and documented is difficult — it is burdensome even for experienced programmers who are well-trained in relevant computational tools such as version control (discussed later), and even harder for the many domain scientists who write code with little formal training in computing and informatics15. Further, modern interactive computer environments (e.g., R, Python), while greatly enhancing code development16, also make it easier to create results that are irreducible. For example, it is all too easy to run interactive code without recording or controlling the seed of a pseudo-random number generator, or generate results in a “contaminated” environment that contains objects whose values are critical but unrecorded. Both these issues can lead to results that are difficult or impossible to reproduce. Finally, even when analysts produce code that is reproducible in principle, sharing it in a way that makes it easy for others to retrieve and use (e.g., via GitHub or Bitbucket) involves technologies that many scientists are not familiar with13,17.\n\nIn light of this, there is a pressing need for easy-to-use tools to help analysts maintain reproducible code, document progress, and disseminate code and results to collaborators and to the scientific community. We have developed an open source R18 package, workflowr, to address this need. The workflowr package aims to instill a particular “workflow” — a sequence of steps to be repeated and integrated into research practice — that helps make projects more reproducible and accessible. To achieve this, workflowr integrates four key features that facilitate reproducible code development: (1) version control19,20; (2) literate programming21; (3) automatic checks and safeguards that improve code reproducibility; and (4) sharing code and results via a browsable website. These features exploit powerful existing tools, whose mastery would take considerable study. However, the workflowr interface is designed to be simple so that learning it does not become another barrier in itself and novice users can quickly enjoy its many benefits. By simply following the workflowr “workflow”, R users can create projects whose results and figures are easily accessible on a static website — thereby conveniently shareable with collaborators by sending them a URL — and accompanied by source code and reproducibility safeguards. The Web-based interface, updated with version control, also makes it easy to navigate through different parts of the project and browse the project history, including previous versions of figures and results, and the code used to produce them. By using workflowr, all this can be achieved with minimal experience in version control systems and Web technologies.\n\nThe workflowr package builds on several software technologies and R packages, without which this work would have been impossible. Workflowr builds on the invaluable R Markdown literate programming system implemented in knitr22,23 and rmarkdown21,24, which in turn build on pandoc, the “Markdown” markup language, and various Web technologies such as Cascading Style Sheets and Bootstrap25. Several popular R packages extend knitr and rmarkdown for specific aims such as writing blogs (blogdown26), monographs (bookdown27), and software documentation (pkgdown28). Analogously, workflowr extends rmarkdown with additional features such as the reproducibility safeguards, and adds integration with the version control system Git19,20. Git was designed to support large-scale, distributed software development, but in workflowr it serves a different purpose: to record, and provide access to, the development history of a project. Workflowr also uses another feature of Git, “remotes”, to enable collaborative project development across multiple locations, and to help users create browsable projects via integration with popular online services such as GitHub Pages and GitLab Pages. These features are implemented using the R package git2r29, which provides an interface to the libgit2 C library. Finally, beyond extending the R programming language, workflowr is also integrated with the popular RStudio interactive development environment30.\n\nIn addition to the tools upon which workflowr directly builds, there are many other related tools that directly or indirectly advance open and reproducible data analysis. A comprehensive review of such tools is beyond the scope of this article, but we note that many of these tools are complementary to workflowr in that they tackle aspects of reproducibility that workflowr currently leaves to the user, such as management and deployment of computational environments and dependencies (e.g., conda, Homebrew, Singularity, Docker, Kubernetes, packrat31, checkpoint32, switchr33, RSuite34); development and management of computational pipelines (e.g., GNU Make, Snakemake35, drake36); management and archiving of data objects (e.g., archivist37, Dryad38, Zenodo); and distribution of open source software (e.g., CRAN, Bioconductor39, Bioconda40). Most of these tools or services could be used in combination with workflowr. There are additional, ambitious efforts to develop cloud-based services that come with many computational reproducibility features (e.g., Code Ocean, Binder, Gigantum, The Whole Tale). Many of these platforms manage individual projects as Git repositories, so workflowr could, in principle, be installed and used on these platforms, possibly to enhance their existing features. Other R packages with utilities to facilitate reproducibility that could complement workflowr include ProjectTemplate41, rrtools42, and usethis43, as well as many of the R packages listed in the “Reproducible Research” CRAN Task View.\n\nOf the available software tools facilitating reproducible research, perhaps the closest in scope to workflowr are the R package adapr44 and the Python-based toolkit Sumatra45. Like workflowr, both adapr and Sumatra use version control to maintain a project development history. Unlike workflowr, both place considerable emphasis on managing and documenting dependencies (software and data), whereas workflowr only records this information. In contrast, workflowr places more emphasis on literate programming — the publishing of text and code in a readable form — and more closely integrates other features such as tracking project development history via Git with literate programming.\n\nThe workflowr R package is available from CRAN and GitHub, and is distributed under the flexible open source MIT license (see Software availability). The R package and its dependencies are straightforward to install while being highly customizable for more dedicated users. Extensive documentation, tutorials, and user support can be found at the GitHub site. In the remainder of this article, we describe the workflowr interface, explain its design, and give examples illustrating how workflowr is used in practice.\n\n\nOperation\n\nIn this section, we give an overview of workflowr’s main features from a user’s perspective. For step-by-step instructions on starting a workflowr project, see the “Getting started with workflowr” vignette.\n\nFor basic usage, only five functions are needed (summarized here, and described in more detail later):\n\nwflow_start() initializes a new project, including the template directory structure (Figure 1A);\n\nwflow_build() renders webpages from R Markdown (Rmd) analysis files, with reproducibility safeguards in place;\n\nwflow_publish() renders the webpages and updates the project development history—it commits the code, calls wflow_build(), then commits the webpages;\n\nwflow_status() reports the status of the project files; and\n\nwflow_git_push() uploads the results from the user’s local repository to a website hosting service.\n\nA) The function wflow_start() populates a project directory with all the files and subdirectories (shown in red) needed to begin a workflowr project. This default directory structure encourages users to organize their files as the project progresses—as the project develops, additional Rmd files may be organized in the \"analyses\" folder. This is only a suggested structure; users can change the names of most files and directories. Required files are shown in boldface. B) All results are organized into a website (all HTML files generated by workflowr are automatically stored in docs/). The use of hyperlinks allows for efficient access to the results. The screenshots above illustrate how a workflowr website can be navigated. Clicking a hyperlink in the main page, index.html, (1) navigates the browser to a webpage containing some results, visualize.html; clicking on the “Home” hyperlink (2) in the navigation bar brings the browser back to the main page. For larger projects, the navigation bar can be used to quickly access different sections of a project.\n\nThe primary output of workflowr is a project website for browsing the results generated by the Rmd analysis files (Figure 1B). The use of websites to organize information is, of course, now widespread. Nonetheless, we believe they are under-utilized for organizing the results of scientific projects. In particular, hypertext provides an ideal way to connect different analyses that have been performed, and to provide easy access to relevant external data (e.g., related work or helpful background information); see Figure 1B and Use cases below.\n\nThe function wflow_start() facilitates project organization by populating a directory with suggested subdirectories, scripts, and configuration files for a data analysis project (Figure 1A). The subdirectories created by default are analysis/, where the Rmd analysis files are stored; docs/, which stores the website HTML files; code/, which is intended for longer-running scripts, compiled code (e.g., C++) and other source code supporting the data analyses; data/, for storing raw data files; and output/, for saving processed data files and other outputs generated by the scripts and analyses. This setup is flexible and configurable; only two of the directories, analysis/ and docs/, are required, and both can be renamed later.\n\nIn addition to creating a default file structure for a data analysis project, wflow_start() also initializes the project development history: it creates a Git repository, and commits the files and directories to this repository. This is all done behind the scenes so no familiarity with Git is needed. We give more details about the Git repository in the Implementation section below.\n\nIn some cases, a user will have an existing project (with files that may or may not be tracked by Git), and would like to incorporate workflowr into the project — wflow_start() also easily accommodates this scenario, with additional arguments to control how the workflowr files are added to the existing project. See the package vignette, “Migrating an existing project to use workflowr,” for more details; it can be accessed by running vignette(\"wflow-03-migrating\") after loading the workflowr package in R.\n\nFinally, wflow_start() changes R’s working directory to the root of the project directory. Although this is a simple step, it is important for correctly resolving file paths. Forgetting to change the working directory is a very common source of errors in data analyses.\n\nIn a workflowr project, analyses are performed using the R Markdown literate programming system21. The user develops their R code inside Rmd files in the analysis/ directory, then calls wflow_build(), which runs the code and renders the results as HTML files in the docs/ directory. The wflow_build() function extends the render_site() command from the rmarkdown package with several reproducibility safeguards:\n\n1. It creates a clean R session for executing the code. This is critical for reproducibility—results should not depend on the current state of the user’s R environment, and all objects necessary to run the code should be defined in the code or loaded by packages.\n\n2. It automatically sets the working directory in a consistent manner (the exact setting is controlled by a configuration file; see Implementation below). This prevents one of the most common failures to reproduce in R—not setting the working directory before running the R script, resulting in incorrectly resolved relative file paths.\n\n3. It sets a seed for the pseudorandom number generator before executing the code. This ensures that analyses that use random numbers always return the same result.\n\n4. It records information about the computing environment, including the operating system, the version of R used, and the packages that were used to produce the results.\n\nFinally, wflow_build() summarizes the results of these reproducibility safeguards in a report at the top of the webpage, along with additional “reproducibility checks”, which alert the user to potential reproducibility issues, such as changes that were not committed to the project development history, and the use of (non-reproducible) absolute file paths (Figure 2).\n\n(A) A button is added to the top of each webpage. Clicking on the button (1) reveals the full reproducibility report with multiple tabs. If any of the reproducibility checks have failed, a red warning symbol (!) is shown. Clicking on the \"Checks\" tab (2) summarizes the reproducibility checks, with icons next to each check indicating a pass or failure. Clicking on an individual item (3) reveals a more detailed description of the reproducibility check, with an explanation of why it passed or failed. In (A), the Rmd file contains changes that have not yet been committed, so one of the reproducibility checks has failed (uncommitted changes are acceptable during active development, but not acceptable when results are published). In this case, the recommendation is given to run wflow_publish() to fix the issue. (B) If all the workflowr reproducibility checks pass, the workflowr button shows a green check mark (✔), and clicking an individual item in the reproducibility report (3) gives more detail on the reproducibility check.\n\nAs a project progresses, many versions of the results will be generated as results are scrutinized, analyses are revised, errors are corrected, and new data are considered. Keeping track of a project’s evolution is important for documenting progress and retracing the development of the analyses. This is sometimes done without version control tools by copying code and results whenever an important change is made. This typically results in a large collection of files with names such as results-v2-final_final.pdf or anova_analyses_before_adding_new_samples.R. This approach is tedious and error-prone, and makes it difficult to communicate changes to collaborators.\n\nThe version control system, Git, provides a more systematic and reliable way to keep track of a project’s development history. However, Git was designed to manage source code for large-scale software projects, and using it for scientific analyses brings some specific challenges. The relative complexity of Git provides a high barrier to entry, discouraging many researchers from adopting it for their projects. And Git is not ideally suited to data analysis projects where one wants to coordinate the tracking of source code, data, and the results generated by the code and data. Using Git commands to identify the version of the code that was used to generate a result can be non-trivial.\n\nThe wflow_publish() function is designed to address these challenges: it takes the steps necessary to coordinate tracking of code and results, and reduces these steps to calling a single, easy-to-use function. The command performs three steps, detailed in Figure 3. These steps are designed to ensure that each new collection of results added to the project development history has been produced by a unique and identifiable version of an Rmd analysis file.\n\nThe function performs a three-step procedure to store the code and results in a project development history, and ensure that the results HTML file is always created from a unique and identifiable versioned Rmd analysis file. (1) The first step commits the changes to the Rmd analysis file. (2) The second step builds the results HTML file from the Rmd file. These two steps ensure that the results were generated from the committed version of the Rmd file. Furthermore, the unique version of the Git repository is inserted directly into the HTML file so that the source code used to generate the results is easily identified and accessed. If the code generates an error, the entire process is aborted and the previous commit made in the first step is undone. (3) The results HTML file, as well as any related figure files, are committed to the Git repository. Thus, the versioning of Rmd analysis files and corresponding HTML results files are coordinated whenever wflow_publish() is used.\n\nEven experienced Git users will benefit from using wflow_publish(). Besides the convenience of a single function, wflow_publish() ensures that:\n\n1. Every commit to an (Rmd) analysis file is associated with a commit to the results file generated by that analysis file.\n\n2. An analysis file is only published and committed if it runs successfully; on failure, wflow_publish() aborts, and neither code nor results are committed to the Git repository (R code that does not work can still be committed to a workflowr project via other methods, e.g., directly using Git, but it will not be associated with a committed results file).\n\nPublishing an analysis is not necessarily final — after calling wflow_publish(), the analysis can be repeatedly updated and re-published using wflow_publish(). Each time wflow_publish() succeeds in committing a new version of the code and results, a link to previously published versions of the analysis are embedded in the webpage so that readers can easily access previous versions and compare with the latest results.\n\nAs a workflowr project grows, it is important to be able to get an overview of the project’s status and identify files that may need attention. This functionality is provided by the wflow_status() command, which gives the status of each Rmd file in the project — either “scratch”, “unpublished”, or “published”, whose definitions are given in Figure 4. The “published” Rmd files, which are those that have been run through wflow_publish(), are further recorded as either “up-to-date” or “modified” depending on whether the Rmd file has been modified since wflow_publish() was run. The wflow_status() function highlights all Rmd files in the “scratch”, “unpublished” or “modified” states, and suggests suitable next steps.\n\nThe function wflow_status() assigns a state to each Rmd file in the workflowr project based on its status in the Git repository’s working tree, and based on the Git status of the associated HTML results file.\n\nThe version-controlled website created by workflowr is self-contained, so it can be hosted by most Web servers with little effort. Once the website is available online, the code and results can be shared with collaborators and colleagues by providing them with the website’s URL. Similarly, the workflowr repository can also serve as a companion resource for a manuscript by referencing the website URL in the paper.\n\nSince a workflowr project is also a Git repository, the most convenient way to make the website available online is to use a Git hosting service. The workflowr package includes functions wflow_use_github() and wflow_use_gitlab() to simplify the setup process on two of the most widely used services, GitHub and GitLab. Once a user has created a Git repository on one of these online platforms, the project can be easily uploaded using wflow_git_push() (there is also a companion function wflow_git_pull(), which is used when multiple people are collaborating on a workflowr project, or when a project is being updated from multiple computers).\n\nThe results files in a workflowr website include links to past versions of analysis and figures, making it easy for collaborators to benefit from the versioning of analyses without knowing anything about Git. For example, if a collaborator wants to download a previous version of a figure generated several months ago, this can be done by navigating the links on the workflowr website.\n\nThe workflowr package is available on CRAN. It works with R versions 2.3.5 or later, and can be installed on any major platform that is supported by R (Linux, macOS, Windows). It is regularly tested on all major operating systems via several continuous integration services (AppVeyor, CircleCI, Travis CI). It is also regularly tested by CRAN using machines running Debian GNU/Linux, Fedora, macOS, Solaris, and Windows.\n\nBecause workflowr uses the rmarkdown package to build the HTML pages, it requires the document conversion software pandoc to be installed. The easiest way for R users to install pandoc is to install RStudio.\n\nInstalling Git is not required because the R package dependency git2r includes libgit2, a minimal Git implementation (nonetheless, installing Git may be useful for occasional management of the Git repository outside regular workflowr usage).\n\nWorkflowr projects are highly customizable. For example, the look of the webpages can be customized, via options provided by the rmarkdown package, by editing the analysis/_site.yml configuration file. Additional settings specific to workflowr, such as setting the seed for the pseudorandom number generator, or setting the working directory for the Rmd files, can be controlled in the _workflowr.yml file.\n\n\nImplementation\n\nHere we give an overview of the workflowr package implementation. All workflowr commands can be invoked from R (or RStudio) so long as the working directory in R is set to the directory containing a workflowr project, or any subdirectory of a workflowr project (this is similar to how Git commands are invoked). To determine the root directory of a workflowr project from a subdirectory, whenever a command is called from the R console, workflowr uses the rprojroot46 R package to search for the RStudio project file stored at the root of the project (the RStudio project file is a required file, so if this file is deleted, the workflowr commands will not work).\n\nThe function wflow_start() populates the project directory using predefined template files (see Figure 1). It uses the glue47 R package to insert relevant variables, e.g., the name of the project, directly into the newly created files. When wflow_start() is called with git = TRUE (which is the default), a Git repository is created in the project directory, and all newly created or modified files are committed to the repository. If the user has never previously created a Git repository on their computer, they may need to first call wflow_git_config() to configure Git.\n\nThe wflow_build() function generates a responsive website from a collection of Rmd files. Both wflow_build() and wflow_publish() support file patterns, also known as “wildcard expansion”; for example, wflow_build(\"analysis/*.Rmd\") will generate webpages for all the Rmd files in the analysis/ directory.\n\nThe wflow_build() function extends the render_site() function from the rmarkdown package. The render_site() function in turn builds on the Bootstrap framework to create a responsive website with a navigation bar. This rendering step includes downloading and linking to the required CSS and JavaScript files. Many website settings, such as the labels and URLs included in the navigation bar, can be adjusted in the analysis/_site.yml configuration file (these options can also be set individually inside the Rmd files, which will override the default options set in analysis/_site.yml). Like other R packages that extend rmarkdown (e.g., bookdown), workflowr provides a custom site generator in the function wflow_site(), which alters the website generation process. For example, one change to this process is that the generated website files (the HTML, CSS, JavaScript and figures) are moved instead of copied from analysis/ to docs/. This reduces unnecessary duplication of files. Most of workflowr’s key features, including the reproducibility report, are implemented in wflow_html(), which we describe next.\n\nIn the rmarkdown package, the rendering of individual webpages from Rmd files is controlled by a separate function, html_document(). The workflowr package provides an analogous function, wflow_html(). This function also extends html_document(), so all features implemented in rmarkdown (e.g., code chunk folding, generating a table of contents from the section headings) are inherited by wflow_html().\n\nMost of the workflowr content is added as a preprocessing step prior to executing the R code in the Rmd file. To achieve this, wflow_html() copies the original Rmd file to a temporary directory, incorporates the additional content, then executes the code. The content embedded into the Rmd file includes a code chunk that calls set.seed(), a code chunk toward the end of the file that calls sessionInfo(), and inline HTML tags for elements such as the reproducibility report (Figure 2) and links to previous versions of figures. There is also a brief postprocessing step to incorporate additional HTML, CSS, and JavaScript elements needed to display the workflowr elements added in the preprocessing step. This postprocessing is done when pandoc converts the generated markdown to the final webpage.\n\nThe process for embedding links to past versions of files — that is, files added to previous commits in a Git repository — requires some additional explanation. Links to past versions are included only if the user has set up a remote repository hosted by either GitHub or GitLab. Clicking on a link to a past version of an Rmd file (or figure file) in a Web browser will load a webpage displaying the R Markdown source code (or figure file) as it is saved in the given commit. For past versions of the webpages, we use an independent service raw.githack.com, which displays the HTML file in the browser like any other webpage (this is because GitHub and GitLab only show the raw HTML code). These links will point to valid webpages only after the remote repository (on GitHub or GitLab) is updated, e.g., using wflow_git_push(). In the current implementation, when an Rmd file (and its corresponding HTML file) is renamed, the webpage does not include links to past versions prior to renaming. So renaming files will limit the ability to browse the project development history.\n\nThe wflow_html() function allows for considerable customization of the workflowr reproducibility report, and other features. The settings in the analysis/_site.yml configuration file are passed to function html_document() in the rmarkdown package, whereas the settings in _workflowr.yml are read by wflow_html(); see help(wflow_html) for a full details on all workflowr settings that can be customized in this file. For example, the default function used to record the session information at the bottom of each webpage, sessionInfo(), can be overridden by adding the YAML field sessioninfo (e.g., the function from the devtools48 package could be used instead by setting sessioninfo: devtools::session_info()).\n\nTo execute the code, wflow_build() first creates a new R session to execute the code. This is implemented using the R package callr49.\n\nBy default, the rmarkdown package renders an Rmd file in the directory where the Rmd file is stored; that is, the R working directory is automatically changed to the directory containing the target Rmd file. By default, wflow_html() overrides the behaviour, and instead executes the R code with respect to the root project directory. This default is intended to improve reproducibility by resolving file paths from a consistent reference point. This execution directory can be controlled by the knit_root_dir option, which is set in the _workflowr.yml configuration file. By default, new projects execute the R Markdown code chunks in the root directory. If this setting is not configured, workflowr reverts to the rmarkdown default. It is also possible to have a different knit_root_dir setting for different files, but this is generally not recommended as it will make the code more difficult to follow.\n\nOne of the steps in wflow_publish(), as we have mentioned, is a call to wflow_build(). It also runs Git commands to commit the source code and rendered HTML files (Figure 3). These Git commands are executed behind the scenes. We have also implemented many checks and extensive error handling to make sure that the Git repository and R environment are in an acceptable state for committing the results. When an issue arises, wflow_publish() attempts to detect the issue as early as possible, then it reverts the Git repository to the initial state and, when possible, suggests how to fix the issue. For example, wflow_publish() will stop if any of the files contain conflicts from a previous merge using Git.\n\nThe wflow_status() function checks the status of each Rmd file in the project by comparing the state of the file in the Git repository’s working tree against the Git status of the corresponding HTML file. In Git terminology, a “scratch” Rmd file in a workflowr project is an uncommitted file in a Git repository; “unpublished” means that the Rmd file is committed to the Git repository but the corresponding HTML is not; a \"published\" Rmd file and its HTML file are both committed to the Git repository; and a \"modified\" Rmd file has changes — these changes can be unstaged, staged, or committed — that were made since the last time the corresponding HTML file was committed (Figure 4).\n\nUsing git2r, it is mostly straightforward to determine the status of each file. The only complicated step is determining whether published Rmd files have been modified. If all changes to an Rmd file have been committed to the Git history, an Rmd file is considered “modified” if it has modifying commits that are more recent than commits modifying the corresponding HTML file.\n\nTo use wflow_git_push(), the remote Git repository must first be configured. The user can configure the remotes manually using the git remote subcommand or using wflow_git_remote(). Alternatively, the workflowr package provides two functions, wflow_use_github() and wflow_use_gitlab(), that simplify the creation and configuration of remote repositories hosted on GitHub and GitLab. These two convenience functions also add a navigation bar link with the URL of the remote source code repository. The wflow_use_gitlab() function takes the additional step of activating the GitLab Pages by creating a file .gitlab-ci.yml with the proper configuration (GitHub Pages must be set up manually; there is currently no way to automate this via the GitHub API).\n\n\nUse cases\n\nWorkflowr was officially released on CRAN in April 2018. As of September 2019, it has been downloaded from CRAN over 7,000 times, and it has been adopted by many researchers. The most common use cases are 1) documenting research development and including the project website in the accompanying academic paper, and 2) developing reproducible course materials to share with students. Here we highlight some successful examples.\n\nHuman dermal fibroblast clonality project\n\nhttps://davismcc.github.io/fibroblast-clonality\n\nA workflowr project accompanying a scientific paper on computational methods for decoding the clonal substructures of somatic tissues from DNA sequencing data50. The webpages describe how to reproduce the data processing and analysis, along with the outputs and plots.\n\nCharacterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis\n\nhttps://github.com/jdblischak/fucci-seq\n\nA workflowr project supporting a paper on measuring cell cycle phase and gene expression levels in human induced pluripotent stem cells51. The repository contains the processed data and the code implementing the analyses. The full results can be browsed on the website.\n\nFlexible statistical methods for estimating and testing effects in genomic studies with multiple conditions\n\nhttps://github.com/stephenslab/gtexresults\n\nA workflowr project containing the code and data used to produce the results from the GTEx data set that were presented in Urbut et al.52.\n\nInvestigations on \"truncated adaptive shrinkage\"\n\nhttps://github.com/LSun/truncash\n\nA workflowr project created by a Ph.D. student created to keep track of his investigations into controlling false discoveries in the presence of correlation and heteroskedastic noise. This repository illustrates the use of workflowr as a scientific notebook — the webpages contain written notes, mathematical equations, source code, and the outputs generated from running the code.\n\nStanford STATS 110\n\nhttps://xiangzhu.github.io/stanford-stats110\n\nA workflowr website for a statistics course taught at Stanford. The website includes working R examples, homework, the course syllabus, and other course materials.\n\nSingle-cell RNA-seq workshop\n\nhttps://github.com/crazyhottommy/scRNA-seq-workshop-Fall-2019\n\nA workflowr website for a workshop on analysis of single-cell RNA-seq data offered by the Harvard Faculty of Arts and Sciences Informatics group as part of a two-week long bioinformatics course. The R examples demonstrate how to use several bioinformatics packages such as Seurat and msigdbr to prepare and analyze single-cell RNA-seq data sets.\n\nIntroduction to GIS in R\n\nhttps://github.com/annakrystalli/intro-r-gis\n\nA workflowr website for a workshop given at the 2018 Evolutionary Biology Conference. The website includes working R demonstrations, setup instructions, and exercises.\n\n\nSummary\n\nOur main aim in developing workflowr is to lower barriers to open and reproducible code. Workflowr provides a core set of commands that can be easily integrated into research practice, and combined with other tools, to make projects more accessible and reproducible. The R package is straightforward to install, easy to learn, and highly customizable.\n\nSince the first official release of workflowr (version 1.0.1, released in April 2018), the core functionality has remained intact, and we expect it to remain that way. The core features of workflowr have been carefully tested and revised, in large part thanks to feedback and issue reports from the user community. Our next aim is to implement several enhancements, including:\n\nCreate a centralized workflowr project website to make it easier for researchers to share and discover workflowr projects.\n\nProvide additional functions to simplify website hosting on other popular platforms such as Netlify and Heroku.\n\nAs workflowr projects grow, it becomes increasingly important to document not only the evolution of the code and results over time, but also how the results interrelate with one another. Therefore, we aim to implement syntax that allows file dependencies to be recorded in the Rmd files, and incorporate checking of dependencies as part of the workflowr reproducibility safeguards.\n\nAs workflowr has been used in a variety of settings, we have also uncovered some limitations. Here we report on some of the more common issues that have arisen.\n\nOne limitation is that Git — hence workflowr — is not well suited to tracking very large files. Therefore, large data files must be left out of the project development history, which reduces reproducibility. One possible workaround is to use Git LFS (Large File Storage) or related tools that allow large data files to be tracked and stored remotely inside a Git repository. This, however, requires considerable expertise to install and configure Git LFS, so it is not a satisfactory solution for some workflowr users. Also note that sensitive or secure data can be added to a workflowr project so long as the storage and access practices meet the data security requirements (workflowr has options to simplify creation and management of projects with security requirements).\n\nSince workflowr builds on Git, users who already have experience with Git can use Git directly to manage their workflowr projects. This provides additional flexibility, but is not without risk; for example, Git commands such as git reset can be used to alter the project development history, and has the potential to break workflowr.\n\nFinally, workflowr records information about the computing environment used to generate the results, but it does not provide any facilities for replicating the environment. This is an area with many recent software advances — there are many widely used tools for managing and deploying computational environments, from container technologies such as Docker to package managers such as Anaconda and packrat. We view these tools as being complementary to workflowr, and one future direction would be to develop easy-to-use functions that configure such tools for use in a workflowr project.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: https://cran.r-project.org/package=workflowr\n\nSource code available from: https://github.com/jdblischak/workflowr\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.324180153\n\nLicense: MIT", "appendix": "Acknowledgments\n\nWe thank the workflowr contributors for helping improve the package. We are also grateful for the many workflowr users for testing the package and providing feedback—thanks especially to Lei Sun, Xiang Zhu, Wei Wang, and many other members of the Stephens lab, past and present. 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Reference Source\n\nMcCarthy DJ, Rostom R, Huang Y, et al.: Cardelino: integrating whole exomes and single-cell transcriptomes to reveal phenotypic impact of somatic variants. bioRxiv. 2018. Publisher Full Text\n\nHsiao CJ, Tung P, Blischak JD, et al.: Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis. bioRxiv. 2019. Publisher Full Text\n\nUrbut SM, Wang G, Carbonetto P, et al.: Flexible statistical methods for estimating and testing effects in genomic studies with multiple conditions. Nat Genet. 2019; 51(1): 187–195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlischak J, Carbonetto P, Li J, et al.: jdblischak/workflowr: workflowr 1.4.0. 2019. Reference Source" }	[ { "id": "55176", "date": "31 Oct 2019", "name": "Peter F. Hickey", "expertise": [ "Reviewer Expertise Statistics", "bioinformatics", "R programming" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe workflowr package, available from CRAN for 1.5 years, has already demonstrated itself to be a valuable contribution to improving the reproducibility of scientific analyses. This paper does a thorough job setting out the rationale, design, and implementation of the workflowr package. It is clear that the authors have spent considerable time thinking about some of the key challenges of this endeavour, learning about best practises, getting feedback from users, and implementing these using 4 key technologies/features:\n\nVersion control.\n\nLiterate programming.\n\nAutomatic checks and safeguards to improve code reproducibility.\n\nSharing code and results via a website.\n\nThe paper is clearly written and I am happy to approve the article in its current form.\nSome minor comments, queries, and corrections are given below:\nFigure caption 1: '\"analyses\" folder' is '\"analysis\" folder' in the figure.\n\np5: Re workflowr executing code in a clean session. My impression was that rendering Rmarkdown documents, at least when done by clicking the 'knit' button in RStudio, was already run in a separate process. But I may be mistaken and perhaps this is different from running `rmarkdown::render_site()`?\n\nRegarding published sites. Not all scientific analyses can be made public, particularly in the early stages. Some discussion of options available for private/protected hosting would be valuable.\n\nI'm a little wary of relying on raw.githack.com for hosting past versions of the webpages. For example, what if the service becomes unavailable? Does or could workflowr support other hosting services?\n\np11: Is the idea of a 'centralized workflowr project website' like that of the homepage https://bookdown.org/ or for an organisation/user to share their personal workflowr projects?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "55119", "date": "04 Nov 2019", "name": "Przemysław Biecek", "expertise": [ "Reviewer Expertise Human-Oriented machine learning", "Human-Centered Artificial Intelligence", "Software engineering" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI like the workflowr package and I like the paper. Nicely written. Reproducibility is a big thing and workflowr lowers the entrance barrier for non technical users. This is a huge benefit. The package has has already some visibility (judging based on GitHub stars) and is being adopted to various applications (based on examples presented in the paper). I recommend to accept the paper.\n\nHere are some comments that authors may consider:\nA. The point that I am missing the most is the comparison against the drake package. These two packages seems to be similar, maybe complementary. Can they be used together? It would be good to show pros and cons/similarities and differences.\n\nB. I like the workflowr package it is a useful tool. What I am missing is the methodology / description of a process / good practices of how the reproducible analysis should look like. This would be very useful for people that look for precise guidelines on how to integrate the workflowr with every day practice. Wet labs researchers are used to \"protocols\" that directly guide step by step what to do during the analysis. Maybe it would be possible to give such protocols for reproducible research with the workflowr package.\n\nJust to give an example, in the Model Development Process (https://arxiv.org/abs/1907.04461) article there is an overview of phases and tasks shared across model development. In which phases the workflowr or similar tools shall be used?\n\nC. Authors have mentioned blogdown and bookdown packages. I think that even a closer match to the reproducibility problem is the package modelDown (see https://joss.theoj.org/papers/10.21105/joss.01444 or GitHub http://github.com/ModelOriented/modelDown).1 The modelDown package takes predictive models and creates a HTML website with information about session info, binary models, training/test data and model explanations. The website is created without any additional effort. ModelDown automates the most boring part of the modeling i.e. model documentation. D. When mentioning tools for archivisation of binary objects, it may be also useful to add the pins package recently developed by RStudio (https://cran.r-project.org/web/packages/pins/index.html). It is more limited than other mentioned packages (do not keep information about meta data) but quickly gains popularity.\n\nE. After the \"Unfortunately, this ideal is not usually achieved in practice; most scientific articles do not come with code that can reproduce their results\" maybe authors could share their thoughts why it is the case. It will be useful to list specific reasons why reproducibility fails. Is it primarily because we do not have proper software, or they software is too complex, or one needs to pay for the proper software, or researchers are not aware of the problem?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "55117", "date": "08 Nov 2019", "name": "Peter Baker", "expertise": [ "Reviewer Expertise Biostatistics", "R", "workflow of data analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce an R package that provides an easy way to set up a workflow for data analysis using R and publish results to a web page.\nThe workflowr package seems to work well and appears to be widely used.\nWhat is relatively novel about this package is that while most workflow tools I have seen in R concentrate on setting up a directory structure, syntax files and producing output and reports from analyses, this package adds both the ability to publish the results to web pages and also to set up a Github or Gitlab project repositories with minimal effort.\nThe article is clear and well written as are the associated vignettes. The underlying functions in the R are also well written but perhaps could employ more error checking and reporting (see below).\nWhile I am unlikely to use this approach myself I think it is an excellent approach for new users since it\nsets up a project skeleton with instructions,\n\nencourages users to document their project and workflow right from the start, and\n\nalso the package provides quite a few helpful vignettes and guides for various scenarios that should be useful for those starting in the area.\n\nHowever, I do have minor reservations with the approach outlined, including\nfor new users, they must not only learn R but also R Markdown which adds an extra level of complexity;\n\nwhile it seems necessary in workflowr, users do not really need to use R Markdown files to produce well documented code (see https://rmarkdown.rstudio.com/articles_report_from_r_script.html) but R Markdown seems better suited to reports and articles;\n\nthe workflow is very R Markdown-centric: experienced users may wish to employ R or other software directly or a build system like Make or drake. While this is relatively straight forward outside of the package, e.g. by adding these to the git repository outside of the package and using employing R directly to update intermediate results the article or documentation do not give any details;\n\nsome functions do not appear to be particularly error-proof for new users, e.g. wflow_git_config will overwrite existing settings without checking or even providing a warning although this may conceivably change in future; and\n\nnew users will undoubtedly run into git merge issues and the version I reviewed (1.4.0) did not seem to cater for such eventualities although this appears to have been addressed according to the change log in the latest version (1.5.0).\n\nMinor comments are:\nI am not sure why only some of the software on page 3 is cited when presumably a reader may benefit from the author's recommendations appropriate to the data analysis workflow area rather than tracking down a general reference for themselves;\n\nwhile it is good that the potential pitfalls of using git directly or using git reset are addressed on page 11, it may also prove useful for readers to address limitations and potential pitfalls in more depth, perhaps not in the article but with reference to other material or vignettes. For example, users at all levels might benefit from knowing where to get help on merge conflicts, whether users with large data sets should consider databases rather than git or whether typical git workflows like branching would work with this package,\n\nit would be nice to see how this package compares to other alternatives like drake but admittedly the scope this article is more an introduction to the workflowr package.\n\nIn summary, this article is clear and well written. The package is an original contribution to the range of software addressing reproducibility and workflow in data analysis projects.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1749
https://f1000research.com/articles/8-1041/v1	10 Jul 19	{ "type": "Systematic Review", "title": "Effects of evidence-based clinical practice guidelines in cardiovascular health care quality improvements: A systematic review", "authors": [ "Anggie Ramírez-Morera", "Mario Tristan", "Juan Carlos Vazquez", "Mario Tristan", "Juan Carlos Vazquez" ], "abstract": "Background: The development of clinical practice guidelines (CPGs) has increasing global growth; however, the certainty of impact on patients and health systems, as well as the magnitude of the impact, is not apparent. The objective of this systematic review was to assess the effectiveness of the application of CPGs for the improvement of the quality of health care in three domains: structure, process and results in the patient for the management of cardiovascular disease. Methods: We followed the methods described by the Cochrane Handbook and present a descriptive analysis because of the high heterogeneity found across the included studies. We searched the Cochrane Central Register of Controlled Trials, MEDLINE and EMBASE databases, as well as the grey literature, between 1990 and June 2016. No language restrictions were applied. Only randomised clinical trials (RCTs) were selected. Three authors independently carried out the data extraction, using a modified version of the Cochrane Effective Practice and Organization of Care form. Results: Of the total of 84 interventions included in the nine RCTs evaluated, three (4%) were related to health care structure, 54 (64%) to the health care delivery process and 27 (32%) to patient outcomes. Regarding the impact of using the CPGs, in 55 interventions (65%), there were no significant differences between control and experimental groups. In four interventions (5%), the result favoured the control group, and the result favoured the intervention group on 25 of the interventions (30%). Conclusions: This systematic review showed that CPGs could be useful to improve the process and structure of health care and, to a lesser extent, to improve the results in patients. However, evidence was weak. There are probably still undiscovered variables that interfere with the use of the CPGs and, therefore, with their impact. Therefore, more studies of good quality are needed. Registration: PROSPERO CRD42013003589.", "keywords": [ "Clinical Practice Guidelines. CPG", "effect", "health care quality" ], "content": "Introduction\n\nThis review refers to the changes in the quality of healthcare services that are direct consequences of the systematic use of clinical practice guidelines (CPGs). Donabedian (1988) states that before evaluating health care, we must first decide how to define \"quality\" and whether it depends only on the actions of physicians or if it also depends on contributions from the patients and the healthcare system.\n\nDefining quality is challenging since it is not easy to characterise coherently and objectively. Health must be analysed from a holistic point of view, and guideline developers must determine the ideal amount of influence health should receive from individual preferences and social components. We must also understand the relationship between structural characteristics and healthcare processes, as well as their results in health services (Donabedian, 1988).\n\nSince the 1990s, increasing numbers of CPGs are being developed. However, it is unknown whether these high-quality recommendations have a beneficial impact on patient health. Despite the high number of recently published CPGs, there are few studies on their effectiveness in improving clinical outcomes, the process and structure quality throughout the healthcare system.\n\nAfter an exhaustive search, only two systematic reviews (SR) we found on this topic, but this SR did not cover the three domains of evaluation of quality and international perspective together.\n\nThis review sought to assess whether the quality of health care improves in patients with the cardiovascular disease when using CPGs vs standard professional medical practice. The primary aim was to assess the impact of the CPGs for the management of cardiovascular diseases on healthcare quality, in terms of patient outcomes, management process, and healthcare structure.\n\n\nMethods\n\nWe designed a methodology aimed to find and analyse studies measuring the impact of CPGs on the improvement of quality in health care services in the three areas proposed by Donabedian (Donabedian, 1988): structure, process, and patient outcomes. From the very beginning of the process when the authors wrote this review protocol, it was clear that these items would not be easy to measure and, as the search and data extraction moved forward, it became harder to synthesise the information delivered by the different studies included. The main obstacle was the inconsistency observed in the different outcome measures used by the studies, which included continuous as well as dichotomous values for different clinical conditions and interventions. The intervention definition read as any planned action taken to modify the clinical practice and use of the clinical guidelines for influencing in the clinical practice. The authors followed the methodological recommendations described in the Cochrane Handbook (Higgins & Green, 2012). This review is registered with PROSPERO (ID: CRD42013003589).\n\nWe did a systematic search using the following electronic databases for primary studies (randomised controlled trials (RCTs)): Cochrane Central Register of Controlled Trials (CENTRAL). The Cochrane Library, including the Cochrane EPOC (Cochrane Effective Practice and Organization of Care) specialised database; MEDLINE; EMBASE; CINAHL; PsycINFO; LILACS; Health Technology Assessment Databases and Web of Science, Science Citation Index, and Social Sciences Citation Index.\n\nThe review authors combined search strategy for indexed terms and developed free text terms. We included searches of grey literature in different sources, such as reports of the world and regional conferences, academic theses and scientific reports not published in indexed journals. We searched for studies published between January 1990 and June 2016, without any language restriction. An advanced search strategy is available as Extended data, Appendix 1 (Ramirez et al., 2019).\n\nWe analysed the studies found through the search strategy, and two authors independently selected the articles according to the following inclusion criteria: RCT measuring the change in health care when using CPGs on cardiovascular disease. The study should measure the change in any of the three health care domains (structure, process, and patient outcomes).\n\nThree authors independently performed data extraction using a modified shorter version of the Cochrane Collaboration EPOC Data Collection Checklist translated into Spanish. Apart from preparing the Spanish version, we eliminated several items not applicable to this review, given that we only included RCTs. We used a standardised digital form for data extraction and analysis. We used a standardised digital form for data extraction and analysis. We used Review Manager software (RevMan 5.3) for the data analysis.\n\nWe assessed the risk of bias (quality) according to the Cochrane Handbook (Higgins & Green, 2012). We found very high variability, so the study results were introduced as a narrative in the Results.\n\nBecause of the variability between the measurements of the effect of the impact of CPGs on the change of quality in the studies included in this review, it was not possible or appropriate to perform a meta-analysis; therefore, it was not possible to measure the statistical heterogeneity.\n\n\nResults\n\nAfter removing duplicates, the search yielded 4279 potential studies. We evaluated the titles and abstracts of the studies and excluded 4051. Of these, we selected 96 studies after assessing the full texts.\n\nFor the analysis, we organised the studies according to the topic or pathology being the core subject of the CPG, and for this report, we only selected RCTs on cardiovascular diseases, because this theme accounts for the higher number of original articles. In total, we selected nine RCTs (Figure 1). Characteristics of the included studies are available as Extended data, Appendix 2 (Ramirez et al., 2019).\n\nWe included nine RCTs analysing cardiovascular diseases (Beaulieu et al., 2004; Berner et al., 2003; Ellis et al., 2000; Guadagnoli et al., 2004; Hand et al., 2014; Jäntti et al., 2007; Kiessling & Henriksson, 2002; Tierney et al., 2003; Tsuyuki et al., 2015). All trials were carried out between 2000 and 2015, five in the United States of America, two in Canada, one in Finland and one in Sweden. Eight articles addressed outpatient care and three inpatient care. The CPGs included in the selected trial looked at the following clinical problems: management of stable angina pectoris (over 65 years), unstable angina, dyslipidemia, acute infarction, heart failure and blood pressure. Besides, the trials included perioperative cardiac evaluations of patients with non-cardiac surgery, cardiopulmonary resuscitation and secondary prevention in patients having coronary artery disease.\n\nA number of the 4,279 studies found in the initial search were not randomised clinical trials, as the authors described in their titles or abstracts. When assessing the full-text articles, we found that many were cluster trials and observational studies with a “before and after” design.\n\nAs for the quality of the evidence, we observed the presence of a high or unclear risk of bias for allocation concealment (selection bias), blinding of participants and personnel (performance bias), and blinding of outcome assessment (detection bias), which can be explained by the nature of the interventions studied. We found several types of systematic errors: random sequence generation (selection bias), incomplete outcome data (attrition bias) and selective reporting (reporting bias). We found that the interventions measured yielded outcomes assessed with moderate to low evidence certainty according to the GRADE classification.\n\nAnalysing the risk of bias in the nine included RCTs studies found the random sequence generation (selection bias) assessment had a low risk of bias (between 50–75%) for allocation concealment (selection bias) 33% low, 33% unknown and 33% high risk of bias. The blinding of participants and personnel (performance bias) obtained almost 50% high risk of bias. The blinding of outcome assessments (detection bias) obtained a 50% low and 50% uncertain risk of bias. Incomplete outcome data (attrition bias) obtained between a 50 and 75% low risk of bias. For the selective reporting (reporting bias), there was a 100% low risk of bias. Finally, other types of bias had a low risk of between 50 and 75% (Figure 2).\n\nThe studies with the lowest and highest risk of bias (Kiessling & Henriksson, 2002 and Jäntti et al., 2007, respectively) were chosen to be evaluated with the GRADE methodology and to build a summary of findings table. The aim of doing this GRADE table was obtaining the rank of possible grades of the evidence certainty found in the 84 interventions from the nine studies included. The results of these studies produced moderate-to-low evidence certainty, according to the GRADE classification (see Table 1 and Table 2).\n\nExplanations\n\n1. For the generation of the random sequence (selection bias) the risk is High: sealed, numbered and opaque envelopes were used for the randomisation of the cases to be treated. It is not clear why people with less work experience or no academic degree were assigned to the ERC 2000. For the allocation concealment (selection bias) the risk is High: It is likely that by the nature of the study the allocation concealment of the selection could not be made. For blinding of participants and staff (performance bias) the risk is High: It is likely that the nature of the study could not prevent participants from knowing which group they belonged to (which CPG they were using). For blinding of the outcome evaluation (detection bias) the risk is Low: A computer automatically collected it. For incomplete results data (attrition bias) the risk is Low: There was no loss of follow-up since it was a single session. For the particular report (notification bias) the risk is Low: A computer automatically collected it. In other risks of bias, the risk of bias is High: The description of the study design was not clear; therefore, we assumed that the study has a high risk of bias.\n\n2. P < 0.001\n\n3. P 0.949\n\nExplanations\n\n1. For the generation of the random sequence (selection bias) the risk of bias is Uncertain: Not described. For allocation concealment (selection bias) the risk is low: general practitioners and patients did not know in which research group they were assigned. For blinding of participants and staff (performance bias) the risk is low: General practitioners were not aware of being involved in the study and a blinded nurse on which group each patient belonged to, was the one who handled the paperwork, protocols of the investigation and had no contact with general practitioners. For blinding of the outcome assessment (detection bias), the risk is Low: The research codes and databases were not disclosed until the authors completed the statistical analysis. For incomplete results data (attrition bias), the risk is Low: The study used the intention-to-treat analysis and indicated that the follow-up for two years was 86%. For the particular report (notification bias) the risk is Low: The research codes and databases were not disclosed until the authors completed the statistical analysis. For Other biases the risk is Low: None known\n\n2. p < 0.05\n\n3. p > 0.05\n\nThe authors grouped the outcomes in simple relative and absolute numbers. There was not a global estimate of the measurements of the effects included in the studies because of the considerable variability of measuring units, as well as the clinical heterogeneity found among the studies included. The measurement of the outcomes reported in the studies was dichotomous, continuous or nominal; the majority were dichotomous and were used to measure the mastery of the process, e.g. the number of patients receiving an adequate treatment following a recommendation versus those who did not.\n\nOf the total of 84 interventions included in the nine RCTs evaluated, three (4%) corresponded to the health care structure domain, 54 interventions (64%) to the domain health care delivery and 27 interventions (32%) to the domain of patient results. Regarding the impact of CPG use, we found that in 55 interventions (65%) there was no significant difference between the control and experimental groups.\n\nIn four interventions (5%) the outcome favoured the control group (comparison of the measure of compliance of the recommendations (the average adjusted by the patient characteristics, the setting, and the number of measures applied per patient)). The measure of effects (odds ratio (OR)) regarding the conditions acute myocardial infarction and heart failure, separated by care provided by a cardiologists or primary care physicians, were as follows: acute myocardial infarction, cared by a cardiologist OR 0.81 (95% CI: 0.79 to 0.83), and cared by a primary care physician OR 0.73 (95% CI: 0.71 a 0.76); heart failure, cared by a cardiologist OR 0.88 (95% CI: 0.86 to 0.90), and care by a primary care physician OR 0.79 (95% CI: 0.76 to 0.81).\n\nThe result favoured the intervention group for 25 interventions (30%). Some of the recommendations were: use of antiplatelet medication during the 24-hour hospital stay, a study of the left ventricle ejection fraction, total cholesterol and LDL measurements, cardiopulmonary resuscitation, and degree of compliance with CPGs recommendations.\n\n\nDiscussion\n\nMethodological efforts have been made to develop trustworthy CPGs. However, it still seems that some of this CPGs are far from the reality of the clinical practice (Institute of Medicine (US) Committee on Standards for Developing Trustworthy Clinical Practice Guidelines, 2011).\n\nIt is essential to emphasise the main findings from the outcome analysis. It is surprising that most of the studies did differentiate between the control and experimental groups regarding the improvements with the use of CPGs. The effects of recommendations of the interventions included in the nine RCTs on the areas of health care structure (4%) and patient outcomes were the least studied (32%). This fact could lead us to assume that researchers have given more importance to the evaluation of using (or not using) the recommendations in the area of the process, instead of their direct impact on the patient health.\n\nThe changes observed in the patient progression were generally modest. In the six studies evaluating patient outcomes (27 interventions), four of them reported a positive result for seven measurements (26%) of the interventions. For example, the study by Ellis et al. (2000) measured the difference in the average change of total cholesterol (mg/dl) and reported a decrease of 7.4 mg/dl in favour of the intervention. It is essential to mention that these measures were mostly surrogate variables. Only one study reported variables for clinical results (Tierney et al., 2003), with 13 interventions, but found no meaningful differences in the results between the groups.\n\nMost of the studies (eight) reported results in the process area and showed favourable results for the intervention in six studies with 16 measurements. This fact reflects the reluctance of health care providers to use CPGs and means that only 30% of these interventions followed the recommendations given by the CPGs.\n\nHowever, most of the studies included in this review used multiple strategies to implement the CPGs (electronic notifications in a digital file, cell phone applications, letters, phone call memos, printed material), and the authors do not tailor to every recommendation (the implementation strategy is usually the same for every recommendation within a CPG). A more specific approach, based on the results of the analysis of the obstacles hindering the adoption of every recommendation separately, might improve the use and effect of the recommendations in practice.\n\n\nConclusions\n\nThere is an imbalance between the number of CPGs developed and the number of high-quality studies evaluating their effectiveness. The study demonstrates weak evidence about the benefit of CPGs for improving the quality of the healthcare process and structure and, to a lesser extent, for improving patient outcomes. There are still undiscovered variables that may interfere with the use of the CPGs and, therefore, with their impact. Therefore, more studies of good quality are still needed.\n\nThe variation in the effects of the recommendations of the CPGs suggests that it would be useful to focus on the analysis of the adherence limitations, as well as on designing implementation strategies adapt every recommendation, instead of considering the CPGs as a whole. Further research is still needed to determine which factors related to the CPGs and their specific recommendations are essential to predict the use of CPGs, and thus achieve better patient outcomes.\n\nThe initial objective of this review was to strengthen the development programs for CPG by evaluating their effects on the quality of health care and to give reliable evidence to sustain the decision-making process related to the construction of CPGs in medium- and low-income countries. Even the research evidence is not strong enough to support the CPGs as a tool to improve the practice for a better quality of care, the results of this review must need to be interpreted with caution. Definitely. It seems the standard application used so far must be reviewed and must incorporate new psychosocial strategies oriented toward driving change in clinical practice and the doctor-patient relationship We need reaching the right hands at the right time. It is necessary to emphasise that the standard implementation used so far must be reviewed, and must incorporate new psychosocial strategies oriented toward driving change in clinical practice and the doctor-patient relationships.\n\nFor an adequate implementation of a CPG, it is necessary to take into account the possible costs and benefits that will be necessary to know the expected results precisely. The health systems that build CPGs must maximise their efforts to get health care personnel to follow the recommendations of the CPGs and make an effort to evaluate their impact.\n\nSince research in this field is so new, and the results of this study were not conclusive, more research is needed to evaluate the change that CPGs could make to the quality of health care, emphasising the less studied areas, such as the structure of health care services and patient outcomes.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: Effects of Evidence-Based Clinical Practice Guidelines in cardiovascular health care quality improvements- A Systematic Review. https://doi.org/10.17605/OSF.IO/9A5FM (Ramirez et al., 2019).\n\nThis project contains the following extended data:\n\nAppendix 1 Advanced Search Strategy\n\nAppendix 2 Characteristics of included studies English version\n\nOpen Science Framework: PRISMA checklist for “Effects of evidence-based clinical practice guidelines in cardiovascular health care quality improvements: A systematic review”. https://doi.org/10.17605/OSF.IO/9A5FM (Ramirez et al., 2019).", "appendix": "Grant information\n\nIHCAI Foundation, the sponsor of the Cochrane Center for Central America and the Spanish-speaking Caribbean, provided the necessary to carry out this review such as access to databases, full-text articles and other resources.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThanks to the professors of the School of Epidemiology of the University of Frontera Temuco, Chile, and the advisor of the Master's thesis, Mario Delgado MD, PhD, for his contribution and suggestions of the proposed protocol of the review.\n\n\nAuthor information\n\nAnggie Ramírez-Morera and Juan Carlos Vazquez are PhD candidates at Autonomous University of Barcelona.\n\n\nReferences\n\nBeaulieu MD, Brophy J, Jacques A, et al.: Drug treatment of stable angina pectoris and mass dissemination of therapeutic guidelines: a randomized controlled trial. QJM. 2004; 97(1): 21–31. PubMed Abstract \| Publisher Full Text\n\nBerner ES, Baker CS, Funkhouser E, et al.: Do local opinion leaders augment hospital quality improvement efforts? A randomized trial to promote adherence to unstable angina guidelines. Med Care. 2003; 41(3): 420–31. PubMed Abstract \| Publisher Full Text\n\nDonabedian A: The quality of care. How can it be assessed? JAMA. 1988; 260(12): 1743–1748. PubMed Abstract \| Publisher Full Text\n\nEllis SL, Carter BL, Malone DC, et al.: Clinical and economic impact of ambulatory care clinical pharmacists in management of dyslipidemia in older adults: the IMPROVE study. Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers. Pharmacotherapy. 2000; 20(12): 1508–1516. PubMed Abstract \| Publisher Full Text\n\nGuadagnoli E, Normand SL, DiSalvo TG, et al.: Effects of treatment recommendations and specialist intervention on care provided by primary care physicians to patients with myocardial infarction or heart failure. Am J Med. 2004; 117(6): 371–9. PubMed Abstract \| Publisher Full Text\n\nHand WR, Bridges KH, Stiegler MP, et al.: Effect of a cognitive aid on adherence to perioperative assessment and management guidelines for the cardiac evaluation of noncardiac surgical patients. Anesthesiology. 2014; 120(6): 1339–49, quiz 1349-53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Green S (editors): Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. Barcelona: The Cochrane Collaboration. 2012; [Accessed February 21, 2019]. Reference Source\n\nInstitute of Medicine (US) Committee on Standards for Developing Trustworthy Clinical Practice Guidelines: Clinical practice guidelines we can trust R. Graham et al., eds., Washington (DC): National Academies Press (US). 2011. PubMed Abstract \| Publisher Full Text\n\nJäntti H, Kuisma M, Uusaro A: The effects of changes to the ERC resuscitation guidelines on no flow time and cardiopulmonary resuscitation quality: a randomised controlled study on manikins. Resuscitation. 2007; 75(2): 338–344. PubMed Abstract \| Publisher Full Text\n\nKiessling A, Henriksson P: Efficacy of case method learning in general practice for secondary prevention in patients with coronary artery disease: randomised controlled study. BMJ. 2002; 325(7369): 877–880. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamirez A, Tristan M, Vazquez JC: Effects of Evidence-Based Clinical Practice Guidelines in cardiovascular health care quality improvements- A Systematic Review. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/9A5FM\n\nTierney WM, Overhage JM, Murray MD, et al.: Effects of computerized guidelines for managing heart disease in primary care. J Gen Intern Med. 2003; 18(12): 967–976. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsuyuki RT, Houle SK, Charrois TL, et al.: Randomized Trial of the Effect of Pharmacist Prescribing on Improving Blood Pressure in the Community: The Alberta Clinical Trial in Optimizing Hypertension (RxACTION). Circulation. 2015; 132(2): 93–100. PubMed Abstract \| Publisher Full Text" }	[ { "id": "51034", "date": "24 Jul 2019", "name": "Yasser Sami Abdel Dayem Amer", "expertise": [ "Reviewer Expertise Pediatrics", "child healthcare", "evidence-based healthcare", "knowledge translation", "quality and safety in healthcare", "healthcare informatics", "and implementation and improvement sciences and research in general" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to review this well-conducted systematic review.\nThe study under review is very interesting for the evidence-based clinical practice guidelines (CPGs)- and for the healthcare quality and safety- research communities.\nMoreover, I would like to congratulate the authors for this work with some minor comments with the aim of improving the content and message of this distinguished study.\nAbstract:\n\nI recommend replacing the phrase 'results in patients' with 'patient outcomes' as it is more commonly used in the field of quality and safety in healthcare. Additionally, the authors have used it for the rest of the MS.\nIntroduction:\n\nI would recommend adding recent definitions of both CPGs and healthcare quality (HCQ) as nowadays clinicians are getting increasingly interested in the fields of evidence-based medicine (EBM) and HCQ either from a research perspective or through engagement in their respective healthcare organizational accreditation. The HCQ definition and description of EB-CPGs are already in the PROSPERO protocol, please include them here as well.\nAdditional relevant studies to cite here: (i) about the relation between EBM and HCQ: Glasziou P, Ogrinc G, Goodman S. Can evidence-based medicine and clinical quality improvement learn from each other?. BMJ Qual Saf. 2011;20 Suppl 1(Suppl_1):i13–i17. doi:10.1136/bmjqs.2010.0465241 (ii) an earlier similar key review: Grimshaw J, Russell I: Effect of clinical guidelines on medical practice: a systematic review of rigorous evaluations. The Lancet. 1993; 342 (8883): 1317-13222\npage 3, left column, para 4: '..After an exhaustive.....only two systematic reviews (SRs)....., but 'these SRs' did not....' Please add citations for these two references (SRs).\n\n'...and international perspective together.' What do you mean by an 'international perspective'; of what; (i.e. of CPGs, of healthcare quality, or of cardiovascular healthcare?), please clarify or re-write.\npage 3, right column, para 4: Regarding the data extraction modified and translated from the EPOC checklist, was it validated and if not, why the authors felt they did not need to? Additionally, please provide the readers who would be interested in the replication of this research with this tool as an online supplementary here. It was indeed mentioned with a link in the PROSPERO protocol (https://fs20.formsite.com/mtristan/form41/index.html, attached on Appendix 3) but the link did not work, giving an error! Moreover, I checked the (Extended data) links in the OSF Home but could not retrieve it.\n\nPlease delete the duplicated sentence; 'We used a standardized digital ..........and analysis'.\npage 3, right column, para 7: The #87 studies that were planned to be included in another analysis were not mentioned here since the final included were 9 rather than 96 studies. Please elaborate on this in this location.\n\nFigure 1. PRISMA flow chart: #87 of studies to include in other 'analyses'.\nTables 1 and 2.: the GRADE quality assessments and the explanations for the certainty of evidence were very well conducted and written.\n\nTable 1: please, if feasible, replace the question in the GRADE SoF table; '.....the results for cardiopulmonary resuscitation?' with '.....the outcomes for cardiopulmonary resuscitation?'\npage 7, left column para 1: Once more, please replace 'patient results' with 'patient outcomes'.\npage 7, left column, para4: Discussion: '..However, it still seems that some of these CPGs are...'\n\npage 7, right column, para 2: As part of discussing 'multiple strategies to implement the CPGs', I would recommend adding insights and citing these relevant keynote articles that discuss multi-faceted CPGI strategies and their importance:\n(i) Grol R, Wensing M. Improving patient care: the implementation of change in health care (in Dutch: Implementatie: effective verbetering van de patiëntenzorg). Amsterdam: Reed Business Education; 2013. (ii) Kastner M, Bhattacharyya O, Hayden L, et al. Guideline uptake is influenced by six implementability domains for creating and communicating guidelines: a realist review. J Clin Epidemiol. 2015;68(5):498–509. doi: 10.1016/j.jclinepi.2014.12.013.3 (iii) Gagliardi A, Brouwers M, Bhattacharyya O, et al. A framework of the desirable features of guideline implementation tools (GItools): Delphi survey and assessment of GItools. Implement Sci. 2014;9:98. doi: 10.1186/s13012-014-0098-84 (iv) Gagliardi A, Brouwers M, Palda V, Lemieux-Charles L, Grimshaw J. How can we improve guideline use? A conceptual framework of implementability. Implement Sci. 2011;6:26. doi: 10.1186/1748-5908-6-26.5\npage 7, right column, para 4: '...... as well as on designing implementation strategies adapt every recommendation...' Do you mean \"designing implementation strategies AND adapt every recommendation....if so add a comma ',' \" or \"designing implementation strategies (by) adapting every recommendation\". Additionally, I suggest to add: '....initiate a pilot implementation of specific recommendation(s) instead of considering implementation of the CPGs as a whole'.\npage 7, right column, para 5: Implications for practice The initial objective of this review was to strengthen the development programs for CPGs (plural)\n\nThe results of this review must need to be interpreted with caution. It reads better either (the results of this review must be interpreted with caution.) or (the results of this review need to be interpreted with caution.).\n\n'It is necessary to emphasise that the standard CPG implementation used so...'\npage 7, right column, para 6: 'For an adequate implementation of a CPG, it is necessary to take into account the possible costs, risks, and benefits\n\nFinally, I would like to congratulate the authors once more on this work and invite all interested researchers to conduct similar research using this robust methodology to study the impact of EB-CPG implementation on all different healthcare specialties in addition to their effects on the interdisciplinary workflows and transitions of care between different specialties and services in healthcare.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "4904", "date": "20 Sep 2019", "name": "Anggie Ramirez", "role": "Author Response", "response": "Dear Yasser Sami Abdel Dayem Amer Thank you for your Review ReportThis response was concomitant between Dr. Ramirez and Dr. Tristan, part of the authors of this systematic review. Thank you for the invitation to review this well-conducted systematic review.The study under review is very interesting for the evidence-based clinical practice guidelines (CPGs)- and for the healthcare quality and safety- research communities.Moreover, I would like to congratulate the authors for this work with some minor comments with the aim of improving the content and message of this distinguished study. Abstract: I recommend replacing the phrase 'results in patients' with 'patient outcomes' as it is more commonly used in the field of quality and safety in healthcare. Additionally, the authors have used it for the rest of the MS. Answer: We agree and we will make the change.Introduction: I would recommend adding recent definitions of both CPGs and healthcare quality (HCQ) as nowadays clinicians are getting increasingly interested in the fields of evidence-based medicine (EBM) and HCQ either from a research perspective or through engagement in their respective healthcare organizational accreditation. The HCQ definition and description of EB-CPGs are already in the PROSPERO protocol, please include them here as well.Additional relevant studies to cite here:(i) about the relation between EBM and HCQ:Glasziou P, Ogrinc G, Goodman S. Can evidence-based medicine and clinical quality improvement learn from each other?. BMJ Qual Saf. 2011;20 Suppl 1(Suppl_1):i13–i17. doi:10.1136/bmjqs.2010.0465241(ii) an earlier similar key review:Grimshaw J, Russell I: Effect of clinical guidelines on medical practice: a systematic review of rigorous evaluations. The Lancet. 1993; 342 (8883): 1317-13222Answer: We agree and we will make the change CPGs for EB-GPGs and added the recommended references the are very useful. page 3, left column, para 4: '..After an exhaustive.....only two systematic reviews (SRs)....., but'these SRs' did not....'Please add citations for these two references (SRs). Answer: We agree, and we will make the change. '...and international perspective together.' What do you mean by an 'international perspective'; of what; (i.e. of CPGs, of healthcare quality, or of cardiovascular healthcare?), please clarify or re-write. Answer: We agree, and we will make the change for: after an exhaustive search, only two systematic reviews (SR) we found on this topic (Worrall 1997, Lugtenberg 2009). Worral (1997) only analysed patient outcomes missing two of the proposed Donadebian Model. Lugtemberg 2009 used the full Donadebian (1988) model only including studies from The Netherlands.page 3, right column, para 4: Regarding the data extraction modified and translated from the EPOC checklist, was it validated and if not, why the authors felt they did not need to? Additionally, please provide the readers who would be interested in the replication of this research with this tool as an online supplementary here.It was indeed mentioned with a link in the PROSPERO protocol (https://fs20.formsite.com/mtristan/form41/index.html, attached on Appendix 3) but the link did not work, giving an error!Moreover, I checked the (Extended data) links in the OSF Home but could not retrieve it. Answer: We agree, and we will make the change. The Some electronic data extraction forms the link is not available now it is a temporary link during the active time of the survey period in the form site server. Now some results of the on line survey are available in this link https://osf.io/9a5fm/?view_only=0ec7b68ed1004bf6b1aa74a19ed1912d as Some electronic data extraction forms/Short version of EPOC Check list.docx. The link in the OSF Home is working. Please delete the duplicated sentence; 'We used a standardized digital ..........and analysis'.Answer: We agree, and we will make the change page 3, right column, para 7: The #87 studies that were planned to be included in another analysis were not mentioned here since the final included were 9 rather than 96 studies. Please elaborate on this in this location. Answer: We agree, and we will make the change. And we add the following:When the first version of the protocol of this Systematic Review was initiated, consider the inclusion of several clinical topics in a single systematic review. We decided to create a series of three systematic reviews on different clinical topics: Cardiovascular Health, Breast Cancer and Child and Mother Health. The initial searches included all those topics with the same inclusion criteria described. After removing duplicates, the search produced 4279 potential studies. After screening by title and abstract, 4051 were excluded. After the full-text evaluation, 96 studies of all clinical subjects were selected. For this SR, only nine studies covering the cardiovascular issue were included.This is the first of series of three systematic reviews on the effects of Evidence-Based Clinical Practice Guidelines (EB-CPGs) on health care quality improvements. The second RD will cover Breast Cancer and the third child and maternal health. We created an interactive https://lnkd.in/dwTxaD8 LIVE GUIDELINES program following the CDC https://lnkd.in/d5r4z6m Figure 1. PRISMA flow chart: #87 of studies to include in other 'analyses'. Answer: We agree, and we will make the changeTables 1 and 2.: the GRADE quality assessments and the explanations for the certainty of evidence were very well conducted and written. Table 1: please, if feasible, replace the question in the GRADE SoF table; '.....the results for cardiopulmonary resuscitation?' with '.....the outcomes for cardiopulmonary resuscitation?' Answer: We agree, and we will make the change page 7, left column para 1: Once more, please replace 'patient results' with 'patient outcomes'. Answer: We agree, and we will make the change page 7, left column, para4:Discussion: '..However, it still seems that some of these CPGs are...' Answer: We agree, and we will make the change page 7, right column, para 2: As part of discussing 'multiple strategies to implement the CPGs', I would recommend adding insights and citing these relevant keynote articles that discuss multi-faceted CPGI strategies and their importance:(i) Grol R, Wensing M. Improving patient care: the implementation of change in health care (in Dutch: Implementatie: effective verbetering van de patiëntenzorg). Amsterdam: Reed Business Education; 2013. (ii) Kastner M, Bhattacharyya O, Hayden L, et al. Guideline uptake is influenced by six implementability domains for creating and communicating guidelines: a realist review. J Clin Epidemiol. 2015;68(5):498–509. doi: 10.1016/j.jclinepi.2014.12.013.3(iii) Gagliardi A, Brouwers M, Bhattacharyya O, et al. A framework of the desirable features of guideline implementation tools (GItools): Delphi survey and assessment of GItools. Implement Sci. 2014;9:98. doi: 10.1186/s13012-014-0098-84(iv) Gagliardi A, Brouwers M, Palda V, Lemieux-Charles L, Grimshaw J. How can we improve guideline use? A conceptual framework of implementability. Implement Sci. 2011;6:26. doi: 10.1186/1748-5908-6-26.5Answer: Thank you very much for the recommended references. We agree that they will give strength to the implementation aspect, one of the most sensitive components for the use of CPG.In relation to the reference Gagliardi 2011, We will not take it in the article because the Research Implementation Guide and Application Network (GiRAnet), is not active and in its link http://www.cebhc.co.za/guideline- implementability-research -y-application-network-giranet-2 /, one link does not work and the other is restricted access.We had access to another article by the same author of 2012, which we share ideas that we have already implemented and this study is in protocol: Gagliardi, A. R., Brouwers, M. C., & Bhattacharyya, O. K. (2012). The guideline implementability research and application network (GIRAnet): an international collaborative to support knowledge exchange: study protocol. Implementation Science, 7, 26. https://doi.org/10.1186/1748-5908-7-26The new paragraph reads as follows:Our findings in this review coincide with some of those captured by Gagliardi (2012) and Kastner (2015) most of the studies included in this review used multiple strategies to implement the EB-CPGs (electronic notifications in a digital file, cell phone applications, letters, phone call memos, printed material), and the authors do not tailor to every recommendation (the implementation strategy is usually the same for every recommendation within a EB-CPG) Cosby (2006) formulates very clearly that although clinicians have the new evidence at hand, using it means a change in the behaviour and habits of the clinical management of patients that is complex to achieve unless appropriate strategies are applied. A more specific approach, based on the results of the analysis of the obstacles hindering the adoption of every recommendation separately, might improve the use and effect of the recommendations in practice. page 7, right column, para 4: '...... as well as on designing implementation strategies adapt every recommendation...'Do you mean \"designing implementation strategies AND adapt every recommendation....if so add a comma ',' \" or \"designing implementation strategies (by) adapting every recommendation\". Additionally, I suggest to add:'....initiate a pilot implementation of specific recommendation(s) instead of considering implementation of the CPGs as a whole'. Answer: We partially agree, and we will make the change. We will not mention or suggest of pilot program implementation because most of them majority yield very little or nothing. page 7, right column, para 5: Implications for practice The initial objective of this review was to strengthen the development programs for CPGs (plural) Answer: We agree, and we will make the change The results of this review must need to be interpreted with caution. It reads better either (the results of this review must be interpreted with caution.) or (the results of this review need to be interpreted with caution.). Answer: We agree, and we will make the change 'It is necessary to emphasise that the standard CPG implementation used so...'Answer: We agree, and we will make the change page 7, right column, para 6: 'For an adequate implementation of a CPG, it is necessary to take into account the possible costs, risks, and benefits Answer: We agree, and we will make the change" } ] }, { "id": "51028", "date": "22 Aug 2019", "name": "Ignacio Marín León", "expertise": [ "Reviewer Expertise Internal Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to review this well-conducted systematic review. The study under review is very interesting and necessary for the Clinical Practice Guidelines (CPGs) community and for the implementation science and healthcare quality communities. I would like to congratulate the authors for this difficult challenge with some minor comments to contribute to improve the content and message of this necessary study\nABSTRACT: Conclusion\n\"There are probably still undiscovered variables that interfere with the use of the CPGs\". That could not be a conclusion from the study, given that the aim of the work was not to study factors and conditions that facilitates the implementation of CPG neither the conditions to change professional behavior. No data on these issues are reported, then no conclusion could be provided. A coherent conclusion could be the need to improve and systematize this kind of implementation studies.\n\nIntroduction: pag 3, para 4, left column\n\"After an exhaustive search, only two systematic reviews (SR) we found on this topic,\"\nIt is needed to provide both references\n\nRESULTS: pag 3, 4 and figure 1. Study identification and selection.\n\"Of these, we selected 96 studies after assessing the full texts\".\nIt is needed, in order to strengthen the transparency and reproducibility of the work, to display in an extended data appendix, the full reference list of rejected originals and reasons to be rejected (96 found minus 9 selected = 87 rejected). It should be clarified what is meant in the figure 1 tab \"# 87 of studies to include in other analyzes\".\n\nPag 4, left column, para 3, rows 4-6:\n\n\"When assessing the full-text articles, we found that many were cluster trials\"\nThere is no clear reasons to reject RCT clusters studies, when the intervention could be randomized to cluster offices, and the outcomes effect measured at individual patient level. It is a well-accepted methodology in implementation science, given that can control for bias and non-measured variables. (Please see 2 references provided: Eccles and Romero).1,2\n\nPag 5, Table 1, Rows 2-4 and Column 5:\n\n\"Certainty of the evidence (GRADE)\"\nIt is difficult to understand why the certainty of evidence for the outcomes \"time without flow\" and \"delay to start CPR\" are classified differently when, as displayed in the explanations, the bias and systematic errors are the same, and there is not a loss of patients to follow up. It is true that both effects measure score different statistical significance, but with data provided the readers feel with the same certainty (either moderate or low) that one effect is significantly different from the control group, and for the other outcomes (delay) there are not differences with the control group.\n\nPag 6, Table 2, Rows 2-4 and Column 5:\n\n\"Certainty of the evidence (GRADE)\"\nThe same reasoning for the difficulty to understand why are classified differently the certainty of evidence for the outcomes \"LDL cholesterol\" and \"Total cholesterol\".\n\nDiscussion: Pag 7, para 2, rows 7-13\n\n\"This fact could lead us to assume that researchers have given more importance to the evaluation of using (or not using) the recommendations in the area of the process, instead of their direct impact on the patient health\".\nAnother explanation could be to emphasize that it use to be easier to measure process of care than patient health outcomes. These health outcomes take a longer period of observations than to measure how professionals adopt CPG recommendations. That can explain the \"preferences\" of researchers.\n\nConclusions\nThe same considerations exposed in the abstract regarding the sentences \"There are probably still undiscovered variables that interfere with the use of the CPGs\".\n\nThat could not be a conclusion from the study, given that was not the aim of the work to study factors and conditions that facilitates the implementation of CPG neither the conditions to change professional behavior. No data on these issues are reported, them no conclusion could be provided.\nA coherent conclusion could be the needs to improve and systematize these kinds of implementation studies.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "4905", "date": "20 Sep 2019", "name": "Anggie Ramirez", "role": "Author Response", "response": "Dear Ignacio Marín León Thank you for your Review Report This response was concomitant between Dr. Ramirez and Dr. Tristan, part of the authors of this systematic review. APPROVED WITH RESERVATIONS Thank you for the invitation to review this well-conducted systematic review. The study under review is very interesting and necessary for the Clinical Practice Guidelines (CPGs) community and for the implementation of science and healthcare quality communities. I would like to congratulate the authors for this difficult challenge with some minor comments to contribute to improving the content and message of this necessary study #1 ABSTRACT: Conclusion \"There are probably still undiscovered variables that interfere with the use of the CPGs\". That could not be a conclusion from the study, given that the aim of the work was not to study factors and conditions that facilitates the implementation of CPG neither the conditions to change professional behavior. No data on these issues are reported, then no conclusion could be provided. A coherent conclusion could be the need to improve and systematize this kind of implementation studies. Answer: We change the text for a better understanding of our meaning. After analyzing many studies, we can have one more hypothesis for further research for more light upon those undiscovered variables that might interfere with the use of the CPGs. #2 Introduction: pag 3, para 4, left column \"After an exhaustive search, only two systematic reviews (SR) we found on this topic,\" It is needed to provide both references Answer: We agree and change for: After an exhaustive search, only two systematic reviews (SR) we found on this topic (Worrall, 1997; Lugtenberg, 2009). Worral (1997) only analysed patient outcomes missing two of the proposed Donadebian Model. Lugtemberg 2009 used the full Donadebian (1988) model only including studies from The Netherlands. #3 RESULTS: pag 3, 4 and figure 1. Study identification and selection. \"Of these, we selected 96 studies after assessing the full texts\". It is needed, in order to strengthen the transparency and reproducibility of the work, to display in an extended data appendix, the full reference list of rejected originals and reasons to be rejected (96 found minus 9 selected = 87 rejected). It should be clarified what is meant in the figure 1 tab \"# 87 of studies to include in other analyses\". Answer: We agree . The Appendix 2: List of studies selected after screening and assessing the full text is added at OSF Home, the link is: https://osf.io/9a5fm/?view_only=0ec7b68ed1004bf6b1aa74a19ed1912d We did not reject the 87 publications, not analyzed. This SR is the first of series that has three systematic reviews. This two new ones will include the topics breast cancer, type 2 diabetes and others. #4 Pag 4, left column, para 3, rows 4-6: \"When assessing the full-text articles, we found that many were cluster trials\" There is no clear reasons to reject RCT clusters studies, when the intervention could be randomized to cluster offices, and the outcomes effect measured at individual patient level. It is a well-accepted methodology in implementation science, given that can control for bias and non-measured variables. (Please see 2 references provided: Eccles and Romero).1,2 Answer: The authors decide when preparing the protocol to include only RCT. #5 Pag 5, Table 1, Rows 2-4 and Column 5: \"Certainty of the evidence (GRADE)\" It is difficult to understand why the certainty of evidence for the outcomes \"time without flow\" and \"delay to start CPR\" are classified differently when, as displayed in the explanations, the bias and systematic errors are the same, and there is not a loss of patients to follow up. It is true that both effects measure score different statistical significance, but with data provided the readers feel with the same certainty (either moderate or low) that one effect is significantly different from the control group, and for the other outcomes (delay) there are not differences with the control group. Answer: Please check the note explanations both have different statistical significance due to this fact the certainty of evidence is downgraded for “Delay to start CPR (s)”. #6 Pag 6, Table 2, Rows 2-4 and Column 5: \"Certainty of the evidence (GRADE)\" The same reasoning for the difficulty to understand why are classified differently the certainty of evidence for the outcomes \"LDL cholesterol\" and \"Total cholesterol\". Answer: Please check the note explanations both have different statistical significance due to this fact the certainty of evidence is downgraded for “Difference in percent change in total cholesterol at 2 years [Mean (mmol / L)]”. #7 Discussion: Pag 7, para 2, rows 7-13 \"This fact could lead us to assume that researchers have given more importance to the evaluation of using (or not using) the recommendations in the area of the process, instead of their direct impact on the patient health\". Another explanation could be to emphasize that it use to be easier to measure process of care than patient health outcomes. These health outcomes take a longer period of observations than to measure how professionals adopt CPG recommendations. That can explain the \"preferences\" of researchers. Answer: You are right. #10 Conclusions The same considerations exposed in the abstract regarding the sentences \"There are probably still undiscovered variables that interfere with the use of the CPGs\". That could not be a conclusion from the study, given that was not the aim of the work to study factors and conditions that facilitates the implementation of CPG neither the conditions to change professional behavior. No data on these issues are reported, them no conclusion could be provided. A coherent conclusion could be the needs to improve and systematize these kinds of implementation studies. Answer: We change the text for a better understanding of our meaning. After analyzing many studies, we can have one more hypothesis for further research for more light upon those undiscovered variables that might interfere with the use of the CPGs." } ] } ]	1	https://f1000research.com/articles/8-1041
https://f1000research.com/articles/8-776/v1	03 Jun 19	{ "type": "Software Tool Article", "title": "Using singscore to predict mutations in acute myeloid leukemia from transcriptomic signatures", "authors": [ "Dharmesh D. Bhuva", "Momeneh Foroutan", "Yi Xie", "Ruqian Lyu", "Joseph Cursons", "Melissa J. Davis", "Dharmesh D. Bhuva", "Momeneh Foroutan", "Yi Xie", "Ruqian Lyu" ], "abstract": "Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.", "keywords": [ "single sample", "gene set scoring", "signature scoring", "AML mutations", "NPM1c mutation", "mutation prediction", "TCGA" ], "content": "Introduction\n\nThe development of microarrays and more recently the rapid uptake of RNA-sequencing technologies have provided a platform to examine the transcriptional profile (or transcriptome) of biological samples1. Transcriptomic analyses have traditionally focused on ‘differential expression’ of genes between sets of samples, however with the rapid growth of publicly available RNA data there has been increasing usage of ‘relative approaches’, which quantify the relative concordance of a sample or samples with a specific gene signature1. While sequencing of genomic mutations has been important for classifying different tumour subsets based upon the presence of mutations or fusion genes, and identifying genetic lesions which may act as drivers of cancer progression, transcriptomic profiling can provide further information on the state or phenotype of cells carrying these mutations.\n\nCancers are a heterogeneous set of diseases with a number of clinical and pathological subtypes. In diseases such as breast cancer the primary clinical classifications relate to the expression of hormone receptors (estrogen receptor: ER; and progesterone receptor: PR) or the overexpression of Erb-B2 receptor tyrosine kinase (HER2), as these features can be directly targeted with therapeutic agents. A common example of transcriptomic or gene expression data informing clinical practice is the use of prediction analysis of microarray 50 (PAM50) signatures for distinguishing the intrinsic breast cancer subtypes [2, Cieślik and Chinnaiyan1]. For many other cancers, subtype classification has largely relied upon identifying sets of recurrent mutations across large patient cohorts, with whole genome or whole exome sequencing studies helping to resolve the clinically significant subtypes [3, Papaemmanuil et al.4].\n\nPerhaps the most well-known ‘relative approach’ is single-sample gene set enrichment analysis (ssGSEA)5, often used through the GenePattern web-tool. Another common approach is gene set variation analysis (GSVA)6 which is available as an R/Bioconductor package that also includes functionality for ssGSEA, an alternative approach known as PLAGE7, and a z-score based approach8. Both ssGSEA and GSVA use a Kolmogorov-Smirnov like random-walk statistic to convert normalised gene ranks to the resulting score; however, this normalisation procedure means that the scores are not truly ‘single-sample’, and variations in the overall sample composition for a study (e.g. variations in the presence or relative frequency of different cancer subtypes) can lead to unexpected changes in sample scores. Furthermore, the resultant scores from these methods can vary in their range and absolute value, making them difficult to interpret without further processing. To overcome this, we have developed a single-sample gene set scoring method singscore9, which simply uses the ranks of genes within a given set, normalised relative to the maximum and minimum theoretical scores for a gene set of a given size.\n\nThrough large-scale efforts such as The Cancer Genome Atlas (TCGA), transcriptomic data are available for thousands of clinical samples, often together with corresponding genomic or epigenomic (often DNA methylation) data. These transcriptomic data can help to characterise the functional effects of corresponding mutations, and provide a window to study the heterogeneity which arises within different subtypes of cancer due to epigenetic and transcriptional regulatory programs which can also influence cell behaviour. Here, we demonstrate that the single-sample gene set scoring method singscore9 can be used to classify TCGA AML samples using transcriptional ‘gene signatures’ for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions that were derived from independent studies. Without any need for parameter fitting or estimation, we show that gene set scoring with singscore can distinguish samples carrying these mutations. The case studies we present demonstrate the application of gene set scoring to examine not only the differences, but also the relative similarities between established subtypes of AML that impact clinical outcome. This workflow is available as a bioconductor workflow package from https://bioconductor.org/packages/release/workflows/html/SingscoreAMLMutations.html10.\n\n\nDescription of the biological problem\n\nAs with most cancers, acute myeloid leukemia (AML) is a heterogeneous disease with a number of classified subtypes. Analysis of TCGA AML genomic data identified a number of subtypes based upon the presence or absence of specific ‘driver mutations’; recapitulating and expanding upon previously identified clinical subsets3. A more recent study which focused primarily on genomic data has further refined the clinically significant AML subtypes4, highlighting a number of co-occurring as well as mutually exclusive mutations.\n\nOf note for this work, one of the most common mutations in clinical AML samples is a frameshift mutation within exon 12 of the nucleophosmin (NPM1) gene4. This mutation leads to aberrant localisation of nucleophosmin with cytoplasmic accumulation rather than localising to the nucleolus, and accordingly this mutation is often referred to as the NPM1c mutation11. As noted by Verhaak et al.12, the NPM1c mutation is associated with dysregulated activity of the homeobox domain (Hox) family of transcription factors which are essential for developmental patterning. The effects of this mutation in disease progression have been further demonstrated in recent work which showed that loss of NPM1c leads to differentiation of AML cells11.\n\nFurther recurrent genetic lesions in AML relevant for this work include lysine methyl transferase 2A (KMT2A; previously known as MLL) fusion genes, partial tandem duplications within KMT2A (KMT2A-PTD), and fusion genes between promyelocytic leukemia and retinoic acid receptor alpha (PML-RARA). Given the role of NPM1c in dysregulating the Hox gene family, it is interesting to note that AML samples with MLL fusion genes also show dysregulated expression of Hox family genes [13, Ross et al.14]; however, samples with MLL-PTD appear to show a relatively distinct phenotype from MLL-fusion samples14. While there is good evidence demonstrating the role of NPM1c mutations and other genetic lesions in blocking AML cell differentiation, the PML-RARA fusion subset is diagnostic for a specific subset of AML known as acute promyelocytic leukemia (APL). This clinically distinct subtype of AML is associated with a specific morphology under the French-American-British (FAB) classification of AML, FAB-M3, with cells showing a distinct morphology due to a differentiation block at the promyelocyte stage15.\n\nIn this workflow we demonstrate the ability of the singscore method for single sample gene set scoring9 to classify tumour ‘driver mutations’ from transcriptomic data. We use a previously identified gene signature for the NPM1c mutation12. We also use signatures for PML-RARA gene fusions and MLL-fusions that were derived using pediatric AML samples but shown to work well for classifying adult AML samples with similar lesions14, although we note that there is evidence of relatively large differences in the mutational profiles of adult and pediatric cancers16. Using these signatures, which are included within the molecular signatures database (MSigDB)17, we demonstrate that a bi-directional scoring approach can classify TCGA AML samples with corresponding mutations with a good precision and recall. A particularly useful feature of gene set scoring is the ability to project samples onto 2D or higher-order landscapes defined by corresponding phenotypic signatures. Accordingly, by comparing scores for both the NPM1c and KMT2A-/MLL-fusion signatures, we show that this classification likely arises through the shared downstream biological effects of Hox family dysregulation. We also compare the NPM1c mutation signature to the PML-RARA signature and show a clear separation of these subtypes reflecting their divergent phenotypes and the mutually exclusive nature of these mutations.\n\n\nDownloading and preparing the data\n\nData from TCGA project is made available through the Genomic Data Commons (GDC). Open access data from the project can be accessed in multiple pre-processed formats. Transcriptomic data can be downloaded either at the count level or as FPKM transformed abundance, before or after upper quantile normalisation. Other pre-processed version can be found from sources such as the cBioPortal and FireBrowse. The GDC data used STAR to perform a two-pass alignment followed by quantification using HTSeq. Data from the GDC can be downloaded using the GDC data transfer tool which allows users to select the specific files of interest using the GDC portal. These files then have to be read, merged, annotated and processed into a data structure that simplifies downstream analysis. Alternatively, all the above mentioned steps, including the download, can be performed using the R package TCGAbiolinks18. The package supports data download using the GDC API and the GDC client. We will use the TCGAbiolinks package to download, annotate and process the data into a SummarizedExperiment R object.\n\nThe following steps need to be performed to prepare the data:\n\n1. Create a query to select the files to download\n\n2. Execute the query and download the data\n\n3. Read the data into R\n\n4. Filter out genes with low expression\n\n5. Normalise the data for compositional bias and transform to account for gene-length biases as outlined in the singscore manuscript9\n\nThe first step in any analysis should be to determine and report the data version and the service used to download the data. The getGDCInfo() function returns the release date of all data on the GDC along with a version.\n\n\n\n\n\nA query then needs to be run, using the GDC to identify the specific files for download. This step is similar to generating a MANIFEST file using the GDC portal. The first parameter of the query specifies the project - available projects can be accessed using getGDCprojects() or from https://portal.gdc.cancer.gov/projects. The TCGA acute myeloid leukemia data is part of the TCGA-LAML project. Following this, the data category, data type and workflow type need to be specified. The query formed below selects files containing the count level transcriptomic measurements. Values for different parameters of the query can be identified from “searching arguments” section of the “query” vignette: vignette(\"query\", package = \"TCGAbiolinks\"). The result of this query will be a dataframe containing filenames and additional annotations related to the files.\n\nRead count level data are selected instead of the processed FPKM data as one of the downstream pre-processing analysis results in filtering out of genes. A general recommendation is to compute FPKM values after filtering genes out so as to ensure counts are normalised by the corresponding library sizes. In cases where count-level data is not available, filtering can be performed directly on FPKM values, provided that the library size is large enough.\n\n\n\n\n\n\n\n\n\nThe GDCdownload function then executes the query on the GDC database and begins downloading the data using the GDC API. The download method should be changed to “client”, if the size of the data is expected to be large, e.g for RNA-seq read data or methylation data. It is good practice to specify the directory for data storage - we store all the data in the “GDCdata” directory in the temporary directory. Users should store their data in a permanent storage to retain the data. The function downloads the data and organises it into the folder based on parameters specified in the query. This allows multiple different levels and types of data to be stored in the same directory structure. Files with counts are stored at TEMPDIR/GDCdata/TCGA-LAML/harmonized/Transcriptome_Profiling/Gene_Expression_Quantification/.\n\n\n\nThe GDCprepare function reads and processes the downloaded data into a RangedSummarizedExperiment object from the SummarizedExperiment package which allows patient annotations, gene annotations and count data to be stored in one object. Patient annotations are downloaded upon calling this function and subsequently mapped and attached to the resulting object. A RangedSummarizedExperiment object is similar to an ExpressionSet object but provides added functionality such as indexing with genomic coordinates and storing multiple data matrices with the same structure. Feature annotations used to annotate the data are stored in an RDA/RDATA file.\n\n\n\nThe object contains data for 56,925 features and 151 samples. The original data files contain 60,483 features, some of which (3,767) could not be mapped to ENSEMBL GRCh38.p12. Feature and sample annotations can be accessed using rowData(se) and colData(se), respectively, and the counts data can be accessed using assay(se). TCGA data usually contains some formalin-fixed paraffin-embedded (FFPE) samples which should be discarded from the analysis as the protocol introduces biological artefacts. This procedure is only performed on solid tumours and not leukemias, therefore, no filtering is required for this data set.\n\n\n\n\n\nThe edgeR package contains methods that assist in the data normalisation and transformation required for filtering and subsequent steps. The methods require a DGEList object therefore we begin by creating a DGEList for the AML data from the SummarizedExperiment. Creation of a DGEList object is initially unsuccessful due to duplicated row names in the data matrix (107 features). The raw count files for individual samples do not have any duplicated features indicating this is introduced by the GDCprepare function. This is further supported by the fact that the counts for a single feature are the same for duplicated entries. An example of such a feature is shown below and we have verified duplicated entries of all other features to be the same.\n\n\n\n\n\nAs such, it is safe to discard duplicated entries.\n\n\n\nGenes with low counts across most samples are discarded from the analysis. This is a standard step in differential expression analysis as inclusion of such genes in the analysis could skew estimates of dispersion. It is also motivated in rank-based analysis, such as with singscore, to avoid rank duplication. Rank duplication reduces the discriminant power of scores as the number of unique ranks is reduced. A commonly used filter is to select only those genes that have CPMs above a certain threshold across a proportion of samples. Filtering is performed on the CPMs rather than raw counts as the former accounts for variation in library sizes, therefore, is unbiased. For instance, a CPM of 1 would equate to read counts between 19 and 50 for samples in the AML data where library sizes vary between 18.6 and 49.7 million reads. Here, we retain genes that have a CPM > 1 across more than 50% of the samples. Other methods to filter out genes with low counts exist and may be preferable in specific applications. Chen et al.19 and Law et al.20 filter genes based on the experimental design whereby the proportion of samples with enough read counts are evaluated per experimental group. As the AML data have many samples, filtering is performed across all samples rather than within sub-groups. Group specific filtering would be recommended for the study of rare groups. The distribution of logCPMs is much closer to the expected log-normal distribution after filtering out genes with low counts as seen in Figure 1.\n\n\n\n\n\n\n\nFiltering results in fewer zeros in the data. Most genes with CPM less than 1, logCPM < 0; (red line) across the majority of samples get discarded, resulting in an approcimately log-normal distribution.\n\nSingscore requires gene expression measurements to be comparable between genes within a sample, therefore, correction for gene length bias needs to be performed21. Transformations such as transcripts per million (TPM) and reads/fragments per kilobase per million (RPKM/FPKM), that normalise by gene length, may be used. Both- TPM and RPKM/FPKM values should produce similar results when applying singscore provided that the library size is large enough, which they are here. RPKM values are generally computed after correcting for compositional biases. The calcNormFactors function in edgeR provides three methods to do so, TMM normalisation being the default. Chen et al.19 and Law et al.20 discuss the implications of normalisation prior to down-stream processing such as differential expression analysis. Normlisation is generally performed for cross-sample analysis where samples need to be comparable. Singscores are invariant to data normalisation as the analysis is contained within the sample of interest. The idea extends to any transformation that preserves the relative ranks of genes within a sample such as a log transformation. Here, we use TMM normalisation solely for visualisation purposes.\n\nData transformation to TPM or RPKM/FPKM requires the lengths for all genes to be calculated. Gene lengths need to be computed based on the alignment and quantification parameters. The TCGA transcriptomic data has been aligned using STAR and quantified using HTSeq (details of the pipeline available at https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/). HTSeq quantifies reads mapping to the exons of each gene, therefore, effective gene lengths can be calculated as the sum of all exons spanning the gene. The GENCODE v22 annotation file was used for quantification therefore the same file needs to be used to compute gene lengths.\n\n\n\nThe rtracklayer R package provides functions to help parse GTF files.\n\n\n\nGenes are also annotated with their biotype for further analysis. The annotation file uses Ensembl IDs with versions as keys to records, which then need to be converted to Ensembl IDs. This is simply achieved by truncating the trailing version number.\n\n\n\n\n\nThe SummarizedExperiment object allows feature annotations to be stored, therefore, information on gene length and biotypes should be added to the existing annotations. Similarly, annotations need to be added to the DGEList object. The column containing lengths should include “length” in its name.\n\n\n\nRPKM/FPKM values can now be calculated with the computed gene lengths after computing the normalisation factors. The SummarizedExperiment object can store multiple levels of the data simultaneously, provided that the number of features and samples remains the same across measurements. As such, FPKM values are appended to the existing object.\n\n\n\n\n\nFor this analysis we have used the curated mutation list from the original TCGA AML publication3 (Supplemental Table 01 at https://gdc.cancer.gov/node/876) rather than variant calls from the standard TCGA pipeline (available through the National Cancer Institute Genomic Data Commons) and readers should note that there are some discrepancies between these. For genetic lesions of interest (NPM1c, KMT2A-MLL, KMT2A-PTD and PML-RARA), patients were identified by the following criteria:\n\nPatient ID: The ‘TCGA Patient ID’ column was extracted directly\n\nNPM1c: TRUE if the ‘NPM1’ column contains the strings ‘p.W287fs’ or ‘p.W288fs’\n\nKMT2A-fusion: TRUE if the ‘MLL-partner’ column contains the string ‘MLL-’ or ‘-MLL’ (note that the official gene symbol for MLL is now KMT2A)\n\nKMT2A-PTD: TRUE if the ‘MLL-PTD’ column contains the string ‘exons’\n\nPML-RARA: TRUE if the ‘PML-RARA’ column contains the string ‘PML-RARA’\n\n\n\n\n\nEnsembl annotations (Ensembl IDs) have higher coverage of the genome which may be useful in applications such as variant calling and similar exploratory analysis22. However, RefSeq annotations (Entrez IDs) may be better suited to RNA-seq analyses which require a stable reference annotation for comparison23. As such, we choose to map Ensembl IDs to Entrez IDs and discard any unmapped features.\n\nMapping can be performed using the Ensembl Biomart service, which can be queried using the biomaRt bioconductor package. This would provide the most up to date annotations. Alternatively, mapping could be performed with the bi-annually updated org.Hs.eg.db annotation R package which provides a stable set of annotations, thereby enhancing reproducibility. Mapping is performed with the mapIds function in the AnnotationDbi R package.\n\n\n\nMultimapped Ensembl IDs are replaced by NAs, then discarded to enforce unique mapping. Similarly, Entrez IDs that map to multiple Ensembl IDs are identified from the mapping, and discarded. Only features with unique Ensembl ID to Entrez ID mappings remain.\n\n\n\n\n\n\n\n\nTranscriptional signatures to predict mutation status\n\nThe signature by Verhaak et al.12 is now used to predict the mutation status of the NPM1c mutation. This is done by quantifying the concordance of genes in the signature with their expression in each sample. As such, high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores. This single value can then be used to predict the mutation status of individual samples if these data were unavailable.\n\nThe signatures of interest are first downloaded from the MSigDB and read into GeneSet objects from the GSEABase R package. We then use the singscore R/Bioconductor package to quantify each sample for the Verhaak signature. Some of the visualisation and diagnostic tools within the singscore package are used to interpret the signatures and scores. Finally, we use a simple logistic regression model on the scores to predict the mutation status.\n\nThe Verhaak et al.12 signature is composed of an up-regulated and a down-regulated gene set. Many signatures are developed in such a manner to improve discrimination of samples. MSigDB stores such signatures using names with suffixes “_UP” and “_DN” representing the independent components of the signature. Here, we form the download links for the signature with the base name “VERHAAK_AML_WITH_NPM1_MUTATED”.\n\n\n\n\n\nThe signatures are then downloaded using the links, resulting in an XML file for each component of the signature. The mapply function is used to run the download function on all pairs of link-output arguments.\n\n\n\nFunctions in the GSEABase package help with reading, parsing and processing the signatures. Signatures from an MSigDB XML file can be read using the getBroadSets function which results in a GeneSet object. Gene symbols, Entrez IDs and affymetrix chip IDs from the original experiment (HG-U133A in this case) are stored in the XML file. Entrez IDs are read from the file as these can be mapped directly to our data. Conversions to other identifiers can be achieved using the mapIdentifiers function from GSEABase and an annotation package that contains the mappings. The advantage of using this function instead of the mapIds function from the AnnotationDbi package is that the former retains the GeneSet object after conversion of IDs.\n\n\n\n\n\nTo make data indexing easier during signature scoring, row names of the SummarisedExperiment object are changed to Entrez IDs which are already part of the row annotations.\n\n\n\nSingscore is a rank based metric of gene set enrichment in single samples. Scores for multiple signatures make use of the same ranked expression per sample. As such, it makes sense to compute the ranks only once and re-use them for scoring different signatures. The implementation separates these two phases of the analysis to reduce the computational cost of scoring. The rankGenes function will compute ranks from expression data in the form of either a numeric matrix, numeric data frame, ExpressionSet object, DGEList object or a SummarizedExperiment object. Users also have to specify what method should be used to break ties. The default is ‘min’ and we recommend this be used for RNA-seq data which may have many genes with zero counts. This will reduce the effect of zeros in the scores, however, appropriate pre-filtering of genes with low counts will still be required.\n\n\n\nSingscores can be computed using three modes, depending on the properties of the gene signature. The first mode of operation is applied when two directed gene sets (expected up- and down-regulated gene sets) form the transcriptomic signature. Many signatures in the MSigDB, including the Verhaak et al.12 signature come in such pairs. This mode can be invoked by passing the up- and down-regulated gene sets to the arguments upSet and downSet respectively. In some cases, only one set of genes forms the signature. If all genes in the gene set are up-regulated or all down-regulated, the second mode of operation applies and is invoked by passing the gene set to the upSet argument. For sets of down-regulated genes, the score would simply be inverted (-score if scores are centered, 1 - score otherwise). Finally, if the user is unsure of the composition of genes in the gene-set, such that, the gene set may contain both up- and down- regulated genes, the final mode of singscore applies. The gene set is passed to the upSet argument similar to the previous mode with the additional argument knownDirection set to FALSE.\n\nBy default, singscores are centered such that the range of scores is [−1, 1] and [−0.5, 0.5] for the first two modes respectively. Negative scores indicate an inverse enrichment of signatures, that is, expected up-regulated genes are in fact down-regulated and vice-versa. Scores from the last mode can not be centered and have the range [0, 1]. In this mode, high scores are obtained when ranks of genes are distant from the median and low scores obtained when ranks converge to the median rank. If scores are centered in this scenario, it would lead to the conclusion that a negative score shows inverted enrichment, which is not the case. Score centering only serves the purpose of easing interpretation for users, a simple linear transformation is applied to achieve it.\n\nScores for the NPM1c mutation signature are computed using the default settings, with the first mode of operation being used due to the presence of an up- and down- regulated gene set. The function returns a data frame reporting the score and dispersion of ranks for the up-regulated gene set, down-regulated gene set and the combination of both. Dispersion of the combined gene set in this mode is simply the mean of the independent dispersion estimates. If any gene names/IDs are present in the signature but missing in the expression data, a warning will be reported.\n\n\n\n\n\nIt should be noted that singscores are composed of two components, an enrichment score and a dispersion estimate of ranks. The quantity of interest in gene set enrichment is the distribution of the expression or ranks of genes in the signature. In an ideal scenario, all expected up-regulated genes would have high expression therefore higher values of ranks. As such, ranks would be distributed on the higher end of the entire rank spectrum. Singscore aims to quantify this distribution of ranks, therefore, computes and reports the average and dispersion of ranks of genes in the signature relative to all other genes. The first quantity is similar to scores computed from all other single sample scoring methods. We determined a two component score to be a more appropriate and accurate representation of the distribution of ranks of signature genes. The default and recommended measure of dispersion is the median absolute deviation (MAD) due to its non-parametric property. Other appropriate measure of dispersion could be the inter-quartile range (IQR) and can be used by passing the IQR function as an argument to the dispersionFun argument.\n\n\n\nThe singscore package provides a set of visualisation tools that enable diagnostics of the gene signature. For instance, these tools may be used to determine the importance of each component for a bidirectional signature (up- and down-regulated gene sets) to the total score, determine the importance of each gene of a signature in discriminating between the classes of interest, and to investigate the relationship between the final score and the dispersion of signature gene ranks. Annotations of interest can be overlayed on each plot. Singscore supports both continuous and categorical annotations, which can either be input as a vector, or as a string specifying a column within the score data frame. We begin by investigating the relationship between the score and dispersion of ranks for the up-regulated gene signature, down-regulated gene signature and the full signature. The plotDispersion functions generates a diagnostic plot with annotations overlaid. Annotations can be discrete or continuous, and can be passed as independent variables, or as a column name when the data is appended to the score data frame. It should be noted that all plotting functions in singscore can be made interactive by setting the isInteractive argument to TRUE.\n\n\n\nFigure 2 shows that the set of down-regulated genes has more discriminant power in separating NPM1c mutated samples from those those without. Moreover, the signature is able to discriminate MLL (KMT2A) fusions and PTDs from the other samples. NPM1c mutations produce higher scores for the down-regulated gene set while MLL fusions and PTDs produce moderate scores. Similar trends are observed with the set of up-regulated genes; however, despite the range of scores increasing, the discriminant power drops moderately. In fact, the signature of up-regulated genes is able to discriminate samples without the genomic changes of interest (‘Other’) better by producing negative scores for most of these samples. Negative scores for the expected up-regulated gene set indicate that these genes are expressed below median expression, therefore, likely down-regulated within corresponding samples. Another observation from these plots is based on the trend of the dispersion (MAD). The dispersion is generally expected to be higher for scores close to zero. Zero scores are achieved in three possible scenarios: when genes are expressed at median expression level; when genes are evenly distributed at both ends of the expression spectrum; or, the more likely scenario whereby the rank of expression of all genes are uniformly distributed. The last two scenarios would result in a high dispersion. To explain these ideas, we select 3 samples and plot the ranks of genes in both signatures. The sample with the highest total score, lowest total score and highest dispersion are chosen.\n\n\n\nScores are plot against the median absolute deviation (MAD) of the ranks of genes in each gene set forming the NPM1c signature. The plots also reveal that scores close to 0 result in higher MADs.\n\nFigure 3 shows that both the up- and down-regulated gene sets contribute to the score of the top scoring sample as these genes are at the extremes ends of the rank spectrum. Similarly, the lowest scoring sample has the ranks of genes in each set inverted. As observed in the previous plot, the up-regulated genes improve discrimination between NPM1c mutated samples and other samples and may be a stronger indicator of wild-type NPM1c samples than the down-regulated genes. Finally, the sample with the maximum dispersion exhibits uniformly distributed down-regulated gene ranks and a bimodal distribution of the up-regulated gene ranks with the modes at the extremes of the spectrum.\n\nSamples with the highest score, lowest score and highest dispersion (MAD) are show from left to right. The barcode plot for ranks of genes in each signature along with the density of the ranks is shown in each plot. Up-regulated genes are represented in green and down-regulated genes in purple. Gaussian distributions are observed at the extremes for the lowest and highest scores. High dispersion results in scores close to 0 because all genes are either evenly distributed (down-regulated gene set) or bi-modally distributed at the extrema (up-regulated gene set).\n\nCombined, these plots show that both the up- and down-regulated genes play an important role in discriminating between NPM1c mutated (and MLL fusion/PTD samples), and wild-type samples. These diagnostic plots help in determining the importance of genes in signatures with respect to the samples of interest and should be used prior to application of signatures. In some cases, signatures may have been developed in specific contexts due to inherent biases in datasets and yet, described or applied in a generalised setting. These diagnostic plots may help in validating their application in specific contexts and possibly assist in characterising the contexts of all existing signatures.\n\nMutation status can be predicted from singscores using a logistic regression model with a “logit” link function. The benefits of this model over one where each gene in the signature is used as a term in the model is the simplicity of the model. The Verhaak et al.12 signature consists of 429 genes which would result in a regression model with 429 predictors. As there are fewer samples than the predictors, some feature selection method would be required which may result in the loss of information. Moreover, automating model development on a larger selection of gene signatures would be limited with gene-level models. Additionally, models trained using singscore would inherit its non-parametric properties to some extent. For instance, such models would be invariant to all data transformations that retain within sample ranks of genes. The main limitation of such models would be the loss of accuracy due to summarisation of the data (information on 429 genes is summarised into a two-component score).\n\nIn any case, our aim here is not to discuss the various models that can be used to predict mutation status, but to exhibit the discriminant power of singscore and transcriptomic signatures. consequently, the data used to train the model are used to evaluate the basic performance properties. We begin by combining the scores with mutation annotations.\n\n\n\nBefore training a model, we can visualise how well scores resulting from the Verhaak et al.12 signature separates the mutants from wild-type samples for some genes of interest. Figure 4 shows that NPM1c signature scores can discriminate between NPM1c wild-type and mutants. Similar observations are made for MLL (KMT2A) fusions and PML-RARA fusions.\n\n\n\nBoxplot of scores from the NPM1c signature split by different types of mutations.\n\nTo quantify the above observations, we fit logistic regression models with each genomic lesion as the response variable and the score as the predictor. Coefficient estimates, standard errors, z-statistics, and p-values of each model are listed below.\n\n\n\n\n\nNPM1c mutations are significantly associated with the score. Samples carrying a MLL gene fusion are also associated with the score reflecting their shared effects on Hox gene dysregulation, although with a lower significance. Interestingly the PML-RARA fusion carries a negative coefficient, likely reflecting the distinct cellular morphology/phenotype of acute promyelocytic leukemia relative to other subsets of AML, as noted above.\n\nThe above statistics give insight on the models trained but not on their performance. Precision and recall provide insight on a models predictive performance. The dcanr package provides functions to compute these metrics along with other measures of performance.\n\n\n\n\n\nPrecision for predictions of all genomic changes other than NPM1c is undefined because all samples are predicted to be wild-types. Consequently, their recall is zero. Precision, recall and the F1 score of the NPM1c mutation model are high as expected. As singscores are two-components scores, performance may be further improved by including the dispersion of ranks. This is observed in Figure 2.\n\nAs observed from the model below, using both components of singscores significantly improves the predictive performance.\n\n\n\n\n\n\nSignature landscapes with multiple signatures\n\nOften, we are interested in the relationship between two dependent or independent phenotypes, for instance, the epithelial and mesenchymal phenotypes. The role of most signatures is to estimate an unobservable molecular phenotype so they may be considered as proxies of phenotypes. As such, we could investigate the relationship between two phenotypes using corresponding signatures. Foroutan et al.24 first introduced the idea of molecular signature landscapes to investigate the relationship between signatures related to the epithelial-mesenchymal transition (EMT) and TGFβ -induced EMT. Subsequently, Cursons et al.25 computed epithelial and mesenchymal phenotype signature singscores and demonstrated an epithelial phenotype shift following miR-200c transfection into a mesenchymal cell line using a signature landscape. Foroutan et al.9 used signature landscapes to stratify breast cancer subtypes along the epithelial-mesenchymal axis and included it as part of the singscore package. Here, we show how such landscapes can be used beyond the current application of the epithelial-mesenchymal axis. We demonstrate how transcriptomic signatures of different mutations can be used to stratify AML samples.\n\nWe now use the Ross et al.14 MLL-fusion signatures to score the TCGA AML samples. Unlike the NPM1c signature, this signature is composed of genes that discriminate samples with MLL-fusion genes. We download and parse the signature as demonstrated with the NPM1c signature.\n\n\n\n\n\nThe gene set is a composition of both up- and down-regulated genes as genes were selected based on their ability to discriminate mutants from wild-types. We use the third mode of singscore, which does not require the direction of genes in the gene set to be known. The knownDirection parameter of the simpleScore function is set to FALSE to induce this mode. Ranks computed previously can be reused to compute scores with the new signature.\n\n\n\nThe plotScoreLandscape function plots a hexbin plot to visualise the two-dimensional distribution of scores. Scores computed using the Verhaak et al.12 signature and the Ross et al.14 MLL-fusions signature are passed as arguments. Both scores should have been computed on the same samples with the order of samples retained. Names of the scores should be passed as arguments to the scorenames argument. The textSize argument can be used to specify the size of all text relative to the plot size. This may prove useful when plots are being generated for scientific posters, publications and presentations, all of which require different image sizes.\n\n\n\nFigure 5 shows a strong positive association between scores from the two signatures (Spearman’s ρ = 0.611) despite only 16 genes being shared across the two signatures (signature sizes are 78 and 429 genes). In such an analysis, we may be interested in projecting new data points onto the landscape as done by Cursons et al.25. Alternatively, we may want to overlay some existing data points to investigate sample stratification using scores. Here, we overlay MLL fusions, MLL PTDs, PML-RARA fusions and NPM1c mutations onto the landscape. First, we build the annotation vector.\n\n\n\nA positive association is revealed between the two signatures in AML.\n\nPoints are projected onto an existing landscape using the projectScoreLandscape function. This functions uses the p_mll_npm1c plot generated using the plotScoreLandscape and overlays new data points onto it. Scores for the new data must be computed with the same signatures that were used to compute the landscape. As we are using existing data, scores computed in earlier sections are re-used. The subSamples can be used to select a subset of samples to project. Here, we select samples with the mutations we are interested in annotating.\n\n\n\nFigure 6 shows that the MLL fusions and MLL PTDs exhibit variation across different axes. MLL PTDs have a lower score than MLL fusions for the MLL fusion signature as expected. These sets of samples do not cluster along the two axes, consistent with observations by Ross et al.14. Overlaying samples annotations onto the plot assists in interpreting different regions of the landscape.\n\nDifferent mutations occupy different regions of the landscape, possessing different types of molecular traits.\n\nIt is evident from the previous analysis that PML-RARA samples differ from all other samples examined here. We repeat the analysis with the PML-RARA signature from Ross et al.14 to verify this distinction. The signature is available on the MSigDB and is named “ROSS_AML_WITH_PML_RARA_FUSION”. We download and parse the signature, score all samples against it, plot a landscape with scores from the Verhaak et al.12 signature, and finally, project samples onto the plot. The signature was constructed in a similar manner to the MLL fusions signature, therefore, samples are scored using the same settings.\n\nFigure 7 shows a completely different landscape from what was observed with the MLL fusion signature. The PML-RARA signature forms a clear separation between the PML-RARA and NPM1c samples such that PML-RARA samples are the only ones with a high score for this signature. Moreover, no association is observed between the two signatures. As discussed above (Description of the biological problem) the PML-RARA fusion is diagnostic of a specific subtype of AML known as acute promyelocytic leukemia, with a highly distinct cell phenotype reflecting a block on differentiation at the promyelocyte stage15. The distinct features of this subtype are correspondingly reflected in the L-shaped landscape for these two signatures and the different mechanisms by which these lesions drive leukemogenesis.\n\nThe L-shaped landscape suggests the molecular mechanisms underlying these mutations are mutually exclusive.\n\n\nSummary\n\nThe singscore package provides an easy interface to apply gene set scoring methods within the R/Bioconductor environment. The TCGAbiolinks package allows relatively easy access to large clinically relevant data sets such as TCGA, together with appropriate annotation functions for interpreting biological data. Diagnostic and plotting functions included with singscore allow the user to investigate gene sets of interest to determine their power for distinguishing differences between samples. Different gene signatures can then be combined to explore how different cellular phenotypes are associated across a large cohort of cancer samples. As demonstrated, when appropriate gene set signatures are used, metrics calculated by singscore can be used for sample classification and this may be useful for further interrogation of large transcriptomic data sets where no genomic data are available.\n\n\nPackages used\n\nThis workflow depends on various packages from version 3.9 of the Bioconductor project, running on R version 3.6.0 (2019-04-26) or higher. The complete list of the packages used for this workflow are shown below:\n\n\n\n\nData availability\n\nAll data analyzed in the workflow is read automatically from public websites as part of the code. Mutation data for the samples in this study are available as part of the R/Bioconductor package SingscoreAMLMutations (v1.0.0).\n\n\nSoftware availability\n\nSoftware available from: https://bioconductor.org/packages/release/workflows/html/SingscoreAMLMutations.html.\n\nSource code available from: https://github.com/DavisLaboratory/SingscoreAMLMutations/.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.310880710.\n\nLicense: Artistic License 2.0.\n\n\nAuthor information\n\nDDB, JC and MJD wrote the article. DDB wrote the code for the workflow and the associated R/Bioconductor workflow package SingscoreAMLMutations. MF, JC, DDB and MJD conceived the idea for the workflow. YX translated the vignette for the package vignette into chinese. YX and RL reviewed the chinese translation. All authors have tested and reviewed the workflow. This work was supervised by JC and MJD.", "appendix": "Grant information\n\nThis work was partially supported by the National Health and Medical Research Council (NHMRC) Project Grant APP1128609. MJD was supported by the NBCF Career Development Fellowship ECF-14-043 and the Betty Smyth Centenary Fellowship in Bioinformatics.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. Thanks to Christoffer Flensburg of the Walter and Eliza Hall Institute for advice on the TCGA AML mutation data.\n\n\nReferences\n\nCieślik M, Chinnaiyan AM: Cancer transcriptome profiling at the juncture of clinical translation. Nat Rev Genet. 2018; 19(2): 93–109. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Lun AT, Smyth GK: From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline [version 2; peer review: 5 approved]. F1000Res. 2016; 5: 1438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaw CW, Alhamdoosh M, Su S, et al.: RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR [version 3; peer review: 3 approved]. F1000Res. 2016; 5: pii: ISCB Comm J-1408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOshlack A, Wakefield MJ: Transcript length bias in RNA-seq data confounds systems biology. Biol Direct. 2009; 4(1): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao S, Zhang B: A comprehensive evaluation of ensembl, RefSeq, and UCSC annotations in the context of RNA-seq read mapping and gene quantification. BMC Genomics. 2015; 16(1): 97. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "49410", "date": "04 Jun 2019", "name": "Marina Lizio", "expertise": [ "Reviewer Expertise Transcriptome analysis", "Gene expression regulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors Bhuva et al. presented the application of a previously published gene set scoring tool, singscore, to discriminate AML subsets based on gene expression signatures, using signatures derived from known specific gene mutations or fusions. The authors show that singscore can effectively separate AML samples based on such gene signatures.\nGiven the ability of the software to discriminate samples within heterogenous diseases, such as leukemias, its application to other cancers and diseases with similar levels of heterogeneity can be beneficial to improve patients' clinical outcomes.\nThe rationale behind the application of the software and its step-wise descriptions are well explained. So is the interpretation of results. I appreciate the authors' efforts to explain all the steps and suggest alternatives, giving the software description the feel of a tutorial.\nI have some small comments on some points I'd like to see addressed:\nTitle. As far as I have understood, singscore doesn't actually predict mutations. Rather, it uses gene signatures associated to certain mutations in order to identify sub-groups within a population. As it is, the title is misleading. Similar rationale about the title of section \"Predicting mutations using the Verhaak signature\", page 16. In the section \"Filter out genes with low counts\", page 7, the authors describe explicitly how to handle duplicated entries caused by the GDCprepare function. Here I would reorganise and shorten the paragraph by first mentioning the issue introduced by the function, then saying edgeR is applied to the data filtered of duplicate entries. Figure 3. The legend below each graph is identical. I suggest to only use one legend for all three plots. The authors comment on several instances about the similarities between scores for signatures of two subgroups of AML samples, NPM1c and KMT2A-/MLL-fusion, and that's due to the presence of Hox genes. I suggest to have the lists of the two gene signatures directly in the article for immediate lookup. This is not essential, as the authors already refer to the articles they take the data from, but I think it will help readers.\nMinor comments:\n\nTypo in the caption of figure 1 (approcimately instead of approximately). Typo in figure 2 caption. I suppose \"scores are plot against...\" should read \"scores are plotted against...\".\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4842", "date": "16 Aug 2019", "name": "Melissa Davis", "role": "Author Response", "response": "We would like to thank Dr Marina Lizio for her very useful review, and we have addressed her comments as outlined below. ML: I have some small comments on some points I'd like to see addressed: 1. Title. As far as I have understood, singscore doesn't actually predict mutations. Rather, it uses gene signatures associated to certain mutations in order to identify sub-groups within a population. As it is, the title is misleading. DDB: This is an excellent point. We have now changed the title to “Using singscore to predict mutation status in AML from transcriptomic signatures” in order to clarify that we are simply predicting the mutation status of samples, not actual mutations. 2. Similar rationale about the title of section \"Predicting mutations using the Verhaak signature\", page 16. DDB: A similar change is made here, the title of this section is now: “Predicting NPM1c mutation status using the Verhaak signature”. 3. In the section \"Filter out genes with low counts\", page 7, the authors describe explicitly how to handle duplicated entries caused by the GDCprepare function. Here I would reorganise and shorten the paragraph by first mentioning the issue introduced by the function, then saying edgeR is applied to the data filtered of duplicate entries. DDB: The issue of duplicated features with the TCGAbiolinks package has been fixed in a recent update (TCGAbiolinks v2.12.2). Consequently, we will be removing the text describing the identification and removal of duplicated features. The text removed is: ------------------------------ Creation of a DGEList object is initially unsuccessful due to duplicated row names in the data matrix (107 features). The raw count files for individual samples do not have any duplicated features indicating this is introduced by the `GDCprepare` function. This is further supported by the fact that the counts for a single feature are the same for duplicated entries. An example of such a feature is shown below and we have verified duplicated entries of all other features to be the same. ```{r} dup_features = rownames(aml_se)[duplicated(aml_se)] ``` ```{r dup_entries} library(SummarizedExperiment) #example check on one feature ID dup_feature = 'ENSG00000011454' dup_data = assay(aml_se[rownames(aml_se) %in% dup_feature, ]) identical(dup_data[1, ], dup_data[2, ]) ``` As such, it is safe to discard duplicated entries. ------------------------------ We also remove the code that discards duplicate features ------------------------------ aml_se = aml_se[!duplicated(rownames(aml_se)), ] ------------------------------ 4. Figure 3. The legend below each graph is identical. I suggest to only use one legend for all three plots. DDB: We appreciate this point, however each plot in this figure is generated by a function in the package which includes the legend. For a normal use case, users would be generating a single plot to diagnose a single sample, which is why we include a legend for the plot. In this workflow, we simply combine the three plots into a single figure. Removing legends from the default plots would overcomplicate the code of this diagnostic procedure in the workflow which is why we decided to leave the legends as they are. If users were interested in manipulating figures in their application, they could add extra options to the ggplot2 object (as we show with adding a new title and modifying the legend to be a single column). 5. The authors comment on several instances about the similarities between scores for signatures of two subgroups of AML samples, NPM1c and KMT2A-/MLL-fusion, and that's due to the presence of Hox genes. I suggest to have the lists of the two gene signatures directly in the article for immediate lookup. This is not essential, as the authors already refer to the articles they take the data from, but I think it will help readers. DDB: The signatures we downloaded from MSigDB have Entrez IDs for genes. The NPM1c and MLL signatures contain 491 unique genes. The reason we did not have the lists in the article was that we would be presenting 491 numbers that would have been difficult to interpret without mapping. Additionally, our intention is for readers to execute the workflow while reading this article. In this scenario, the geneIds() function could be used to list the gene identifiers forming the signatures once they have been downloaded and parsed into R. Identifiers could be mapped to symbols using the mapIds() function in the AnnotationDbi package and an annotation such those provided by the org.Hs.eg.db R/Bioconductor package. We hope that the ease with which these data can be accessed in the application will make the lists accessible for readers. ML: Minor comments: 1. Typo in the caption of figure 1 (approcimately instead of approximately). DDB: Thanks for helping us identify this, we have fixed it now. 2. Typo in figure 2 caption. I suppose \"scores are plot against...\" should read \"scores are plotted against...\". DDB: This has also been changed. Many thanks for the useful and constructive review. Best wishes, Mr Dharmesh Bhuva Bioinformatics Division The Walter and Eliza Hall Institute Dr Melissa Davis Bioinformatics Division The Walter and Eliza Hall Institute" } ] }, { "id": "51547", "date": "05 Aug 2019", "name": "Luiza Handschuh", "expertise": [ "Reviewer Expertise Genomics", "transcriptomics", "biology of acute myeloid leukemia." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction The article presents a new package, which was developed from a previous one (‘singscore’) elaborated by the same authors. The package ‘singscore’ is a single-sample gene set scoring method which is valuable for analysis of transcriptomes of samples collected through the long time and not sequenced in the same run, experiment, platform or laboratory. Here, the authors apply the method for classification of TCGA acute myeloid (AML) samples using transcriptional ‘gene signatures’ identified by other authors (Verhaak and Ross) as typical for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions.\nAML is a heterogeneous and multi-clonal disease. The transcriptome picture of AML is very complex and can result from different mutations, genomic rearrangements, and aberrant regulation of gene expression at different levels (see my last review paper1). Sometimes, the gene expression profiles can overlap between samples with different genetic lesions. The examples are HOX-gene-based discriminative signatures determined by Verhaak and Ross, not limited to AML with mutated NPM1, but specific also for AML cases with 11q23 abnormalities and KMT2A (MLL) gene rearrangements. The authors are aware of this fact and underline it in the article, too. On the other side, samples with the same mutation can present varying expression profiles because of additional features which also affect the transcriptome. Therefore, interpretation of the results must be done with caution. For me, the authors interpret the results a little bit too optimistically. The presented method is not sufficient to determine the mutation of interest, but can be used as a supplementary approach, a screening tool or (in future) a personalized medicine tool which could classify patients based on transcriptomic profiles, associated with specific treatment response.\nTesting package I have used the package in R with TCGA data and confirm the code works, it is fast and generates the same results and plots as presented in the article. Moreover, I have tested the whole procedure on my own RNA-seq dataset (not published yet but used for supplementary expression analysis of NPM1 alternative transcripts, see the article2). My dataset contains 28 AML samples, including 8 with NPM1 mutation (verified with three independent approaches). The data load was more tricky as I started from the csv table with counts and I had to convert the DESeqDataSet object into the RangedSummarizedExperiment object. The best discrimination was achieved with the Verhaak signature, but only with the down-regulated genes. All samples with NPM1 mutation were clearly separated from others, however, 2 additional samples, without NPM1 mutation, were grouped together with NPM1c. It is possible they have KMT2A (MLL) rearrangements, but I cannot verify it now. Analyzing the plots, up-regulated genes and all genes form the signature are not as efficient, separating only 2 or 3 NPM1c samples from the rest. In the article, the authors also admit that the set of down-regulated genes has more discriminant power, but they claim up-regulated genes also contribute to the discrimination.\nI have no samples with PML-RARA fusion in my dataset and the status of KMT2A (MLL) gene was unknown in my patients, so I could not compare the efficiency of signatures other than NPM1c. APL with PML-RARA is the most distinctive AML subtype which is easily separated from other AML samples based on transcriptome profile, so I would expect good results. This example shows the package works well for samples with very specific gene expression profile.\nInterpretation of the results Considering the result interpretation, I have some doubts. The singscores are composed of two components, an enrichment score and a dispersion estimate of ranks. I am informed that “high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores”. This is logical. I also know the maximal ranges ([−1, 1] for signatures involving up- and down-regulated genes). However, which value is really high? For example, is 0.2 enough (it looks like from the plots in the article and from my own data, too) or maybe I should expect much more, e.g. 0.7? Similarly, which value of dispersion should I expect? I suppose, the lowest, but which is low enough or which range is optimal? What is difficult to understand is that “despite the range of scores increasing, the discriminant power drops moderately” (for up-regulated genes from Verhaak signature, when compared to down-regulated genes). I see the same paradox in my data – the scores are higher for up-reg. genes, which are much less efficient in discrimination between NPM1c+ and NPM1c- samples. It looks like scores only do not reflect the trends observed from score vs dispersion plots.\nPossible improvements Although the package is generally useful and well described, the following improvements will make it more friendly for less advanced R users, e.g.\nInstruction how to prepare and load the data other than those deposited in the GDC database.\n\nAnnotation of samples on the plots with unique sample IDs (when a sample is misclassified, a user does not know which one it is; it will also be helpful to localize a sample on various plots, e.g. generated with a signature of up- and down-regulated genes) – it is written in the article that the ‘singscore’ package supports different types of annotations (“Annotations of interest can be overlayed on each plot”) but I found only color code annotation whereas I would like to have color code for mutation type and additional text labels with sample IDs on the same plot.\n\nA command which lists the samples typed as strong candidates for having particular mutation, ordered according to the calculated metrics.\n\nGenerating a threshold line between samples with and without mutation.\nIn future, it would be also good to include other signatures, e.g. the signature of 369 genes identified by Alcalay et al. in 2005 3, discriminating AML patients with NPMc+ from patients with NPMc-, even in cases with rare chromosomal abnormalities.\nWhat I would expect the most from a package designed to identify mutations in transcriptomes, is a direct identification of mutations in RNAseq data. The results of mutation typing based on gene expression profile will be strongly supported when a particular mutation will be covered by RNAseq reads. From my own experience, I know it is possible (for NPM1 mutation detection, see 4). For genes with high and middle expression level, the coverage can be even higher than that obtained from genome- or exome-level data. And in the case when DNA data are unavailable, it would be really fantastic. Because it demands completely different data processing, it may be considered for future versions of the package.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "4971", "date": "15 Oct 2019", "name": "Melissa Davis", "role": "Author Response", "response": "We thank the reviewer for their time and effort directed towards reviewing our manuscript and for the helpful feedback they have provided. Where appropriate we have modified our manuscript to address the reviewer’s comments. Below we include a point by point response to the reviewer’s comments and where appropriate we list corresponding changes to the manuscript. IntroductionLH: The article presents a new package, which was developed from a previous one (‘singscore’) elaborated by the same authors. The package ‘singscore’ is a single-sample gene set scoring method which is valuable for analysis of transcriptomes of samples collected through the long time and not sequenced in the same run, experiment, platform or laboratory. Here, the authors apply the method for classification of TCGA acute myeloid (AML) samples using transcriptional ‘gene signatures’ identified by other authors (Verhaak and Ross) as typical for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions.DDB: We note that the purpose of the initial manuscript may have been unclear, in part because it is listed under the F1000 Software Tool Article section. The purpose of this article is to present a workflow demonstrating the use of singscore, and it is a R/Bioconductor workflow illustrating the use of singscore, and so is not intended as a new package or tool.https://www.bioconductor.org/packages/release/workflows/https://www.bioconductor.org/packages/release/workflows/html/SingscoreAMLMutations.htmlSome of the review comments below assume that this manuscript presents a specific software package for detecting NPM1 mutations, so we have clarified the manuscript as outlined below in order to make the purpose and intention more clear (note that the title of the manuscript has changed from ‘predicting mutations’ to ‘predicting mutation status’ based upon feedback from reviewer 1). This work arose from an observation in another project that the Verhaak signature scored with singscore appeared to correlate strongly with mutation status, and we thought this would be an interesting example that might also help researchers to investigate the links between genetic lesions and corresponding transcriptional changes, an area in which the reviewer clearly has expertise. LH: AML is a heterogeneous and multi-clonal disease. The transcriptome picture of AML is very complex and can result from different mutations, genomic rearrangements, and aberrant regulation of gene expression at different levels (see my last review paper1). Sometimes, the gene expression profiles can overlap between samples with different genetic lesions. The examples are HOX-gene-based discriminative signatures determined by Verhaak and Ross, not limited to AML with mutated NPM1, but specific also for AML cases with 11q23 abnormalities and KMT2A (MLL) gene rearrangements. The authors are aware of this fact and underline it in the article, too. On the other side, samples with the same mutation can present varying expression profiles because of additional features which also affect the transcriptome.LH: Therefore, interpretation of the results must be done with caution. For me, the authors interpret the results a little bit too optimistically. The presented method is not sufficient to determine the mutation of interest, but can be used as a supplementary approach, a screening tool or (in future) a personalized medicine tool which could classify patients based on transcriptomic profiles, associated with specific treatment response.DDB: We agree with the reviewer that gene set scoring of transcriptomic data should not be the only method used to identify patient samples carrying genetic lesions. The Bioconductor workflow we present is intended to provide an example of applying singscore to study mutation/fusion based gene sets as we believe that singscore provides a relatively flexible and intuitive approach for investigating different gene sets across large data sets. We feel that a particularly useful feature is the ability to combine different signatures/gene sets (including phenotype/cell-cycle signatures etc) to explore how these transcriptional changes are associated over different samples, demonstrated in Figures 5-7.We have modified the “Description of biological relevance” section to address the reviewer’s comments around the complexity of AML genomic lesions and corresponding transcriptomic changes. The review highlighted is particularly relevant for this work and accordingly we mention the complexity of AML and direct the reader towards this resource by extending the first paragraph (new text underlined):.. A more recent study which has focused primarily on genomic data has further refined the clinically significant AML subtypes [Papaemmanuil (2016), NEJM], highlighting a number of co-occurring as well as mutually exclusive mutations. As the identification of putative driver fusions/mutations continues, work has also been directed towards how these lesions interact with each other and other features (e.g. cellular proliferation, changes due to phenotypic plasticity, or variation in post-transcriptional regulators such as microRNAs) to drive transcriptional changes as discussed in a recent review [Hanschuch (2019), J. Oncol.]. We have also added a paragraph at the end of this section which discusses some of the limitations for our approach and provides more context in which it could be applied:While we demonstrate that singscore is capable of inferring mutation status from the transcriptional profile of AML samples, we note that it is best used to supplement alternative data which can provide a more definitive resolution of these lesions. Processing of raw RNA‑seq data will directly identify the presence of gene‑fusion products or mutations within protein-coding regions, although for many large data sets the quantified transcript abundance data are much easier to obtain without access agreements. The method can also be applied to legacy microarray data sets where genome and RNA sequencing data are unavailable. As such singscore provides a useful approach to supplement established methods for the study of genetic lesions in cancer. By exploring associations between different genomic and phenotypically relevant signatures, it may also help to better characterise true driver mutations which exert consistent effects on the transcriptome of AML samples and other cancers. Testing packageLH: I have used the package in R with TCGA data and confirm the code works, it is fast and generates the same results and plots as presented in the article. Moreover, I have tested the whole procedure on my own RNA-seq dataset (not published yet but used for supplementary expression analysis of NPM1 alternative transcripts, see the article2). My dataset contains 28 AML samples, including 8 with NPM1 mutation (verified with three independent approaches). The data load was more tricky as I started from the csv table with counts and I had to convert the DESeqDataSet object into the RangedSummarizedExperiment object. The best discrimination was achieved with the Verhaak signature, but only with the down-regulated genes. All samples with NPM1 mutation were clearly separated from others, however, 2 additional samples, without NPM1 mutation, were grouped together with NPM1c. It is possible they have KMT2A (MLL) rearrangements, but I cannot verify it now. Analyzing the plots, up-regulated genes and all genes form the signature are not as efficient, separating only 2 or 3 NPM1c samples from the rest. In the article, the authors also admit that the set of down-regulated genes has more discriminant power, but they claim up-regulated genes also contribute to the discrimination.LH: I have no samples with PML-RARA fusion in my dataset and the status of KMT2A (MLL) gene was unknown in my patients, so I could not compare the efficiency of signatures other than NPM1c. APL with PML-RARA is the most distinctive AML subtype which is easily separated from other AML samples based on transcriptome profile, so I would expect good results. This example shows the package works well for samples with very specific gene expression profile.DDB: We thank the reviewer for the exceptional effort and time invested to test our workflow on independent data - we hope results from this analysis have been informative for identifying other features within their data. Our workflow includes guidance for users who wish to import data from other sources such as those used by the reviewer. Interpretation of the resultsLH: Considering the result interpretation, I have some doubts. The singscores are composed of two components, an enrichment score and a dispersion estimate of ranks. I am informed that “high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores”. This is logical. I also know the maximal ranges ([−1, 1] for signatures involving up- and down-regulated genes). However, which value is really high? For example, is 0.2 enough (it looks like from the plots in the article and from my own data, too) or maybe I should expect much more, e.g. 0.7? Similarly, which value of dispersion should I expect? I suppose, the lowest, but which is low enough or which range is optimal? What is difficult to understand is that “despite the range of scores increasing, the discriminant power drops moderately” (for up-regulated genes from Verhaak signature, when compared to down-regulated genes). I see the same paradox in my data – the scores are higher for up-reg. genes, which are much less efficient in discrimination between NPM1c+ and NPM1c- samples. It looks like scores only do not reflect the trends observed from score vs dispersion plots.DDB: Interpretation of singscores is intentionally left to be problem specific as it generally requires some domain-specific knowledge of the biological system and corresponding signature genes – ideally the computational biologist or bioinformaticians working on each project can provide some guidance.The basic interpretation of the score is the normalised mean rank of genes within the signature relative to all other genes in the sample. At the extrema this interpretation is relatively simple - near 1, a higher value would indicate that genes in the signature are expressed at higher levels relative to other genes. For scores towards zero, however, the interpretation can be much more difficult – a score of zero (on the range [-1,1]) could indicate the signature genes are tightly clustered around the sample-wide mean abundance, or it could indicate a highly-dispersed almost uniform distribution across the entire abundance range (with a mean near the mean of all genes). By exploring the singscores together with dispersion estimates this information is summarised, assisting in estimation of effect size variability. Interpretation of scores depends on the context of the experiment and the typical behaviour of the gene set. A “high” score is best determined relative to other samples. This is achieved either by comparing scores from other samples in large datasets such as TCGA, or better, across a set of samples from a given experiment with known conditions. Other methods normalise the data before computing scores, and we note that a recent paper has applied z-score normalisation to results from singscore for comparison to ssGSEA [Cui et al (2019) Oncogene, DOI: 10.1038/s41388-019-1026-9]. All singscores for samples remain the same and do not have to be recomputed upon addition of new samples, and interpretation will improve as more samples are added to the study. For example, Gaussian mixture modelling could be used to separate the NPM1c scores based on our expectation that there are two groups. This could be switched with other unsupervised classification algorithms such as hierarchical clustering or k-means clustering. We have added an example analysis to the manuscript to demonstrate how scores can be interpreted in an unsupervised setting, under the section “Transcriptional signatures to predict mutation status/Unsupervised classification of mutations”.There may be some cases where sample annotation is not available. In such scenarios, we are unable to build regression models to interpret scores. A higher singscore would provide stronger evidence for the signature but the magnitude is difficult to interpret without a reference. One approach to deal with this situation is to compare scores to those from other datasets where the mutations status is known. An alternative approach would be to compare scores within the dataset using unsupervised learning methods. Here we demonstrate the use of three clustering methods (Gaussian mixture decomposition, k-means clustering, and hierarchical clustering) to stratify samples, and as we have done previously [wang et al (2012) Journal of clinical bioinformatics] use the adjusted Rand index (ARI) to compare classifications. As expected, supervised (GLM) classification results in the best prediction. This is followed by clustering based on the score using Gaussian mixture decomposition. Any other classification algorithm along with prior knowledge could be used to decompose scores into groups. The important characteristic of singscores is that they maintain the discriminant power of gene signatures therefore can be coupled with supervised, semi-supervised or unsupervised algorithms to perform stratification.```library(mclust) #Gaussian mixture modelm1 = Mclust(scoredf$Score, G = 2, verbose = FALSE)#k-means clusteringm2 = kmeans(scoredf[, 5:6], centers = 2, nstart = 100)#hierarchical clusteringm3 = hclust(dist(scoredf[, 5:6])) mutation_inference = cbind( 'GLM' = prediction, 'mclust' = m1$classification, 'k-means' = m2$cluster, 'hclust' = cutree(m3, k = 2))apply(mutation_inference, 2, adjustedRandIndex, scoredf$NPM1c.Mut)```Possible improvementsLH: Although the package is generally useful and well described, the following improvements will make it more friendly for less advanced R users, e.g.LH: Instruction how to prepare and load the data other than those deposited in the GDC database.DDB: We have noted in text that the rank matrix can be computed using either a SummarizedExperiment object, DGEList object, ExpressionSet object, numeric matrix or a numeric data frame. As such, a numeric matrix with sample names as column names and genes as row names would suffice. Scoring should be performed on length bias corrected measurements such as RPKM/FPKM or TPM and not CPM or raw counts.Transcriptional signatures to predict mutation status/Score TCGA AML samples using the Verhaak signature - extract from text: “The `rankGenes` function will compute ranks from expression data in the form of either a numeric matrix, numeric data frame, ExpressionSet object, DGEList object or a SummarizedExperiment object”LH: Annotation of samples on the plots with unique sample IDs (when a sample is misclassified, a user does not know which one it is; it will also be helpful to localize a sample on various plots, e.g. generated with a signature of up- and down-regulated genes) – it is written in the article that the ‘singscore’ package supports different types of annotations (“Annotations of interest can be overlayed on each plot”) but I found only color code annotation whereas I would like to have color code for mutation type and additional text labels with sample IDs on the same plot.DDB: Sample labels can be added to landscape plots but were not supported in other visualisations. We have added functionality to the latest version of the singscore package (v1.5.1) to allow labelling of samples in the score vs. dispersion plots. We have modified the text to clarify, and modified Figure 6 to label samples where classification uncertainty (NMP1c vs WT) is high to demonstrate this feature. The changes below have been made to the section: “Transcriptional signatures to predict mutation status/Diagnostics of the Verhaak signature”.… Sample annotations of interest (e.g. clinical annotations) can be colour coded on each plot. … Figure 6:```select_aml = !mutated_gene %in% 'Other' #label samples with an mclust NPM1c classification uncertainty of > 0.3label_samples = substr(rownames(verhaak_scores), 6, 12) #sample ID from barcodeslabel_samples[m1$uncertainty < 0.3] = NA #project mutations onto the landscapep1 = projectScoreLandscape( p_mll_npm1c, verhaak_scores, rossmll_scores, subSamples = select_aml, annot = mutated_gene[select_aml], sampleLabels = label_samples[select_aml])p1 + theme(legend.box = 'vertical')```LH: A command which lists the samples typed as strong candidates for having particular mutation, ordered according to the calculated metrics.DDB: As discussed in an earlier comment, we recommend such analyses to be problem specific. Generally, a higher score would indicate a stronger effect of genes in the signature relative to WT samples therefore samples with higher scores would be stronger candidates for mutations. Alternatively, the partitioning created using Gaussian mixture modelling could be used as a guide for separation and samples with a score much higher than the threshold would be the strongest candidates for the mutation.LH: Generating a threshold line between samples with and without mutation.DDB: See above discussion/recommendation.LH: In future, it would be also good to include other signatures, e.g. the signature of 369 genes identified by Alcalay et al. in 2005 3, discriminating AML patients with NPMc+ from patients with NPMc-, even in cases with rare chromosomal abnormalities.LH: What I would expect the most from a package designed to identify mutations in transcriptomes, is a direct identification of mutations in RNAseq data. The results of mutation typing based on gene expression profile will be strongly supported when a particular mutation will be covered by RNAseq reads. From my own experience, I know it is possible (for NPM1 mutation detection, see 4). For genes with high and middle expression level, the coverage can be even higher than that obtained from genome- or exome-level data. And in the case when DNA data are unavailable, it would be really fantastic. Because it demands completely different data processing, it may be considered for future versions of the package.DDB: As we have outlined at the start of this review there may have been some misunderstanding around the purpose of this Workflow package/paper. We agree with the reviewer that direct detection of mutations/fusions from RNA-seq data is the best approach, and we now note this in the ‘Description of biological relevance section’ as stated above. The other gene signatures mentioned above could be incorporated in a workflow as singscore supports analysis and comparison of multiple gene sets." } ] } ]	1	https://f1000research.com/articles/8-776
https://f1000research.com/articles/8-551/v1	25 Apr 19	{ "type": "Research Article", "title": "Effectiveness of a mobile health intervention on infant and young child feeding among children ≤ 24 months of age in rural Islamabad over six months duration", "authors": [ "Subhana Akber", "Hana Mahmood", "Razia Fatima", "Ahmed Wali", "Ashraful Alam", "Syed Yahya Sheraz", "Aashifa Yaqoob", "Hina Najmi", "Saleem Abbasi", "Humaira Mahmood", "Michael J. Dibley", "Tabish Hazir", "Hana Mahmood", "Razia Fatima", "Ahmed Wali", "Ashraful Alam", "Syed Yahya Sheraz", "Aashifa Yaqoob", "Hina Najmi", "Saleem Abbasi", "Humaira Mahmood", "Michael J. Dibley", "Tabish Hazir" ], "abstract": "Background: Childhood development is highly influenced by feeding practices at the infancy and young age of children. Unfortunately, according to the National Nutrition Survey (2011), the prevalence of exclusive breastfeeding in Pakistan was 21% at four months, and 13% at six months of age with 51.3% of mothers initiating semisolid foods to their children at the recommended 6-8 months of age. The latest Pakistan Demographic & Health Survey (PDHS 2018) however; indicates that only 48% of infants are exclusively breastfed which has been improved from 38% as reported in the past five years but still more improvement is anticipated. Methods: A quasi-experimental study design was employed for this post-intervention survey assessing effectiveness of mobile health (mhealth) regarding infant & young child feeding (IYCF) among pregnant and lactating mothers in Tarlai, Islamabad from May to June 2018. A total of 135 mothers who were earlier included in the intervention phase were recruited after obtaining verbal & written consent. The data was entered in EpiData (3.1) and analyzed in SPSS version 21. Results: The mean age of these pregnant and lactating mothers was 30.5 years ± 4.5 SD with the majority of mothers in the age group of 25 to 29 years. After the intervention, the overall knowledge of mothers regarding IYCF nutrition was raised to 69.6% among 94 mothers as compared to 74 (54.8%). Overall attitude regarding IYCF was found to be positive among 86 (63.7%) of the mothers, whereas 88 (65.2%) of the mothers had good IYCF related practices. Conclusion: Our post-intervention survey signifies the effectiveness of mhealth in raising knowledge, attitude, and practices of mothers regarding IYCF in rural Islamabad. However, implementation of mhealth in masses requires future research specifically to address the cost-effectiveness of such interventions in maternal & child health programmes.", "keywords": [ "Mobile health", "mhealth", "IYCF nutrition", "Operational research", "Islamabad", "Pakistan" ], "content": "Introduction\n\nChildhood under-nutrition is a major public health problem which has been contributing extensively to childhood mortality and morbidity1. Globally; 45% of child mortality results due to‘undernutrition’ which highlights the right of every child to good nutrition. According to World Health Organization (2018), globally more than 100 million children were found to be stunted, and nearly 52 million were found to be wasted in 2016 alone. Adequate nutrition is required for optimal growth and development of children2. Evidence indicates that under-nutrition leads to severe cognitive and behavioural disabilities throughout life if not managed in early infancy3–5. The magnitude of malnutrition is extensive in the South Asian region leading to high rates of stunting, wasting, and disease burden5. One of the major causes of these high indicators of undernutrition is poor infant and young child feeding (IYCF) practices. The WHO and United Nations International Children’s Emergency Fund (UNICEF) recommends early initiation of breastfeeding within an hour after birth, and exclusive breastfeeding for the first 6 months during infancy with timely and appropriate initiation of complementary feeding6. Despite this recommendation, the recognized adverse effects of malnutrition and undernutrition among infants and children have been significantly reported in various studies7–9.\n\nRegarding infant and young child feeding practices, around 40% of infants from 0–6 months of age had been exclusively breastfed, worldwide. Whereas, only a few children acquire adequate nutrition along with proper complementary feeding which is appropriate for their age group they belong to 6. In developing countries, these sub-optimal feeding practices of infants and young children contribute to the prevailing burden of malnourishment. According to the Pakistan Demographic Health Survey (PDHS, 2018), almost 38% of children under five years of age are stunted, 23% are underweight, and 7% are wasted. Although the national findings of PDHS (2018) indicate that improved nutritional status of children has resulted in decline of stunted children from 45% in 2012–201310. However, the findings are still alarming. The suboptimal IYCF practices could be attributed to a lack of knowledge, lower socio-economic status, and relatively low levels of education of mothers or caregivers11.\n\nEmphasis has been laid on implementing effective innovative interventions to improve nutrition among children particularly in poor resource countries. One of the most effective strategies laid down by WHO to improve IYCF practices is effective counselling on proper nutritional practices through community health workers12,13. In Pakistan, these community health workers are referred to as Lady Health Workers (LHWs), recruited under the National Program for Family Planning and Primary Health Care14. With support from WHO, the government of Pakistan launched the ‘Lady Health Workers Programme’ in 1994, which was mainly aimed to provide an effective grassroot level system for accessing primary health care14. This program was aspired to bridge the communities for accessing primary healthcare through LHWs. Moreover; among the various roles and responsibilities under this program, the LHWs are also expected to provide nutritional counselling. However, the deliverables by LHWs somehow are affected due to being overburdened15.\n\nThus, it is imperative that a facilitating system if is provided to these LHWs which could result to lower their existing burden of responsibilities. Among some of the innovative strategies of providing health service, mobile health or mhealth, is gaining momentum in low- and middle-income countries. As defined by WHO mhealth is the “provision of health services and information via mobile and wireless technologies”16. The innovation and use of information and technology through mhealth has been vastly employed to address access, resource utilization, and coverage gaps. Many developing countries including those in South Asia have been employing mhealth approach through the Community Health Workers or peer counsellors to improve healthcare as an innovative strategy17,18. A study conducted in Bangladesh demonstrated gaps in IYCF related service delivery which prompted the need of healthcare messages, including information related to emergency and medical care, to be delivered through mobile phones. The potential benefits and necessity of mhealth led the technology to embrace community-based nutrition services to improve the service delivery and coverage related to IYCF nutrition. Mass-scale behavioural interventions that actively included social mobilization at the community-level, media campaigns, and counselling by trained workers have also been found useful18–20. Evidence from India suggest that IYCF related nutrition among children can be improved using counselling strategies aimed at the parents21. Limited evidence is available from Pakistan indicating effectiveness of mhealth related to IYCF nutrition. Therefore, considering the current situation in Pakistan, we planned to pilot this technology with counselling by LHWs. For this intervention we first conducted a formative study in collaboration with Lady Health Worker (LHW) programme, which aided in development and implementation of a mhealth based program to counsel women on proper nutritional practices related to infant and young children (IYCF) in a rural periphery located in Islamabad. In this case, a user-friendly audio-visual android-based mobile application was developed for LHWs who were then trained and supervised on its use.\n\nOur mhealth intervention was deployed on pregnant and lactating mothers from July 2016 to December 2016 in rural Islamabad. The aim was to test the feasibility and acceptance of mhealth intervention among the target population. A pre-intervention survey was conducted one month prior to the intervention in June 2016 followed by a post intervention survey conducted in April to June 2018 to determine the effectiveness of mhealth intervention in improving infant and young child feeding (IYCF) nutrition related knowledge, attitude and practices among pregnant and lactating mothers in rural Islamabad (Both surveys are available as Extended data22). The specific objective of this post-intervention study was to compare the pre and post mhealth intervention related knowledge, attitude & practices of pregnant and lactating mothers regarding IYCF.\n\n\nMethods\n\nA quasi-experimental study design was employed to determine the effectiveness of mhealth in improving knowledge, attitude, and practices of pregnant and lactating mothers regarding IYCF in rural Islamabad.\n\nIslamabad is the federal capital territory of Pakistan. According to census conducted in 2017, the total population of Islamabad is more than 2 million23. The rural population of Islamabad comprises of 991,747 individuals with approximately 165,490 number of households24. This study was conducted in Tarlai Kalan which is a rural union council in Islamabad which comprises of around 37,500 households.\n\nAs mentioned, our intervention was deployed to pregnant and lactating women residing in Tarlai, Islamabad (See Figure 1). The study area is covered by the Lady Health Workers (LHWs) who are considered as the first level healthcare providers in this community. Upon availability and approval from the district health office, ten LHWs were randomly selected and trained on IYCF. Out of these ten LHWs, five were selected on basis of their best performance during the IYCF training. The sampling frame was based on these five selected LHW-wise households. Pregnant or lactating mothers residing within the catchment area of Tarlai, Islamabad and have children of ≤2 years of age were recruited and registered. Their husbands were also invited to participate and after obtaining consent the post-intervention study was conducted. Non-residents, non-consenting cases and mothers with serious co-morbidities were excluded from the study.\n\nA user-friendly audio-visual android based mobile application was developed which contained the formulated messages related to IYCF. The content of messages was prepared and translated into local language ‘Urdu’ after extensive review by experts, which were mainly based on WHO and UNICEF guidelines13,25.\n\nIntervention Design: Based upon the results of phase one study (pre-intervention survey), it was decided that biweekly voice and text messages on appropriate IYCF practices were to be disseminated to the recruited pregnant and lactating mothers, along with their mothers in law and husbands. The project team was then trained by a mobile application developer to create their application along with the message content for the voice and text messages.\n\nMessages: A team of public health professionals, paediatricians, health informatics professionals, academicians and program managers formulated the messages based on WHO and UNICEF guidelines. These messages were initially drafted in English and then translated into Urdu which was identified as preferred language in phase one. The messages were drafted for pregnant and women in their third trimester for lactating mothers of children between 0-24 months of age. An age and stage model was employed such that the messages were to be disseminated according to the week of pregnancy or age of the child as the case may be. Once these messages were created, they were incorporated into a specialized message scheduling system whereby separate audio and text based message libraries were created to be sent to recipients.\n\nMobile Application: The application was created over a period of 3 months with testing to optimise the user experience. The application had two modules which were registration and follow up. Each module further included two sections, one for the pregnant women from their third trimester and one for children 0–12 months. The questions within each module were drafted in Urdu and included logical checks and errors based on the responses to avoid errors in data entry. The LHWs were to first register pregnant women or mothers of children of 0–12 months of age using the registration form. The included data consisted of name of the mother and the child, age, gender of the child, date of birth, last menstrual period (in case of pregnant women), address, phone number and dietary habits. From the next visit onwards, they were instructed to use the follow up forms to collect monthly data on their dietary intake, supplement intake (in case of pregnant women) and any associated illnesses or problems. A laptop/desktop-based dashboard was also created to import data collected in the field by the LHWs. The same dashboard also had the capacity to monitor the LHW activity through display activity times, number of forms sent, and time spent with each mother. Through the same dashboard the project manager had the capacity to create mobile application users. After creation of the mhealth application, it was installed into android phones and following pretesting by the team members it was then modified.\n\nOnce the application was ready and the message library was created, the project team sought written permission from the Federal District Health Office (DHO) to recruit the LHWs of Union Council of Tarlai Kalan for the intervention. The DHO assigned an assistant district coordinator who assisted the project team in recruiting the LHWs. Upon availability of the LHWs, a three-day training workshop was scheduled in a health house. A health house is a household of LHWs. The agenda of the training was to first educate the LHWs on IYCF, explain the objective of the intervention and train them on using the application. A pre- and post-test on knowledge on IYCF was also conducted during the training. This was followed by a field visit to test the use of the application in the field. After obtaining the national identity card copies of the selected LHWs, they were then provided with android-based smart phones along with the SIMS and mhealth application installed. Consent to be a part of the was also obtained for participating in the project, along with their National Identity card copies (this is required by the Pakistan Telecommunication Authority (PTA) for provision of sim cards).\n\nUpon selection, the participating LHWs were then requested to provide a list of eligible pregnant and lactating mothers for the project within their catchment area. These comprised of three groups including all pregnant women in their third trimester, children from 0–6 months of age and those mothers who had children of 7–12 months of age. Before including their names in the study, the LHWs were advised to describe the purpose of the mhealth project to the respondents or the caretakers (which were assumed as those individuals who were responsible for the care of infant or the child at their homes). Only those individuals or caretakers who had consented to be a part of the intervention were included. On average, around 10–15 individuals’ names were provided under each group by each LHW. Once the lists were provided, three individuals under each group for each LHW were selected and assigned to be included in the intervention through a lottery / draw method. These respondents were randomly picked out of all the recruited participants whose names were included in a draw box to receive the mhealth intervention. The participants consisted of primary participants who were pregnant women and mothers of children of 0–12 months of age followed by secondary participants who were mothers in law/grandmothers and husbands/fathers of the primary participants. The purpose of including the other family members was sociocultural. As in the first phase of the project; it was indicated that for a successful delivery of mhealth intervention, involving husbands and mothers in law will be very important. This was so that the participants would own the intervention and consider themselves as participants in the study.\n\nOnce the final list of participants was created, a one-day inaugural session was organized for them near to their place of their residence so as to brief them about the intervention. A pre-intervention survey was conducted after which they were provided with mobile phones along with SIM cards upon obtaining their National Identity Card copies and their written consent for participating in the study. Once all the information was obtained from the participants, it was then incorporated in the messaging system according to the stage of pregnancy and age of the child for the purpose of dissemination of IYCF nutrition related knowledge. From the next day, mhealth intervention was initiated as all the LHWs registered their participants and they were then followed up every month in their routine visits. They were also counselled and provided with information on using the mobile application and audio-visual aids which was generated based upon their responses to the questions being asked regarding IYCF.\n\nAt the same time, a weekly voice and text message was sent to the mobile phones of the participant to educate them on appropriate IYCF practices. The voice message was sent on Tuesday mornings to the phones of the females and evenings for the husbands/fathers, whereas the text message was sent on Thursday at the same time. The content of both the voice and text messages was the same to avoid confusion. Every month the pregnant women and mothers of children were also called up through our call centre to inquire about the routine LHW visit, whereby they were asked about the visit and whether if they had received the voice and text messages. They were also asked about the content of sent messages which they had received. A monitoring dashboard was used to monitor the LHW activity, whereby the project manager observed whether the visits were actually made using GPS coordinated data. Similarly, the time when the visit was made was also noted along with the time spent in each household. This intervention lasted for six months starting from July to December 2016 followed by a short post intervention research consisting of a focus group discussion with the mothers.\n\nA structured, post intervention questionnaire was used for data collection which was developed on the same lines as that of the pre-intervention survey. The initial version of questionnaire was developed through extensive review of literature and experts review19,25. The questionnaire’s Part A comprised of socio-demographic characteristics of the study participants which included age and education level of the women, number of children, family size, place of birth, and mode of delivery. Part B and C contained questions related to breastfeeding, exclusive breastfeeding, and complementary feeding.\n\nThe study variables related to knowledge, attitude and practices regarding IYCF were timely initiation of breastfeeding after birth, advisable duration of breastfeeding and exclusive breastfeeding, complementary feeding initiation and continuation, and practices related to prelacteal feeding. The data was collected through telephonic interviews which were indicated as a preference in our formative study. Only if the woman was unreachable via the phone were they then visited at their house for the interview which was facilitated by the respective LHW of the respondents’ catchment area.\n\nThe collected data was double-entered in EpiData software version 3.1. It was analyzed using SPSS version 21. The total sample size was 135 eligible women which were recruited based on the sampling frame created earlier for the intervention phase. Both descriptive and inferential statistics are reported in frequencies and percentages, including the percentage difference for pre and post knowledge, attitude, and practices related to IYCF nutrition.\n\nEthical clearance was obtained from Hospital Ethics Committee of Pakistan Institute of Medical Sciences (PIMS), Islamabad26. Informed consent (both written and verbal) was obtained from all study participants prior to their recruitment in the study. These women and their husbands were approached and were explained about the study purpose. Their queries to the study were addressed and they were provided with necessary information to contact in case of withdrawing from the study. They were also ensured about their privacy and confidentiality to be protected.\n\n\nResults\n\nTable 1 shows baseline characteristics of the 135 mothers, out of which 49 (36.3%) women belong to the age group of 25 to 29 years of age. The mean age of these pregnant and lactating mothers was 30.5 years ± 4.5 SD. Out of 135 women, 71 (52.6%) had 3 children and on average had 7 family members. Most women i.e. 59 (43.7%) had their education up to Matriculation and Intermediate followed by primary level of education for 48 (35.6%) of the women. The occupation status of 128 (94.8%) of these women was ‘unemployed’. The birth place of children as reported by 77 (57.3%) of women was a government facility, and the mode of delivery of 86 (63.7%) women was reported as ‘normal’. The common source of water was ‘tap water’ in the households according to 50 (37%) of women.\n\n* No Formal Education, LHW=Lady Health Worker\n\nThe findings of our survey elucidated that the mhealth intervention was effective in improving the overall knowledge of mothers regarding IYCF from 74 (54.8%) in 2016 to 94 (69.6%) in 2018 after the intervention (Table 4). However, the overall pre-intervention knowledge of 34 pregnant mothers regarding breastfeeding, exclusive breastfeeding and complementary feeding was 75.6%which decreased to 46.7% among 21of these women even after the mhealth intervention. This emphasizes the need of increasing awareness among pregnant women, in particular (Table 2).\n\nThe overall attitude regarding IYCF among 59 (43.7%) of the mothers before intervention, and among 86 (63.7%) of the mothers after intervention, was found to be positive. Whereas; overall practices of 22 (16.3%) mothers before intervention and 88 (65.2%) of the mothers after intervention were found adherent to good practices (Table 4). A noticeable percentage increase in knowledge related to prelacteal feeding considered as harmful and the benefits of colostrum was 28.2% and 23%, respectively (Table 2).\n\nA percentage difference of 46.4 was observed in attitude of mothers towards consistency of food consumed by their children, which was 12.1% before intervention, and was found to be adequate among 58.5% of the mothers after intervention. Furthermore, practices regarding complementary feeding and additional foods during the first six months of infancy were 0.0% before the intervention which was significantly raised to 66.7% among these mothers (Table 3). In addition, 55 (40.7%) of the mothers reported to be visited ‘once’ by LHW, followed by 49 (36.3%) of the mothers who were visited ‘twice’ on a monthly basis (Table 1). Pre- and post-intervention findings are available as Underlying data22.\n\n\nDiscussion\n\nFor child survival, growth and development, a key strategy is to improve infant and young child feeding (IYCF) related practices which is becoming an essential component of child health programs in various countries27. The results of our post-mhealth intervention survey regarding infant and young child feeding (IYCF) conducted in a rural territory in Islamabad yielded to be effective in improving the knowledge, attitude, and practices of pregnant and lactating mothers. Based on findings of our earlier research conducted on the same study population we found that community-based nutritional intervention such as ‘mhealth’ offer new opportunities for effective and efficient service delivery, resource utilization, and improving access to healthcare28.\n\nImproving infant and young child feeding (IYCF) practices in poor resource setting can be effectively contextualized through information technology involving mhealth. Specific socio-cultural barriers hindering the access of mothers to information related to IYCF must be overcome in order to reduce the prevailing burden of preventable malnutrition. A study by Akter et al., concludes that healthcare services can be augmented through the use of mobile phone-based technology such as mhealth29. It offers enormous opportunities for improving health indicators related to maternal, new born and child health specifically in rural settings. It was found in one study that mhealth or SMS-based health education could provide an essential chance to educate pregnant and lactating mothers about antenatal care (ANC) visits, child birth, and education related to family planning30. This indicates that there is a potential capacity to implement mhealth based IYCF which may render opportunities for scaling up the intervention in rural Islamabad. The findings of our survey elucidate that specific focus should be placed on the components of knowledge related to breastfeeding and exclusive breastfeeding during early infancy. However; relevance and quality of mhealth to other components of maternal and child health must be rigorously studied to promote the proliferation of mobile phones as a source of acquiring health information in developing countries.\n\nDespite the improvement in overall knowledge, attitude and practices of women related to IYCF in our study, certain important components related to breastfeeding showed steady findings. The knowledge of women regarding advisable duration of breastfeeding, early initiation of breastfeeding after birth, and timely complementary feeding initiation with additional food to be given to in early 6 months of infancy showed no significant change after mhealth intervention. This could be attributed to a prolong washout period after the deployment of mhealth intervention among the mothers or it can possibly subject to recall bias. Despite this more than a quarter of women in our study still practised and considered prelacteal feed such as honey and water as advantageous for the infant. This was found to be consistent to the findings of research studies conducted in Myanmar, Ethopia & India where prelacteal feeding was perceived as a cultural practice and was related to maternal beliefs31–34.\n\nThere seems to be a paucity of relevant available literature on assessing the effectiveness of mhealth, particularly in the context of infant and young child nutrition particularly in Pakistan28, which signifies its importance in implementing such interventions in poor resource settings. In contrast to our earlier research findings on testing the acceptance of mhealth among women residing in Tarlai Kalan Islamabad, a study from Sri Lanka demonstrated that women preferred to interact with healthcare providers on their maternity and child health needs35. In our study, the majority of women favoured the use of mobile phones to access information related to infant and young child feeding. On the basis of which, we therefore recommend scaling up of the health intervention in poor resource settings. Our study findings reflect that extensive mobile coverage has emerged as an innovative tool in rural Islamabad, and has acted as a facilitator which can effectively reach the underserved communities for providing health as well as education regarding infant and young child nutrition.\n\n• Overall, the strength of the deployed intervention lies in an increase in the practices of mothers related to IYCF nutrition\n\n• This was a novel intervention, the first of its kind in Pakistan\n\n• Additionally, we have managed to incorporate the intervention within the existing LHW program rather than having to create a new intervention. This would enable the intervention to be scaled up feasibly\n\n• One of our study limitations is that we conducted telephonic interviews which can introduce potential biases in responses of the mothers unlike in face-face interview approach.\n\n\nConclusion\n\nOur study indicates that community-based nutritional interventions using mhealth are innovative and effective in increasing IYCF related knowledge, attitude and practices among mothers. Cost-effectiveness of such behaviour change approaches and interventions should be assessed for future implementation in maternal and child health related programmes.\n\n\nSoftware availability\n\nThe source code of android phone-based application developed for the Lady Health Workers (LHWs) under the project “Sehatmnd Kl” is the property of Maternal, Neonatal and Child Health Research Network (MNCHRN) and cannot be made public.\n\nAll content used in the app to provide information to the recruited mothers is available as Extended data22.\n\n\nData availability\n\nOpen Science Framework: Effectiveness of mhealth on IYCF. https://doi.org/10.17605/OSF.IO/VRHA522\n\nThis project contains the following underlying data:\n\nData Set Epi Data.zip (Data entry sheet on Epi Data 3.1)\n\nPost Analysis.sav (Output file of data analysis on SPSS version 21)\n\nPre & Post Scoring _Pregnant.sav (SPSS file of pre & post entered data)\n\nOpen Science Framework: Effectiveness of mhealth on IYCF. https://doi.org/10.17605/OSF.IO/VRHA522\n\nThis project contains the following extended data:\n\nFinalized__0-6__months[1].pdf (Pre-intervention survey for 0–6 month infants)\n\nFinalized___7-12_months[1].pdf (Pre-intervention survey for 0–6 month infants)\n\nFinalized_Pregnancy_survey[1].pdf (Pre-intervention survey for mothers in final trimester)\n\nIntervention Application.zip (content from Android app)", "appendix": "Grant information\n\nThe study was funded by the Department of Foreign Affairs and Trade (DFAT), Australia. This research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership led by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR). The training model is based on a course developed jointly by the International Union against Tuberculosis and Lung Disease (The Union, Paris, France) and Médecins Sans Frontières (MSF, Geneva, Switzerland). The specific SORT IT programme that resulted in this publication was implemented by the National Tuberculosis Control Programme of Pakistan, through the support of the Global Fund to Fight AIDS, Tuberculosis and Malaria (The Global Fund, Geneva, Switzerland). The publication fee was covered by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nWe would like to thank the South Asian Infant Feeding Research Network and the University of Sydney for supporting the development of this mHealth intervention. We also would like to acknowledge Dr. Baseer Achakzai from Ministry of National Health Services, Regulations & Coordination, Pakistan as well as the LHWs, District Health Officer and Assistant District Coordinator from Islamabad. Also, we would like to acknowledge International Research Force for facilitation in data collection process.\n\n\nReferences\n\nAhmed T, Hossain M, Sanin KI: Global burden of maternal and child undernutrition and micronutrient deficiencies. Ann Nutr Metab. 2012; 61 Suppl 1: 8–17. PubMed Abstract \| Publisher Full Text\n\nWeise AS: Global Nutrition Targets 2025: Stunting policy brief. WHO. World Health Organization; 2014. Reference Source\n\nPrado EL, Dewey KG: Nutrition and brain development in early life. Nutr Rev. 2014; 72(4): 267–84. PubMed Abstract \| Publisher Full Text\n\nNelson CA, Luciana M: Handbook of developmental cognitive neuroscience. MIT Press; 2008; 923. Reference Source\n\nWiggins RC, Fuller G, Enna SJ: Undernutrition and the development of brain neurotransmitter systems. Life Sci. 1984; 35(21): 2085–94. PubMed Abstract \| Publisher Full Text\n\nInfant and young child feeding. Reference Source\n\nAlam M, D’Este C, Banwell C, et al.: The impact of mobile phone based messages on maternal and child healthcare behaviour: a retrospective cross-sectional survey in Bangladesh. BMC Health Serv Res. 2017; 17(1): 434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiks GA, Tariku A, Wassie MM, et al.: Mother's Infant and Young Child Feeding (IYCF) knowledge improved timely initiation of complementary feeding of children aged 6-24 months in the rural population of northwest Ethiopia. BMC Res Notes. 2018; 11(1): 593. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLatif S, Rana R, Qadir J, et al.: Mobile Health in the Developing World: Review of Literature and Lessons from a Case Study. IEEE Access. 2017; 5: 11540–56. Publisher Full Text\n\nPakistan Demographic and Health Survey 2017-18 Key Indicators Report. 2018. Reference Source\n\nManikam L, Sharmila A, Dharmaratnam A, et al.: Systematic review of infant and young child complementary feeding practices in South Asian families: the Pakistan perspective. Public Health Nutr. 2018; 21(4): 655–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization, UNICEF: Infant and young child feeding counselling: an integrated course. Who. 2006; 1–265. Reference Source\n\nWorld Health Organization (WHO): Global Strategy for Infant and Young Child Feeding. World Health Organization; 2003. Reference Source\n\nHafeez A, Mohamud BK, Shiekh MR, et al.: Lady health workers programme in Pakistan: challenges, achievements and the way forward. J Pak Med Assoc. 2011; 61(3): 210–5. PubMed Abstract\n\nJalal S: The lady health worker program in Pakistan--a commentary. Eur J Public Health. 2011; 21(2): 143–4. PubMed Abstract \| Publisher Full Text\n\nUSAID: mHealth Compendium. 2012. Reference Source\n\nAlam M, Khanam T, Khan R: Assessing the scope for use of mobile based solution to improve maternal and child health in Bangladesh: A case study. Proc 4th ACM/IEEE Int Conf Inf Commun Technol Dev.. 2010; 3. Reference Source\n\nMobile Solutions Aiding Knowledge For Health Improvement – Lata Medical Research Foundation (Reg. No. E-1559, Nagpur, India). 2017. Reference Source\n\nWorld Health Organization.WHO \| Global Strategy for Infant and Young Child Feeding. WHO. World Health Organization; 2010. Reference Source\n\nKhan NUZ, Rasheed S, Sharmin T, et al.: How can mobile phones be used to improve nutrition service delivery in rural Bangladesh? BMC Health Serv Res. 2018; 18(1): 530. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAvula R, Oddo VM, Kadiyala S, et al.: Scaling-up interventions to improve infant and young child feeding in India: What will it take? Matern Child Nutr. 2017; 13(Suppl 2): e12414. PubMed Abstract \| Publisher Full Text\n\nKhan SA, Mahmood H, Fatima R, et al.: Effectiveness of mhealth on IYCF. 2019. Publisher Full Text\n\nPROVINCE WISE PROVISIONAL RESULTS OF CENSUS - 2017.. Reference Source\n\nPOPULATION ADMIN UNIT NO OF HH POPULATION AND HOUSEHOLD CENSUS 2018. Reference Source\n\nUNICEF: Programming Guide Infant and Young Child Feeding. Nutr Sect UNICEF. 2011; 173. Reference Source\n\nPakistan Institute of Medical Sciences - PIMS. Reference Source\n\nSave the Children UK: Strengthening Infant and Young Child Feeding Programming and Planning for Emergency Preparedness and Response. Proceedings of an international workshop. Organised and funded by Save the Children UK in co-operation with UNICEF’s IYCN and Emergencies Units. Reference Source\n\nMildon A, Sellen D: Behaviour Change Communication Using Mobile Phones: Implications for Infant and Young Child Feeding Interventions. FASEB J. Reference Source\n\nAkter S, Ray P: mHealth - an Ultimate Platform to Serve the Unserved. Yearb Med Inform. 2010; 19(1): 94–100. PubMed Abstract \| Publisher Full Text\n\nUddin J, Biswas T, Adhikary G, et al.: Impact of mobile phone-based technology to improve health, population and nutrition services in Rural Bangladesh: a study protocol. BMC Med Inform Decis Mak. 2017; 17(1): 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHmone MP, Dibley MJ, Li M, et al.: A formative study to inform mHealth based randomized controlled trial intervention to promote exclusive breastfeeding practices in Myanmar: incorporating qualitative study findings. BMC Med Inform Decis Mak. 2016; 16(1): 60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMane SS, Chundi PR: Infant and young child feeding practices among mothers in Hyderabad, Telangana. Int J Community Med Public Heal. 2017; 4(10): 3808. Publisher Full Text\n\nSriram S, Soni P, Thanvi R, et al.: NATIONAL JOURNAL OF MEDICAL RESEARCH KNOWLEDGE, ATTITUDE AND PRACTICES OF MOTHERS REGARDING INFANT FEEDING PRACTICES.2013; 3(2); 147–150. Reference Source\n\nTekaly G, Kassa M, Belete T, et al.: Pre-lacteal feeding practice and associated factors among mothers having children less than two years of age in Aksum town, Tigray, Ethiopia, 2017: a cross-sectional study. BMC Pediatr. 2018; 18(1): 310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeerasinghe MC, Senerath U, Godakandage S, et al.: Use of Mobile Phones for Infant and Young Child Feeding Counseling in Sri Lankan Tea Estates: A Formative Study. The Qualitative Report. 2016; 21(5). Reference Source" }	[ { "id": "47680", "date": "08 May 2019", "name": "Zohra S. Lassi", "expertise": [ "Reviewer Expertise Maternal and child health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments:\nAbstract-Results: After the intervention, the overall knowledge of mothers regarding IYCF nutrition was raised to 69.6% among 94 mothers as compared to 74 (54.8%). Please complete the sentence, 74 (54.8%) in the comparison group? Introduction - Second Paragraph - first line: Needs a reference. Introduction - Second Paragraph - third line: Reference 6 should be hyper-script. Introduction - Second Paragraph: Developing countries should be replaced with low- and middle-income countries. Introduction - Second Paragraph: The sentence \"According to the Pakistan Demographic Health Survey (PDHS, 2018), almost 38% of children under five years of age are stunted, 23% are underweight, and 7% are wasted\" needs a reference. The words developing countries and LMICs should be used consistently. In the current form, both have been used interchangeably. The IYCF acronym was introduced in the first paragraph of the Introduction, and still used in full several times in the Introduction. Abstract - methods: The date of study mentioned was May to June 2018. However in the Introduction, it was mentioned that it was piloted in 2016 and evaluated between April to June 2018. Please check the dates and provide the correct one. Under mobile applications - Second Paragraph: Please add \"study\" in the sentence: \"Consent to be a part of the was also obtained for participating in the project,\" Please provide the name of the smart phone application that was created? It would be more informative, if the interface of the application is attached to the paper and the type of options available for the participants to receive information on IYCF. Discussion: Please indicate if there is agreement of the results with the earlier studies?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4765", "date": "29 Jul 2019", "name": "Subhana Khan Akber", "role": "Author Response", "response": "Responses to Reviewers Reviewer 1: Zohra Lassi, The Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia Abstract-Results: After the intervention, the overall knowledge of mothers regarding IYCF nutrition was raised to 69.6% among 94 mothers as compared to 74 (54.8%). Please complete the sentence, 74 (54.8%) in the comparison group? Added as suggested Introduction - Second Paragraph - first line: Needs a reference. Provided at the end of completing the sentences at line 4 Introduction - Second Paragraph - third line: Reference 6 should be hyper-script. Revised as suggested Introduction - Second Paragraph: Developing countries should be replaced with low- and middle-income countries. Revised as suggested Introduction - Second Paragraph: The sentence \"According to the Pakistan Demographic Health Survey (PDHS, 2018), almost 38% of children under five years of age are stunted, 23% are underweight, and 7% are wasted\" needs a reference. Reference number 10 is provided at line number 10. The words developing countries and LMICs should be used consistently. In the current form, both have been used interchangeably. Revised as suggested The IYCF acronym was introduced in the first paragraph of the Introduction, and still used in full several times in the Introduction. Revised as suggested Abstract - methods: The date of study mentioned was May to June 2018. However in the Introduction, it was mentioned that it was piloted in 2016 and evaluated between April to June 2018. Please check the dates and provide the correct one. Revised as suggested Under mobile applications - Second Paragraph: Please add \"study\" in the sentence: \"Consent to be a part of the was also obtained for participating in the project,\" Added as suggested Please provide the name of the smartphone application that was created? The name of application was “mhealth” and is mentioned in the text. It would be more informative, if the interface of the application is attached to the paper and the type of options available for the participants to receive information on IYCF. Regarding the interface of the application, we have mentioned under heading of software availability that “the source code of android phone-based application developed for the Lady Health Workers (LHWs) under the project “Sehatmnd Kl” is the property of Maternal, Neonatal and Child Health Research Network (MNCHRN) and cannot be made public.” Discussion: Please indicate if there is agreement of the results with the earlier studies? The text has been highlighted under the heading of discussion." } ] }, { "id": "47677", "date": "21 May 2019", "name": "Ejaz Ahmad Khan", "expertise": [ "Reviewer Expertise Epidemiology", "systematic analyses", "environmental health", "nutrition", "immunization", "communicable and non-communicable disease", "injuries", "research methods" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: This is a good effort by the authors to introduce mHealth in overcoming the barriers to achieving optimum IYCF. The article is not written very well. There are numerous English language comprehension and spelling (perhaps typo) mistakes. The authors leave the reader confused about their sampling frame at several places, and text is some times extremely haphazard. The tables are cited irrespective of their order as they appear in the article. Discussion lacks critical points from results discussed. There is an element of publication bias and misinterpretation of technical terms such as Recall Bias (contrary to how it is defined in text books of epidemiology). About 30% of the references are more than five years old.\n\nSpecific comments:\nAbstract:\nThe abstract reports only the positive findings without highlighting the negative findings. It also concludes for effectiveness, whereas, no test of significance was applied, and just uses the difference in percentages as proof of significant effectiveness.\n\nMethods:\nTen can be written as 10, as numbers below 10 are written in words only. For intervention part, the authors had messages translated to Urdu from English just once. However, the standard is to back translate it in English so that they are exactly the same as they were before the initial translation. The paragraph on \"intervention design\" needs rephrasing as the text is confusing. Similarly, the paragraph on \"messages\" also needs rephrasing, so that the reader easily understand what the authors wanted them to know. Sampling frame creates big confusion. In the start it is 0-24 months of age children. But then it changes to 0-12 months in the later text (mobile application part). Further, in the flow diagram, this age group is further subdivided into 0-6 and 7-24 months of age. It is unclear how the application designed only for 0-12 months of age could have catered for age group beyond 12 months. The flow diagram has repeatedly wrong spellings of \"participants\". In paragraph three, following the heading \"mobile application\" again contradicts the 7-24 months of age and states 7-12 months of age. age 6, paragraph two, the text has not been able to explain to the reader whether the SIM and mobile were acquired for the LHWs or for all the participants. This should be clearly stated and differentiated.\n\nAdditional points for methods:\nFor practices, why were there non-direct or indirect observations? The acquired knowledge of the participants during the intervention can lead to over reporting of good practices to please the researchers (Hawthorne effect). Furthermore, did the researchers take into an account the effect of other sources of knowledge (confounders), which could supplement or negatively impact their intervention, and how did they take care of them and controlled those factors?\n\nResults:\nResults are divided into descriptive and inferential statistics. However, no test of significance was applied for the inferential statistics. Just on the bases of difference of percentages in pre- and post- is insufficient to claim significant effectiveness. The results should be written in order of the objectives of the study as they were achieved. The tables are cited without proper and ascending sequence.\n\nDiscussion:\nInstead of discussing the mHealth intervention in length, depicting their inclination towards supporting their positive results only, the authors should do impartial and unbiased discussion. They should also discuss the implication of negative results, why some practices also increased while a few declined, what could be the underlying factors (supported by literature), claiming that the women favored the use of mobile phone to access information was not supported by any result given in the text.\nReferences:\nAbout 30% of references were of more than five years old.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4766", "date": "29 Jul 2019", "name": "Subhana Khan Akber", "role": "Author Response", "response": "Reviewer 2: Ejaz Ahmad Khan, Department of Epidemiology and Biostatistics, Health Services Academy (HSA), Islamabad, Pakistan General comments: This is a good effort by the authors to introduce mHealth in overcoming the barriers to achieving optimum IYCF. The article is not written very well. There are numerous English language comprehension and spelling (perhaps typo) mistakes. The authors leave the reader confused about their sampling frame at several places, and text is sometimes extremely haphazard. The tables are cited irrespective of their order as they appear in the article. Discussion lacks critical points from results discussed. There is an element of publication bias and misinterpretation of technical terms such as Recall Bias (contrary to how it is defined in text books of epidemiology). About 30% of the references are more than five years old. The comprehension mistakes have been corrected. Sentences regarding sampling frame have been rephrased. Tables have been cited correctly and in ascending order. We mentioned that we were handicapped on finding useful literature related to mhealth in our context for the discussion section. Recall bias is a systematic error introduced in the study due to providing inaccurate answers to questions, similarly mothers who were interviewed were found indecisive to answer the questions at some points which can be attributed to long wash out period after intervention. There are 12 references that are more than five years old, due to limited available data so we searched literature that is available in past 10 years. Specific comments: Abstract: The abstract reports only the positive findings without highlighting the negative findings. It also concludes for effectiveness, whereas, no test of significance was applied, and just uses the difference in percentages as proof of significant effectiveness. Negative findings have been reported in the results and tables. Since it was not a trial therefore by effectiveness we mean the success of deploying mhealth intervention for IYCF. Hence, differences were calculated pre-post to assess the increase in knowledge, attitude and practices among the study participants. Methods: Ten can be written as 10, as numbers below 10 are written in words only. Revised For intervention part, the authors had messages translated to Urdu from English just once. However, the standard is to back translate it in English so that they are exactly the same as they were before the initial translation. The messages were back translated into English language, the text has been revised. The paragraph on \"intervention design\" needs rephrasing as the text is confusing. Similarly, the paragraph on \"messages\" also needs rephrasing, so that the reader easily understand what the authors wanted them to know. Rephrased Sampling frame creates big confusion. In the start it is 0-24 months of age children. But then it changes to 0-12 months in the later text (mobile application part). Further, in the flow diagram, this age group is further subdivided into 0-6 and 7-24 months of age. It is unclear how the application designed only for 0-12 months of age could have catered for age group beyond 12 months. The flow diagram has repeatedly wrong spellings of \"participants\". In paragraph three, following the heading \"mobile application\" again contradicts the 7-24 months of age and states 7-12 months of age. The mobile application part is the intervention phase where mothers having children of 0-12 months of age were recruited in 2016 but messages were sent according to the age groups defined which were from 0-6 months and 7-12 months. Later on in the post-intervention survey which was conducted in 2018, we included the same population but the age of the children was set 0-24 months so to match the ages of children. Spelling errors have been corrected. In the paragraph three, the age group refers to the pre-intervention phase. Age 6, paragraph two, the text has not been able to explain to the reader whether the SIM and mobile were acquired for the LHWs or for all the participants. This should be clearly stated and differentiated. Revised. The text has been mentioned in paragraph two, line number 11. Additional points for methods: For practices, why were there non-direct or indirect observations? The acquired knowledge of the participants during the intervention can lead to over reporting of good practices to please the researchers (Hawthorne effect). Furthermore, did the researchers take into an account the effect of other sources of knowledge (confounders), which could supplement or negatively impact their intervention, and how did they take care of them and controlled those factors? Practice-based knowledge questions were asked from the study participants without observation. We did not took into account Hawthorne effect or other confounding factors though we have mentioned that this evaluation was conducted after 2016 in 2018 and have mentioned in limitations about potential biases. Results: Results are divided into descriptive and inferential statistics. However, no test of significance was applied for the inferential statistics. Just on the bases of difference of percentages in pre- and post- is insufficient to claim significant effectiveness. For effectiveness we referred to it the success of using mhealth for dissemination of knowledge regarding IYCF. The results should be written in order of the objectives of the study as they were achieved. The tables are cited without proper and ascending sequence. Revised and table have cited correctly in ascending order. Discussion: Instead of discussing the mHealth intervention in length, depicting their inclination towards supporting their positive results only, the authors should do impartial and unbiased discussion. They should also discuss the implication of negative results, why some practices also increased while a few declined, what could be the underlying factors (supported by literature), claiming that the women favored the use of mobile phone to access information was not supported by any result given in the text. Decrease of some practices has been ascribed to prolong wash period mentioned in the text paragraph 3 line number 6. The discussion might be read as narrow due to limited evidence on use of mhealth for IYCF knowledge and quality of mhealth for MNCH programs. But negative results have been discussed and supported by literature in paragraph two line numbers 3 & 5, paragraph three line number 10. The use of mobile phone to access information was been demonstrated by the results of our formative study. References: About 30% of references were of more than five years old. There are 12 references that are more than five years old, due to limited available data so we searched literature that is available in past 10 years." } ] }, { "id": "47681", "date": "23 May 2019", "name": "Alexandre Delamou", "expertise": [ "Reviewer Expertise Public Health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have provided my comments directly in a PDF document because the manuscript pages are not numbered to facilitate the reviewer's work.\nMy main comments are:\nThe intent of the authors is good. They made a lot of effort to well describe the intervention and conduct pre- and post-intervention surveys.\n\nHowever:\nThe design does not optimistic conclusions. I think the authors should be very modest and discuss some key limitations: the selection was not randomly done, there was no comparator to your study (cluster randomized trial). Also, the questionnaire was quantitative and you did not capture the qualitative aspect of the intervention (feasibility, acceptability, sustainability, etc). The introduction is long but the authors did not use the space to justify the study in Pakistan. Just saying that \"there is insufficient information\" is not the good justification. There are many typos in the text, I have flagged some but the text needs some corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4767", "date": "29 Jul 2019", "name": "Subhana Khan Akber", "role": "Author Response", "response": "Reviewer 3: Alexandre Delamou, Department of Public Health, Gamal Abdel Nasser University of Conakry, Conakry, Guinea I have provided my comments directly in a PDF document because the manuscript pages are not numbered to facilitate the reviewer's work. Suggested changes revised in the manuscript. PDF file is sent for review comments. My main comments are: The intent of the authors is good. They made a lot of effort to well describe the intervention and conduct pre- and post-intervention surveys. However: The design does not optimistic conclusions. I think the authors should be very modest and discuss some key limitations: the selection was not randomly done, there was no comparator to your study (cluster randomized trial). Also, the questionnaire was quantitative and you did not capture the qualitative aspect of the intervention (feasibility, acceptability, sustainability, etc). We opted for quasi-experimental (pre-post) design and convenience-based sampling was done for the selection of participants form the sampling frame. The qualitative component, we covered in the formative study where we tested the acceptance of the intervention mentioned in the introduction section, last paragraph. The introduction is long but the authors did not use the space to justify the study in Pakistan. Just saying that \"there is insufficient information\" is not the good justification. Revised in the introduction. There are many typos in the text, I have flagged some but the text needs some corrections. Corrections have been made in the text." } ] } ]	1	https://f1000research.com/articles/8-551
https://f1000research.com/articles/8-296/v1	15 Mar 19	{ "type": "Research Article", "title": "Evaluation of methods to assign cell type labels to cell clusters from single-cell RNA-sequencing data", "authors": [ "J. Javier Diaz-Mejia", "Elaine C. Meng", "Alexander R. Pico", "Sonya A. MacParland", "Troy Ketela", "Trevor J. Pugh", "Gary D. Bader", "John H. Morris", "J. Javier Diaz-Mejia", "Elaine C. Meng", "Alexander R. Pico", "Sonya A. MacParland", "Troy Ketela", "Trevor J. Pugh", "Gary D. Bader" ], "abstract": "Background: Identification of cell type subpopulations from complex cell mixtures using single-cell RNA-sequencing (scRNA-seq) data includes automated computational steps like data normalization, dimensionality reduction and cell clustering. However, assigning cell type labels to cell clusters is still conducted manually by most researchers, resulting in limited documentation, low reproducibility and uncontrolled vocabularies. Two bottlenecks to automating this task are the scarcity of reference cell type gene expression signatures and the fact that some dedicated methods are available only as web servers with limited cell type gene expression signatures. Methods: In this study, we benchmarked four methods (CIBERSORT, GSEA, GSVA, and ORA) for the task of assigning cell type labels to cell clusters from scRNA-seq data. We used scRNA-seq datasets from liver, peripheral blood mononuclear cells and retinal neurons for which reference cell type gene expression signatures were available. Results: Our results show that, in general, all four methods show a high performance in the task as evaluated by receiver operating characteristic curve analysis (average area under the curve (AUC) = 0.94, sd = 0.036), whereas precision-recall curve analyses show a wide variation depending on the method and dataset (average AUC = 0.53, sd = 0.24). Conclusions: CIBERSORT and GSVA were the top two performers. Additionally, GSVA was the fastest of the four methods and was more robust in cell type gene expression signature subsampling simulations. We provide an extensible framework to evaluate other methods and datasets at https://github.com/jdime/scRNAseq_cell_cluster_labeling.", "keywords": [ "single cell", "RNA-seq", "scRNA-seq", "bioinformatics", "benchmark", "evaluation", "labeling", "cell type" ], "content": "Introduction\n\nDuring the last five years a number of single-cell sequencing technologies have been developed to identify cell subpopulations from complex cell mixtures (Bakken et al., 2017). For instance, recent advances in single-cell RNA-sequencing (scRNA-seq) enable the simultaneous measurement of expression levels of hundreds to thousands of genes across hundreds to thousands of individual cells. The resulting expression matrices of genes by cells are used (see below) to identify cell subpopulations with characteristic gene expression profiles and other biological properties (i.e. cell types).\n\nA typical computational pipeline to process scRNA-seq data involves the following steps: i) quality control of sequencing reads, ii) mapping reads against a reference transcriptome, iii) normalization of mapped reads to correct batch effects and remove contaminants, iv) data dimensionality reduction with principal component analysis or alternative approaches, v) clustering of cells using principal component values, vi) detection of genes differentially expressed between clusters, vii) visualization of cell clusters in t-SNE or alternative plots, and viii) assignment of cell type labels to cell clusters. A number of computational tools, including Cell Ranger (Zheng et al., 2017a) and Seurat (Butler et al., 2018), allow automation of steps i to vii (Duò et al., 2018; Freytag et al., 2018; Innes & Bader, 2018). However, assignment of cell type labels to cell clusters is still conducted manually by most researchers. The typical procedure involves manual inspection of the genes expressed in a cluster, combined with a detailed literature search to identify if any of those genes are known gene expression markers for cell types of interest. This manual approach has several caveats, including limited documentation and low reproducibility of cell type gene marker selection, use of uncontrolled and non-ontological vocabularies for cell type labels, and it can be time-consuming. For these reasons, computational tools that allow researchers to systematically, reproducibly and quickly assign cell type labels to cell clusters derived from scRNA-seq experiments are needed.\n\nIn this study we used three scRNA-seq datasets from liver cells (MacParland et al., 2018), peripheral blood mononuclear cells (PBMCs) (Zheng et al., 2017a) and retinal neurons (Shekhar et al., 2016b) (Table 1) to compare four methods that can be used for assigning cell type labels to cell clusters: CIBERSORT (Newman et al., 2015b), GSEA (Subramanian et al., 2005), GSVA (Hänzelmann et al., 2013) and ORA (Fisher, 1935; Goeman & Bühlmann, 2007) (Table 2). We chose these four methods to represent different categories of algorithms, ranging from first-generation enrichment analysis (ORA) to second-generation approaches (GSEA and GSVA) and machine learning tools (CIBERSORT). Although ORA and GSEA were not originally developed to process RNA-seq data, they have been extensively used in transcriptomic studies for gene set enrichment analyses. GSVA was developed to analyze microarray and bulk RNA-seq data, and CIBERSORT was developed to estimate abundances of cell types in mixed cell populations from bulk RNA-seq data. We adapted all four methods to assign cell type labels to cell clusters from scRNA-seq data based on known sets of cell type marker genes. We evaluated these methods using two types of inputs: a matrix with the average expression of each gene x from all the cells in each cell cluster y (Ěxy) from scRNA-seq measurements, which we assume corresponds to the profile of a cell type or state, and known cell type gene expression signatures, represented as gene sets or continuous gene expression profiles (Figures 1A-C).\n\nTwo inputs are needed by automated cell type detection methods (A–C). (A) a matrix with the average expression of each gene x for each cell cluster y (Exy). (B, C) cell type gene marker signatures can be provided as either gene sets (lists of gene identifiers, B) or numeric gene expression profiles (C). Gene sets can be manually compiled from literature and are used for methods like GSEA, GSVA or ORA. Whereas gene-expression profiles are measurements from microarrays, bulk- or single-cell RNA-sequencing (scRNA-seq) experiments, and are used by methods like CIBERSORT. (D, E) Automated cell type detection methods produce a matrix of cell type likelihoods for each cell cluster. (F) Some authors of scRNA-seq studies assign cell type labels manually to cell clusters using empirical expertise or orthogonal experiments such as fluorescence activated cell sorting. These assignments can be used as references to benchmark automated cell type detections. (G) Top cell type predictions (red rectangles in E) are contrasted against annotation references (F) to assess the performance of cell type detection methods by receiver operating characteristic (ROC) curve and precision-recall (PR) curve analyses. (H) Robustness of cell type detection methods can be analyzed by gradually subsampling gene markers from cell type gene expression signatures (B or C) and repeating procedures of (D–G) to obtain distributions of the area under the curve (AUC) for ROC (ROC AUC) and PR (PR AUC) curves, which are shown as violin plots. We hypothesized that some detection methods may be more robust than others to the proportion of gene markers subsampled from cell type gene expression signatures.\n\nDetails of the four methods compared in this study, and their computing times to classify cell clusters of indicated datasets. (b) refers to CIBERSORT ‘binary’ analysis mode, (c) refers to CIBERSORT ‘continuous’ analysis mode.\n\nCIBERSORT uses gene expression profiles as training data for a machine learning algorithm to estimate abundances of known cell types in a mixed cell population and was originally developed to identify composition of known immune cell types in bulk RNA-seq sample measurements. In our evaluation, we used Ěxy matrices instead of bulk RNA-seq data. GSEA uses a Kolmogorov–Smirnov (KS) like statistic to determine whether a gene set shows statistically significant, concordant differences between biological states. It was originally developed to analyze microarray gene expression data and has been applied to multiple genomic data types. GSVA transforms a gene by sample matrix to a gene set by sample matrix, and evaluates gene set enrichment for each sample. Like GSEA, GSVA uses a KS like statistic but GSVA bypasses explicitly modeling phenotypes within the enrichment scoring step. GSVA was originally developed to process microarray and bulk RNA-seq measurements. ORA uses the Fisher’s exact test to detect an overrepresentation of members of a gene set in a subsample of highly expressed genes, compared against both the total number of gene set members and the total number of genes measured in the sample.\n\nMethods explicitly developed to assign cell type labels to cell clusters of scRNA-seq data have been reported (Alavi et al., 2018; Alquicira-Hernandez et al., 2018; Crow et al., 2018). However, to our knowledge they are in beta, or implemented as web-servers to process cell types for which we could not find reference cell type annotations (Figure 1F) that we would require to include in our evaluation. For this reason, we included only the four methods described above, and we provide execution and benchmark scripts that will be useful to extend our comparisons to other methods in the future.\n\n\nMethods\n\nFor the liver dataset (MacParland et al., 2018) (NCBI GEO: GSE115469) we followed the authors’ reported procedure to obtain cell clusters, and obtained the Ěxy matrix for each cluster using the function AverageExpression(use.raw = T) from Seurat v2 (Butler et al., 2018). For the PBMCs dataset (Zheng et al., 2017a), Fresh 68k PBMCs DonorA gene expression matrix files were obtained from 10X (Zheng et al., 2017b) (NCBI Sequence Read Archive: SRX1723926). Normalization, data dimensionality reduction, cell clustering and Ěxy matrix calculations were conducted with Seurat with the following functions: FilterCells(low.thresholds = 200,-Inf, high.thresholds = 0.05,10000); FindClusters(reduction.type = \"pca\", dims.use = 1:10, resolution = 0.4); AverageExpression(use.raw = T). For the retinal neurons dataset (Shekhar et al., 2016b) (NCBI GEO: GSE81905) the gene expression matrix and cell cluster assignments were obtained from (Shekhar et al., 2016a) and the Ěxy matrix calculation was conducted with AverageExpression(use.raw = T) from Seurat.\n\nA gene expression signature is defined simply as a set of genes characteristically and detectably expressed in a cell type. These are typically inferred from small-scale experiments that need to be manually identified in the literature, or by comparing the transcriptome of a given cell type against all other available cell type gene expression profiles, usually from the same experiment. The liver cell type gene set signatures were manually curated by us (author S.A.M.) and were originally used to manually annotate cell types in the liver dataset (MacParland et al., 2018). We provide these gene sets on Zenodo (Diaz-Mejia, 2019a). For the PBMC dataset, we used a blood cell type gene expression profile signature compiled by the CIBERSORT developers called LM22, containing 547 genes and 22 cell types (Newman et al., 2015a). Reference cell type assignments to the PBMCs by fluorescence-activated cell sorting (FACS) were obtained from (Zheng et al., 2017c). The PBMC cell clusters we obtained with Seurat were mapped using cell barcode identifiers against the FACS assignments, and cell type names were manually matched to the LM22 signature. For the retinal neuron dataset (Shekhar et al., 2016b), known cell type markers reported by the authors were used as cell type gene set signatures.\n\nCIBERSORT requires as input a cell type gene expression signature in the form of gene expression profiles (i.e. a matrix of genes in rows and cell types in columns). For the PBMC dataset, we used two versions of the LM22 signature for CIBERSORT. First, we used the original LM22 signature (Newman et al., 2015b) with continuous valued gene expression measurements, which we called CIBERSORT ‘continuous’. Second, for each cell type of the LM22 signature, a value of ‘1’ was assigned to 5% of genes with highest expression values in their column or a value of ‘0’ otherwise, and we called this approach CIBERSORT ‘binary’. The same 5% of genes was used to create cell type gene set signatures as inputs for GSEA, GSVA and ORA. For the liver dataset, we transformed the cell type gene set signature into a binary matrix of genes in rows and cell types in columns for CIBERSORT ‘binary’ analysis mode. To do this, each gene included in each cell type gene set m was assigned a value of ‘1’ in the column corresponding to m in the matrix, whereas other genes absent in m but present in other cell type gene sets were assigned a value of ‘0’. Similarly, for the retinal neuron dataset the ‘previously known markers’ for bipolar cell types provided in Table S2 of Shekhar et al. (2016b) were transformed into a binary matrix of genes by cell types for CIBERSORT ‘binary’ analysis.\n\nCell type gene set signatures (Figure 1B) were subsampled by randomly removing between 10 and ~99% of genes from each signature in increments of 10%, keeping a minimum of one gene. Each subsampling of gene sets was transformed into a binary matrix of genes by cell types for CIBERSORT ‘binary’ as indicated above. Cell type gene expression profile signatures (Figure 1C) were subsampled in two stages: first we selected the top 5% highest expressed genes for each cell type, then we randomly replaced the gene expression value of 10 to 100% of those genes from each cell type, in increments of 10%, by the minimum value of the cell type column. This resulted in subsampled gene expression profile signatures with identical size to the original profile signatures, but with values of the top highly expressed genes randomly replaced by the minimum score of each cell type. For percentage values between 10 to 100%, 1,000 subsampling replicates were generated for each cell type gene expression signature, and each replicate was processed as indicated by Figures 1D-G. Violin plots were used to show the resulting ROC and PR AUC distributions.\n\nThe enrichment scores (ES) from CIBERSORT and GSVA were directly used as ranks for the benchmark comparisons against gold standard references, whereas the P-values from GSEA and ORA were first -log 10 transformed and the resulting values were used as ranks for the benchmark analyses. For ORA, the universe of genes used was the intersection of genes present in the cell type gene expression signature and the Ěxy matrix of each dataset. All methods were implemented locally using Java, R and Perl (Table 2), using the following libraries and programs: for CIBERSORT we used CIBERSORT.jar v1.01 and R(Rserve) 1.8.6, for GSEA we used gsea-3.0.jar, for GSVA we used R(GSVA) v1.30 and R(GSA) v1.3, and for ORA we used R(fisher.test) R v3.5.1.\n\nWe implemented wrapper scripts to execute each of the four methods tested, including a stopwatch to time the cell type prediction task. Other tasks, such as input and output preparation, were excluded from computing time values reported in Table 2. All computing time measurements were made using a 3.1-GHz Intel Core i5 CPU with 2 cores and 16 GB RAM.\n\nThe scripts used to run and benchmark cell type labeling methods described in this study are available on GitHub and archived at Zenodo (Diaz-Mejia, 2019b). An earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/562082).\n\n\nResults\n\nWe benchmarked the performance and computing time of four cell type labeling methods, namely: CIBERSORT, GSVA, GSEA and ORA (Table 2), using average gene expression profiles of scRNA-seq cell clusters and known cell type gene expression signatures. We used three scRNA-seq datasets: liver cells (MacParland et al., 2018), PBMCs (Zheng et al., 2017a) and retinal neurons (Shekhar et al., 2016b) (Table 1). Each method used two inputs: an Ěxy matrix with the average gene expression for each cell cluster (Figure 1A) and a cell type gene expression signature, represented as either a gene set or a gene expression profile. Three of the four methods tested (GSVA, GSEA and ORA) used cell type gene set signatures (Figure 1B), whereas CIBERSORT used cell type gene expression profiles either with continuous or binarized values (Figure 1C). Each method produced a matrix of cell type predictions (Figure 1D, E) which was compared to manually annotated cell type references (Figure 1F) to conduct receiver operating characteristic (ROC) and precision-recall (PR) curve analyses (Figure 1G). The robustness of each method was assessed by randomly subsampling 10% to 100% of the genes from the cell type gene expression signatures and repeating the cell type detection and ROC and PR curve analyses for each subsample (Figure 1H).\n\nIn general, we observed that all four methods showed high ROC AUC values for all three analyzed scRNA-seq datasets. An average ROC AUC = 0.97 was found for the liver dataset (Figure 2A), average ROC AUC = 0.92 for the PBMC dataset (Figure 2B) and average ROC AUC = 0.94 for the retinal neuron dataset (Figure 2C). Since CIBERSORT takes as input a cell type gene expression signature in the form of gene expression profiles (Figure 1C), and the only available signatures for the liver and retinal neuron datasets were in the form of gene sets, we transformed the gene sets into binary matrices and used them as inputs for CIBERSORT (Methods). Notably, the binary matrix approach, which we called CIBERSORT ‘binary’, produced the highest performance among all tested methods for the liver (ROC AUC = 1, Figure 2A) and retinal neurons datasets (ROC AUC = 0.95, Figure 2C). The CIBERSORT ‘binary’ approach performance was almost identical to that of the original LM22 cell type gene expression signature with continuous values, which we called CIBERSORT ‘continuous’, for the PBMC dataset (ROC AUC = 0.91 and 0.92, Figure 2B). GSVA was the top performer using the PBMC dataset (ROC AUC = 0.95, Figure 2B), closely followed by GSEA (ROC AUC = 0.94) and the two versions of CIBERSORT (‘binary’ ROC AUC = 0.92 and ‘continuous’ ROC AUC = 0.91), while ORA’s performance was slightly lower (ROC AUC = 0.86) (Figure 2B).\n\nReceiver operating characteristic (ROC) and precision-recall (PR) curve analyses of four automated cell type detection methods (CIBERSORT, GSEA, GSVA and ORA) (Table 2) using three scRNA-seq datasets (Table 1). ROC curve analyses for datasets from: (A) human liver cells, (B) human PBMCs, and (C) mouse retinal neurons. PR curve analyses for the same datasets: (D) human liver cells, (E) human peripheral blood mononuclear cells (PBMCs), and (F) mouse retinal neurons. The ROC area under the curve (AUC) and PR AUC are shown for each method using each dataset. For the PBMCs dataset, two analyses were conducted with CIBERSORT, one using the original LM22 cell type gene expression signature with continuous gene expression values, that we called CIBERSORT ‘continuous’ (CIBER(c)), and another where the LM22 profiles were thresholded and binarized, which we called CIBERSORT ‘binary’ (CIBER(b), see Methods). The same thresholded signature was used to create cell type gene sets for GSEA, GSVA and ORA (Methods). For the liver and retinal neuron datasets, only gene set signatures were available and they were transformed into binary matrices for CIBERSORT ‘binary’ (CIBER(b)).\n\nThe analysis of ROC AUC robustness showed that, in general, performance of all methods decayed as a function of removing genes from cell type gene expression signatures. However, GSVA tolerated removal of up to 90% of the genes from the PBMC signature to maintain ROC AUCs ≥ 0.8. ORA tolerated removal of up to 60% of genes at the same ROC AUC cutoff (Figure 3B), whereas GSEA and the two versions of CIBERSORT gave ROC AUCs < 0.8 when ≥30% of the genes were removed from the PBMC cell type signatures. For the liver dataset, GSVA and GSEA tolerated removal of up to 60% of genes from the liver signature to maintain ROC AUCs ≥ 0.8, whereas CIBERSORT ‘binary’ and ORA tolerated removal of up to 50% of the genes at the same ROC AUC cutoff (Figure 3A). For the retinal neuron dataset, GSVA and ORA tolerated removal of up to 50% of the genes from the signature to maintain ROC AUCs ≥ 0.8, whereas GSEA and CIBERSORT ‘binary’ tolerated removal of 30% and 20%, respectively, for the same ROC AUC cutoff (Figure 3C).\n\nThe cell type gene expression signatures used for ROC curve analyses in Figure 2 were randomly subsampled 1,000 times, keeping 10 to 100% of genes from the original signatures each time. Automated cell type detection was repeated for each subsample and violin plots representing the distribution of resulting ROC AUCs are shown for datasets from: (A) human liver cells, (B) human peripheral blood mononuclear cells (PBMCs), and (C) mouse retinal neurons. For the PBMC dataset, two analyses were conducted with either the original LM22 cell type gene expression signature with continuous gene expression values (CIBER(c)) or with a thresholded and binarized version (CIBER(b)). For the liver and retinal neuron datasets only binary matrices for CIBER(b) were used.\n\nWhen benchmarking the four methods compared in this study, we classified each cell cluster positively into a single cell type and negatively into the remaining cell types of their corresponding dataset signature. This produced a skewed distribution with few positive predictions and several negative predictions. To ameliorate this imbalance, we used PR curve analyses in addition to ROC curve analyses. In general, the PR AUCs were smaller than the ROC AUCs (Figure 2, top vs. bottom panels). Some methods clearly separated from the rest using PR curve analyses. For instance, GSEA showed the lowest PR AUC values for both the liver and retinal neurons datasets (PR AUCs = 0.51 and 0.28), compared with CIBERSORT (PR AUCs = 0.98 and 0.5), ORA (PR AUCs = 0.90 and 0.53), and GSVA (PR AUC = 0.89 and 0.56) (Figure 2D, F). GSEA also displayed the lowest AUC in the ROC curve analyses for the liver and retinal neurons datasets, and the performance differences between GSEA and the other methods were more pronounced using PR curve analyses. In contrast, the two versions of CIBERSORT for the PBMC dataset ranked very close to the other three methods using ROC curve analyses (all ROC AUCs were > 0.9, Figure 2B), but they were relatively low using PR curve analyses (CIBERSORT ‘continuous’ PR AUC = 0.22 and CIBERSORT ‘binary’ PR AUC = 0.24), compared with GSVA (PR AUC = 0.56), ORA (PR AUC = 0.42) and GSEA (PR AUC = 0.34) (Figure 2E).\n\nThe PR AUC robustness analysis showed that all methods’ performance decayed as a function of removing genes from cell type gene expression signatures. Interestingly, using the liver dataset all four methods showed higher PR AUCs than for the PBMC and retinal neuron datasets (Figure 4A-C). GSVA and ORA tolerated removal of up to 60% of genes from the liver dataset signatures to maintain PR AUCs ≥ 0.5. CIBERSORT ‘binary’ tolerated removal of 50% of genes for the same PR AUC cutoff (Figure 4A), whereas GSEA PR AUCs were < 0.5 using either the full liver cell type signature or any subsampling of it. For the retinal neuron dataset, CIBERSORT ‘binary’, GSVA and ORA tolerated removal of up to 20% of the genes from the signature to maintain average PR AUC ≥ 0.5, whereas for GSEA the average was < 0.5 at any fraction of genes in the signature. For the PBMC dataset, GSVA was the only method showing PR AUC > 0.5 with the full signature (Figure 2E) and it tolerated removal of up to 20% of genes from the signature to maintain average PR AUC > 0.5 (Figure 4B).\n\nThe same procedure described in Figure 3 for ROC AUCs was used here for PR AUCs. Please see Figure 3 legend for details.\n\nAs shown in Table 2, the computing times of method implementations varied from 0.73 s for GSVA processing the retinal neurons dataset, up to 700 s for CIBERSORT ‘continuous’ processing the PBMC dataset. For all three datasets, GSVA was the fastest method to process cell type classification tasks. ORA ranked second with computing times between 3 and 5 times longer than GSVA. GSEA showed computing times between 77 and 134 times longer than GSVA, and CIBERSORT showed computing times between 37 and 777 times longer than GSVA. The size of the cell type gene expression signatures used for CIBERSORT influenced the speed of the classification task. For CIBERSORT ‘continuous’ we used the original LM22 signature, which contained 547 genes for the PBMC dataset, whereas the thresholded binary matrix used for CIBERSORT ‘binary’ had 248 genes, and it took 169 s, or 24% of the time that took CIBERSORT ‘continuous’ for the same task. For comparison, we created a second ‘continuous’ signature by restricting the original LM22 signature to the 248 genes present in the thresholded binary matrix. This ‘reduced continuous’ signature approach showed a performance (ROC AUC = 0.92, PR AUC = 0.32) which was similar to the full CIBERSORT ‘continuous’ (ROC AUC = 0.92, PR AUC = 0.24) and ‘binary’ modes (ROC AUC = 0.91, PR AUC = 0.22), and the computing time was reduced substantially to 189 s, or 27% of the time that took CIBERSORT ‘continuous’ for the same task.\n\n\nDiscussion\n\nThe size and volume of scRNA-seq datasets are continually increasing, and several methods are available to normalize scRNA-seq measurements and cluster cells. In contrast, cell type labeling of cell clusters is still conducted manually by most researchers. This is in part due to a scarcity of reference cell type gene expression signatures and also because most methods to address this challenge are only available via web servers with limited number of cell types (Alavi et al., 2018; Alquicira-Hernandez et al., 2018; Crow et al., 2018), making it difficult for users to adapt them for their needs. In this study we used three scRNA-seq datasets to benchmark four methods that can address these challenges. Although three of the four tested methods (GSEA, GSVA and ORA) were not explicitly developed to identify cell types, their extensive use in gene set enrichment tasks and their wide portability motivated us to test them as cell type classifiers. CIBERSORT is implemented both as a webserver and a local distribution that can be licensed by developers, allowing users to benchmark it with relatively low programmatic effort.\n\nIn general, our results show that for the three scRNA-seq datasets tested (liver, PBMCs and retinal neurons) all four tested methods achieved good performance by ROC curve analyses. However, ROC curves tend to overestimate methods’ performance when the ratio of positive to negative predictions is highly skewed. For this reason, we decided to also conduct PR curve analyses. GSVA was consistently one of the top performers by both ROC and PR curve analyses for the three datasets, and its performance was more robust in analyses where we subsampled genes from cell type gene expression signatures. This is particularly important at this stage of the scRNA-seq field, as only limited information on cell type gene expression signatures is available. Notably, despite its relative simplicity, ORA showed a performance comparable to GSVA. CIBERSORT’s performance was good, particularly for the liver dataset by both ROC and PR analyses, albeit lower than that of GSVA or ORA in the PBMC dataset, and it was comparable using the retinal neuron dataset. CIBERSORT’s computing times were orders of magnitude higher those of GSVA and ORA. Our results showed that CIBERSORT ‘binary’ performed as well as CIBERSORT ‘continuous’ by both ROC and PR curve analyses and used only one quarter of the computing time. In the present implementation, GSEA performed worse than the other three methods, particularly in the PR curve analyses.\n\nThe size of current publicly available scRNA-seq datasets is currently typically on the order of thousands of cells clustered into dozens of cell clusters. In our tests, each of the four tested methods completed the cell type prediction tasks in seconds or minutes. However, bigger datasets from the Human Cell Atlas (Rozenblatt-Rosen et al., 2017) and other sources are expected to have millions of cells (e.g. 1.3 million brain cell from E18 mice, NCBI GEO: GSE93421) grouped into thousands of clusters, for which the fastest method implementations will be preferred. In this sense, we found that GSVA is the best option since its computing time for the tested datasets was fastest (one to two orders of magnitude faster than GSEA and CIBERSORT). ORA also offers a good option for cell cluster labeling as its ROC and PR curve benchmarks were comparable to GSVA and its computing times were only 3 to 5 times longer than those of GSVA. One extra requirement for ORA compared with the other three methods is that the Ěxy matrix profiles need to be thresholded. In this study we used an arbitrary cutoff, based on the overall distribution of gene expression values, but future analyses could evaluate iterative thresholding.\n\nOne of the limitations of this study is that we included only three scRNA-seq datasets (liver, PBMCs and retinal neurons). This was due to the lack of reference cell type annotations needed for the ROC and PR curve analyses. As more scRNA-seq datasets become available and authors provide gold standard annotations of their cell types, those annotations could be used as references to benchmark methods with other scRNA-seq datasets. This is exemplified by the LM22 signature, which was constructed by Newman et al. (2015b) from microarray gene expression measurements to predict cell types from bulk RNA-seq data, and we have shown here that LM22 could also be used to detect cell types from scRNA-seq data. Thus, in the future, we envision that methods to detect differentially expressed genes can be used as part of pipelines to produce cell type gene expression signatures. As with any classification task, researchers would need to control for circularity between training, test and validation cell-annotation data and also will need to evaluate generalizability.\n\nOne of the challenges that we faced while adapting the LM22 signature to detect cell types in the scRNA-seq cell clusters generated by Zheng et al. (2017a) was that, even though both datasets correspond to PBMCs, the granularity of their cell type labels was different. For instance, the LM22 signature contains six T-cell types, including three CD4+ (naïve, memory resting, and memory activated), follicular helper, regulatory and gamma delta, whereas the dataset of Zheng et al. (2017a) contained labels for four T-cell related cell types: CD4+/CD25 T Regulatory, CD4+/CD45RO+ Memory, CD4+/CD45RA+/CD25- Naive T and CD4+ T Helper2. Thus, even though these two datasets both classify PBMCs, they cannot be easily related one-to-one. This could be addressed with an ontology analogous to the Gene Ontology (Ashburner et al., 2000) but dedicated to cell type annotations (Bakken et al., 2017; Bard et al., 2005). Fortunately, the Cell Ontology is being developed for this purpose. This is particularly important as an increasing number of signatures are expected to arise from initiatives like the Human Cell Atlas (Rozenblatt-Rosen et al., 2017).\n\n\nData availability\n\nThe datasets used in this study were processed from the below underlying source data:\n\nSingle-cell RNA-sequencing data from human liver cells. Accession number, GSE115469. https://identifiers.org/geo/GSE115469.\n\nSingle-cell RNA-sequencing data from human peripheral blood mononuclear cells. Accession number, SRX1723926. https://identifiers.org/insdc.sra/SRX1723926.\n\nSingle cell RNA-sequencing of retinal bipolar cells. Accession number, GSE81905. https://identifiers.org/geo/GSE81905.\n\nZenodo: Supplementary data for \"Evaluation of methods to assign cell type labels to cell clusters from single-cell RNA-sequencing data\". http://doi.org/10.5281/zenodo.2575050 (Diaz-Mejia, 2019a).\n\nThis project contains the three processed scRNA-seq datasets—from liver cells (MacParland et al., 2018), peripheral blood mononuclear cells (Zheng et al., 2017a) and retinal neurons (Shekhar et al., 2016b)—examined in this study.\n\n\nSoftware availability\n\nR and Perl scripts used to run and benchmark cell type labeling methods available from: https://github.com/jdime/scRNAseq_cell_cluster_labeling.\n\nArchived code at time of publication: http://doi.org/10.5281/zenodo.2583161 (Diaz-Mejia, 2019b).\n\nLicense: MIT license.", "appendix": "Grant information\n\nJJDM, ECM, ARP, and JHM are funded by grant number 2018-183120 from the Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation. ARP, GDB and JHM are supported by the National Resource for Network Biology, P41GM103504 (NIGMS).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are thankful to Jeff Liu and Brendan Innes from the Bader lab for advice processing the liver dataset and implementing GSVA; to Danielle Croucher and Laura Richards from the Pugh lab for feedback collecting benchmark datasets; and to Rene Quevedo from the Pugh lab for help implementing R scripts.\n\n\nReferences\n\nAlavi A, Ruffalo M, Parvangada A, et al.: A web server for comparative analysis of single-cell RNA-seq data. Nat Commun. 2018; 9(1): 4768. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlquicira-Hernandez J, Nguyen Q, Powell JE: scPred: scPred: Cell type prediction at single-cell resolution. bioRxiv. 2018. 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Publisher Full Text\n\nMacParland SA, Liu JC, Ma XZ, et al.: Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat Commun. 2018; 9(1): 4383. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNewman AM, Liu CL, Green MR, et al.: Robust enumeration of cell subsets from tissue expression profiles. LM22 signature. 2015a. Reference Source\n\nNewman AM, Liu CL, Green MR, et al.: Robust enumeration of cell subsets from tissue expression profiles. Nat Methods. 2015b; 12(5): 453–457. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRozenblatt-Rosen O, Stubbington MJT, Regev A, et al.: The Human Cell Atlas: from vision to reality. Nature. 2017; 550(7677): 451–453. PubMed Abstract \| Publisher Full Text\n\nShekhar K, Lapan SW, Whitney IE, et al.: Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics.2016a; Reference Source\n\nShekhar K, Lapan SW, Whitney IE, et al.: Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics. Cell. 2016b; 166(5): 1308–1323.e30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubramanian A, Tamayo P, Mootha VK, et al.: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–15550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng GX, Terry JM, Belgrader P, et al.: Massively parallel digital transcriptional profiling of single cells. Nat Commun. 2017a; 8: 14049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng GXY, Terry JM, Belgrader P, et al.: Fresh 68k PBMCs (Donor A). 2017b. Reference Source\n\nZheng GXY, Terry JM, Belgrader P, et al.: Single Cell RNA-seq Secondary Analysis of 68k PBMCs. 2017c. Reference Source" }	[ { "id": "45814", "date": "20 Mar 2019", "name": "Saskia Freytag", "expertise": [ "Reviewer Expertise bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDiaz-Mejia et al have produced a nice research article on assessing methods for assigning cluster labels to cell clusters from scRNA-seq. I think this work is of great importance, but I felt that some crucial cluster labelling methods were not compared. I hope the authors can update the article with some of the suggestions:\n\nThe biggest suggestion for improvement is the choice of methods that the authors compare. The authors chose to adapt 4 methods originally developed for bulk RNA-seq in order to label clusters. While their approach is commendable, none of their adapted methods reflect the current standard practice in the field. Additionally, their claim that methods for cluster labelling in scRNA-seq are too immature or implemented as web-servers is not true. The website scRNA-tools.org lists 29 methods in this category. Many of these methods, such as scMCA, MetaNeighbour and scmap, are well-established and frequently used in the field. Furthermore, many of these methods recommend using annotated scRNA-seq datasets as references instead of bulk data. Hence, it would be great if the authors could include some of these tools in their analysis.\n\nI am confused as to which classifier parameter was varied in order to generate the ROCs. Were these comparable across the different methods?\n\nIt would be interesting to see what the effect of varying the cluster resolution is to the ability of the methods to accurately label the populations. Do you obtain more diverse labelling when there are more clusters?\n\nLM22 is a great reference dataset, but recently a new dataset has become openly accessible. This dataset, generated by Monaco et al1, characterizes 29 human immune cell types by RNA-seq and flow cytometry. It would be interesting to see if the use of this dataset leads to an improvement.\n\nI think it would be helpful for the reader if the authors could summarize their results. The sheer number of comparisons made, means that the reader can feel overwhelmed at the end. A figure summarizing the various results for each method in each dataset could help clarify the message.\n\nThank you for making your code publicly available.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4843", "date": "27 Aug 2019", "name": "J. Javier Diaz-Mejia", "role": "Author Response", "response": "R1-Q1) The biggest suggestion for improvement is the choice of methods that the authors compare. The authors chose to adapt 4 methods originally developed for bulk RNA-seq in order to label clusters. While their approach is commendable, none of their adapted methods reflect the current standard practice in the field. Additionally, their claim that methods for cluster labelling in scRNA-seq are too immature or implemented as web-servers is not true. The website scRNA-tools.org lists 29 methods in this category. Many of these methods, such as scMCA, MetaNeighbour and scmap, are well-established and frequently used in the field. Furthermore, many of these methods recommend using annotated scRNA-seq datasets as references instead of bulk data. Hence, it would be great if the authors could include some of these tools in their analysis.R1-A1) We thank the reviewer for their comments. We have added MetaNeighbor to the methods compared. The implementation of MetaNeighbor required considerable communication with one of the method developers (M. Crow) who kindly guided us on which parts of the MetaNeighbor source code we needed to modify to make one of its variants (MetaNeighborUS) compatible with the type of task in our study. As we detail in the Methods section ‘Implementation of tested methods and transformation of enrichment metrics for ROC and PR analyses’, the original goal of MetaNeighbor is to quantify cell type replicability across scRNA-seq datasets (that the authors call ‘studies’); whereas in our comparison, we are “comparing” all the clusters in one scRNA-seq dataset against known cell type specific gene sets or gene expression profiles. Similar to MetaNeighbor, Scmap projects cells from a scRNA-seq experiment on to the cell types or individual cells identified in a different experiment. We would need to apply similar workarounds and modify its code to use it in our study. Although we acknowledge that adding more methods to our comparison would make our results more complete, these other methods were not designed for the specific task we evaluate and would require code modifications to work on our input data. However, we provide an extensible framework, code and datasets that others can use for additional benchmarks. We now clarify this point in the paper.R1-Q2) I am confused as to which classifier parameter was varied in order to generate the ROCs. Were these comparable across the different methods?R1-A2) Sorry for the confusion. For a set of cell clusters, a given method was used to score each cluster against all cell type gene sets resulting in a matrix of cell type prediction scores per cluster. All scores in this matrix were combined into one column to capture all cell type prediction scores across all clusters and this set of prediction scores was varied to generate the ROC and PR curves. This is now clarified in Figure 1 and the text.R1-Q3) It would be interesting to see what the effect of varying the cluster resolution is to the ability of the methods to accurately label the populations. Do you obtain more diverse labelling when there are more clusters?R1-A3) Presumably yes. However, there is a methodological barrier that prevents us from investigating this aspect of the data using our current evaluation design. Authors of the analyzed datasets provided gold standard annotations only at a single resolution per dataset, and we use these. Reclustering the original data to test other resolutions would require gold standards to be created for those resolutions (ideally by the original authors). However, we agree with the reviewer that studying the influence of cell cluster resolution is an interesting question. As the field moves towards increasing the number of scRNA-seq datasets annotated following standard ontology-based cell type annotations that consider a hierarchy of cell types at multiple granularities, this question could be addressed. We have added this to our discussion.R1-Q4) LM22 is a great reference dataset, but recently a new dataset has become openly accessible. This dataset, generated by Monaco et al1, characterizes 29 human immune cell types by RNA-seq and flow cytometry. It would be interesting to see if the use of this dataset leads to an improvement.R1-A4) Thanks for the pointer. We decided to keep the LM22 dataset because only six of the 22 cell types represented in it could be mapped into the PBMC data we analyzed. The Monaco dataset does not improve this number. Only five of the 17 cell types represented in the Monaco signature for RNA seq data are present in the PBMC data we analyzed. Furthermore, the ROC AUC and PR AUC values obtained using the LM22 and the Monaco signature are comparable to each other (Supplementary Table 2).R1-Q5) I think it would be helpful for the reader if the authors could summarize their results. The sheer number of comparisons made, means that the reader can feel overwhelmed at the end. A figure summarizing the various results for each method in each dataset could help clarify the message.R1-A5) Thanks for raising this point. We have now included summary Figure 6. R1-Q6) Thank you for making your code publicly available.R1-A6) Thanks. We have updated our GitHub repository with the MetaNeighbor implementation and modifications to our main wrapper to make it easier to incorporate new methods." } ] }, { "id": "45811", "date": "22 Mar 2019", "name": "Jimmy Tsz Hang Lee", "expertise": [ "Reviewer Expertise Bioinformatics", "single-cell RNA-seq", "clustering", "network inference" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDiaz-Mejia et al. test the ability of four different algorithms to correctly annotate a set of clusters identified in single-cell RNA-seq data. They find that GSVA tends to be the most accurate and fastest method, interestingly they find ORA and GSVA are much more robust to small numbers of marker genes than GSEA or CIBERSORT. This is a very useful and timely study, as manual annotation of cell-types is currently the main bottleneck when analyzing single-cell RNA-seq data.\nComments:\nIt was unclear to me how the accuracy of the classification methods was evaluated. What was the gold standard truth used for each dataset? Were clusters assigned to (a) the single cell-type for which they had the greatest score or (b) all cell-types where their score exceeded some threshold, or (c) to the single cell-type for which they had the greatest score provided that score was above some threshold or another approach? This is crucial to interpreting the PR and ROC curves presented in the results. Based on the first sentence of the “Precision-Recall curve analysis” section: I inferred you to be using method (c), but using such a method should not necessarily lead to recall values of 1 as clusters which are more similar to an incorrect cell-type than to the correct cell-type would never become true positives. Thus, I had inferred you to be using method (b) based on Figure 2. It would be very helpful to add a section to the Methods explaining precisely how the accuracy was evaluated. In addition, I suggest adding figures/tables for the accuracy of each classification approach (% of clusters correctly assigned) when all clusters are simply assigned to the cell-type for which they have the highest score, since I expect this to be the most common approach users of these classifications would take. The main weakness of the paper, as the authors admit, is the small number of datasets used to test the classification methods, particularly since the variability in performance between datasets was high. It would be useful to show reproducibility of the results in additional datasets. We acknowledge identifying marker gene lists for many different tissues can be very time consuming, there are datasets similar to those the authors have already have markers for that they could use. E.g. mouse retina: Shekhar et al. 20161, PBMCs: Gierahn et al. 2017 (Seq-Well)2. Alternatively, they could do cross-comparisons using the two mouse cell atlas (Tabula Muris3 and Mouse Cell Atlas4). Or use datasets such as Pollen et al., 20145 where gold-standard cell-type identity is known by design. The authors show that performance degrades when small numbers of marker genes are used by the classifiers. Is it the case that more marker genes is always better or does performance also degrade if too many genes are used?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4844", "date": "27 Aug 2019", "name": "J. Javier Diaz-Mejia", "role": "Author Response", "response": "R2-Q1) It was unclear to me how the accuracy of the classification methods was evaluated. What was the gold standard truth used for each dataset? Were clusters assigned to (a) the single cell-type for which they had the greatest score or (b) all cell-types where their score exceeded some threshold, or (c) to the single cell-type for which they had the greatest score provided that score was above some threshold or another approach? This is crucial to interpreting the PR and ROC curves presented in the results.Based on the first sentence of the “Precision-Recall curve analysis” section: I inferred you to be using method (c), but using such a method should not necessarily lead to recall values of 1 as clusters which are more similar to an incorrect cell-type than to the correct cell-type would never become true positives. Thus, I had inferred you to be using method (b) based on Figure 2. It would be very helpful to add a section to the Methods explaining precisely how the accuracy was evaluated.R2-A1) Apologies for the confusion around this point. We have now clarified how the ROC and PR curves were computed in Figure 1 and the text, as described for reviewer 1, above. We combine all cell type gene set prediction scores for a method across all clusters into one column and vary the prediction score threshold to compute the ROC and PR curves. A cluster is only allowed to be correctly labeled using one cell type, as enforced by our gold standard cluster annotation data (the set of cell types an author used to label their given cell clusters). So this matches strategy (c).R2-Q2) In addition, I suggest adding figures/tables for the accuracy of each classification approach (% of clusters correctly assigned) when all clusters are simply assigned to the cell-type for which they have the highest score, since I expect this to be the most common approach users of these classifications would take.R2-A2) Percent of clusters correctly assigned is now included in Figure 6C and Supplementary Table 1. It is useful to have a range of performance indicators to capture different performance facets.R2-Q3) The main weakness of the paper, as the authors admit, is the small number of datasets used to test the classification methods, particularly since the variability in performance between datasets was high. It would be useful to show reproducibility of the results in additional datasets.We acknowledge identifying marker gene lists for many different tissues can be very time consuming, there are datasets similar to those the authors have already have markers for that they could use. E.g. mouse retina: Shekhar et al. 20161, PBMCs: Gierahn et al. 2017 (Seq-Well)2. Alternatively, they could do cross-comparisons using the two mouse cell atlas (Tabula Muris3 and Mouse Cell Atlas4). Or use datasets such as Pollen et al., 20145 where gold-standard cell-type identity is known by design.R2-A3) We thank the reviewer for suggesting these datasets. We already used Shekhar et al. 2016 in version 1 of our paper. In the current version, we added Gierahn et al. (2017) as the authors provide cell type labels for the cell clusters, and used the LM22 cell type signatures as input for the prediction methods. We also added the Tabula Muris dataset. We contacted one of the Tabula Muris authors (Angela Pisco), who kindly gave us access to a set of cell type signatures curated by experts on tissues of the Tabula Muris dataset. From the 20 tissues provided in the Tabula Muris data we could map 11 of them into the dataset of cell type signatures and that is what we used as ‘Tabula Muris 11’ in the current version of our paper. We also investigated the influence of cell type signatures using the PBMC datasets (10X and Seq-Well) using either the full LM22 signature database, that we call ‘PBMC-22’, or only the six cell types expected to occur in the data, that we call ‘PBMC-6’. Altogether, we provide analysis using eight dataset variants, up from the three in our initial manuscript.R2-Q4) The authors show that performance degrades when small numbers of marker genes are used by the classifiers. Is it the case that more marker genes is always better or does performance also degrade if too many genes are used?R2-A4) In general, having more marker genes is better, but not always. We approached this question by examining the influence of the number of genes in each gene set (x-axis) and asking what rank does the corresponding gold standard positive receive (y-axis). As can be seen in Figure 6E, the most common scenario is that the fewer the number of genes in the signatures, the more chances that the prediction is incorrect (i.e. assigned a rank lower than the top-rank). However, there are a few exceptions, like ORA in the 11-20 genes bin, where we found more incorrect predictions than having 6-10 genes, or CIBERSORT, which had higher error rate in the 31-50 genes category, than in the 11-20 or 21-30 categories. Thus, it is possible to use too many genes, but it is not always clear how many genes this will be and the performance drop is not great for most of the cases we have data for. We have added this analysis to the paper." } ] }, { "id": "45813", "date": "01 Apr 2019", "name": "Lindsay Cowell", "expertise": [ "Reviewer Expertise computational immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors address an important problem, which is the need for systematic and reproducible approaches for assigning cell type labels based on single cell transcriptome data. They use three data sets with gold standard cell type annotations available and compare the performance of four computational tools on these data sets. The authors measure performance using ROC curves and plots of precision versus recall. They also assess performance over subsamples of the data used as reference gene expression patterns for cell types (either cell type-specific gene sets or cell type-specific expression profiles). In general, they found that all four methods perform reasonably well, although ORA and GSVA perform more consistently well across the three data sets. I do have some questions about the details of how the work was done. The answers to these questions are important for interpreting the results, reproducing the work, or extending it to include additional tools.\nPresumably the approach to creating the cell clusters, and how dense versus diffuse the clusters are, can have an impact on performance and confidence in the output? How exactly were clusters mapped to cell types? From Figure 1E, it appears that each of the four tools generates a numerical vector for each cell type that contains a score for each cluster, presumably corresponding to the likelihood that that cluster is of the corresponding cell type.\nIs a cluster always assigned to the cell type corresponding to its highest score? (presumably yes). In the example, each cell type and each cluster has only a single high score with all other scores being very small. What is the distribution of scores typically? Do clusters sometimes have multiple high scores? Were ties ever observed? Can multiple clusters map to the same cell type? Must a cluster be assigned to a cell type? Or could some remain unassigned?\n\nHow were the performance curves generated? What parameter was varied?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4845", "date": "27 Aug 2019", "name": "J. Javier Diaz-Mejia", "role": "Author Response", "response": "R3-Q1) Presumably the approach to creating the cell clusters, and how dense versus diffuse the clusters are, can have an impact on performance and confidence in the output?R3-A1) We agree that cluster density and other structure in the data will likely impact automatic cluster annotation performance. Investigating the relationship between a given structure in the data (e.g. density vs. sparseness) and performance would require simulations that may not be realistic. Thus, we limited our analysis to published data with available gold standards. We have now added this point to the discussion.R3-Q2) [a] How exactly were clusters mapped to cell types? From Figure 1E, it appears that each of the four tools generates a numerical vector for each cell type that contains a score for each cluster, presumably corresponding to the likelihood that that cluster is of the corresponding cell type.[b] Is a cluster always assigned to the cell type corresponding to its highest score? (presumably yes).[c] In the example, each cell type and each cluster has only a single high score with all other scores being very small. What is the distribution of scores typically? Do clusters sometimes have multiple high scores? Were ties ever observed?[d] Can multiple clusters map to the same cell type?[e] Must a cluster be assigned to a cell type? Or could some remain unassigned?R3-A2)[a] Correct, each tool generates a numerical vector as the reviewer describes.[b] Yes, a cluster is always assigned to the cell type corresponding to its highest score.[c] In the methods that we compared, each cell cluster vs. each cell type receives only one score. As can be observed in our new Figure 6E, most cell clusters which were incorrectly classified (i.e. that were not the top-1 ranked prediction) still had top-ranks (ticker distribution in the violin plots closer to the top-1 ranks), which indicates that some clusters can have multiple high scores. We found that 118 out of all 1,276 (9.2%) cell cluster labeling predictions we ran showed ties in the top-score: 65 of the 118 ties (68%) corresponded to METANEIGHBOR ‘binary, 24 (20%) to ORA, 15 (13%) to METANEIGHBOR ‘continuous’, 10 (8%) to GSEA, and 4 (3%) to GSVA. None of the CIBERSORT analyses showed ties.[d] Yes, multiple clusters can map to the same cell type and this is particularly the case for the newly incorporated Tabula Muris dataset, where 130 cell clusters map to 53 cell types. This doesn’t affect our evaluation because a method is not penalized for predicting that multiple clusters have the same cell type annotation.[e] Yes, a cluster must be assigned a cell type in our case because all clusters have a cell type assignment in our gold standards. In the case of the newly incorporated PBMC-SeqWell data (Gierahn et al., 2017), some of the cell clusters were labeled as ‘Removed_’ by the authors, and they didn’t classify those clusters into cell types, thus we did not include these in our analysis.As mentioned above in response to reviewers 1 and 2, we’ve updated the Methods section “Implementation of tested methods and transformation of enrichment metrics for ROC and PR analyses” to clarify all of these points.R3-Q3) How were the performance curves generated? What parameter was varied?R3-A3) As mentioned above in response to reviewers 1 and 2, we’ve updated the Methods section “Implementation of tested methods and transformation of enrichment metrics for ROC and PR analyses” to clarify this. For each dataset, we combine all cell type gene set prediction scores for a method across all clusters into one column and vary the prediction score threshold to compute the ROC and PR curves." } ] } ]	1	https://f1000research.com/articles/8-296
https://f1000research.com/articles/8-1743/v1	11 Oct 19	{ "type": "Research Article", "title": "Adhesive systems effect over bond strength of resin-infiltrated and de/remineralized enamel", "authors": [ "Alessandra Buhler Borges", "Amjad Abu Hasna", "Amanda Guedes Nogueira Matuda", "Stephanie Ribeiro Lopes", "Ana Paula Valente Pinho Mafetano", "Aline Arantes", "Angélica Ferreira Duarte", "Daphne Camara Barcellos", "Carlos Rocha Gomes Torres", "Cesar Rogério Pucci", "Alessandra Buhler Borges", "Amjad Abu Hasna", "Amanda Guedes Nogueira Matuda", "Stephanie Ribeiro Lopes", "Ana Paula Valente Pinho Mafetano", "Aline Arantes", "Angélica Ferreira Duarte", "Daphne Camara Barcellos", "Carlos Rocha Gomes Torres" ], "abstract": "Background: The purpose of this study was to evaluate the effect of different bonding agents on bond-strength to demineralized enamel after remineralizing treatments and resin infiltration. Methods: Buccal enamel of 120 bovine incisors was polished and then were divided into five experimental groups: SE (sound enamel); DE (demineralized enamel); AS (demineralized enamel immersed in artificial saliva for eight weeks); NaF (demineralized enamel treated with 0.05% sodium fluoride solution (one minute) for eight weeks); Ic (demineralized enamel infiltrated with a low-viscosity resin (Icon-DGM). These groups were subdivided according to adhesive system used: self-etching adhesive Adper Easy One (3M/ESPE) and etch-and-rinse adhesive Single Bond (3M/ESPE). The composite resin blocks were fabricated using a Teflon matrix. A thermomechanical cycling machine was used to carry out the artificial aging of the specimens and thus were sectioned into sticks. The microtensile tests were performed using a universal testing machine at a cross-head speed of 1 mm/min. Data (in MPa) were subjected to two-way ANOVA and Tukey’s tests (5%). Results: Significant differences were found for both factors tested and interactions (p<0.05). Tukey’s test results of µTBS (mean ± SD) were: etch-and-rinse SE (28.79±3.93); DE (30.41±7.22); AS (29.03±3.33); NaF (29.81±4.06)a; Ic (29.47±5.5); and self-etching SE (30.37±6.96); DE (14.62±4.47); AS (9.79±2.32); NaF (9.36±2.31); Ic (30.78±8.68).\n\nConclusions: Resin infiltration did not affect the bond strength of demineralized enamel for both adhesive systems tested. For etch-and-rinse adhesive, no differences were observed for the tested groups. For self-etching adhesive, only the resin-infiltrated group showed similar bond strength to sound enamel. Both etch-and-rinse and self-etching adhesive systems can be used in resin-infiltrated enamel, if a composite restoration needs to be further performed. In enamel that has undergone the de/remineralization process, the use of a total-etch adhesive might be preferable for the restorative procedure.", "keywords": [ "Enamel", "Adhesives", "Remineralization", "Demineralization", "Resin infiltration." ], "content": "Introduction\n\nDental caries is a disease that results from the interaction of microbial factors, diet, host factors, and time. When an imbalance occurs in the de/remineralization process on the tooth surface, demineralization takes over and the initial carious lesions occur (Fejerskov et al., 2015). The submicroscopic changes of initial enamel demineralization include mineral loss from the body of the lesion, with enlargement of intercrystalline spaces and a reduction in subsurface microhardness, whereas the surface remains comparatively highly mineralized (Featherstone, 2004; Rocha Gomes Torres et al., 2011). The enamel pores cause the characteristic whitish appearance and promote diffusion routes for acids and dissolved minerals (Paris et al., 2007b; Torres et al., 2012).\n\nInitial enamel carious lesions can regress or even disappear with remineralization treatment (Featherstone, 2004; Fejerskov et al., 2015; Rocha Gomes Torres et al., 2011). Non-cavitated caries lesions can be repaired by saliva, if there is control of diet and plaque. Additionally, treatment with fluoride is an alternative noninvasive method used for remineralizing carious processes because fluoride improves the acid resistance of enamel and interferes with bacterial metabolism and enzymatic processes. Thus, less invasive treatments have long been adopted to control the progression of initial enamel carious lesions (Anusavice, 1997; Anusavice, 1998).\n\nThe resin infiltration technique is a micro-invasive alternative treatment to prevent the progression of non-cavitated carious lesions. The purpose of low viscosity light-activated resin infiltration is to seal the carious lesion microporosities in order to create a diffusion barrier within the lesion, preventing acids from penetrating into the lesion without resulting in any material on the enamel surface (Paris et al., 2007b). A number of studies have shown the effectiveness of this technique (Meyer-Lueckel et al., 2012; Paris et al., 2006; Paris et al., 2010; Paris et al., 2007). The resin matrix is able to strengthen the enamel structure, increasing surface microhardness and thereby preventing enamel surface breakdown (Anusavice, 1997; Anusavice, 1998; Paris et al., 2007; Paris et al., 2007b; Torres et al., 2012).\n\nThe purpose of this study was to evaluate the micro-tensile bond strength (μTBS) between composite resin and demineralized enamel that has been remineralized by different treatments or resin infiltrated, comparing two adhesive systems: self-etching and etch-and-rinse.\n\n\nMethods\n\nIn this study, 120 bovine incisors were obtained from a slaughterhouse (Mantiqueira - Sao Jose dos Campos - SP - Brazil). Teeth were sectioned 2 mm from the cement-enamel junction to standardize the specimens. The crowns were embedded in acrylic resin and the enamel of the buccal surfaces was worn using abrasive papers (600 grit, FEPA P, Extec, Enfield, CT, USA) coupled to a circular polishing machine (PA-10, Panambra, São Paulo, Brazil) under water-cooling, to expose a standardized area of 6 x 6 mm.\n\nAll specimens were subjected to demineralization (artificial caries), except the control group (sound enamel), and were separately immersed in a solution composed of 50mM acetate buffer solution containing 1.28 mM Ca(NO3)2·4H2O, 0.74 mM NaH2PO4·2H2O, and 0.03 ppm fluorine at a pH of 5.0 for 16 hours at 37ºC. The total volume of solution used was calculated using 2 mL/mm2 of the enamel area (Queiroz et al., 2008).\n\nArtificial saliva was prepared according to the formulation of (Göhring et al., 2004) and consisted of hydrogen carbonate (22.1 mmol/L), potassium (16.1 mmol/L), sodium (14.5 mmol/L), hydrogen phosphate (2.6 mmol/L), boric acid (0.8 mmol/L), calcium (0.7 mmol/L), thiocyanate (0.2 mmol/L), and magnesium (0.2 mmol/L), with a final of pH 7.0.\n\nThe specimens were divided into four groups (n=30) according to the caries treatment used Table 1.\n\nIn the resin infiltration (Ic) group, the infiltration procedure was carried out in accordance with the manufacturer’s instructions: (1) Icon-Etch (15% hydrochloric acid) was applied for two minutes and then the specimens were rinsed with water and air dried for 30 seconds; (2) Icon-Dry (ethanol) was applied for 30 seconds and air dried; (3) Icon-Infiltrant was applied two times, the first time for three minute and the second time for one minute; (4) both applications were light cured for 40 seconds; (5) specimens were polished with aluminum oxide abrasive papers (4000 grit; FEPA-P, Extec) for 20 seconds, to remove the surplus material.\n\nTwo adhesive systems, namely the etch-and-rinse technique, using two layers of Adper Single Bond/SB total-etch adhesive (3M ESPE, St. Paul, MN, USA), and the one-step self-etching technique, using Adper Easy One one-step self-etching adhesive system (3M ESPE), were used in each group (n=15), as used in the study of (Gupta et al., 2017).\n\nComposite resin blocks (Filtek Z350, 3M ESPE) (4mm high) were built on the treated surfaces of enamel using a Teflon mold. Every 2mm portion was light cured for 40 seconds. The bonded specimens were stored in distilled water (37ºC for 24 h).\n\nThe specimens were then subjected to thermo-mechanical wear (37000 ER machine, ERIOS, São Paulo, SP, Brazil). Mechanical cycling was performed with load of 60 N and 100,000 cycles. The load was applied on the composite resin restoration, perpendicular to the enamel surface. Simultaneously, thermal cycling was performed with distilled water at temperatures of 5ºC, 37ºC and 55ºC for 30 seconds at each temperature, totaling 10,000 cycles (Bedran-de-Castro et al., 2004).\n\nParallel sections measuring approximately 1mm2 were used, as in the study of (Farias de Lacerda et al., 2016), and μTBS tests were performed in a universal testing machine (DL-1000, EMIC, São José dos Pinhais, PR, Brazil), with a 10 kg load cell, at a cross-head speed of 0.5 mm/min, in accordance with the ISO 11405 Standard. The μTBS data were expressed in megapascal (MPa).\n\nQualitative analysis was performed with stereomicroscopy (Discovery V20, Germany) at 20× magnification for failure mode analysis of each specimen. Failure mode was classified as:\n\n(1) Predominant cohesive in composite (composite resin failure)\n\n(2) Predominant cohesive in enamel (enamel failure)\n\n(3) Adhesive in the interface enamel/composite (failure only in the interface)\n\n(4) Mixed (mixed failure between composite resin -enamel and cohesive in composite or cohesive in enamel)\n\nSticks that presented cohesive failure were discarded. The mean value for the sticks originating from each tooth was calculated and used for the statistical analysis. Data were analyzed by two-way ANOVA (enamel treatment, adhesive system) followed by the Tukey’s test (α = 5%) using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).\n\nSpecimens were sectioned perpendicularly to the bonding interface for SEM analysis. The sections were polished with 2000 and 4000 mesh abrasives. Phosphoric acid etchant was applied for five seconds and rinsed off with water for 10 seconds. Specimens were dehydrated, sputter-coated with gold-palladium and examined by SEM (Inspect S50, FEI, Hillsboro, Oregon, USA) operated at 15 kV (Farias de Lacerda et al., 2016).\n\n\nResults\n\nTable 2 presents the results of the Tukey’s test for the factor ‘adhesive system’. It can be observed that etch-and-rinse showed significantly higher values than self-etching (Abu Hasna, 2019).\n\nMean values with different letters show significant difference.\n\nTable 3 shows the results of the Tukey’s test for the factor ‘enamel treatment’. Positive control and resin infiltration groups showed significantly higher values than the negative control, 0.05% fluoride solution and artificial saliva groups (p<0.05).\n\nMean values with same letters show there was no statistically significant difference.\n\nAll groups showed significantly higher μTBS values than self-etching adhesive bonded to demineralized enamel that had been remineralized by saliva or sodium fluoride and bonded to negative control (demineralized enamel that received no remineralizing treatment) (Figure1).\n\nThe letters denote significant differences among the groups. SE, sound enamel; DE, demineralized enamel; AS, artificial saliva; NaF, sodium fluoride; Ic, resin infiltration; SEtch, self-etching; E&R, etch-and-rinse.\n\nThe adhesive failures were predominant in all experimental groups (Figure 2).\n\nSE, sound enamel; DE, demineralized enamel; AS, artificial saliva; NaF, sodium fluoride; Ic, resin infiltration.\n\nFigures 3 to Figure 6 show SEM images obtained from the interfaces of the self-etching treated.\n\nThe porous enamel subsurface remains visible because demineralized enamel could not be completely infiltrated by the self-etching adhesive system.\n\nThe image shows reduced enamel penetration of the self-etching adhesive system.\n\nIt is observed a poor adhesive penetration, with a fracture line between the adhesive and the treated enamel.\n\nDemineralized enamel was permeated with the infiltrant resin and no distinction between the infiltrant and the adhesive is observed.\n\n\nDiscussion\n\nThis study showed that the etch-and-rinse adhesive system presented superior μTBS means in comparison with the self-etching system. The enamel etch-and-rinse method is based on selective demineralization of the hydroxyapatite crystals founded in tooth enamel, resulting in a highly roughened surface with elevated energy. These features offer better wetting capacity of the resinous monomers that, when polymerized, result in prolongations named tags that ‘anchor’ the resin to the tooth (Gwinnett, 1981; Torres et al., 2009). In dentistry, the substance most commonly used for this purpose is phosphoric acid at a concentration of 35-37%. Because of its high ionization potential, it results in a final pH of 0.6. As a consequence of the high availability of H+ ions, its application for short periods, such as the 15 seconds usually recommended, is capable of producing a suitable enamel etching pattern (Barkmeier et al., 1986), resulting in exceptional micromechanical interlocking by the tags created (Torres et al., 2009).\n\nAcidic adhesive agents, called self-etchants, were introduced with the objective of promoting simultaneous demineralization and impregnation of the substrate. The methacrylated phosphoric ester present in the Adper Easy One adhesive system is used for this purpose. The self-etching adhesive systems were selected depending on their pH values (Poggio et al., 2014; Van Meerbeek et al., 2003), and Adper Easy One is classified as mild (pH >2.0). Therefore, the concentration of this adhesive solution in an aqueous solution and the number of ionizable radicals are lower in comparison with those in phosphoric acid, and consequently, the etching capacity of this adhesive system is more restricted. As a result, this one-step adhesive does not demineralize enamel to the same extent as phosphoric acid does, promoting a less microretentive surface and usually lower μTBS values (Poggio et al., 2014).\n\nMoreover, according to (Erickson et al., 2009), phosphoric acid promotes a particular morphology of the resin-enamel that permits fairly extensive resin penetration, creating a three-dimensional structure (i.e. scalloped), and the transition from resin to sound enamel is distributed over a variety of microns. This interface can be more resistant to crack propagation when compared with the relatively planar interface promoted by self-etch adhesives.\n\nThe groups treated with artificial saliva and sodium fluoride showed the lowest μTBS values when associated with self-etching Adper Easy One. Saliva contains calcium (Ca) and phosphate (Pi) in supersaturated concentrations, and these ions are continually deposited or re-deposited on the enamel surface that has suffered loss of these ions (Cury & Tenuta, 2009). The fluoride ions can also remain incorporated into remineralizing enamel, mainly in surface lesions, changing the carbonated apatite to a fluoroapatite-like form that is more acid tolerant and makes more acid resistant hard tissues (Cury & Tenuta, 2009; Lee et al., 2010; Rocha Gomes Torres et al., 2011). A previous study showed that both saliva exposure for eight weeks and daily sodium fluoride treatment resulted in increased surface microhardness of demineralized specimens (Rocha Gomes Torres et al., 2011). It is therefore hypothesized that the increased acid resistance of remineralized enamel impaired the conditioning effect of the moderate self-etching system tested, thus promoting a less microretentive surface, as shown in Figure 2, and consequently, lower μTBS values. Additionally, inactive lesions have thick surface layers compared with active lesions (Neuhaus et al., 2013). These remineralizing treatments may have promoted thick surface layers of inactive lesions, inhibiting penetration of both the acidic and resinous monomers of Adper Easy One into the lesion.\n\nWhen the etch-and-rinse system was used, no difference was observed between sound and demineralized enamel bonding, as also shown previously (Wiegand et al., 2011). Nevertheless, the demineralized enamel presented lower μTBS values than the sound specimens when the self-etching system was applied, in accordance with a previous study (Jia et al., 2012). Similar to the groups remineralized with artificial saliva and fluoride, this result might be explained by differences in lesion structures, in particular with regard to the surface layer. The surface layer of an initial enamel carious lesion has a higher mineral content in comparison with the underlying body of the lesion (Cury & Tenuta, 2009; Torres et al., 2012). Therefore, this surface layer can form a barrier, hampering infiltration into the lesion body. In order to increase surface layer porosity, acid etching has been considered to make the underlying body of lesion accessible (Paris et al., 2007b). The etching capacity of Adper Easy One may not be effective in degrading the surface layer, due to the higher pH and lower acidic capacity of this mild self-etch adhesive, as also reported previously with a self-etching system (Mueller et al., 2006). This would result in a shallow inter-crystallite infiltration of the adhesive and a lack of inter-prismatic resin tag formation (Erickson et al., 2009), with remaining non-infiltrated porosities (Figure 3), which could be a possible explanation for the less effective bonding of Adper Easy One to demineralized enamel.\n\nThe groups infiltrated with the low-viscosity resin and bonded with Adper Easy Bond or Single Bond adhesives showed similar μTBS values to the positive control group (sound enamel). The resin infiltrant contains monomers with high penetration coefficients and adequate hardening (Paris et al., 2013). The satisfactory μTBS values associated with infiltrated groups may have been optimized due to the affinity between the monomers present in the infiltrant and the monomers of the adhesive systems. According to the results of the present study, resin infiltration is compatible with both total-etch and self-etch adhesives, and therefore, restorative treatment can be indicated on tooth surfaces treated with resin infiltration, since it does not negatively interfere in the composite bond to enamel. Previous studies also showed that the application of an etch-and-rinse adhesive after resin infiltration did not alter enamel μTBS (Wiegand et al., 2011), or even increase the adhesion of a self-etching adhesive (Jia et al., 2012).\n\nIn order to infiltrate a caries lesion, resin infiltration requires the application of 15% hydrochloric acid to promote erosion of the surface layer and allow the resin to penetrate into the porous spaces of the lesion body (Paris et al., 2006; Torres et al., 2012). An appropriate acid etching pattern enhances resin infiltration into the more porous lesion body structures, both in natural caries lesions (Paris et al., 2006) and also in artificial lesions (Belli et al., 2011), optimizing the μTBS to the substrate, as was observed in this study, in which the infiltrated groups reestablished the μTBS to the levels achieved in the sound enamel (Wiegand et al., 2011).\n\nIn order to increase the penetration coefficient of the resin on porous enamel subsurface, a high content of TEG-DMA monomer is added to the infiltrant low-viscosity resin (Paris et al., 2007a). On the other hand, this monomer has been related to increased susceptibility to degradation of the resin over time (Munksgaard & Freund, 1990). In this study, thermo-mechanical artificial aging was performed; nevertheless, this did not seem to influence the μTBS of the infiltrated/bonded groups, as also observed in a previous study (Jia et al., 2012), since these groups exhibited μTBS means comparative to the positive control group.\n\nAccording to the above, resin infiltration is compatible with both total-etch and self-etch adhesives; thus, restorative treatment can be indicated on tooth surfaces treated with resin infiltration, since it does not negatively interfere with the composite bond to enamel.\n\nThe low-viscosity resin infiltration treatment did not affect enamel μTBS values both for the single-step self-etching and the conventional two-step self-etching adhesive systems. The demineralization and remineralization treatments reduced enamel μTBS values of the self-etching adhesive tested.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Adhesive systems effect over bond strength of resin-infiltrated and de/remineralized enamel. https://doi.org/10.7910/DVN/V3WF3M (Abu Hasna, 2019).\n\nThis project contains the following underlying data:\n\n- Raw Data 1.tab (raw bond strength values for all groups with self-etching adhesive system)\n\n- Raw Data 2.tab (raw bond strength values for all groups with etch-and-rinse)\n\n- Figure 3.tiff – Figure 6.tiff (unedited scanning electron microscopy images)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAbu Hasna A: \"Replication Data for Adhesive systems effect over bond strength of resin-infiltrated and de/remineralized enamel\". Harvard Dataverse, V5. UNF:6:VkbKa1jJBbxb0div9BAH6Q== [fileUNF] 2019. http://www.doi.org/10.7910/DVN/V3WF3M\n\nAnusavice KJ: Chlorhexidine, fluoride varnish, and xylitol chewing gum underutilized preventive therapies? Gen Dent. 1998; 46(1): 34–8, 40. PubMed Abstract\n\nAnusavice KJ: Efficacy of nonsurgical management of the initial caries lesion. J Dent Educ. 1997; 61(11): 895–905. PubMed Abstract\n\nBarkmeier WW, Shaffer SE, Gwinnett AJ: Effects of 15 vs 60 second enamel acid conditioning on adhesion and morphology. Oper Dent. 1986; 11(3): 111–116. PubMed Abstract\n\nBedran-de-Castro AK, Pereira PN, Pimenta LA, et al.: Effect of thermal and mechanical load cycling on microtensile bond strength of a total-etch adhesive system. Oper Dent. 2004; 29(2): 150–156. PubMed Abstract\n\nBelli R, Rahiotis C, Schubert EW, et al.: Wear and morphology of infiltrated white spot lesions. J Dent. 2011; 39(5): 376–385. PubMed Abstract \| Publisher Full Text\n\nCury JA, Tenuta LM: Enamel remineralization: controlling the caries disease or treating early caries lesions? Braz Oral Res. 2009; 23(Suppl 1): 23–30. PubMed Abstract \| Publisher Full Text\n\nErickson RL, Barkmeier WW, Latta MA: The role of etching in bonding to enamel: a comparison of self-etching and etch-and-rinse adhesive systems. Dent Mater. 2009; 25(11): 1459–1467. PubMed Abstract \| Publisher Full Text\n\nFarias de Lacerda AJ, Ferreira Zanatta R, Crispim B, et al.: Influence of de/remineralization of enamel on the tensile bond strength of etch-and-rinse and self-etching adhesives. Am J Dent. 2016; 29(5): 289–293. PubMed Abstract\n\nFeatherstone JD: The caries balance: the basis for caries management by risk assessment. Oral Health Prev Dent. 2004; 2(Suppl 1): 259–264. PubMed Abstract\n\nFejerskov O, Nyvad B, Kidd E: Dental Caries: The Disease and its Clinical Management. 3rd ed. 2015. Reference Source\n\nGöhring TN, Zehnder M, Sener B, et al.: In vitro microleakage of adhesive-sealed dentin with lactic acid and saliva exposure: a radio-isotope analysis. J Dent. 2004; 32(3): 235–240. PubMed Abstract \| Publisher Full Text\n\nGupta A, Tavane P, Gupta PK, et al.: Evaluation of Microleakage with Total Etch, Self Etch and Universal Adhesive Systems in Class V Restorations: An In vitro Study. J Clin Diagn Res. 2017; 11(4): ZC53–ZC56. PubMed Abstract \| Free Full Text\n\nGwinnett AJ: Acid etching for composite resins. Dent Clin North Am. 1981; 25(2): 271–289. PubMed Abstract\n\nJia L, Stawarczyk B, Schmidlin PR, et al.: Effect of caries infiltrant application on shear bond strength of different adhesive systems to sound and demineralized enamel. J Adhes Dent. 2012; 14(6): 569–574. PubMed Abstract \| Publisher Full Text\n\nLee YE, Baek HJ, Choi YH, et al.: Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. J Dent. 2010; 38(2): 166–171. PubMed Abstract \| Publisher Full Text\n\nMeyer-Lueckel H, Bitter K, Paris S: Randomized controlled clinical trial on proximal caries infiltration: three-year follow-up. Caries Res. 2012; 46(6): 544–548. PubMed Abstract \| Publisher Full Text\n\nMueller J, Meyer-Lueckel H, Paris S, et al.: Inhibition of lesion progression by the penetration of resins in vitro: influence of the application procedure. Oper Dent. 2006; 31(3): 338–345. PubMed Abstract \| Publisher Full Text\n\nMunksgaard EC, Freund M: Enzymatic hydrolysis of (di)methacrylates and their polymers. Scand J Dent Res. 1990; 98(3): 261–267. PubMed Abstract \| Publisher Full Text\n\nNeuhaus KW, Schlafer S, Lussi A, et al.: Infiltration of natural caries lesions in relation to their activity status and acid pretreatment in vitro. Caries Res. 2013; 47(3): 203–210. PubMed Abstract \| Publisher Full Text\n\nParis S, Hopfenmuller W, Meyer-Lueckel H: Resin infiltration of caries lesions: an efficacy randomized trial. J Dent Res. 2010; 89(8): 823–826. PubMed Abstract \| Publisher Full Text\n\nParis S, Meyer-Lueckel H, Cölfen H, et al.: Penetration coefficients of commercially available and experimental composites intended to infiltrate enamel carious lesions. Dent Mater. 2007a; 23(6): 742–748. PubMed Abstract \| Publisher Full Text\n\nParis S, Meyer-Lueckel H, Cölfen H, et al.: Resin infiltration of artificial enamel caries lesions with experimental light curing resins. Dent Mater J. 2007b; 26(4): 582–588. PubMed Abstract \| Publisher Full Text\n\nParis S, Meyer-Lueckel H, Kielbassa AM: Resin infiltration of natural caries lesions. J Dent Res. 2007; 86(7): 662–666. PubMed Abstract \| Publisher Full Text\n\nParis S, Meyer-Lueckel H, Mueller J, et al.: Progression of sealed initial bovine enamel lesions under demineralizing conditions in vitro. Caries Res. 2006; 40(2): 124–129. PubMed Abstract \| Publisher Full Text\n\nParis S, Soviero VM, Schuch M, et al.: Pretreatment of natural caries lesions affects penetration depth of infiltrants in vitro. Clin Oral Investig. 2013; 17(9): 2085–2089. PubMed Abstract \| Publisher Full Text\n\nPoggio C, Scribante A, Della Zoppa F, et al.: Shear bond strength of one-step self-etch adhesives to enamel: effect of acid pretreatment. Dent Traumatol. 2014; 30(1): 43–48. PubMed Abstract \| Publisher Full Text\n\nQueiroz CS, Hara AT, Paes Leme AF, et al.: pH-cycling models to evaluate the effect of low fluoride dentifrice on enamel de- and remineralization. Braz Dent J. 2008; 19(1): 21–27. PubMed Abstract \| Publisher Full Text\n\nRocha Gomes Torres C, Borges AB, Torres LM, et al.: Effect of caries infiltration technique and fluoride therapy on the colour masking of white spot lesions. J Dent. 2011; 39(3): 202–207. PubMed Abstract \| Publisher Full Text\n\nTorres CR, Barcellos DC, Pucci CR, et al.: Influence of methods of application of self-etching adhesive systems on adhesive bond strength to enamel. J Adhes Dent. 2009; 11(4): 279–286. PubMed Abstract\n\nTorres CRG, Rosa PCF, Ferreira NS, et al.: Effect of caries infiltration technique and fluoride therapy on microhardness of enamel carious lesions. Oper Dent. 2012; 37(4): 363–369. PubMed Abstract \| Publisher Full Text\n\nWiegand A, Stawarczyk B, Kolakovic M, et al.: Adhesive performance of a caries infiltrant on sound and demineralised enamel. J Dent. 2011; 39(2): 117–121. PubMed Abstract \| Publisher Full Text\n\nVan Meerbeek B, De Munck J, Yoshida Y, et al.: Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent. 2003; 28(3): 215–235. PubMed Abstract" }	[ { "id": "55147", "date": "04 Nov 2019", "name": "Heleine Maria Chagas Rêgo", "expertise": [ "Reviewer Expertise dentistry", "operative dentistry", "adhesive systems", "restorative materials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article establishes a guide to the protocol that should be used to improve the bond strength of adhesive systems to the de/remineralized enamel surface. The subject is relevant, once that the use of the adhesive technique is a common procedure in the dental office and any change on the dental subtract can affect the effectives and longevity of the restauration.\n\nAbstract\n\nThe results and conclusion in the abstract are clear and related to the objective of the study.\n\nIntroduction The authors should have explained that the choice of treatment for caries lesions is not only related to the progress and location of the injury, but also to the patient's caries risk. It would have been interesting if they had addressed the importance of studying the bond strength of the adhesive systems to the enamel surface and the differences (possible vantages and disadvantages) of the adhesive systems that were used in the study, this would provide a stronger support for the relevance of the study and a link with the discussions and possible results.\nMethodology Good study design with clear and detailed methodology.\n\nReferences should have been cited to support the use of bovine tooth considering its similarity to the human tooth. It would have been relevant to establish the correlation between the number of thermo-mechanical cycles used and the correspondent period of activity in the oral cavity.\n\nResults The results of the Tukey’s test are clear and showed that the etch-and-rinse adhesive system performed better than the self – etch adhesive system, and that the use of the resin infiltration was effective to maintain a good bond strength for both types of adhesive systems used, been equivalent to the control group. The authors should have used SEM images of the control groups of the two adhesive systems, as this should be the standard to be achieved, thus clarifying the effectiveness of the resin infiltration in maintaining the bond strength of the treated enamel.\nDiscussion The discussion is consistent with the results of the study and the authors were able to justify the good performance of the resin infiltration and the limitations regarding the use of the self - etching adhesive system when compared to the etch-and-rinse adhesive system.\nReview the last paragraph of the discussion; Instead: '…and the conventional two-step self-etching adhesive systems…' I believe that it should be: '…and the conventional two-step total-etching adhesive systems…'\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "55146", "date": "05 Nov 2019", "name": "Talal Al-Nahlawi", "expertise": [ "Reviewer Expertise Restorative dentistry", "operative dentistry", "and Endodontics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Abstract was well written and fully comprehensive, however this phrase should be corrected: ...; NaF (demineralized enamel treated with 0.05% sodium fluoride solution (one minute) for eight weeks);... Should mention that this immersion was done DAILY for one minute and lasts 8 weeks.\nThe introduction presents the studied issue through a brief literature review. The objective was clear in the bottom of the introduction. However, I suggest for the authors to include the null hypothesis of the this study.\n\nThe methods: As a suggestion, this paragraph (The specimens were then subjected to thermo-mechanical wear (37000 ER machine, ERIOS, São Paulo, SP, Brazil). Mechanical cycling was performed with load of 60 N and 100,000 cycles. The load was applied on the composite resin restoration, perpendicular to the enamel surface.) may be rewritten as the following: (The specimens were then subjected to thermo-mechanical cycling (37000 ER machine, ERIOS, São Paulo, SP, Brazil) with load of 60 N and 100,000 cycles applied on the composite resin restoration, perpendicular to the enamel surface.)\nThe discussion, it may be interested to discuss the use of NaF solution instead of using AgF and KF and the immersion for 1 minute instead of for 3 minutes as in the study of Zhi et al1. Even more, the discussion of more recent studies could be interested as the study of Alqahtani et al2.\nThe study was well written, the objective is interested and has a relevance in this area. The results were promising and the study and the authors collaborated in the understanding of the behavior of many materials in the de/remineralization process on the tooth surface. The conclusions of this paper drawn adequately supported by the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1743
https://f1000research.com/articles/8-1739/v1	10 Oct 19	{ "type": "Research Article", "title": "Immunohistochemical expression of p53 in Type I and II epithelial ovarian cancer among Sudanese women: a cross-sectional study", "authors": [ "Aisha Osman Mohamed", "Nazik Elmalaika Husain", "Rawia Eljaili Elmassry", "Lubna Alnageeb", "Mohammed Elhassan", "Mohammed Siddig Abdelaziz", "Nazik Elmalaika Husain", "Rawia Eljaili Elmassry", "Lubna Alnageeb", "Mohammed Elhassan", "Mohammed Siddig Abdelaziz" ], "abstract": "Background: Epithelial ovarian cancer (EOC) represents the leading cause of death from gynecologic malignancies worldwide. In Sudan, ovarian cancer represents the fourth most frequent tumors among females. TP53 somatic mutations is a defining feature of ovarian high-grade serous carcinoma. However, p53 sequencing is not feasible in most low- and middle-income countries, like Sudan, and its frequency varies greatly. The study aimed to determine the frequency of p53 overexpression and its relationship with tumor types I and II and tumor grade among Sudanese women with EOC. Methods: In this cross-sectional, hospital-based study a total of 114 paraffin-embedded tissue blocks previously diagnosed as epithelial ovarian cancer were collected from six governmental hospitals in Khartoum state, Sudan, in the period 2013-2016. Immunohistochemistry was performed on tissue microarray slides to measure the protein expression of p53 in the EOC. Results: Overexpression of p53 was detected in 35.1% (n=40/114) of EOC samples, with a higher frequency in women with Type II 53.7% (n= 29/54) than type I 18.5% (n= 10/54) (P= 0.000). Also, a high frequency of p53 overexpression was evident in 49.2% (n= 30/61) of high-grade carcinoma compared with 16.7% (n= 1/6) of non-graded borderline tumors, and in 19.1% (n= 9/47) of low-grade tumors (P= 0.003). A high-grade serous carcinoma harbor p53 overexpression in 53.7% (n= 29/54) and none of low-grade serous carcinoma harbor p53 overexpression. Our result showed a significant association between p53 overexpression and tumor types and grades (P = 0.000 and 0.003, respectively) Conclusion: p53 over-expression was detected in one-third of Sudanese women with EOC. It was more common in type II EOC and high-grade serous, but negative in low-grade serous tumors. Our result showed a significant association between p53 over-expression and tumor type and grade, and can help discriminate between high- and low-grade serous carcinomas.", "keywords": [ "Epithelial ovarian cancer", "cross-sectional study", "Immunohistochemistry", "p53overexpression", "Sudan" ], "content": "Introduction\n\nOvarian cancer is a fatal disease, the mortality rate ranks the highest of all gynecological malignancies1. It is considered the third most common cancer in the female reproductive system (following uterine cervix and corpus) and the leading cause of death from gynecologic malignancies in the United States as estimated by the American Cancer Society for the year 20191,2. Ovarian cancer refers to a group of morphologically and genetically heterogeneous neoplasms3,4. In Sudan, ovarian cancer represents the fourth most frequent tumor type in women5.\n\nBased on clinicopathological and molecular studies, epithelial ovarian cancer (EOC) is classified as type I or type II. Type I tumors are genetically quite stable, typically present at a low stage, and reveal distinct, morphologic differences than type II tumors6. These include different histotypes: low-grade serous, endometrioid, clear-cell, and mucinous ovarian carcinoma. Type I tumors are characterized by distinct molecular genetics profiles, such as mutations in KRAS, BRAF, PIK3CA, PTEN and ERBB2, but not TP536. Type II tumors are generally high-grade serous (about 90% of all EOCs). They are highly aggressive, develop rapidly, present in an advanced stage in most cases, genetically unstable and express a mutated TP537–12. TP53 mutations have an important role in the prognosis and treatment of ovarian cancer13. Mutations in TP53 are found in high grade and rarely in low grade serous ovarian cancers. TP53 encodes the 53 kDa nuclear protein, their mutations leading to gain or loss of function of its protein product. TP53 mutation leads either to overexpression of p53 protein or complete lack of expression, while wild-type p53 is associated with focal expression14,15.\n\nImmunohistochemical staining for p53 was considered as an essential biomarker for clinical trials targeting mutant p53 and used in the diagnostic workup of carcinomas of multiple sites, including ovarian cancers16. It is used as a substitute for TP53 mutational analysis, these mutations were global in high-grade serous ovarian cancer (HGSOC) (over 96% were mutated), so were used to discriminates between high- and low-grade serous carcinomas17–20. Access to TP53 sequencing is not feasible in many low- and middle-income countries; pathologists there used p53 immunohistochemistry, which is quick, easy to perform, inexpensive and can approach 100% specificity for the presence of TP53 mutation. Its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumor18–20. To our knowledge, there are no published reports about the frequency of p53 immunostaining in type I and II EOC in Sudan. The study aimed to determine the frequency of p53 overexpression and its relationship with tumor types I and II and tumor grades among Sudanese women with EOC.\n\n\nMethods\n\nA cross-sectional, hospital-based study was implemented. All 114 available formalin-fixed paraffin-embedded tissue blocks (convenience sampling) previously diagnosed as epithelial ovarian cancer were collected during the period 2013–2016, in six governmental hospitals in Khartoum state, Sudan (The National Public Health Laboratory, Maternity Hospital, Military Omdurman Hospital, Alribat, Bahri, and Omdurman Teaching Hospital). Well-preserved tissue blocks with adequate tissue left for tissue microarray (TMA) procedure were included. Inadequate tissue blocks, and cases with missing tissue blocks were excluded. Slides from the original paraffin blocks were stained with hematoxylin and eosin (H&E), were reviewed according to 2014 WHO classification of ovarian tumors21, and were graded and typed according to the Kurman model8.\n\nAvailable paraffin-embedded blocks from tumors were used for the construction of a tissue microarray (TMA). Representative areas of the tumor were identified and TMA blocks were constructed using two cores from each case. Sections were obtained from each TMA and were placed on negatively charged slides for immunohistochemistry.\n\nImmunohistochemistry was performed to measure the protein expression of p53 monoclonal antibodies in ovarian carcinoma cases, as follow: Sections were cut into widths of 3–4 µm and placed on clean, electrostatically charged glass slides. Sections were dried by placing on a hot plate at 60°C for 15 minutes. Sections were dewaxed in two changes of xylene for two minutes. Sections were then hydrated through an ethanol series (100%, 90%, 70%, 50%) and water two minutes for each. Slides were retrieved using the water bath heat-retrieval technique22 and then treated with 3% hydrogen peroxide for 10 minutes. After that, sections were washed in phosphate buffer saline (PBS) (pH 7.4) for five minutes and treated with a 10% casein solution for 10 minutes. Sections were treated with ready-to-use primary antibody of mouse monoclonal antibody to p53 protein (clone DO7 IgG2b; catalog no AM239-5M; BioGenex, CA) for 30 minutes at room temperature in a humidity chamber, then rinsed in PBS before being treated with Super Sensitive polymer –HRP IHC Detection System (catalog no QD420-YIKE; BioGenex, CA) by incubated with enhancer reagents 15 min at room temperature, followed by a polymer-HRP reagent conjugated to anti-mouse and anti-rabbit secondary antibody for 15 minutes at room temperature, and rinsed in PBS. The entire antibody-enzyme complex is then made visible by incubation with a chromogenic substrate 3,3 diaminobenzidine for 7 minutes then washed in PBS for five minutes. For the staining step, sections were counter-stained in Mayer's hematoxylin for one minute washed and blued in running tap water before they were dehydrated through ascending concentrations of ethanol (50%, 70%, 90%, 100%). Sections were finally cleared in xylene and mounted using DPX. A known p53-positive breast cancer tumor was used as positive control. As the negative control, tumor specimens were immunostained under the same conditions without the primary antibody. Both the quantity of nuclear positivity and the staining intensity were measured in the immune slides examined under a light microscope (Olympus BX41, Japan). The intensity of staining was reported as negative, weak, moderate, or strong (0, 1+,2+,3+) in comparison with the positive controls (internal or external) and it indicated the average staining intensity of the tumor nuclei on the entire slide. An IHC score of p53 staining intensity was categorized as 0 for none, (no brownish color seen using x40 magnification), +1 for weak (brownish color seen using x20 and x40), +2 for moderate (brownish color seen using x10 magnification) and +3 for strong staining (brown color visible using x4 magnification).\n\nThe percentage of positive tumor cells was quantified by counting cells manually in at least 100 cells in 10 high power fields, averaged and categorized as ≥75% of cells considered as high overexpression, ≤75–50% considered as moderate expression and less than 50% considered as focal expression. Only 75–100% positive tumor cells with moderate and strong staining intensity considered as positive results.\n\nThe data were analyzed using the statistical package for social sciences (SPSS version 24) to describe the variables. Pearson’s Chi-square test was used to determine a statistically significant association between p53 expression and clinicopathological variables.\n\nThe study was approved by the ethical committees of Alzaiem Alazhari University and the Ministry of Health, Sudan. Informed consent from patients was waived by the committees, since patients' identity was anonymized, and only laboratory numbers were used.\n\n\nResults\n\nAccording to the histopathological diagnosis, all 114 cases were EOC. They were classified as type I, type II and borderline, in 47.4% (n=54), 47.4% (n=54) and 5.3% (n=6) of cases, respectively. Grade of samples was classified as high in 53.5% of cases (n=61), low in 41.2% of cases (n=47) and non-graded tumors in 5.3% of cases, respectively (n=6). EOC histological subtypes were high-grade serous (HGS) in 54 cases (47.4%), low-grade serous (LGS) in six cases (5.3%), mucinous carcinoma (MC) in 22 cases (19.3%), endometroid carcinoma (EC) in 16 cases (14%), clear cell carcinoma (CCC) in seven cases (6.1%), malignant Brenner tumor in three cases (2.6%) and borderline in six cases (5.3%). Details of samples for each patient, alongside all unprocessed images, are available as Underlying data23.\n\nPositive p53 immunostaining was seen in 35.1% (40/114) of ovarian epithelial carcinoma. From the 40 positive cases, staining intensity was as follows: 25 cases exhibited strong staining (+3), and 15 cases were moderate staining (+2). While the remaining 74 cases were considered as negative results (complete absence of p53 expression seen in 45 cases and +1 staining in 29 cases) (Figure 1–Figure 4).\n\nMagnification: main image, x40; inset, x10.\n\nMagnification, x40).\n\nMagnification, x40.\n\nMagnification, 40X.\n\nOverexpression of p53 was associated significantly with the histological subtype (p = 0.004) as seen in Table 1. p53 expression was significantly associated with tumor grade (p = 0.003): it was positive in 49.2% (30/61) of high-grade tumors, 16.7% (1/6) of the non-graded borderline tumors, and 19.15% (9/47) of the low-grade tumors. The association between p53immunohistochemical expression and epithelial ovarian cancer type showed a highly significant association (p = 0.000). p53 was positive in 53.7% (29/54) of Type II and 18.5% (10/54) of Type I tumors (Table 2).\n\n\nDiscussion\n\nImmunohistochemical staining for p53 was used as a surrogate for TP53 mutational analysis to discriminate between high (over 96% was mutated) and low-grade serous carcinomas17–20.\n\nIn the present study, p53 positivity was observed in 35.1% (40/114) of EOC cases, and higher positivity was showed in HGS (53.7%; 29/54); a significant association was found between p53 expression and histopathological diagnosis and with tumor grade and tumor type. This result agreed with those of Tan et al., who found that 64.18% (43/67) of HGS samples analyzed overexpressed p5313. Harris et al., found that in 274 cases, 68% of tumors were characterized as p53 mutant (n= 186) and p53-mutant tumors were more likely to be HGS (72%) with significant association between p53 expression and histology and grade of tumor10. Markowska et al., found that positive expression of p53 protein was observed in 27.8% of EOC24. Yemelyanova et al. found out of the 57 tumors, 36 contained functional mutations and 23 cases (63%) with mutant Tp53 were positive for p53 IHC and 70% of HGS were p53 mutant16. Kobel et al. showed p53 marker overexpressed in 69% (118/171) of HGS cases18. Kobel et al. also showed that abnormal Tp53 expression was detected in 904 out of 912 (99.1%) of HGS25. Our results also agreed with those of Matsuo et al., who found that for 121 cases of high-grade serous p53 positivity was (71.4%)26, and Zhang et al., found that strong staining of p53 was observed in (70.8%) of serous carcinomas and showed that the tumor type was closely associated with the expression of p5327. Mozes et al., found p53 overexpression in 83.6% of HGS28. Bell et al. reported that Tp53 mutated in 96% of HGS29. Moreover, Oaknin et al. reported that expression of p53 markers in the various histological types of ovarian carcinoma (HGS 94%, LGS 0%, CCC 12%, EC 15% and MC 61%)17 were nearly to our results (HGS 53.7%, LGS 0%, CCC 14.3%, EC 12.5% and MC 31.8%). Lim et al., reported that p53 was not expressed in EC in 30 samples, but they agreed with our result in that they found HGS overexpressed p53 in 50% of cases examined30. Alexander et al. reported that p53 was positive for HGS in 68% of cases (52/76) and differed from our result in that they found positivity in LGS (24%; 18/76)19. Mackenzie et al., reported a higher percentage of p53 in mucinous carcinoma (68 %)31.\n\nFurthermore, our result agreed also with Sallum et al., who reported that p53 positivity was found in 68.2% of HGS and significant association was found between p53 expression and tumor type15. Sundov et al.,32 and Tan et al.,13 also found that immunoexpression of p53 was significantly associated with tumor grade. And it disagreed with Brachova et al., who showed that p53 protein expression insignificantly associated with tumor grade33.\n\nThe present study showed that p53 marker was overexpressed in (53.7%) of type II, and (18.5%) of type I. This result agreed with Carter et al., 2018, who reported that p53 was highly expressed in type II EOC (68.8%) than type I (33.3%)4. HGSOC is the most frequent type of ovarian cancer and has been associated with a poor clinical outcome34. According to The Cancer Genome Atlas report, mutations in TP53 are the most common events in EOC, especially in HGSCs35.\n\nThe results of some of these studies may be conflicting primarily because of the indiscriminate grouping of TP53 mutations, which can result in either loss of function or gain of function36. GOF mutations can convert p53 protein from a tumor suppressor to an oncogene, leading to expression of a mutant p53 protein at a high level, while LOF mutants leading to loss of p53 protein expression37. TP53 mutations are classified according to their function as oncomorphic, loss of function and unclassified. Around 21% of all ovarian cancer patients harbor oncomorphic TP53 mutations, which had the highest p53 protein levels and contribute to chemoresistance and cancer progression, and the tumors with unclassified TP53 mutations express the mutated p53 protein at a fairly high level33. The differences found between the studies in the frequency of p53 expression may be due to the differences in scoring of p53 expression and interpretation of results: some studies scored this as overexpression (OE), complete absence (CA), cytoplasmic (CY) or normal/wild type (WT)18,20. Some authors consider complete absence of p53 expression as a mutant also because not all TP53 mutations alter the expression of the protein15,17,25. Complete absence of p53 expression does not indicate TP53 mutation, as a lack of immunoexpression may be found in normal cells16. So, we believe that true overexpression (more than 75% of the cells stained positive) was the most important type of mutation to consider due to their importance in clinical practice, as they are chemoresistant mutations33 and could also be interpreted easily in the immune-slide without any confusion.\n\n\nStudy limitations\n\nThe limitations of our study were related to the relatively small sample size. Many cases were found in the lab records but lacking tissue blocks, and some blocks contain less amount of tissue for TMA.\n\n\nConclusion\n\nOur study showed that the overexpression of p53 tumor marker is associated with EOC, histological subtype and tumor grade, and found that high-grade serous tumors had a higher percentage of p53 expression in more than 50% of cases, while low grade serous was negative in 100% of the cases. We recommended the use of p53 immunohistochemical staining in the pathologic workup of ovarian carcinomas. Careful attention to laboratory protocols and practical works, including adequate controls, and training in interpretation is needed to make this a reliable test informing diagnosis and subsequent management of ovarian carcinoma.\n\n\nData availability\n\nFigshare: p53 data.csv, https://doi.org/10.6084/m9.figshare.990130723.\n\nThis project contains the following underlying data:\n\np53 analysis in ovarian cancer (spreadsheet containing details of analysis for each patient sample).\n\nUncropped, unprocessed images taken during this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe acknowledge departments of histopathology and cytology at the six governmental hospitals for providing the work facilities. And gratefully thankful to the histopathology departments in the Universities of Alzaeim Alazhari and Sudan University of Science and Technology for hosting the practical work.\n\n\nReferences\n\nZhang J, Silva EG, Sood AK, et al.: Ovarian Epithelial Carcinogenesis. In: Zheng WFO, Quick C, Shen D, Guo D, editor. Gynecologic and Obstetric Pathology. Singapore: Springer; 2019; 2: 121–39. Publisher Full Text\n\nSiegel RL, Miller KD, Jemal A: Cancer statistics, 2019. CA Cancer J Clin. 2019; 69(1): 7–34. PubMed Abstract \| Publisher Full Text\n\nSadlecki P, Walentowicz P, Bodnar M, et al.: Determination of BRAF V600E (VE1) protein expression and BRAF gene mutation status in codon 600 in borderline and low-grade ovarian cancers. Tumour Biol. 2017; 39(5): 1010428317706230. PubMed Abstract \| Publisher Full Text\n\nCarter JH, Deddens JA, Mueller G, et al.: Transcription factors WT1 and p53 combined: a prognostic biomarker in ovarian cancer. Br J Cancer. 2018; 119: 462–70. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim D, Murali R, Murray MP, et al.: Morphological and Immunohistochemical Reevaluation of Tumors Initially Diagnosed as Ovarian Endometrioid Carcinoma With Emphasis on High-grade Tumors. Am J Surg Pathol. 2016; 40(3): 302–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMackenzie R, Kommoss S, Winterhoff BJ, et al.: Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms. BMC Cancer. 2015; 15: 415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSundov D, Caric A, Mrklic I, et al.: P53, MAPK, topoisomerase II alpha and Ki67 immunohistochemical expression and KRAS/BRAF mutation in ovarian serous carcinomas. Diagn Pathol. 2013; 8: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrachova P, Mueting SR, Carlson MJ, et al.: TP53 oncomorphic mutations predict resistance to platinum‑ and taxane‑based standard chemotherapy in patients diagnosed with advanced serous ovarian carcinoma. Int J Oncol. 2015; 46(2): 607–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabidi-Galy SI, Papp E, Hallberg D, et al.: High grade serous ovarian carcinomas originate in the fallopian tube. Nat Commun. 2017; 8(1): 1093. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVang R, Levine DA, Soslow RA, et al.: Molecular Alterations of TP53 are a Defining Feature of Ovarian High-Grade Serous Carcinoma: A Rereview of Cases Lacking TP53 Mutations in The Cancer Genome Atlas Ovarian Study. Int J Gynecol Pathol. 2016; 35(1): 48–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuller PA, Vousden KH: Mutant p53 in cancer: new functions and therapeutic opportunities. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "80715", "date": "22 Mar 2021", "name": "Daniel R Barnes", "expertise": [ "Reviewer Expertise Statistics", "Genetic Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGENERAL COMMENTS/OVERVIEW In this study, the authors present their findings from analysing p53 expression in a sample of 114 tissue microarrays obtained from Sudanese women diagnosed with ovarian cancer. They report statistically significant associations of differences in p53 expression and ovarian cancer histotype, grade and type.\nThe paper is generally well written, with all experiments and analyses well described.\nMAJOR CONCERNS Table 1: The chi-square test used to test the association between ovarian cancer subtype and negative/positive p53 expression is likely to be inappropriate, stemming from the small table cell counts (and even some zeroes). Here I feel that, although it is unlikely to make any difference to the reported association P-value and conclusions, a Fisher exact test is the correct statistical test.\nTable 2: Similar to the comment on the statistical analysis for data presented in Table 1, a Fisher exact test would be more appropriate to analyse these data. The P-value presented for the association between cancer type and p53 status was “0.000”. Could the authors present the P-value in scientific notation if it is P<0.001?\nTable 2: What was the magnitude of these associations? Could the authors fit logistic regression models to these data to estimate odds ratios of a p53 negative/positive expression by cancer type and cancer grade?\nDiscussion, first and second paragraphs: The authors discuss the findings of many other studies in lengthy detail, showing numbers and percentages. It is somewhat difficult to read. A more general overview with fewer details and providing references should suffice.\nThe authors rightly acknowledge the study sample size as a limitation in the discussion. Could they expand on what could be done in the future to increase sample sizes to strengthen evidence of associations and solidify the conclusions drawn.\nThe authors do not write a section on the strengths of this work. One point I feel that the authors should promote is the fact that this study investigated cancer genetics in an underrepresented population. The authors could make more of the fact that their study was conducted in women of African ancestry.\n\nMINOR CONCERNS Abstract, Results: The authors state a P-value of “P=0.000”, which could be more informative. Could the authors either state P Introduction, first paragraph, first sentence: Could the authors clarify that the ovarian cancer mortality rate is highest worldwide (in agreement with what they say in the abstract).\nIntroduction, first paragraph, last sentence: Could the authors also state what the three cancers are that occur more frequently than ovarian cancer.\nIntroduction, second paragraph: The authors mention several genes involved in type I and type II tumours. I was interested to see that BRCA1 and BRCA2 were not mentioned. Would the authors be able to comment on the role of BRCA1 and BRCA2 in type I and type II ovarian cancers?\nMethods, Immunohistochemistry, last paragraph (p4): The authors write “averaged and categorized as ≥75% of cells considered as high overexpression, ≤75–50% considered as moderate expression…”. The boundary at 75% is not clearly defined as they have greater than or equal to 75% in one category and less than or equal to 75% in the other category. Could they correct this to show which category includes 75%?\nMethods, Statistical analysis: Was there a prior P-value specified for determining what constitutes a statistically significant association? Were there any multiple testing corrections made or considered?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "179339", "date": "03 Jul 2023", "name": "Francis Jacob", "expertise": [ "Reviewer Expertise Molecular cancer research", "cell plasticity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of the manuscript entitled «Immunohistochemical expression of p53 in Type I and II epithelial ovarian cancer among Sudanese women: a cross-sectional study» investigate a cohort of tissue samples embedded in a tissue microarray derived from women with epithelial ovarian cancer. The study supports existing literature indicating that immunohistochemical (IHC) staining of p53 is a reliable method for supporting the pathological diagnosis of high-grade serous ovarian cancer. The manuscript appears in concise and generally well-written format.\nMajor comments\nIt is important for researchers to provide specific details regarding the ethical approval obtained for their study. Merely stating the approval without further information may not be sufficient for readers and reviewers to assess the ethical considerations and compliance of the study. Including the ethical approval number helps to establish transparency and demonstrates that the study has undergone appropriate ethical review and received the necessary permissions to conduct the research. This information allows readers to verify the ethical aspects of the study and ensures that the research adheres to the principles of ethical conduct.\n\nA table summarizing all clinicopathological data should be provided. If available, this includes age, FIGO stage, sampling site, and treatment status (if NACT or relapsed patients were included). Are all samples investigated are derived from chemo-naïve patients?\n\nWhat is the final concentration of the antibody applied on the TMA? In addition, also considering that IHC of p53 may be of diagnostic purpose, did the authors investigate the reproducibility of the p53 staining, e.g., on representative tissues?\n\nThe majority of high-grade serous ovarian cancer is mutated in p53. While the mutations may result in the dominant negative overexpression of the protein, there are also some cases with loss of p53 due to pathogenic mutation. The authors focus on overexpression but also report on cases with an absence of p53 (Figure 4). While comparing the expression between tumor and “normal” cells in the same tissue sample, absence of p53 in tumor may also indicate that the tumor harbors a pathogenic mutation. Here, the question arises whether samples with absence of p53 expression are wildtype or mutated?\n\nThe authors provide a rather limited study limitation. Expanding the limitations section and providing suggestions for addressing these limitations can demonstrate a comprehensive understanding of the study's strengths and weaknesses. It also helps guide future researchers in designing more robust studies and advancing the field. Apart from sample size limitations, authors may also consider representativeness of the cohort, potential biases (e.g. confounding factors that could influence the study results), and recommendations for future research (e.g. multi-center collaborations, statistical power, and validity of the findings).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1739
https://f1000research.com/articles/8-154/v1	05 Feb 19	{ "type": "Research Note", "title": "Evaluation of the scholastic performance of students in 12 programs from a private university in the south-west geopolitical zone in Nigeria", "authors": [ "Roseline O. Ogundokun", "Marion O. Adebiyi", "Oluwakemi C. Abikoye", "Tinuke O. Oladele", "Adewale F. Lukman", "Abidemi E. Adeniyi", "Adekanmi A. Adegun ", "Babatunde Gbadamosi", "Noah O. Akande", "Marion O. Adebiyi", "Oluwakemi C. Abikoye", "Tinuke O. Oladele", "Adewale F. Lukman", "Abidemi E. Adeniyi", "Adekanmi A. Adegun ", "Babatunde Gbadamosi", "Noah O. Akande" ], "abstract": "Cumulative grade point average (CGPA) is a system for calculation of GPA scores and is one way to determine a student's academic performance in a university setting. In Nigeria, an employer evaluates a student's academic performance using their CGPA score. For this study, data were collected from a student database of a private school in the south-west geopolitical zone in Nigeria. Regression analysis, correlation analysis, and analysis of variance (F-test) were employed to determine the study year that students perform better based on CGPA. According to the results, it was observed that students perform much better in year three (300 Level) and year four (400 Level) compared to other levels. In conclusion, we strongly recommend the private university to introduce program that will improve the academic performance of students from year one (100 level).", "keywords": [ "Academics", "Performance", "Students", "Science and Engineering", "Private University", "Programmes" ], "content": "Introduction\n\nIn the white-collar job market now, there is high competition among young graduates. Academic performance is one indicator that highlights university students’ qualification and this is mostly measured using the cumulative grade point average (CGPA). Most employers use CGPA to screen out candidates searching for jobs, and candidates with a higher CGPA are selected (Yogendra & Andrew, 2017). Therefore, the performance of students in universities should be a concern not only to administrators and educators but also to corporations in the labor market.\n\nStudents have to place greater effort in their study to obtain a good grade in order to fulfil the demands of an employer and this makes academic achievement the main factor considered by employers in the recruitment of workers, especially newly graduated students (Yogendra & Andrew, 2017). The objective of the present study is to determine the study year that students perform better academically across 12 programs in a private university in the south-west geopolitical zone in Nigeria.\n\n\nMethods\n\nPrimary data was extracted from Covenant University’s student database (John et al., 2018). The dataset contains the cumulative grade point averages (CGPA) from the first to the fourth year of study and the overall CGPA of students.\n\nIBM Statistical Package for Social Sciences (IBM 20) was used to analyze the data of the scholastic performance of students in 12 programs at the College of Science and Engineering within the year 2010 to 2014. The statistical methodology includes regression analysis, analysis of variance (ANOVA), and descriptive statistics (Lukman et al., 2018).\n\nApproval to use the data was obtained from the Ethical Committee of Landmark University, which is affiliated with Covenant University.\n\n\nResults\n\nA total of 12 programs were assessed, which included 2490 students. The frequency distribution of the number of students who attended the twelve (12) programs and their graduation years are depicted in Table 1 and Table 2, respectively. The descriptive statistics are provided in Table 3. The results show that the mean performance of all the students at each of the level is not too different from each other. Figure 1 shows a histogram of the cumulative CGPA of students for the years 2010–2014. The distribution of the data is skewed to the right which shows that a high number of the students have a CGPA that is between 2 and 5. The number of students with a CGPA that is less than 2 is low.\n\nTable 4 shows the correlation matrix of the variables. The variables include CGPA 100 level, CGPA 200 level, CGPA 300 level, CGPA 400 level, CGPA 500 level and the overall CGPA. A strong positive and significant relationships exist between CGPA in the different level and the overall CGPA. The coefficient of determination (R2) in Table 5 shows that the cumulative grade point average in each level explained about 98.1% of the variations in the response variable (the overall CGPA). The F-test shows that the overall regression model is significant (P-value=0.000<0.05). It was also observed that each of the variables has a positive and significant impact on the overall CGPA. The performance of the students in 200 level is more significant (See Table 5). The maximum variance inflation factor shows that none of the variables is correlated (See Table 5). Results show that overall performance of each student depends on their academic performance in each level.\n\n*.Correlation is significant at the 0.01 level (2-tailed).\n\nP-value in the parenthesis.\n\n\nConclusion\n\nIn this report, we have analyzed the performance of students in 12 programs at a private university in Nigeria. From the various analysis carried out, it was observed that a large number of students graduated in 2013, and from the 12 programs students of electrical and electronic engineering have the highest percentage of graduate students. The descriptive statistics show that the mean performance of all the students at each of the level is not too different from each other. The performance of the student at each level is pivotal to their overall CGPA. In conclusion, we strongly recommend the private university to introduce program that will improve the academic performance of students from year one (100 level).\n\n\nData availability\n\nZenodo: Dataset on the academic performance of students in 12 programmes from a private university, http://doi.org/10.5281/zenodo.1482513 (Oluwaseun et al., 2018).", "appendix": "Grant information\n\nThis research received funding from Landmark University, Omu-Aran, Kwara State, Nigeria.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nJohn TM, Badejo JA, Popoola SI, et al.: The role of gender on academic performance in STEM-related disciplines: Data from a tertiary institution. Data Brief. 2018; 18: 360–374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLukman AF, Adebimpe O, Onate CA, et al.: Data on expenditure, revenue, and economic growth in Nigeria. Data Brief. 2018; 20: 1704–1709. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOluwaseun OR, Oluwabunmi AM, Abikoye CO, et al.: Dataset on the academic performance of students in 12 programmes from a private university [Data set]. Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1482513\n\nYogendra N, Andrew A: A Study on The Factors Influencing on Grade Point Average (GPA) With Special Reference to Third Year Commerce and Management Students of Eastern University, Sri Lanka. J Stud Man Plann. 2017; 3(8): 409–425. Reference Source" }	[ { "id": "47348", "date": "01 May 2019", "name": "Robert G. Carroll", "expertise": [ "Reviewer Expertise Medical Education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the grade point average variances in students enrolled in a private university during a 5 year period. Analysis revealed that grade point averages were not significantly different based on program of study. The cumulative grade point average, a number derived from the grade point average in each year, was significantly correlated with each of the component year grade point averages.\nTables 1 and 2 include a column for cumulative percent, which does not add any useful information, and can be deleted. The rightward shift in figure 1 may be explained by dismissal of students with low GPAs - but no indication of dismissal/attrition is indicated in the manuscript. The conclusion in the abstract that students perform much better in level 3 and 4 do not appear supported by table 3, and is not mentioned in the Conclusion section of the paper .\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4946", "date": "10 Oct 2019", "name": "Roseline Ogundokun", "role": "Author Response", "response": "This paper describes the grade point average variances in students enrolled in a private university during a 5 year period. Analysis revealed that grade point averages were not significantly different based on program of study. The cumulative grade point average, a number derived from the grade point average in each year, was significantly correlated with each of the component year grade point averages. Tables 1 and 2 include a column for cumulative percent, which does not add any useful information, and can be deleted. The rightward shift in figure 1 may be explained by dismissal of students with low GPAs - but no indication of dismissal/attrition is indicated in the manuscript. The conclusion in the abstract that students perform much better in level 3 and 4 do not appear supported by table 3, and is not mentioned in the Conclusion section of the paper .[Response] [Response]All these suggestions have been attended to as suggested by the authors and the necessary revision had been made in the manuscript." } ] }, { "id": "50772", "date": "05 Jul 2019", "name": "Semiu Akanmu", "expertise": [ "Reviewer Expertise Empirical methods in software engineering and information systems." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCiting relevant literature:\nI find the in-text citations extremely scanty. The introduction which should weave the justification(s) of the study in specific terms and how it intends to attend to either a practical problem or/and a theoretical problem is inadequate. Explanations provided in the Introduction section generally addressed why \"the performance of students should be of concern\", not specifically why an investigation of the relationship between the study year and student grade should be conducted. Perhaps, and this should be justified in the literature, it is (a) to be able to design a level-specific intervention program that would support academic performance, (b) to serve as foundation for future studies that would identify factors that contribute to the positive or negative influence of a study level on the student grade.\nThere is a slight mention of this in the concluding sentence, but more emphasis on the \"why this study?\" should be made known at the Introduction section.\nIn the same vein, there should be clearer definitions of GPA and CGPA (on level basis and as overall), their relationship, probably in mathematical terms.\nDesign of the study, methodology and data analysis:\nThe concluding part of the Introduction section reads that \" The objective of the present study is to determine the study year that students perform better academically across 12 programs\" which best informs the choice of descriptive and the correlation analysis. Regression is therefore unnecessary because it accounts for influence of a variable on the other, and it must be with adequate attention to all latent and confounding variables.\nI also did not see ANOVA, though mentioned in the methods, in the data analysis. If it would be needed, there should thorough reading of its justification and how best it suits this work. I will suggest reading Julie Pallant's Guide to Using SPSS for further insights.\nAlso, Variance Inflation Factor (VIF) is wrongly interpreted. Therefore, this statement \"The maximum variance inflation factor shows that none of the variables is correlated\" cannot be correct because (a) it contradicts an earlier statement that \"A strong positive and significant relationships exist between CGPA in the different level and the overall CGPA\", and (b) does not explain variance, which is square of the standard deviation, in view of understanding the variables' multi-collinearity.\nNote that correlation is synonymous to relationship, and it does not mean Variance in statistical terms.\nDiscussion and Conclusion:\nThe conclusion, since it ordinarily relied heavily on the data analysis and literature cannot be said to be adequate. There are no past related studies to situate the findings of this study and establish the fresh insights it attempts bring forth. This requires a Discussion section. A better analysis and inferences would then address the concluding section.\nRequired corrections:\nReview 5 -10 past related studies to partly support your introduction, especially the justification of the study in specific terms, and as literature to situate your findings in a discussion section.\n\nStick to \"finding relationship\", thus, descriptive correlation analyses would be sufficient. These, however, must be done thoroughly with report on data treatment and cleansing, and justification of the suitability of the statistical method.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4947", "date": "10 Oct 2019", "name": "Roseline Ogundokun", "role": "Author Response", "response": "I find the in-text citations extremely scanty. The introduction which should weave the justification(s) of the study in specific terms and how it intends to attend to either a practical problem or/and a theoretical problem is inadequate. Explanations provided in the Introduction section generally addressed why \"the performance of students should be of concern\", not specifically why an investigation of the relationship between the study year and student grade should be conducted. Perhaps, and this should be justified in the literature, it is (a) to be able to design a level-specific intervention program that would support academic performance, (b) to serve as foundation for future studies that would identify factors that contribute to the positive or negative influence of a study level on the student grade.[s1] [Response]All these suggestions have been attended to by the authorsThere is a slight mention of this in the concluding sentence, but more emphasis on the \"why this study?\" should be made known at the Introduction section. In the same vein, there should be clearer definitions of GPA and CGPA (on level basis and as overall), their relationship, probably in mathematical terms.[Response] These have been included in the study (manuscript) The concluding part of the Introduction section reads that \" The objective of the present study is to determine the study year that students perform better academically across 12 programs\" which best informs the choice of descriptive and the correlation analysis. Regression is therefore unnecessary because it accounts for influence of a variable on the other, and it must be with adequate attention to all latent and confounding variables.[s1] [Response]These have been attended to as suggested by the reviewer I also did not see ANOVA, though mentioned in the methods, in the data analysis. If it would be needed,[s1] [Response]This reflects in the regression table (F-test). The Anova in regression is used to assess the overall significance of the model." } ] }, { "id": "44048", "date": "09 Aug 2019", "name": "Raheela Asif", "expertise": [ "Reviewer Expertise Educational Data Mining" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature?\nLiterature review doesn't (directly) lead to the work of this study. Authors should provide an extensive and detailed literature review, which demonstrates their extensive knowledge in the research field and the recognition of the existing achievements in the area.\nIs the study design appropriate and is the work technically sound?\nThe authors should also explain the main differences between their research and the previous achievements.\nThe final conclusions provided in the paper are not clearly formulated and correspond to the presented research results. Further steps of the performed research should also be identified by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4948", "date": "10 Oct 2019", "name": "Roseline Ogundokun", "role": "Author Response", "response": "Literature review doesn't (directly) lead to the work of this study. Authors should provide an extensive and detailed literature review, which demonstrates their extensive knowledge in the research field and the recognition of the existing achievements in the area.[s1] [Response] An extensive and detailed literature review that demonstrates extensive knowledge in this field had been attended to as advised by the reviewer.The authors should also explain the main differences between their research and the previous achievements. The final conclusions provided in the paper are not clearly formulated and correspond to the presented research results. Further steps of the performed research should also be identified by the authors.[s1] [Response]These have been attended to as suggested by the reviewer" } ] } ]	1	https://f1000research.com/articles/8-154
https://f1000research.com/articles/8-1737/v1	10 Oct 19	{ "type": "Case Report", "title": "Case Report: Germ cell tumor presenting as cecal mass", "authors": [ "Venkata Satish Pendela", "Anisleidys Munoz", "JulieAnn Warner", "Roopa Yarlagadda", "Anisleidys Munoz", "JulieAnn Warner", "Roopa Yarlagadda" ], "abstract": "Extra gonadal germ cell tumors most frequently occur in the anterior mediastinum, retro-peritoneum, and pineal and suprasellar regions. The infrequency of its occurrence inside gastrointestinal tract makes it an arduous diagnostic challenge. A 23 year old male with no significant past medical history presented to the emergency department with increasing abdominal pain, diarrhea, episodic vomiting for 3 weeks. Review of systems was positive for melena and shortness of breath on exertion. Fullness and irregularity along with tenderness was noted around the right iliac region. CT scan (computed tomography) of the abdomen revealed a cecal mass with multiple metastases to liver, lungs and abdominal lymph nodes. Colonic endoscope was performed but it could not be advanced beyond the cecal mass. Biopsies from the mass were reported as poorly differentiated metastatic carcinoma. During the course of hospitalization, he developed symptomatic small bowel obstruction with perforation. Colonic resection was performed and histology showed Germ-Cell Tumor. Beta HCG level was 118789 IU/L suggestive of a non-seminomatous germ cell tumor. Ultrasound of the scrotum, MRI brain (magnetic resonance imaging) and CT scan of the chest did not reveal a primary tumor. Chemotherapy was started with Bleomycin, Etoposide and Cisplatin after which beta human chorionic gonadotropin (HCG) levels dropped dramatically. His hospital course got complicated with neutropenic sepsis with shock which progressed to multi-organ dysfunction and unfortunately, he succumbed to the disease burden. This case demonstrates one of the rare presentations of extragonadal germ cell tumors and the diagnostic challenges associated with it. Very few cases have been reported in the literature, and none of them presented as a cecal mass. Early recognition of this presentation will help in reducing the tumor burden and the mortality associated with it, as germ cell tumors are highly susceptible to chemotherapy.", "keywords": [ "germ cell tumor", "cecal mass", "chemotherapy" ], "content": "Introduction\n\nExtra gonadal germ cell tumors (EGCTs) are rare and account for 1–3% of all the gonadal tumors1–3. Several mechanism have been described, including possible migration of pluripotent stem cells, which convert to germ cells during embryonic development. The usual sites of EGCTs are mediastinum, retro-peritoneal organs, and pineal gland1–4. Involvement of gastrointestinal tract is rare and is very unlikely in the absence of a primary elsewhere. Shogbesan et al. reported an EGCT presenting as duodenal mass with primary tumor identified in the testis. There are a few cases reported in literature without an identifiable primary in the gonads; none of which presented as a cecal mass1,2.\n\n\nCase presentation\n\nA 23 year old Caucasian male who worked part-time as a waiter at a restaurant, with no significant past medical history presented during the fall of 2018 to the emergency department with increasing abdominal pain, diarrhea, and episodic vomiting for 3 weeks. Review of systems was positive for melena and shortness of breath on exertion. Family history was only relevant for an aunt who died from lung cancer. He had a five pack year smoking history and smokes marijuana socially. On admission, he was tachycardic and febrile, and exam revealed cachexia with bitemporal wasting. Fullness and irregularity in the right ileac fossa with associated tenderness was noted on palpation of the abdomen. See Figure 1 for full timeline.\n\nCT – computed tomography, HCG - human chorionic gonadotropin, MRI – magnetic resonance imaging.\n\n\nInvestigations\n\nComputed tomography (CT) of the abdomen revealed a cecal mass with multiple metastases to liver, lungs and abdominal lymph nodes (Figure 2, Figure 3). Colonoscopy was performed, but the endoscope was unable to be advanced beyond the cecal mass. The tumor was poorly differentiated, and urothelial carcinoma was in the differential due to the positive GATA3 immunohistochemical staining. Liver biopsy reported an undifferentiated metastatic carcinoma. Beta HCG (human chorionic gonadotropin) level was 118,789 IU/L. After the elevated beta HCG level was detected, the colonic specimen was stained with the markers for germ cell tumors (beta HCG and PLAP) which was positive. Ultrasound of the scrotum was done to look for a primary tumor, which was negative. Magnetic resonance imaging (MRI) of the brain and a CT scan of the chest did not reveal any growth in the pineal gland or mediastinum, respectively.\n\n\nTreatment\n\nChemotherapy was started with Bleomycin (30 units/wk IV on days 1, 8 and 15 regimen), Etoposide (100 mg/m2/day IV ) and Cisplatin ( 20 mg/m2/day IV on days 1–5); given the high Beta HCG levels and deteriorating clinical status. Within 7 days of starting this regimen, beta HCG levels dropped dramatically to 31,163 IU/L. During the course of hospitalization the patient developed symptomatic small bowel obstruction with perforation, which was confirmed with a CT scan. Colonic resection was performed. The resected mass was identified to be germ cell tumor as it picked up beta HCG, SALL4, GATA3, OCT 4 as well as PLAP stains on histo-path exam (Figure 4, Figure 5).\n\n\nOutcome and follow-up\n\nThe patient responded to chemotherapy initially with a drop in HCG levels; however, hospital course was complicated with tumor lysis syndrome and neutropenic sepsis, which progressed to multi-organ dysfunction. He required mechanical ventilatory support along with vasopressors for blood pressure support. As a result of deterioration, the family opted for comfort care measures. The patient expired 36 hours later. Even though his cancer burden was responding to chemotherapy, he unfortunately succumbed from complications of the treatment.\n\n\nDiscussion\n\nEGCTs are considered metastatic from an occult or “burned out” gonadal cancer if a primary testicular tumor is not apparent. An ultrasound of the scrotum should always be performed to rule out a testicular tumor. A gonadal biopsy to rule out such tumor is not recommended5. The patient in the present study was found to have an EGCT in the cecum and colon. Similar to GCTs, EGCTs are sensitive to radiotherapy and chemotherapy.\n\nCurrently, common drugs used for chemotherapy include cisplatin, etoposide and bleomycin6–9. Our patient received these medications along with surgery which helped in reducing his tumor burden. The International Germ Cell Cancer Collaboration Group (IGCCCG) places the patients into poor prognosis category if they are found to have a primary tumor or visceral metastases elsewhere apart from the lungs and retro peritoneum. The 5-year survival rate in such patients is 48%4,10–12.\n\nThis case demonstrates one of the rare presentations of extra-gonadal germ cell tumors and the diagnostic challenges associated with it. Early diagnosis entails high index of suspicion, especially in young males presenting with tumor of unknown origin. Early recognition of this presentation will help in reducing the tumor burden and the mortality associated with it. It also allow for early treatment, which usually entails surgery (for accessible tumors) along with chemotherapy. However, our knowledge about these tumors and their presentations is still limited, and as seen in this patient, treatment can result in complications well. This limitation warrants the need for studies on EGCTs and their management.\n\n\nConclusion/learning points/take home messages\n\nGonadal tumors should be among the differentials in patients presenting with abdominal mass, especially in younger adults.\n\nThe importance of this case is to identify a constellation of symptoms, obscurities, diagnostic difficulties, adverse effects of treatment of an extra gonadal germ cell tumor.\n\nThe authors would encourage testing with Beta HCG in young males presenting with a tumor of unknown origin.\n\nThe authors would also encourage a multidisciplinary approach with surgery and early initiation of chemotherapy in such patients to control the disease burden.\n\nThis report also increases awareness that one should be watchful for complications after implementing treatment with chemotherapy, as early detection of complication may help prevent negative outcomes.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the parent of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nShogbesan O, Abdulkareem A, Jehangir A, et al.: Gastrointestinal Involvement of Testicular Germ Cell Tumor: A Case Report and Literature Review. Case Rep Gastrointest Med. 2017; 2017: 4789259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKucukoner M, Inal A, Kaplan MA, et al.: Germ Cell Tumor Located in Gastrointestinal System: A Report of Two Cases. World J Oncol. 2012; 3(3): 134–137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmoll HJ: Extragonadal germ cell tumors. Ann Oncol. 2002; 13(suppl_4): 265–272. PubMed Abstract \| Publisher Full Text\n\nHanna N, Timmerman R, Foster RS, et al.: Extragonadal Germ Cell Tumors. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Reference Source\n\nShinagare AB, Jagannathan JP, Ramaiya NH, et al.: Adult extragonadal germ cell tumors. AJR Am J Roentgenol. 2010; 195(4): W274–W280. PubMed Abstract \| Publisher Full Text\n\nDueland S, Stenwig AE, Heilo A, et al.: Treatment and outcome of patients with extragonadal germ cell tumours--the Norwegian Radium Hospital's experience 1979-94. Br J Cancer. 1998; 77(2): 329–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukui N, Kohno Y, Ishioka J, et al.: Treatment outcome of patients with extragonadal nonseminomatous germ cell tumors: the Saitama Cancer Center experience. Int J Clin Oncol. 2013; 18(4): 731–734. PubMed Abstract \| Publisher Full Text\n\nDechaphunkul A, Sakdejayont S, Sathitruangsak C, et al.: Clinical Characteristics and Treatment Outcomes of Patients with Primary Mediastinal Germ Cell Tumors: 10-Years' Experience at a Single Institution with a Bleomycin-Containing Regimen. Oncol Res Treat. 2016; 39(11): 688–694. PubMed Abstract \| Publisher Full Text\n\nDaugaard G, Rørth M, von der Maase H, et al.: Management of extragonadal germ-cell tumors and the significance of bilateral testicular biopsies. Ann Oncol. 1992; 3(4): 283–289. PubMed Abstract \| Publisher Full Text\n\nKhurana K, Gilligan TD, Stephenson AJ: Management of poor-prognosis testicular germ cell tumors. Indian J Urol. 2010; 26(1): 108–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBusch J, Seidel C, Zengerling F: Male Extragonadal Germ Cell Tumors of the Adult. Oncol Res Treat. 2016; 39(3): 140–144. PubMed Abstract \| Publisher Full Text\n\nHartmann JT, Nichols CR, Droz JP, et al.: Prognostic variables for response and outcome in patients with extragonadal germ-cell tumors. Ann Oncol. 2002; 13(7): 1017–1028. PubMed Abstract \| Publisher Full Text" }	[ { "id": "67751", "date": "23 Jul 2020", "name": "Joost Blok", "expertise": [ "Reviewer Expertise Testicular germ cell tumor" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a case of a young man presenting with a metastasized extragonadal germ cell tumor. The case is impressive and has educational value. The report is well-written but I have the following questions/suggestions:\nWhat was the timeline (days since presentation) for the various diagnostic procedures? E.g. at how many days after presentation was beta HCG checked?\n\nWhat was the level of other markers for germ cell tumor (AFP, LD)?\n\nWas the histology of the GCT nonseminoma or seminoma? Histology of liver biopsy showed undifferentiated carcinoma, which suggest nonseminoma (this would also be in line with the high levels of HCG). Was the histology of liver biopsy/colonic resection revised? Any presence of subtypes of nonseminoma (e.g. embryonal carcinoma, choriocarcinoma).\n\nI would suggest to revise the first and third learning point. The first learning point can be stated more firmly. My suggestion: Testicular or extragonadal germ cell tumor should always be considered in young males presenting with an abdominal mass. The third learning point can be moved up to the second spot. I would suggest to rewrite it as follows: The diagnostic workup of young males presenting with an abdominal tumor or tumor of unknown origin should always include workup for germ cell tumor (beta-HCG, AFP, LD, scrotal palpation and testicular ultrasound).\n\nI believe the first sentence of the Discussion is not correct. An EGCT originates outside the gonads and therefore does not originate from a (burned out or occult) testicular tumor. Please include a statement that although it is a cancer of the germ cells, it can also originate in the retroperitoneum or mediastinum.\n\nThe Discussion would benefit from a more extensive description of (E)GCT. What is the survival rate for EGCT? Is it better or worse compared to testicular GCT? I think it is also important to note that GCT is the most common cancer type in young men, but that the extragonadal subtype is rare (of course the testicular subtype being much more common). Is the incidence of GCT and EGCT known for this age category?\n\nI would also suggest a more extensive discussion of tumor lysis syndrome and neutropenic sepsis. How common is this? How should this be treated according to the guidelines? How often succumb patients to this condition?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "70467", "date": "09 Sep 2020", "name": "Shi-Ming Tu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an unfortunate 23 yo diagnosed with an extragonadal GCT (EGGCT) arising from the cecum and metastatic to the liver, lungs, and abdominal LN. His HCG was 118,789. He received and responded to standard BEP x1, but developed and succumbed to complications as a result of treatment. Specifically, he developed neutropenic sepsis, bowel perforation, and multiorgan failure. This case has important educational value.\nThe first sentence in Discussion is incorrect: “EGGCTs are considered metastatic from an occult or “burned out” gonadal cancer.” One of the acceptable criteria for a diagnosis of primary EGGCT (according to Abell MR et al., Cancer, 1965)1 are: - Absence of any detectable tumor or subsequent appearance of a tumor in either testis; - An encapsulated lesion without nodal involvement; - The lesion is located high in the retroperitoneum with adjacent lymph node involvement but without involvement of the lower aortic, iliac, or pelvic lymph nodes.\n\nPathology is incomplete. Please clarify if the report stated that the tumor was choriocarcinoma or adenocarcinoma with choriocarcinomatous differentiation. Of note, a patient with EGGCT arising from the GU tract (kidney) with choriocarcinomatous differentiation was curable with a modified chemotherapy regimen (Msaouel P et al., Clin Genitourin Cancer 2017).2\n\nMost extragonadal GCT arising from the GI tract are yolk sac tumors, which produces AFP. To be complete, what were his AFP and LDH levels before chemotherapy?\n\nOften enough when patients are very debilitated and especially when they have a tumor that is likely to respond quickly to chemotherapy and when the tumor regresses before the bowel heals, they may develop bowel perforation. When they also become neutropenic at the same time, this is when catastrophic sepsis occurs. To ensure response but avoid neutropenia and other complications, it is prudent to give gentle upfront chemotherapy (such as BOP, published by Shamash J et al., BJUI, Nov 2019)3 in such cases.\n\nNeeds to better define and confirm tumor lysis syndrome in the setting of a patient with a solid tumor rather than hematological malignancy, who has received nephrotoxic chemotherapy and is septic with multiorgan dysfunction.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1737
https://f1000research.com/articles/8-1736/v1	10 Oct 19	{ "type": "Study Protocol", "title": "Creation of a rectal cancer registry in Italy by the Advanced International Mini-Invasive Surgery (AIMS) academy clinical research network", "authors": [ "Giulio M. Mari", "Pietro Achilli", "Dario Maggioni", "Jacopo Crippa", "Andrea T.M. Costanzi", "Mauro A. Scotti", "Vittorio Giardini", "Mattia Garancini", "Eugenio Cocozza", "Giacomo Borroni", "Ilaria Benzoni", "Mario Martinotti", "Luigi Totaro", "Matteo Origi", "Michele Mazzola", "Giovanni Ferrari", "Antonio Ziccarelli", "Roberto Petri", "Vincenzo Bagnardi", "Giacomo Pugliese", "Antonello Forgione", "Raffaele Pugliese", "AIMS Academy Clinical Research Network", "Pietro Achilli", "Dario Maggioni", "Jacopo Crippa", "Andrea T.M. Costanzi", "Mauro A. Scotti", "Vittorio Giardini", "Mattia Garancini", "Eugenio Cocozza", "Giacomo Borroni", "Ilaria Benzoni", "Mario Martinotti", "Luigi Totaro", "Matteo Origi", "Michele Mazzola", "Giovanni Ferrari", "Antonio Ziccarelli", "Roberto Petri", "Vincenzo Bagnardi", "Giacomo Pugliese", "Antonello Forgione", "Raffaele Pugliese" ], "abstract": "Background: The management of rectal cancer is multimodal and involves a multidisciplinary team of cancer specialists with expertise in medical oncology, surgical oncology, radiation oncology and radiology. It is crucial for highly specialized centers to collaborate via networks that aim to maintain uniformity in every aspect of treatment and rigorously gather patients’ data, from the first clinical evaluation to the last follow-up visit. The Advanced International Mini-Invasive Surgery (AIMS) academy clinical research network aims to create a rectal cancer registry. This will prospectively collect the data of patients operated on for non-metastatic rectal cancer in high volume colorectal surgical units through a well design pre-fashioned database for non-metastatic rectal cancer, in order to take all multidisciplinary aspects into consideration. Methods/Design: The protocol describes a multicenter prospective observational cohort study, investigating demographics, frailty, cancer-related features, surgical and radiological parameters, and oncological outcomes among patients with non-metastatic rectal cancer who are candidates for surgery with curative intent. Patients enrolled in the present registry will be followed up for 5 years after surgery. Discussion: Standardization and centralization of data collection for neoplastic diseases is a virtuous process for patient care. The creation of a register will allow the control of the quality of treatments provided and permit prospective and retrospective studies to be carried out on complete and reliable high quality data. Establishing data collection in a prospective and systematic fashion is the only possibility to preserve the enormous resource that each patient represents.", "keywords": [ "Rectal surgery", "registry", "network" ], "content": "Introduction\n\nThere are nearly 125,000 new cases of rectal cancer diagnosed every year in Europe, representing one of the leading causes of cancer-related morbidity and mortality world-wide1. Five decades ago, the prognosis of rectal cancer was poor, with locoregional cancer recurrence rates of up to 40% and 5-year survival rates of <50% for locally-advanced tumors2. These disappointing outcomes were improved by innovations in surgical technique, multimodality therapy and education3.\n\nTotal mesorectal excision (TME) remains the cornerstone in the treatment of non-metastatic rectal cancer. To achieve high quality TME, which is the key-factor for proper oncological resection, surgeons must respect well-known embryological planes, which were made famous as the boundaries of Heald’s Holy plane4.\n\nWhile the surgical principles for rectal cancer have changed little during the last decade, the novelties in the radiological study of the disease, as well as in the administration of neoadjuvant and adjuvant therapy, have largely modified the treatment of this neoplasm5. Nowadays, the management of rectal cancer is multimodal and involves a multidisciplinary team of cancer specialists with expertise in medical oncology, surgical oncology, radiation oncology and radiology. Therefore, it is becoming crucial for highly specialized centers to design pre-fashioned databases for non-metastatic rectal cancer in order to take all multidisciplinary aspects into consideration. Previous attempts to establish rectal cancer registries, such as The Norwegian Rectal Cancer Project6 and The Spanish Rectal Cancer Project7, were based on the need to first extend adequate oncological treatment, and second to increase the use of minimally invasive surgery.\n\nThe Advanced International Mini-Invasive Surgery (AIMS) academy clinical research network aims to create a rectal cancer registry that will prospectively collect data of patients operated on for non-metastatic rectal cancer in high volume colorectal surgical units, maintaining uniformity in every aspect of the treatment and rigorously gathering patients data, from the first clinical evaluation to the last follow-up visit.\n\nThe aim of the AIMS academy clinical research network rectal cancer registry is to prospectively collect data from different minimally-invasive colorectal units in Northern Italy, with standardization of the pre-operative, intra-operative and post-operative management for patients operated on for non-metastatic rectal cancer with curative intent.\n\nThe primary outcome is to prospectively collect short and long term oncological outcomes. The second outcome is to collect information on the compliance of patients to oncological treatments, both in neoadjuvant and adjuvant settings and their quality of life.\n\n\nProtocol\n\nThis protocol describes a multicenter prospective observational cohort study, investigating demographics, cancer-related features and oncological outcomes among patients who are non-metastatic rectal cancer candidates for surgery with curative intent. Patients enrolled in the present registry will be followed up for 5 years after surgery. All participating centres are public tertiary non-academic hospitals of northern Italy.\n\nThe study was approved by the Comitato Etico Scientifico Milano Area 3 (protocol number 295-052019). The study protocol has been registered as an observational study at ClinicalTrials.gov: NCT04045236 (first received, 3 August 2019). All participating centres received approval from local ethics committees.\n\nPatients receiving the diagnosis of non-metastatic rectal cancer and the indication for a curative treatment will be enrolled in the registry. The target population will consist of all patients enrolled in the participating centers from the start of the rectal cancer registry on. Patients will be identified through their medical record numbers. One investigator in each center will obtain written informed consent from each patient and keep the patients updated on data collection.\n\nInclusion criteria: 1) histologically proved adenocarcinoma of the rectum; 2) patient aged > 18 years old; 3) indication for surgical resection with curative intent.\n\nExclusion criteria: need for emergency surgery, palliative operation or metastatic disease at presentation.\n\nDemographic information and anamnesis with a focus on oncologic history will be recorded at the first outpatient visit, together with a complete clinical examination. Data regarding the symptoms of presentation will be collected and categorized as haemorrhagic framework, alteration of bowel habits or pain. Every patient will undergo a pre-operative staging (see Extended data: CRF1) with chest-abdominal computerized tomography scan with intravenous contrast, complete colonoscopy, pelvic magnetic resonance (MR) imaging and endorectal ultra sound (EUS) examination. Blood sample with serum level of CEA, CA 19.9 and a full nutritional panel will be collected and analysed. Charlson Comorbidities Index adjusted for age8 will be calculated for every patient, while those >70 years old will be assessed for frailty risk using the modified Frailty Index (mFI) described by Robinson et al.9 (see Extended data: CRF1).\n\nAll data regarding radiation doses received, total amount of chemotherapy administered and number of cycles, toxicities or adverse reaction and possible reasons for not completed treatment schedule will be collected for all the patients with locally advance rectal cancer, who received neo adjuvant chemo-radiotherapy. Radiological restaging after neoadjuvant treatment comprises MR imaging, EUS and colonoscopy. Radiological response to neoadjuvant treatment will be measured following a uniform score system among all centers involved10. Endoscopic assessment of tumour regression will be also recorded11 (see Extended data: CRF2).\n\nIntraoperative analysed parameters will be included in the registry (see Extended data: CRF3), with special attention to technical aspects of surgical procedures, such as level of inferior mesenteric artery ligation, type of energy device used, number and type of cartridge, and size of circular stapler and all other variables detailed in the Clinical Trials registration.\n\nHistopathological examination will be performed according to WHO 2010 guidelines12. Macroscopic evaluation of the resected specimen will be classified according to the Quirke score13, while pathologic regression grade will be estimated according to a five-point scoring system14. Mismatch repair status will be reported when analysed (see Extended data: CRF3).\n\nPost-operative complications will be reported according to the Clavien-Dindo scale15. Length of stay and eventual post-discharge complications will be evaluated and recorded. Application of an ERAS protocol will be considered only for at least 80% of ERAS colorectal items satisfied (see Extended data: CRF4)16.\n\nIndication to adjuvant treatment will be defined within a multidisciplinary setting. Regarding adjuvant chemotherapy, all data of interest such as number of cycles, toxicities and possible early interruption of the treatment will be collected as previously shown in the neoadjuvant setting. Oncological follow-up will be performed according to National Comprehensive Cancer Network guidelines17. One investigator in each center will carry out the follow up. Functional follow up will be done yearly according to the Low Anterior Resection Syndrome Score (see Extended data: CRF5)18.\n\nData will be collected daily using a pre-fashioned REDCAP database by one physician for each hospital and referred to a research fellow (GMM) who will monitor the included data for all institutions. Pre-fashioned CRFs are available as Extended data. There will be regular contact between the study coordinators and the participating centers through scheduled meetings every three months. A data manager (GP) will regularly control the quality of the data provided.\n\nAll researchers will be able to access the data uploaded. Data will be hosted by the AIMS Academy. All researchers will be able to use collected data to write scientific articles or to plan surgical audits.\n\nThe registry has been enrolling patients since January 2019.\n\n\nDiscussion\n\nThe primary aim of this registry is to prospectively collect data from different minimally invasive colorectal units in Northern Italy with a standardization of the pre-operative, intra-operative and post-operative management for patients operated on for non-metastatic rectal cancer with curative intent.\n\nStandardization and centralization of data collection for neoplastic diseases is a virtuous process for patient care. The creation of a register allows the control of the quality of treatments provided and permits prospective and retrospective studies to be carried out on complete and reliable high quality data.\n\nIn the last few years, the need to raise the quality of care for rectal cancer patients has been reported in numerous studies19, as well as the clear association between hospital volumes and outcomes after rectal surgery20. Speaking a common language in such a complex field is no longer a benefit but rather the only way to face new challenges waiting for us in the near future.\n\nDue to the aging population, the number of frail patients affected by rectal cancer is expected to grow21. As a consequence, the identification of frail patients and the need to search for tailored management able to prevent adverse complications and to improve clinical outcomes of this population will play a fundamental role in oncological surgery in the years to come22. Thus, all methods available to screen a patient for frailty, such as the Mini Cog test, the Katz Index of Independence in Activities of Daily Living (ADL), and the Timed Up and Go (TUG) test must become an integrated part of daily clinical work23,24.\n\nThe accuracy achieved by pre-operative MR imaging during the last decade25 has lead to important results in both preoperative staging and radiologic response evaluation after neo-adjuvant therapy26. Indeed, radiologic restaging is increasingly involved in the therapeutic decision-making process27. Thus, structuring a synoptic and uniform MR report must become a prerogative in management of rectal cancer patients.\n\nAs mentioned in the study protocol section, intra-operative data concerning which type of devices or staplers used during surgery will be recorded in the registry, allowing us to look for possible correlations with clinical outcomes. A well-structured prospective analysis among high volume units will help to define the real complication rate after rectal cancer surgery, which has been usually derived retrospectively and therefore potentially underestimated28.\n\nRegarding surgical expertise among the centers involved, continuous monitoring of the integrity of the resected specimens should definitely increase the overall quality of surgery.\n\nDetailed analysis of the compliance to adjuvant chemotherapy for locally advanced rectal cancer is extremely important considering recent data reported in the literature29,30. The unexpected low level of compliance reported in these previous case series has questioned the traditional administration of adjuvant chemotherapy, searching for new strategies for locally advanced rectal tumours, such as total neoadjuvant chemotherapy3.\n\n\nConclusions\n\nThe creation of a registry for patients operated on for non-metastatic rectal cancer is a necessary requirement. Establishing data collection in a prospective and systematic fashion is the only possibility to preserve the enormous resource that each patient represents.\n\n\nData availability\n\nNo underlying data is associated with this article.\n\nZenodo: Rectal cancer AIMS Academy clinical research network registry, http://doi.org/10.5281/zenodo.346362731\n\nThis project contains the following extended data:\n\n- CRF1: Patient’s information and cancer staging form.\n\n- CRF2: Neo-adjuvant chemoradiotheraphy and cancer restaging form.\n\n- CRF3: Surgery, surgical outcomes and pathological examination form.\n\n- CRF4: Adjuvant chemotherapy form.\n\n- CRF5: Oncological follow-up form.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nAIMS Academy: www.aimsacademy.org\n\n\nReferences\n\nEuropean cancer information system: Estimates of cancer incedence and mortality in 2018, for all countries. European commission. Reference Source\n\nSlaney G: Results of treatment of carcinoma of the colon and rectum. Mod Trends Surg. 1971; 3: 69–89. PubMed Abstract\n\nZaborowski A, Stakelum A, Winter DC: Systematic review of outcomes after total neoadjuvant therapy for locally advanced rectal cancer. Br J Surg. 2019; 106(8): 979–987. PubMed Abstract \| Publisher Full Text\n\nHeald RJ, Moran BJ, Ryall RD, et al.: Rectal cancer: The Basingstoke experience of total mesorectal excision, 1978-1997. Arch Surg. 1998; 133(8): 894–9. PubMed Abstract \| Publisher Full Text\n\nAl-Sukhni E, Milot L, Fruitman M, et al.: Diagnostic accuracy of MRI for assessment of T category, lymph node metastases, and circumferential resection margin involvement in patients with rectal cancer: a systematic review and meta-analysis. Ann Surg Oncol. 2012; 19(7): 2212–23. PubMed Abstract \| Publisher Full Text\n\nWibe A, Møller B, Norstein J, et al.: A national strategic change in treatment policy for rectal cancer--implementation of total mesorectal excision as routine treatment in Norway. A national audit. Dis Colon Rectum. 2002; 45(7): 857–66. PubMed Abstract \| Publisher Full Text\n\nOrtiz H, Ciga MA, Armendariz P, et al.: Multicentre propensity score-matched analysis of conventional versus extended abdominoperineal excision for low rectal cancer. Br J Surg. 2014; 101(7): 874–882. PubMed Abstract \| Publisher Full Text\n\nCharlson M, Pompei P, Ales KL, et al.: A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987; 40(5): 373–383. PubMed Abstract \| Publisher Full Text\n\nRobinson TN, Wu DS, Pointer L, et al.: Simple frailty score predicts postoperative complications across surgical specialties. Am J Surg. 2013; 206(4): 544–550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeets-Tan RGH, Lambregts DMJ, Maas M, et al.: Correction to: Magnetic resonance imaging for clinical management of rectal cancer: Updated recommendations from the 2016 European Society of Gastrointestinal and Abdominal Radiology (ESGAR) consensus meeting. Eur Radiol. 2018; 28(6): 2711. PubMed Abstract \| Publisher Full Text\n\nSohn DK, Han KS, Kim BC, et al.: Endoscopic assessment of tumor regression after preoperative chemoradiotherapy as a prognostic marker in locally advanced rectal cancer. Surg Oncol. 2017; 26(4): 453–459. PubMed Abstract \| Publisher Full Text\n\nFt B, F C, Rh H, et al.: WHO classification of tumours of the digestive system. J Clin Ultrasound. 2014.\n\nQuirke P, Dixon MF, Durdey P, et al.: Local recurrence of rectal adenocarcinoma due to inadequate surgical resection. Histopathological study of lateral tumour spread and surgical excision. Lancet. 1986; 2(8514): 996–9. PubMed Abstract \| Publisher Full Text\n\nRyan R, Gibbons D, Hyland JMP, et al.: Pathological response following long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Histopathology. 2005; 47(2): 141–146. PubMed Abstract \| Publisher Full Text\n\nDindo D, Demartines N, Clavien PA: Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004; 240(2): 205–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGustafsson UO, Scott MJ, Hubner M, et al.: Guidelines for Perioperative Care in Elective Colorectal Surgery: Enhanced Recovery After Surgery (ERAS®) Society Recommendations: 2018. World J Surg. 2019; 43(3): 659–695 . Publisher Full Text\n\nNCCN guidelines version 4.2018: Colon Cancer. © National Comprehensive Cancer Network, Inc. All Rights Reserved. 2014. Reference Source\n\nEmmertsen KJ, Laurberg S: Low anterior resection syndrome score: development and validation of a symptom-based scoring system for bowel dysfunction after low anterior resection for rectal cancer. Ann Surg. 2012; 255(5): 922–928. PubMed Abstract \| Publisher Full Text\n\nMerchea A, Ali SM, Kelley SR, et al.: Long-Term Oncologic Outcomes of Minimally Invasive Proctectomy for Rectal Adenocarcinoma. J Gastrointest Surg. 2018; 22(8): 1412–1417. PubMed Abstract \| Publisher Full Text\n\nHagemans JAW, Alberda WJ, Verstegen M, et al.: Hospital volume and outcome in rectal cancer patients; results of a population-based study in the Netherlands. Eur J Surg Oncol. 2019; 45(4): 613–619. PubMed Abstract \| Publisher Full Text\n\nClegg A, Young J, Iliffe S, et al.: Frailty in elderly people. Lancet. 2013; 381(9868): 752–762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFagard K, Leonard S, Deschodt M, et al.: The impact of frailty on postoperative outcomes in individuals aged 65 and over undergoing elective surgery for colorectal cancer: A systematic review. J Geriatr Oncol. 2016; 7(6): 479-491. PubMed Abstract \| Publisher Full Text\n\nGhignone F, Van Leeuwen BL, Montroni I, et al.: The assessment and management of older cancer patients: A SIOG surgical task force survey on surgeons’ attitudes. Eur J Surg Oncol. 2016; 42(2): 297–302. PubMed Abstract \| Publisher Full Text\n\nMontroni I, Rostoft S, Spinelli A, et al.: GOSAFE - Geriatric Oncology Surgical Assessment and Functional rEcovery after Surgery: early analysis on 977 patients. J Geriatr Oncol. 2019; pii: S1879-4068(19)30168-7. PubMed Abstract \| Publisher Full Text\n\nKennedy ED, Simunovic M, Jhaveri K, et al.: Safety and Feasibility of Using Magnetic Resonance Imaging Criteria to Identify Patients with “good Prognosis” Rectal Cancer Eligible for Primary Surgery: The Phase 2 Nonrandomized QuickSilver Clinical Trial. JAMA Oncol. 2019; 5(7): 961–966. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiddiqui MRS, Bhoday J, Battersby NJ, et al.: Defining response to radiotherapy in rectal cancer using magnetic resonance imaging and histopathological scales. World J Gastroenterol. 2016; 22(37): 8414–8434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhoday J, Balyasnikova S, Wale A, et al.: How Should Imaging Direct/Orient Management of Rectal Cancer?. Clin Colon Rectal Surg. 2017; 30(5): 297–312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin Z, Jiang ZL, Chen DY, et al.: Short- and long-term outcomes of laparoscopic versus open surgery for rectal cancer. Medicine (Baltimore). 2018; 97(50): e13704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBosset JF, Calais G, Mineur L, et al.: Fluorouracil-based adjuvant chemotherapy after preoperative chemoradiotherapy in rectal cancer: Long-term results of the EORTC 22921 randomised study. Lancet Oncol. 2014; 15(2): 184–9010. PubMed Abstract \| Publisher Full Text\n\nMari GM, Maggioni D, Crippa J, et al.: Compliance to Adjuvant Chemotherapy of Patients Who Underwent Surgery for Rectal Cancer: Report from a Multi-institutional Research Network. World J Surg. 2019; 43(10): 2544–2551. PubMed Abstract \| Publisher Full Text\n\nMari G, Achill P: Rectal cancer AIMS Academy clinical research network registry. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3463627" }	[ { "id": "55012", "date": "17 Oct 2019", "name": "Giovanni Cesana", "expertise": [ "Reviewer Expertise Colorectal surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the creation of a national registry for non metastatic rectal cancer. The main issue is a high quality standardization of rectal cancer multimodal treatment, staging and follow up. I think that the core of the project is well explained and clarified. Additional materials are clear and quite user friendly. The methods are well described and expanded enough. The need for well designed databases on rectal cancer is clearly expressed by the literature. I believe that this registry represents a high quality attempt in the direction of a centralized and appropriate treatment for rectal cancer.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "55011", "date": "05 Nov 2019", "name": "Abe Fingerhut", "expertise": [ "Reviewer Expertise Surgery and clinical research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis registry is unique and essential to reseach in Italy. The Advanced International Mini-Invasive Surgery (AIMS) academy clinical research network has a major role in this multicenter prospective observational cohort study, I agree that standardization and centralization of data collection for neoplastic diseases is essential for patient care. The creation of a register will allow the control of the quality of treatments provided and permit prospective and retrospective studies to be carried out on complete and reliable high quality data.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "55725", "date": "14 Nov 2019", "name": "Fabian Grass", "expertise": [ "Reviewer Expertise Colorectal surgery", "minimally-invasive surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Northern Italian research group presents a study protocol detailing specifics of a rectal cancer registry, which consists of a multicentric collaboration aiming to gather reliable, prospectively collected data for rectal cancer research. The authors need to be congratulated for their initiative. Multi-institutional collaborations are needed in the rapidly evolving field of multi-modal treatment of locally advanced rectal cancer. The authors emphasise new challenges of an ageing, frail population. Radiotherapy- or surgery-sparing strategies may represent interesting alternatives to conventional treatment schemes.\nOverall the protocol is clear and concise. I would like to suggest expanding on the following points:\nMulti-institutional collaborations are challenging, since dealing with a heterogeous patient- and provider population, surgical and perioperative care. How do the authors account for that?\n\nStandardization: Do all centers use similar treatment protocols? How standardized is the surgical approach? Do all centers perform robotic proctoectomy? How experienced are participating surgeons? Are all participating centers teaching facilities?\n\nI think the authors may expand on complication assessment. Who is assessing complications, are these institution-specific abstractors or do centers dispose of surveillance tools, i.e. for surgical site infections?\n\nHow do the authors ascertain data quality? National data registries ideally need (independent) audit and validation. Who is auditing data accuracy?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1736
https://f1000research.com/articles/8-1735/v1	10 Oct 19	{ "type": "Research Article", "title": "Seroprevalence of human cytomegalovirus among pregnant women who had undergone abortion(s) attending El-Damazin Hospital for Obstetrics and Gynecology, Sudan: A cross-sectional study", "authors": [ "Salih Elbushra", "Mohammed Seed Ahmed", "Albadawi Abdelbagi Talha", "Tarig A. Gamar", "Elhadi A. Ahmed", "Salih Elbushra", "Mohammed Seed Ahmed", "Albadawi Abdelbagi Talha", "Tarig A. Gamar" ], "abstract": "Background: Human cytomegalovirus (HCMV) is a major cause of congenital infections. It is more widespread in developing countries and communities with low socioeconomic status. The infection can cause pregnancy loss or spontaneous abortion. Tests are available for the detection of HCMV IgG and IgM antibodies. Many pregnant women in Blue Nile State, Sudan, have suffered from recurrent pregnancy loss, and currently there is no available data concerning the prevalence of HCMV in Blue Nile state. This study aimed to determine HCMV antibodies (IgG and IgM) among pregnant women, who had undergone abortion(s), attending El-Damazin Hospital for Obstetrics and Gynecology.\n\nMethods: This was a descriptive, cross-sectional hospital-based study. 270 pregnant women, who had undergone abortion(s) and who attended El-Damazin Hospital for Obstetrics and Gynecology, were included in the study from September to December 2018. Personal and clinical data were collected directly from each participant into a predesigned questionnaire. Serum samples were separated and stored at -20˚C until used. Samples were analyzed for HCMV IgG and IgM using enzyme-linked immune-sorbent assay (ELISA). Results: Participants were categorized into three age groups: 15-25 years (33.7%; 91/270); 26-40 years (62.2%; 168/270); and >41 years (4.1%; 11/270). The majority of the participants had IgG antibodies to HCMV (74.8%; 202/270), while only 13.3% (36/270) had IgM antibodies to HCMV. Most abortion cases were documented in the first trimester (85.6%; 231/270) and this had a significant relationship with IgG level (P=0.003). Low socioeconomic status was recorded in 84.8% (229/270) of participants and showed significant correlation with IgG level (P=0.025), whereas illiteracy was reported in 41.9% (113/270) of participants and did not have a significant relationship.\n\nConclusions: Seroprevalence of HCMV in this study population was 74.8% for IgG antibodies. There was an association between HCMV IgG level and first trimester abortion and low socioeconomic status among the studied women.", "keywords": [ "HCMV", "Abortion", "IgG", "First trimester", "Socioeconomic", "El-Damazin", "Sudan." ], "content": "Introduction\n\nCytomegalovirus (CMV) is the most ubiquitous member of the herpes virus family. Human cytomegalovirus (HCMV) is the most common cause of congenital malformation resulting from viral intrauterine infection in developed countries1. CMV infects a high percentage of individuals during their life and after recovery of disease it hides in leukocytes. Although this virus is not considered as hazardous to health, in pregnant women it is a major factor that threatens the health of neonates2. Primary CMV infection occurs in 0.15%–2.00% of all pregnancies and may be transmitted to the fetus in 40% of cases3.\n\nSeroprevalence of HCMV in adults ranges from 55% in developed countries to as high as over 90% in developing countries like China4. In Sudan, age is significantly associated with CMV IgM detection, and history of miscarriage was associated with CMV IgG positive women5. Additionally in Sudan, a study conducted in 2013 by Elamin and Omer at Khartoum Teaching Hospital reported the seroprevalence rate among pregnant women with recurrent abortion as 55.3% and 3.2% for HCMV IgG and IgM antibodies, respectively6. Another recent study in Sudan conducted at Omdurman Maternity Hospital reported a sero-frequency rate of HCMV among pregnant women as 74.4% for CMV IgG and 14.4% for HCMV IgM7.\n\nHCMV can produce maternal infection and exhibits a high tropism for cervical mucosa and is considered as the most implicated virus in recurrent spontaneous abortion (RSA)8. Many pregnant women in Blue Nile State, Sudan, have suffered from recurrent pregnancy loss, and currently there is no available data concerning the prevalence of HCMV in Blue Nile state; therefore, this study aimed to determine HCMV antibodies (IgG and IgM) among pregnant women, who had undergone abortion(s) in this state.\n\n\nMethods\n\nThis was a descriptive cross-sectional hospital-based study aimed to detect the seroprevalence of CMV among pregnant women, who had undergone RSA, attending El-Damazin Hospital for Obstetrics and Gynecology in Blue Nile State, Sudan, between March 2017 and February 2019.\n\nPermission to carry out the study was obtained from the College of Graduate Studies, Faculty of Medical Laboratory Sciences, University of Gezira and Ministry of Health, Blue Nile State, Sudan. All women examined were informed about the purpose of the study before collection of the specimens and written consent for participation was taken.\n\nA total of 270 blood samples, taken for the purpose of this study, were collected under aseptic conditions from the participants and sera were separated in sterile containers and stored at -20°C until tested. The inclusion criterion were all pregnant women attending El-Damazin Hospital for Obstetrics and Gynecology, Sudan to undergo an abortion.\n\nSample size was calculated using the following formula9: N=(1.96)2P(1−p)d2\n\nDemographic and clinical data were collected directly from each woman into a predesigned questionnaire (Extended data), including personal information (age, education (no education = illiterate), occupation, socioeconomic status (determined using household income: low, <US$57 per month; moderate, US$57–200 per month; high, >US$200 per month), nationality, number of abortions, duration of pregnancy) and laboratory data.\n\nThe laboratory work was carried out in the Regional Public Health Laboratory and Sudanese Chinese Friendship Hospital in El-Damazin using Stat Fax microplate reader (Model: 3200) and Stat Fax washer (Model: 2600) and commercially available ELISA kits (BIOS Microwell Diagnostic System, Chemux Bioscience, Inc., USA for CMV IgM, Lot No: 18-D-055; Fortress Diagnostics Ltd, UK for CMV IgG Lot No: CG-1807-1). Positive and negative results for IgG and IgM were recorded according to calculated cut-off values. For CMV IgG, the cut-off value was obtained by subtracting the blank absorbance from the mean absorbance of calibrator 2.\n\nFor CMV IgM the cut-off value was obtained by multiplying the optical density (OD) of the calibrator by factor (F) printed on label of calibrator. CMV IgM index for each sample obtained by dividing OD of sample over the cut- off.\n\nData analysis was done using Statistical Package for Social Sciences (SPSS version 24; IBM SPSS). Pearson Chi-squared test was used to test for statistical significance (P value), which was taken as significant when p < 0.05.\n\n\nResults\n\nA total of 270 women were enrolled in the study. The majority of the women were aged between 26 and 40 years. Low socioeconomic status was recorded in 84.8% (229/270) of participants, and illiteracy and women obtaining primary education was observed in 80% of participants. Most women were observed to be in the first trimester of pregnancy (85.5%; 231/270) (Table 1). In total, 27.8% (75/270) of the women had a history for 1-7 abortions, while 72.2% (195/270) had no history.\n\nThe seroprevalence of HCMV IgG and IgM among the 270 women was 74.8% (202/270) and 13.3% (36/270), respectively (Figure 1).\n\nHCMV IgG detection was significantly correlated to socioeconomic status and gestation stage, but was not correlated to age group and education level (Table 2).\n\n\nDiscussion\n\nCMV is globally distributed, with 40–100% of the global population positive for CMV antibodies10,11, particularly among low economic individuals12. In Sudan, there are only a few published data (Western and Central Sudan, and Khartoum) concerning epidemiology of HCMV among pregnant women. In our study area, which it located in the South of Sudan, there are no findings about the seroprevalence of HCMV in pregnant women who have had abortions.\n\nThe relationship between seroprevalence of HCMV and socioeconomic and education level among the present study population is significant, which may explain poor health status and susceptibility to certain diseases. Numerous studies have evaluated socioeconomic and education level for seroprevalence of HCMV, and most of these studies confirm the strong association between the socioeconomic disparities and high seropositivity13–17\n\nHCMV IgG level in this study was significantly correlated to abortion in the 1st trimester gestation, which has also been shown by other studies1,18. HCMV infection is considered a significant public health problem because it can cause disease in those with weakened immune systems, which has been confirmed by a study in Sudan in which a high frequency (98.3%)of seroprevalence of HCMV among pregnant women was reported19.\n\nIn the present study, the sero-prevalence of HCMV among the participants was 74.8% for IgG and 13.3% for IgM; these findings are in total agreement with another study in Sudan among pregnant women7, but is in contrast to other study in Iran in which the frequency for HCMV IgG and IgM was 14.28% and 28.25%, respectively20. Larger and smaller frequencies of HCMV IgG level have also been reported in Egypt21 and Iran22, respectively. The IgM level found in this study is similar to findings reported in Poland (13%) by Fowler and Boppana23, and similar results were obtained by other authors in Iraq24 and India25. Higher results have been reported in Egypt (32.6%) for HCMV IgM18, and lower results have been reported in Turkey26 and Korea27 (1% and 1.7%, respectively). Many factors may contribute to HCMV transmission and prevalence, such as socioeconomic and lifestyle factors, and it should be notes that most immunocompetent carriers of HCMV remain asymptomatic28\n\nIn this study, 11.9% of the study population revealed primary infection with HCMV, i.e. positive results for both IgG and IgM. This frequency is larger than that recorded previously in a hospital in Khartoum29.\n\n\nConclusions\n\nSeroprevalence of HCMV in Blue Nile State, Sudan, among pregnant women who had undergone abortion(s) was 74.8% for IgG and 13.3% for IgM. HCMV prevalence in pregnant women -was most prevalent for women in the first trimester- with low economic status.\n\n\nData availability\n\nFigshare: HCMV seroprevalence, Blue Nile State, Sudan, https://doi.org/10.6084/m9.figshare.9895715.v130\n\nThis project contains the following underlying data:\n\n- ELISA antibody titre data\n\n- Demographic and clinical data for participants\n\nFigshare: HCMV seroprevalence, Blue Nile State, Sudan, https://doi.org/10.6084/m9.figshare.9895715.v130\n\nThis project contains the following extended data:\n\n- Questionnaire in English.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nMunro SC, Hall B, Whybin LR, et al.: Diagnosis of and screening for cytomegalovirus infection in pregnant women. J Clin Microbiol. 2005; 43(9): 4713–4718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAriani S, Chaichi LMA: Study on the IgG and IgM antibodiesrate of virus HSV, CMV and rubella in the women with recurrent pregnancy loss history. Indian Journal of Fundamental and Applied Life Sciences. 2014; 4(3): 212–222. Reference Source\n\nde Vries JJ, Korver AM, Verkerk PH, et al.: Congenital cytomegalovirus infection in the Netherlands: birth prevalence and risk factors. J Med Virol. 2011; 83(10): 1777–82. PubMed Abstract \| Publisher Full Text\n\nZhang S, Zhou YH, Li L, et al.: Monitoring human cytomegalovirus infection with nested PCR: comparison of positive rates in plasma and leukocytes and with quantitative PCR. Virol J. 2010; 7: 73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltayeb MA, Mokhtar SI, Adam ME, et al.: Detection of primary CMV infection in Sudanese pregnant women by IgG avidity test. Asian Pac J Trop Dis. 2016; 6(10): 816–818. Publisher Full Text\n\nElamin BK, Omer MOMA: Seroprevalence of Cytomegalovirus infection among Sudanese women eith recurrent pregnancy loss (RPL). International Journal of Development Research. 2015; 5(5): 4300–4302. Reference Source\n\nMohammed NSAEH, ELhag WI: Serofrequency of Cytomegalovirus among pregnant ladies attending antenatal care at Omdurman Maternity Hospital, Sudan. Ameri J Resea Communi. 2015; 3(3): 108–115. Reference Source\n\nBrok HP, Boven L, van Meurs M, et al.: The human CMV-UL86 peptide 981-1003 shares a crossreactive T-cell epitope with the encephalitogenic MOG peptide 34-56, but lacks the capacity to induce EAE in rhesus monkeys. J Neuroimmunol. 2007; 182(1–2): 135–52. PubMed Abstract \| Publisher Full Text\n\nHall P: Theoretical comparison of bootstrap confidence intervals. Ann Stat. 1988; 16(3): 927–53. Reference Source\n\nFreeman RB Jr: The 'indirect' effects of cytomegalovirus infection. Am J Transplant. 2009; 9(11): 2453–2458. PubMed Abstract \| Publisher Full Text\n\nMarshall BC, Koch WC: Antivirals for cytomegalovirus infection in neonates and infants: focus on pharmacokinetics, formulations, dosing, and adverse events. Paediatr Drugs. 2009; 11(5): 309–321. PubMed Abstract \| Publisher Full Text\n\nNeirukh T, Qaisi A, Saleh N, et al.: Seroprevalence of Cytomegalovirus among pregnant women and hospitalized children in Palestine. BMC Infect Dis. 2013; 13: 528. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowd JB, Aiello AE, Alley DE: Socioeconomic disparities in the seroprevalence of cytomegalovirus infection in the US population: NHANES III. Epidemiol Infect. 2009; 137(1): 58–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWujcicka W, Gaj Z, Wilczynski J, et al.: Impact of socioeconomic risk factors on the seroprevalence of cytomegalovirus infections in a cohort of pregnant Polish women between 2010 and 2011. Eur J Clin Microbiol Infect Dis. 2014; 33(11): 1951–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence GM, Friedlander Y, Calderon-Margalit R, et al.: Associations of social environment, socioeconomic position and social mobility with immune response in young adults: the Jerusalem Perinatal Family Follow-Up Study. BMJ Open. 2017; 7(12): e016949. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasha J, Iwasenko JM, Robertson P, et al.: Congenital cytomegalovirus infection is associated with high maternal socio-economic status and corresponding low maternal cytomegalovirus seropositivity. J Paediatr Child Health. 2014; 50(5): 368–72. PubMed Abstract \| Publisher Full Text\n\nManicklal S, Emery VC, Lazzarotto T, et al.: The \"silent\" global burden of congenital cytomegalovirus. Clin Microbiol Rev. 2013; 26(1): 86–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForner G, Abate D, Mengoli C, et al.: High Cytomegalovirus (CMV) DNAemia Predicts CMV Sequelae in Asymptomatic Congenitally Infected Newborns Born to Women With Primary Infection During Pregnancy. J Infect Dis. 2015; 212(1): 67–71. PubMed Abstract \| Publisher Full Text\n\nNahla K, Ali Y, Enan K: Studies on congenital infections in Sudan: Seroprevalence of cytomegalovirus. J Sci Tech. 2011; 12(4): 82–90. Reference Source\n\nFalahi S, Ravanshad M, Kenar Koohi A, et al.: SeroPrevalence of CMV in women’s with spontaneous abortion in kowsar hospital, Ilam during 2007-2008. Pathobiology Research. 2010; 12(4): 39–43. Reference Source\n\nHassan A, Fattouh M, Saad M, et al.: Assessment of ELISA and PCR in detection of Cytomegalovirus viremia in pregnant women. International Journal of Advanced Research. 2014; 2(6): 247–253.\n\nBagheri L, Mokhtarian H, Sarshar N, et al.: Seroepidemiology of cytomegalovirus infection during pregnancy in Gonabad, east of Iran: a cross-sectional study. J Res Health Sci. 2012; 12(1): 38–44. PubMed Abstract\n\nFowler KB, Boppana SB: Congenital cytomegalovirus (CMV) infection and hearing deficit. J Clin Virol. 2006; 35(2): 226–231. PubMed Abstract \| Publisher Full Text\n\nKhalf MS: The seroprevalence of IgM among Iraqi aborted women infected with human cytomegalovirus. Iraqi Academic Scientific Journal. 2012; 11(1): 123–129. Reference Source\n\nKakru M, Shaheen R, Nazir A: Seroprevalence of Cytomegalovirus (CMV) in Kashmir valley-a preliminary study. JK-Practitioner. 2004; 11(4): 261–262. Reference Source\n\nUyar Y, Balci A, Akcali A, et al.: Prevalence of rubella and cytomegalovirus antibodies among pregnant women in northern Turkey. New Microbiol. 2008; 31(4): 451–455. PubMed Abstract\n\nSeo S, Cho Y, Park J: Serologic screening of pregnant Korean women for primary human cytomegalovirus infection using IgG avidity test. Korean J Lab Med. 2009; 29(6): 557–562. PubMed Abstract \| Publisher Full Text\n\nLachmann R, Loenenbach A, Waterboer T, et al.: Cytomegalovirus (CMV) seroprevalence in the adult population of Germany. PLoS One. 2018; 13(7): e0200267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim AM, Mohammed AB, Elhag WI: Serofrequancy Rate of Cytomegalovirus Infection among Sudanese Aborted Women at Ibrahim Malik Teaching Hospital (Khartoum). Afr J Med Sci. 2017; 2(12). Reference Source\n\nAhmed E, Elbushra S, Ahmed MS, et al.: HCMV seroprevalence, Blue Nile State, Sudan. figshare. Dataset. 2019; 13: 39. http://www.doi.org/10.6084/m9.figshare.9895715.v1" }	[ { "id": "92377", "date": "22 Sep 2021", "name": "Luiz Fernando Almeida Machado", "expertise": [ "Reviewer Expertise Microbiology", "General Virology", "Molecular Epidemiology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of the manuscript is to determine the seroprevalence of IgG and IgM antibodies to HCMV in women who have suffered a miscarriage in a hospital in Sudan.\nThe study has a series of limitations that do not allow estimating the prevalence of HCMV, as it only detected antibodies and not HCMV DNA. In addition, the main focus was on IgG, which could indicate previous contact with the virus, and not IgM, which could indicate a recent virus infection.\n\nAnother important point is that only a qualitative serological test was performed. To get an idea of the prevalence of HCMV infection, it would be interesting to quantify IgG, as well as the IgG avidity test, in addition to HCMV DNA research (the latter being an option only, as it requires a more refined infrastructure).\n\nWhat is the relationship between abortion and IgM? The authors do not comment on the presence of IgM in the results and conclusion.\n\nAnother interesting point would be to verify the prevalence of anti-HCMV antibodies in a control group of women who had live children during the study period, to see if there is a difference in the prevalence between the two groups. Did women who had live children also not have a high prevalence of IgG? Probably yes.\n\nThe authors could make a table making the correlation between abortion and the presence of IgM as well.\n\nWhy does Table 2 only correlate with IgG and not IgM? Although the prevalence of IgM is lower than IgG, the rate is very high when compared to studies1,2,3.\n\nWhat are the limitations of the study? Did the women in the study receive prenatal care?\n\nAuthors should establish some exclusion criteria in the study, such as a history of toxoplasmosis, syphilis, and rubella, which are also important causes of abortion in the first trimester of pregnancy.\nAll this information is important so that it is not prematurely concluded that the presence of IgG in women of low economic status is a risk factor for abortion in the first trimester of pregnancy. Therefore, my opinion is that the study should be further developed in order to be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "94002", "date": "27 Sep 2021", "name": "Irena Tabain", "expertise": [ "Reviewer Expertise Microbiology", "Virology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review the manuscript.\nCytomegalovirus is the most common congenital infection, which mostly follows maternal primary infection1.\nFirstly, only one abbreviation for cytomegalovirus should be used.\n\nThe study has several limitations:\n\nIt would be important to know the CMV seroprevalence in women with healthy and alive babies.\n\nThe control group is missing.\n\nUnfortunately, the authors presented only the correlation between IgG and parameters and IgM correlation is missing. Since PCR detection is not used, correlation of presented parameters with IgM is important.\n\nDiscussion and conclusions should be supported with results.\n\nAnd finally, it should be noted that other causes of obstetric complications are excluded (e.g. toxoplasmosis, rubella).\n\nConclusions are not adequate, but this information is important, and therefore, the study protocol needs an annex, and the annex study carried out and then updated data could be presented.\nBest regards!\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/8-1735
https://f1000research.com/articles/8-755/v1	29 May 19	{ "type": "Case Report", "title": "Case Report: Pancytopenia as an indicator for lysosomal storage disease (Gaucher's Disease)", "authors": [ "Alberto Ortega-Rosales", "Carlos Burneo-Rosales", "Gilda Romero-Ulloa", "Gabriela Burneo-Rosales", "Carlos Burneo-Rosales", "Gilda Romero-Ulloa", "Gabriela Burneo-Rosales" ], "abstract": "Introduction: Lysosomal storage disorders are a rare group of diseases with genetic origin in which Gaucher Disease (GD) stands out as the most frequent. GD type 1 is the most common form of this condition, and patients with this pathology present with unexplained cytopenias, in addition to hepatosplenomegaly, bone involvement, and in other cases neurological disorders. A case of a patient is presented, whose results showed thrombocytopenia and leukopenia in addition to hepatosplenomegaly. In Latin America, there are very few reported cases of this clinical entity, and information on this disease is very limited. Case: We present a case of a patient diagnosed with GD, who presented with thrombocytopenia and leukopenia in addition to hepatosplenomegaly, with the aim of emphasizing the importance of early recognition of this pathology, especially in patients with unexplained cytopenia’s or hepatosplenomegaly’s. In suspicion of GD, enzymatic quantification of β-glucocerebrosidase was performed, showing its deficit in addition to alteration in the GBA gene. Unfortunately, enzymatic replacement could not be done because the Cerazyme (imiglucerase for injection) is not available in Ecuador. Nevertheless, the patient was treated with analgesic (1g of paracetamol generally three times a day) and vitamin supplements (Dayamineral). Currently the patient is waiting for transfer to a foreign institution; she continues with bicytopenia and hepatosplenomegaly, her conditions are expected to be remit once the enzymatic treatment has been administered.\n\nConclusion: We believe that the timely recognition of this disease will allow the initiation of enzymatic replacement therapy in an effective manner, in order to reduce morbidity and improve the clinical aspects of the patient.", "keywords": [ "Gaucher disease", "lysosomal storage disorders", "cytopenia", "β-glucocerebrosidase." ], "content": "Introduction\n\nLysosomal deposit disorders constitute a rare group of recessive hereditary monogenic diseases, resulting from the abnormal accumulation of non-degraded material in the lysosomes of different cells of the organism, as a result of the total or partial functional loss of specific lysosomal enzymes or cofactors involved in the degradation of these materials; the resulting lysosomal dysfunction leads to cell dysfunction and clinical anomalies1. The most common lysosomal storage disease is Gaucher disease (GD), with an estimated frequency of 1:40.000 to 1:86.000 inhabitants in the general population, except for the Jewish Ashkenazi ethnic group were it is estimated to be 1:850 births2, characterized by the defective function of the catabolic β-glucocerebrosidase enzyme, which leads to an accumulation of glucocerebroside in the lysosomes of different cells3, causing pancytopenia, hepatosplenomegaly, bone involvement and, sometimes neurological alteration4. The phenotype is variable and there are three clinical subtypes, which are classified by the absence or presence and progression of neurological involvement: type 1 or non-neuropathic form; type 2, the acute neuropathic form of infantile onset; and type 3, the neuronopathic form of juvenile onset5.\n\nIn Ecuador, as in Latin America, there are very few reported cases of this clinical entity, and information on this disease is very limited6. In symptomatic patients, GD is usually a progressive disease that, if left untreated, can cause irreversible organ damage, severe morbidity, reduced quality of life and even premature death. Currently there is an effective treatment for GD in enzymatic replacement, which reverses or prevents many of the clinical manifestations of this disorder7.\n\nDespite the availability of accurate and minimally invasive diagnostic tests, patients are often misdiagnosed as a result of the lack of experience with GD and the prolonged delay in accurate diagnosis, and this is likely to be translated into relative rarity of this disease8. Therefore, greater knowledge of the clinical and demographic characteristics of this clinical entity can improve early recognition, reduce the rate of inaccurate or delayed diagnoses for patients with GD, allowing timely treatment aimed at reducing morbidity and preventing irreversible sequels6.\n\n\nDescription of the case\n\nA 29-year-old housewife from Loja-Ecuador, of mixed ethnicity, with no relevant family or personal history, presented to the outpatient service of the Isidro Ayora General Hospital, Loja, Ecuador, in October 2018.\n\nShe presented with a pulsating holocraneal headache of slight intensity. Concomitantly, she presented with heartburn which responded positively to self-medication with omeprazole; additionally, she mentioned she was experiencing progressive loss of weight, fatigue and pain in the lower limbs and hands. Physical examination showed ecchymosis and petechiae scattered throughout the body. In the abdomen, hepatosplenomegaly was palpable. Routine laboratory results are summed up in the table below (Table 1).\n\nViral serology tests for hepatitis B, C and human immunodeficiency virus were negative. An ultrasound of the upper abdomen was performed, where there was evidence of a distended liver without occupant injuries and splenomegaly [Figure 1]. Hepatosplenomegaly was observed in the CT-Scan in addition to incipient degenerative osteoarthritis at the dorsal spine. It was decided to perform a bone marrow biopsy, demonstrating abundant cells between the bone trabeculae, consisting of hematopoietic tissue, with a marked decrease in megakaryocytes of hypolobular nuclei, and myeloid-erythroid relationship conserved. In the inter and paratrabecular spaces, abundant clusters of macrophages of the typical broad cytoplasm are observed like “crumpled paper” with small, regular nuclei displaced to the periphery [Figure 2]. The morphological picture is suggestive of lysosomal storage disease, and with the suspicion of GD, quantification of the enzymatic activity of β-glucocerebrosidase was performed, confirming its deficit with a result of 0.27 µmol/L/h (normal range: 2.3 – 12 nmol/h/Ml). Sequential analysis of the GBA gene showed the presence of an apparently homozygous pathogenic alteration in the GBA gene.\n\nUnfortunately, enzymatic replacement could not be performed because Cerazyme (imiglucerase for injection) is not available in Ecuador. Nevertheless, the patient was treated with analgesic (1g of paracetamol generally three times a day) and vitamin supplements (Dayamineral). Currently the patient is waiting for transfer to a foreign institution. Fortunately, the finding of this disease was incidental, and still does not show serious symptoms. At the time of writing the patient is regularly monitored at the Isidro Ayora General Hospital, Loja until the enzymatic medication can be obtained. Currently, the patient persists in good general conditions, without worsening clinical condition. It should be mentioned that she continues with bicytopenia and hepatosplenomegaly, her conditions are expected to be remit once the enzymatic treatment has been administered.\n\n\nDiscussion\n\nThis clinical case is a representative example of the clinical, biochemical and genetic characteristics of GD type 19, characterized by the variability in the signs, symptoms, severity and progression of the disease10. The most common signs and symptoms are: splenomegaly [95%], hepatomegaly [87%], radiological bone disease [81%], thrombocytopenia [50%], anemia [40%], bone pain [27%]11.\n\nGD is caused by mutations in the GBA gene, located on chromosome 1 (1q21), leading to markedly decreased activity of the lysosomal enzyme, β-glucocerebrosidase, which hydrolyzes the glycolipid glucocerebroside in ceramide and glucose12. In the case studied, it was possible to observe a decrease in the enzymatic activity of β-glucocerebrosidase, in addition to alteration of the gene GBA. The consequence of this deficiency is attributed to the accumulation of glucosilcerebroside in macrophages, inducing its transformation in Gaucher cells, which under optical microscopy are usually presented as enlarged cells with eccentric nuclei and condensed chromatin and cytoplasm with heterogeneous appearance as “crumpled paper”13, similar characteristics to the bone marrow biopsy performed in our patient. In our patient, the basic aspects that guided the diagnosis of GD were the incidental finding of a bicitopenia in a routine laboratory examination, in addition to the presence of unexplained hepatosplenomegaly. Without considering the GD in the differential diagnosis of the patient with this type of symptomatology, the definitive diagnosis can be missed or delayed, because the signs and symptoms frequent other more common conditions, including the malignant neoplasms of hematological origin14. Therefore, it is essential to strengthen the knowledge about GD, allowing to reduce the threshold for diagnostic tests and reduce the rate of erroneous diagnoses, in addition to promoting the earliest start of treatment when it is indicated.\n\nGD is usually diagnosed by demonstrating the characteristic “Gaucher cells” in the bone marrow. Microscopically, these cells show a large size, carrying an eccentric nucleus and a cytoplasm that resembles a wrinkled paper13. However, the presence of this type of histological pattern has occasionally been described in several malignant hematological malignancies15, determination of the enzymatic activity of reduced β-glucocerebrosidase or absence is therefore the gold standard for the diagnosis of all GD variants. In the present study a clearly reduced activity of this enzyme was evident, in addition to an apparently homozygous pathogenic alteration of the GBA gene, allowing us to establish a definitive diagnosis of GD.\n\n\nConclusion\n\nGD should be considered in the differential diagnosis of patients with unexplained pancytopenia or hepatosplenomegaly. Early recognition will allow the initiation of enzymatic replacement therapy in order to reduce morbidity and improve the clinical aspects of the patient.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSchultz ML, Tecedor L, Chang M, et al.: Clarifying lysosomal storage diseases. Trends Neurosci. 2011; 34(8): 401–410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinari S, Castaman G: Clinical manifestations and management of Gaucher disease. Clin Cases Miner Bone Metab. 2015; 12(2): 157–164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDandana A, Ben Khelifa S, Chahed H, et al.: Gaucher Disease: Clinical, Biological and Therapeutic Aspects. Pathobiology. 2016; 83(1): 13–23. PubMed Abstract \| Publisher Full Text\n\nStirnemann J, Belmatoug N, Camou F, et al.: A Review of Gaucher Disease Pathophysiology, Clinical Presentation and Treatments. Int J Mol Sci. 2017; 18(2): pii: E441. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRigante D, Cipolla C, Basile U, et al.: Overview of immune abnormalities in lysosomal storage disorders. Immunol Lett. 2017; 188: 79–85. PubMed Abstract \| Publisher Full Text\n\nNalysnyk L, Rotella P, Simeone JC, et al.: Gaucher disease epidemiology and natural history: a comprehensive review of the literature. Hematology. 2017; 22(2): 65–73. PubMed Abstract \| Publisher Full Text\n\nCharrow J, Dulisse B, Grabowski GA, et al.: The effect of enzyme replacement therapy on bone crisis and bone pain in patients with type 1 Gaucher disease. Clin Genet. 2007; 71(3): 205–11. PubMed Abstract \| Publisher Full Text\n\nMistry PK, Sadan S, Yang R, et al.: Consequences of diagnostic delays in type 1 Gaucher disease: the need for greater awareness among hematologists-oncologists and an opportunity for early diagnosis and intervention. Am J Hematol. 2007; 82(8): 697–701. PubMed Abstract \| Publisher Full Text\n\nGiraldo P, Pérez-López J, Núñez R, et al.: Patients with type 1 Gaucher disease in Spain: A cross-sectional evaluation of health status. Blood Cells Mol Dis. 2016; 56(1): 23–30. PubMed Abstract \| Publisher Full Text\n\nBohra V, Nair V: Gaucher's disease. Indian J Endocrinol Metab. 2011; 15(3): 182–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaplan P, Andersson HC, Kacena KA, et al.: The clinical and demographic characteristics of nonneuronopathic Gaucher disease in 887 children at diagnosis. Arch Pediatr Adolesc Med. 2006; 160(6): 603–608. PubMed Abstract \| Publisher Full Text\n\nHruska KS, LaMarca ME, Scott CR, et al.: Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat. 2008; 29(5): 567–583. PubMed Abstract \| Publisher Full Text\n\nChen M, Wang J: Gaucher disease: review of the literature. Arch Pathol Lab Med. 2008; 132(5): 851–853. PubMed Abstract\n\nThomas AS, Mehta AB, Hughes DA: Diagnosing Gaucher disease: an on-going need for increased awareness amongst haematologists. Blood Cells Mol Dis. 2013; 50(3): 212–7. PubMed Abstract \| Publisher Full Text\n\nAsh Image Bank: Waldenström macroglobulinemia with pseudo-Gaucher cells. Blood. 2010; 116(18): 3388. PubMed Abstract \| Publisher Full Text" }	[ { "id": "50548", "date": "09 Jul 2019", "name": "Ellen Sidransky", "expertise": [ "Reviewer Expertise Genetics", "Gaucher disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhile this case does not report any new information, it fulfills an important role in highlighting rare disease cases worldwide, raising awareness and further informing health care practitioners. However there are several errors that must be corrected.\n\nAbstract\nThe English grammar is at times difficult to read, improving translation would increase readability. Cerezyme (Imiglucerase) is one drug for enzyme replacement therapy (ERT), there are two others as well as substrate reduction therapy (SRT). While more work needs to be done characterizing Gaucher disease in Latin America, currently the ICGG does contain almost 16% of its population from Latin American cases of Gaucher disease (GD) and describing the information as very limited is not entirely accurate. It may certainly be accurate in describing Ecuadorean cases of GD.\nDescription of the case\nGive the patient’s values its own column in Table 1. The case is described well, and the figures add depth to the presentation. Referring to this case as an “incidental diagnosis\" is not valid as the patient had numerous manifestations. Likewise, it cannot be said that she does not show “serious symptoms”, as it is stated that she had bruising fatigue, pain, weight loss etc. Were there other diagnoses that the medical team considered? The differential list can be of value to those reading the case report. The genotype is not provided!\nDiscussion\n“Glycolipid glucosylceramide into ceramide and glucose” GD should not usually be diagnosed by bone marrow biopsy! Bone marrow biopsy is a painful procedure and is not needed to diagnose GD. Enzymatic and genetic testing is the preferred method of diagnosis, as stated in the manuscript. The author states that genetic analysis was performed, but the genetic mutations are not included in the case report. This would be very helpful and should be included.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-755
https://f1000research.com/articles/8-872/v1	17 Jun 19	{ "type": "Research Article", "title": "Screening tuberculosis patients for diabetes mellitus in a routine program setting in Kampala, Uganda: a cross-sectional study", "authors": [ "Joseph Nsonga", "John Paul Dongo", "Frank Mugabe", "Gerald Mutungi", "Richard Walyomo", "Christopher Oundo", "Sarah Zalwango", "Daniel Okello", "Simon Muchuro", "Riitta A Dlodlo", "Yan Lin", "Joseph Nsonga", "John Paul Dongo", "Frank Mugabe", "Gerald Mutungi", "Richard Walyomo", "Christopher Oundo", "Sarah Zalwango", "Daniel Okello", "Simon Muchuro", "Riitta A Dlodlo" ], "abstract": "Background: Uganda is located in East Africa and is among the countries with the lowest income globally. The ten health centres in this project serve populations in the under-privileged communities of Kampala. The objective of the study was to implement diabetes mellitus (DM) screening among tuberculosis (TB) patients in a routine program setting with limited resources and high human immunodeficiency virus (HIV) prevalence. Methods: A descriptive cross-sectional observational study was conducted in ten health centres in Kampala, Uganda. As part of a project to implement DM screening in a routine setting, TB patients were screened for DM by trained health workers. A fasting blood glucose (FBG) value ≥7.0mmol/l was considered to indicate DM. For this study, aggregate data was collected and analysed using SPSS for Windows, version 13.0. Results: Among 4,590 TB patients registered, 4,016 (88.0%) were screened with random blood glucose (RBG). Of those with RBG ≥6.1mmol/l, 1,093 (83.3%) were screened with FBG. In total, 92 (2.3%) patients were diagnosed with DM and 66 (71.8%) of them were newly diagnosed. The proportion of TB patients screened with FBG in the health centres varied from 58.2% to 100%. The proportion of patients screened with FBG and the prevalence of DM were significantly higher in private health centres compared with public health centres. The health centres in peri-urban areas screened more patients with RBG than those in urban areas. Health centres without DM services screened a larger number of patients with RBG and FBG than those with DM services. Conclusions: It appears feasible to implement screening TB patients for DM in routine program settings with limited resources and high HIV prevalence. Its introduction requires close collaboration between TB and DM services. The challenges identified need government attention and certain institutional and service-related factors need to be better managed at times.", "keywords": [ "Tuberculosis", "Diabetes Mellitus", "Screening", "Uganda" ], "content": "Introduction\n\nAlthough significant progress has been achieved in tuberculosis (TB) care and prevention during the past decades, TB remains a major public health problem and is responsible for more deaths than any other single infectious disease worldwide1. In 2017 globally, 10.0 million people developed TB and 1.6 million died from it1. Uganda is among the 30 high TB and human immunodeficiency virus (HIV) burden countries, with an estimated TB prevalence of 253 per 100,000 population (95% CI: 196-317) and 40% HIV co-infection rate2,3.\n\nAlong with socio-economic development, urbanization, dietary and lifestyle changes, the prevalence of diabetes mellitus (DM) is escalating in most low- and middle-income countries. Available data suggests that an estimated 425 million people worldwide live with DM and by 2045 this number will grow to 629 million4. Although estimates for sub-Saharan Africa are based on limited data with substantial uncertainty, DM appears to be increasing rapidly in many urban centres in the region5. In Uganda, some risk factors for DM, such as obesity, were greater among persons of a high socio-economic status in rural areas, though the prevalence of DM in the adult population of the entire country was relatively low (1.4% in a nationwide cross-sectional survey in 2014)6,7.\n\nDM is a well-known risk factor for TB and increases its risk 2-3-fold8,9. Systematic reviews and meta-analyses suggest that DM patients with TB experience worse treatment outcomes compared with patients without DM, with delayed sputum smear conversion, increased risk of drug resistance, treatment failure, death and relapse after successful completion of anti-TB treatment8–12. On the other hand, TB patients with previously known DM are at an increased risk of hyperglycemia or worse glycaemic control13,14. Therefore, early detection of DM among TB patients and proper management of both conditions are well known measures to improve treatment outcomes in these patients.\n\nTo address this problem in low- and middle-income countries, the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (The Union) launched a ‘Collaborative Framework for the Care and Control of Diabetes and Tuberculosis’ in 2011, and recommended bi-directional screening for TB and DM within routine health services15.\n\nThe Union, together with national authorities, implemented the first bi-directional screening of DM and TB within routine health services in China and India, where it was found to be feasible in these settings16–18. In Uganda, there is no policy on routine screening for DM among TB patients. Whether a similar approach can be implemented in a resource limited setting with a high HIV prevalence remained unknown. To further understand this, a project was implemented from 2016 to 2017 to screen TB patients for DM, supported by the World Diabetes Foundation (WDF). Objectives of this study were to: a) assess whether DM screening can be implemented among TB patients in a routine programmatic health setting in Uganda; b) identify challenges for DM screening among TB patients and solutions to overcome them; and c) identify institutional and health service related factors that may have an impact on DM screening among TB patients.\n\n\nMethods\n\nSetting and sites. Kampala is the capital city of Uganda and had an estimated population of 1,607,182 in 2017. In the same year, the Kampala Capital City Authority (KCCA) notified a total of 7,260 TB cases, resulting in a case notification rate of 452/100,000 population3. Treatment success was 84.6%. HIV prevalence was 6.9% nationwide (Uganda Population-based HIV Impact Assessment UPHIA 2016–2017)3.\n\nHealth services in the city are provided by the KCCA. Persons with presumed TB are investigated in health centres and diagnosed TB patients receive care in TB clinics. DM services are available mainly in specialised DM clinics at tertiary level health facilities.\n\nTen health centres, both public and private, in Kampala were selected based on their location in the five administrative divisions of KCCA and representative of the health delivery system in Kampala. These were selected from different communities based on sufficient numbers of registered TB patients, the availability of TB and DM diagnostic and treatment facilities within the same catchment area and willingness of the staff to participate in the project.\n\nProject training, screening algorithm and monitoring. The National TB and Non-Communicable Disease (NCD) Programs’ staff, Kampala Capital City Authority (KCCA) and The Union Uganda Office staff attended an inception meeting and training of trainers in February-March 2016. Participants reviewed the association between DM and TB, the need for screening and agreed to use the screening algorithm shown in Figure 1. Cascade training sessions for 40 staff from the selected health centres were then conducted. The Union and KCCA officials performed quarterly monitoring, compiled periodic reports and provided overall supportive supervision for the activities. At the same time, they recorded any challenge encountered during the implementation.\n\nPatients. Patients aged 15 years and over who were seeking health services in the health centres and were newly registered with any form of TB between 1st April 2016 to 31st December 2017 were included in the screening project.\n\nBlood glucose measurement in TB patients. A blood sample was collected for random blood glucose (RBG) using capillary blood immediately after the TB registration in the health centres. A OneTouch glucometer (model SN SAFTM7K8, Lifescan, Johnson & Johnson, New Brunswick, USA) was used with a detection value ranging from 1.7 to 19.1mmol/l. Patients with an RBG value ≥6.1mmol/l were invited to return on the following day, after an overnight fast of at least 10 hours, for measurement of fasting blood glucose (FBG). The FBG cut-off thresholds were based on the World Health Organization (WHO) recommendations as follows: i) FBG ≥7.0 mmol/l (≥126 mg/dl) indicates presence of DM; ii) FBG of 6.1 – 6.9 mmol/l (from 110 mg/dl to less than 126 mg/dl) indicates impaired glucose tolerance; and iii) FBG <6.1 mmol/l (<110 mg /dl) indicates normal blood glucose levels. Health workers provided information on the importance of fasting before returning for a confirmatory test (FBG) to the patients through health education. Reminder phone calls were also provided to all patients who screened positive for RBG to return for confirmatory test.\n\nThis was a descriptive cross-sectional observational study using routine project data from ten health centres in Kampala, Uganda.\n\nDistrict TB and Leprosy Supervisors (DTLs) collected routine programmatic data every six months from the unit TB and DM registers in the 10 implementing health facilities. Final data collection was finished in December 2017. The Union staff reviewed the data and worked together with the health centre staff to trace the missing data (mainly the number of patients with known DM). The DTLs collected aggregated data, which was verified by The Union staff, and double-entered the data into a spreadsheet. Comparisons of characteristics between patients attending different health centres were carried out using the chi square test with odds ratios (OR) and their 95% confidence intervals. Levels of significance were set at 5%. Statistical analysis was carried out using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL, USA).\n\nThis project and the research proposal were approved by the National Tuberculosis and Leprosy Program, the Non-Communicable Diseases Program, Ministry of Health of the Government of Uganda, and Kampala Capital City Authority. As the information collected formed part of routine programmatic health service delivery and no individual patient data was collected, review by an ethics committee and patient consent were not considered necessary.\n\n\nResults\n\nThe characteristics of the selected health centres are described in Table 1. Of the 4,590 TB patients notified, 4,016 (88.0%) were screened with RBG19. Of those with RBG ≥6.1mmol/l, 1,093 (83.3%) were screened with FBG. Of the 92 TB patients diagnosed with DM, 66 (71.7%) were newly diagnosed with DM and all, except four patients who died shortly after the TB registration, were referred to DM services. The prevalence of DM in the screened TB patients was 2.3% (Table 2).\n\nUrban = central business area; Peri-urban = slightly away from central business area; Rural = furthest away from central business area. TB, tuberculosis; DM, diabetes mellitus\n\n Percentage of newly diagnosed DM among total number of DM\n\nTB, tuberculosis; DM, diabetes mellitus; RBG, random blood glucose; FBG, fasting blood glucose\n\nThe number of registered TB patients in reporting periods one, two, three and four were 372, 1,576, 1,364 and 1,278, respectively. The proportion of patients screened with RBG (number of screened with RBG/number of registered TB patients) were 83.1%, 83.8%, 91.6% and 89.0%, respectively, over the four reporting periods, but the absolute number of TB patients screened with RBG in the first reporting period was 7.7%. Of the number of TB patients with RBG ≥6.1mmol/l, the proportion of screened with FBG (number of screened with FBG/number of RBG ≥6.1mmol/l) were 81.4%, 89.8%, 95.7% and 63.9% over the four reporting periods.\n\nThe proportion of patients screened with RBG and FBG varied from 78.9% to 94.5%, and from 58.2% to 100.0%, respectively, among the health centres (Table 3). The proportion of patients screened with RBG was significantly higher in the health centres located in peri-urban areas compared with the centres in urban areas (OR 1.50, 95% CI 1.12-1.99, P=0.006); and in those without DM services compared with those with DM services (OR 1.35, 95% CI 1.11-1.63, P=0.002) (Table 4). Of the patients with RBG ≥6.1mmol/l, the proportion screened with FBG was significantly higher in private health centres (OR 2.16, 95% CI 1.26-3.69, P=0.005) and those without DM services (OR 1.96, 95% CI 1.44-2.67, P<0.001) than that in public health centres and in those with DM services.\n\n* Percentage of newly diagnosed DM among total number of DM\n\nTB = tuberculosis; DM = diabetes mellitus; RBG = random blood glucose; FBG = fasting blood glucose\n\n* Percentage of newly diagnosed DM among total number of DM\n\nTB = tuberculosis; DM = diabetes mellitus; RBG = random blood glucose; FBG = fasting blood glucose\n\nA total of 92 TB patients were diagnosed with DM, accounting for a prevalence of 2.3%. The prevalence of DM in TB patients was higher in the private health centres (OR 2.26, 95% CI 1.40-3.62, P=0.001) and in those with DM services (OR 0.60, 95% CI 0.39-0.93, P=0.022).\n\n\nDiscussion\n\nThis is the first study to report on screening TB patients for DM in a primary health care setting in Uganda. We included both public and private facilities in urban, peri-urban and rural parts of Kampala to assess the feasibility of integrating DM screening into routine TB service.\n\nThe overall prevalence of DM in the screened TB patients was 2.3% (92/4,016), which was higher than that in general population and our finding mirrored the findings of previous studies6,17,18,20, although it was lower than that reported by a study among hospitalized TB patients at a large referral hospital in an urban area of Uganda21. However, using only FBG to diagnose DM can underestimate the prevalence of DM by as much as 50% when compared with the gold standard test, the oral glucose tolerance test, which is more accurate but unavailable at primary health care level22. In addition, some of the facilities raised the FBG screening threshold to 7.0 mmol/l during the first 6–8 months due to misinterpretation of the screening algorithm. We therefore believe that the actual prevalence of DM among TB patients in Uganda might be higher than we have observed in this study. Of the 92 DM patients detected, 66 (71.7%) had not been diagnosed before this study. This strongly demonstrated the need for integrating DM screening into routine TB service5,23.\n\nIn the first six months, a small number of TB patients were screened with RBG, due to the long engagement of stakeholders followed by the cascade training. One health centre (Nsambya) did not send staff to attend the training and did not screen any patients in the first reporting period due to staff turn-over, a situation that should have been identified by training organizers, and this challenge required KCCA attention. The number of screened TB patients increased rapidly over the rest of the reporting periods, suggesting that the procedure was gradually accepted by staff, and we echo a similar finding in China17. The proportion of patients screened with FBG declined over the last six-month period due to gluco-strips stock-out at the government medical stores. It was reassuring that the health centre staff largely followed the screening algorithm, although there were certain differences. Kisugu Health Centre ensured FBG screening for everyone with RBG ≥ 6.1mmol/l due to strong commitment of the staff. On the contrary, Naguru Health Centre managed to screen only 58.2% of the patients who needed an FBG test even though they had the same number of health workers, although they did have a slightly higher number of TB patient visits. It is necessary to understand reasons for missed opportunities in the DM screening cascade.\n\nPrivate health centres screened higher proportions of patients with FBG and, subsequently, identified more DM patients than public facilities. Possible reasons for this could be that patients with a higher socio-economic status sought care in private health centres, which have well-motivated staff thanks to better remuneration. Similarly, staff in peri-urban and rural facilities screened higher proportions of patients with RBG and FBG than in urban areas. Murchison Bay is a health centre in peri-urban area operated by the Ministry of Internal Affairs with prisoners being the main source of patients. Given that most patients were confined in prison, they were available for RBG and FBG. In urban health centres there is more frequently congestion and longer waiting times than in rural centres. This may have caused some patients to miss their FBG tests.\n\nAn intriguing finding that also requires a further study was that health centres with available DM services at time of the study, a feature that tends to reflect better health services, did not screen more patients with RBG and FBG compared with those without DM services. This was unexpected and not in line with the findings of other studies16. Low screening at Naguru Health Centre could have contributed to this finding.\n\nThere were some service-related barriers identified in the project implementation: 1) lack of a sustainable supply of glucose testing strips, which caused insufficient screening for RBG and FBG; 2) lack of community support mechanisms to help trace patients who failed to return for FBG testing; 3) insufficient number of staff in the health centres caused by staff turn-over; 4) routine DM service was not available in some health centres.\n\nThis study had several limitations. Diagnosis of DM relied on FBG alone and we were unable to include oral glucose-tolerance tests, a more accurate test, as part of the screening cascade. This may have resulted in an underestimated prevalence of DM in TB patients21. Blood samples for RBG and FBG were taken from capillary blood and measured with glucometers without quality assurance, rather than using venous blood specimens and tested with biochemical analyser. This may have caused a testing bias24. RBG or FBG were measured only once immediately after TB registration, without any confirmatory test at the end of anti-TB treatment, and glycosylated haemoglobin (HbA1c) tests were not carried out, which is influenced less by infection25. Therefore, we do not know whether any TB patient may have been diagnosed as having DM due to infection-induced hyperglycaemia. However, a previous study suggested that taking one blood sample for FBG immediately after TB diagnosis would be appropriate for majority of TB patients14. Finally, we did not collect individual patient data and perform multivariate analysis and were unable to identify and control potential confounding factors.\n\nThe strength of this study is that we implemented DM screening among a large number of TB patients in routine health service using mainly existing resources. Only training, meetings, support supervision, mentorship and provision of glucometers were supported. There were no staff or patient incentives. This is a key point for sustainability and scale up of integration of DM and TB services. Some challenges identified in the study need government attention and this indicates the need for collaboration between TB and non-communicable disease programs.\n\n\nConclusion\n\nThis study revealed that prevalence of DM in TB patients is higher than that in the general population in Uganda. Screening TB patients for DM in a primary health care setting in a resource-limited country with high HIV-associated TB epidemic appeared feasible. This study identified certain service-related factors that may be barriers and need to be addressed by relevant authorities. Inclusion of bi-directional screening in both TB and DM clinics could be considered. Every year, 44,000-47,000 persons with TB are registered in Uganda and if all were screened for DM, at least 1,748 additional persons with DM could be detected and enrolled to DM care. As early diagnosis and management for the two diseases will improve treatment outcomes and well-being of people, this is an important public health intervention.\n\n\nData availability\n\nFigshare: Screening tuberculosis patients for diabetes mellitus in a routine program setting in Kampala, Uganda: a cross-sectional study. https://doi.org/10.6084/m9.figshare.8248625.v119\n\nThis project contains the following underlying data:\n\n- TB_DM DATA _Uganda 2016_2017.xlsx (raw data collected from health centres over the four reporting periods)\n\n- Screening tuberculosis patients for diabetes mellitus in a routine program setting in Kampala, Uganda a cross-sectional study.docx (table of data processes)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis project was funded by the World Diabetes Foundation [WDF15-1211].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank all the staff and patients at the 10 health centres for their participation and collaboration. This study was funded by the World Diabetes Foundation as part of a grant (WDF 15-1211). Thanks are also extended to the Ministry of Health, National TB and Leprosy Program, National Non-Communicable Disease Program, Kampala Capital City Authority, Uganda Diabetes Association and Non-Communicable Disease Alliance.\n\n\nReferences\n\nWorld Health Organization. WHO Report: Global Tuberculosis Control 2018. 2018. Reference Source\n\nMinistry of Health: The Uganda National Tuberculosis Prevalence Survey, 2014-2015. Kampala, Uganda. 2017. Reference Source\n\nMinistry of Health: National Tuberculosis and Leprosy Program Annual Report 2016-2017. Kampala, Uganda. 2017. Reference Source\n\nInternational Diabetes Federation: IDF Diabetes atlas. 8th Edition. International Diabetes Federation, Brussels. 2017. Reference Source\n\nAtun R, Davies J, Gale EAM, et al.: Diabetes in sub-Saharan Africa: from clinical care to health policy. Lancet Diabetes Endocrinol. 2017; 5(8): 622–67. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker MA, Harries AD, Jeon CY, et al.: The impact of diabetes on tuberculosis treatment outcomes: a systematic review. BMC Med. 2011; 9: 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Q, Ma A, Han X, et al.: Hyperglycemia is associated with increased risk of patient delay in pulmonary tuberculosis in rural areas. J Diabetes. 2017; 9(7): 648–655. PubMed Abstract \| Publisher Full Text\n\nLiu Q, Li W, Xue M, et al.: Diabetes mellitus and the risk of multidrug resistant tuberculosis: a meta-analysis. Sci Rep. 2017; 7(1): 1090. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTabarsi P, Baghaei P, Marjani M, et al.: Changes in glycosylated haemoglobin and treatment outcomes in patients with tuberculosis in Iran: a cohort study. J Diabetes Metab Disord. 2014; 13(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin Y, Yuan Y, Zhao X, et al.: The change in blood glucose levels in tuberculosis patients before and during anti-tuberculosis treatment in China. Glob Health Action. 2017; 10(1): 1289737. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe International Union Against Tuberculosis and Lung Disease, World Health Organization: Collaborative Framework for care and control of Tuberculosis and Diabetes. Geneva. 2011; WHO/HTM/TB/2011.15. Reference Source\n\nLin Y, Li L, Mi FL, et al.: Screening patients with diabetes mellitus for tuberculosis in China. Trop Med Int Health. 2012; 17(10): 1302–1308. PubMed Abstract \| Publisher Full Text\n\nLi L, Lin Y, Mi F, et al.: Screening of patients with tuberculosis for diabetes mellitus in China. Trop Med Int Health. 2012; 17(10): 1294–1301. PubMed Abstract \| Publisher Full Text\n\nIndia Tuberculosis-Diabetes Study Group: Screening of patients with tuberculosis for diabetes mellitus in India. Trop Med Int Health. 2013; 18(5): 636–45. PubMed Abstract \| Publisher Full Text\n\nNsonga J: Screening tuberculosis patients for diabetes mellitus in a routine program setting in Kampala, Uganda: a cross-sectional study. 2019. http://www.doi.org/10.6084/m9.figshare.8248625.v1\n\nLawson L, Muc M, Oladimeji O, et al.: Tuberculosis and diabetes in Nigerian patients with and without HIV. Int J Infect Dis. 2017; 61: 121–125. PubMed Abstract \| Publisher Full Text\n\nDavis K, Richard S, Edrisa M, et al.: Overt diabetes mellitus among newly diagnosed Ugandan tuberculosis patients: a cross sectional study. BMC Infect Dis. 2013; 13: 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMugusi F, Swai AB, Alberti KG, et al.: Increased prevalence of diabetes mellitus in patients with pulmonary tuberculosis in Tanzania. Tubercle. 1990; 71(4): 271–276. PubMed Abstract \| Publisher Full Text\n\nLonnroth K, Roglic G, Harries AD: Improving tuberculosis prevention and care through addressing the global diabetes epidemic: from evidence to policy and practice. Lancet Diabetes Endocrinol. 2014; 2(9): 730–39. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization, International Diabetes Federation: Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia. Geneva, Switzerland: WHO, 2006. Reference Source\n\nWorld Health Organization: Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus: abbreviated report of a WHO consultation. Geneva, Switzerland: WHO; 2011; Accessed September 14 2011. Reference Source" }	[ { "id": "49993", "date": "19 Jun 2019", "name": "Razia Fatima", "expertise": [ "Reviewer Expertise Tuberculosis Research & Epidemiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written and addresses the Global issue of DM screening among TB Patients which is one the common co morbidity recognized with TB that may affect the TB outcome. The points which need to be explained are:\nThe selection criteria of the centres and how public vs private centres were selected moreover was the urban rural representation was also considered or it was random? Moreover it was stated in abstract that health centres without DM screened larger number of patients while in methods it was stated the DM services were mainly in tertiary care hospitals which might create assumptions of large screening in tertiary care hospitals contrary to what was stated in abstract. Table 1 also shows the DM services to be already present in 3 out of 7 centres so this need to be better explained. The average number of daily patients screened was also less in these 3 centres so it might mean that tertiary care where already DM services are present were not part of study?\n\nIn discussion it was mentioned using only FBG to diagnose DM can underestimate the prevalence of DM by as much as 50% when compared with the gold standard test, the oral glucose tolerance test, which is more accurate but unavailable at primary health care level, so how this should spell out in the policy implication? When the program should plan to do this DM screening of TB patients, more focus should be on the availability of gold standard for FBG using Glucometer, this should also be explained.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4940", "date": "10 Oct 2019", "name": "Yan Lin", "role": "Author Response", "response": "Reviewer 1The article is well written and addresses the Global issue of DM screening among TB Patients which is one the common co morbidity recognized with TB that may affect the TB outcome.The points which need to be explained are: The selection criteria of the centres and how public vs private centres were selected moreover was the urban rural representation was also considered or it was random? Response: Thank you for this valuable advice. As described in the site and setting, selection of the health centers was based on their location in the five administrative divisions of KCCA and representative of the health delivery system in Kampala as well as willingness of the staff to participate this study. It was however not random selected. We have rephrased the sentence as “These were high volume health facilities selected from different communities based on their ability to manage sufficient numbers of TB patients registered in the previous year and a daily TB patient visits ≥20/day, availability of diagnostic and treatment facilities and willingness of the staff to participate in this study.” Moreover it was stated in abstract that health centres without DM screened larger number of patients while in methods it was stated the DM services were mainly in tertiary care hospitals which might create assumptions of large screening in tertiary care hospitals contrary to what was stated in abstract. Table 1 also shows the DM services to be already present in 3 out of 7 centres so this need to be better explained. The average number of daily patients screened was also less in these 3 centres so it might mean that tertiary care where already DM services are present were not part of study? Response: Thank you for the question. The ten health centers recruited in this study are primary health care institutions. There was no any tertiary hospital involved in this study. Of the ten health centers, the health center without DM service screened large number of patients (both RBG and FBG) than those with DM service. In discussion it was mentioned using only FBG to diagnose DM can underestimate the prevalence of DM by as much as 50% when compared with the gold standard test, the oral glucose tolerance test, which is more accurate but unavailable at primary health care level, so how this should spell out in the policy implication? When the program should plan to do this DM screening of TB patients, more focus should be on the availability of gold standard for FBG using Glucometer, this should also be explained. Response: Excellent advice! Thank you. We have added a sentences in the Conclusions as “RBG and FBG are practical tools to be used for screening in routine program settings even though they may underestimate the actual situation\"." } ] }, { "id": "53770", "date": "17 Sep 2019", "name": "Daniel C. Oshi", "expertise": [ "Reviewer Expertise Chronic communicable diseases control", "social determinants of disease", "substance abuse", "lifestyles and behavioural change." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comment: The manuscript is on a very important public health issue, which is at the intersection between communicable and non-communicable diseases control. It is especially relevant because of the contextual issues of high TB and HIV burden in Uganda, the increasing burden of DM as a result of changing lifestyles and nutritional patterns, and limited resources in the country.\n\nAbstract: The abstract is well written and captures the essence of the paper.\n\nIntroduction: The Introduction section is well written, supported with key epidemiological facts and figures on TB and DM, globally, in the Sub-Saharan African region and in Uganda. The authors concisely made a strong case for the rationale of the study, from the epidemiological and clinical perspectives and aptly noted the absence of policy on routine screening for TB and DM in health care settings as well as the paucity of evidence to guide the formulation of such a policy. Having created gaps in knowledge and policy that their paper will contribute toward addressing, the authors stated the objectives of their study.\n\nMethods: This section is, on the whole, well written, although it would have been useful to see a justification for the choice of only Kampala Capital City, especially because the research aimed to produce evidence for policy formulation for programmatic screening for TB and DM in the whole country, not just in Kampala Capital City.\n\nResults: This section is also well written. However, there are two issues of concern here. First, since this study was not just an academic exercise but a public health project, the authors should state measures they took, if any, to ensure that patients newly diagnosed with DM and referred to DM clinics actually reached and accessed care at DM clinics. Initial default is a major challenge in such referrals because some of the referred patients fail to go for the needed care. Second, it would have been useful to show the socio-demographic and clinical characteristics of all the patients versus those diagnosed with DM. This would have made this paper (the analysis, results and discussions) much stronger.\n\nDiscussion: This is well written and acknowledged the limitation pointed out above about the non-collection and analysis of patient-level data.\n\nConclusion: One key conclusion of the study is “Screening TB patients for DM in a primary health care setting in a resource-limited country with high HIV-associated TB epidemic appeared feasible.” This conclusion is apparently not supported by the geographical coverage of the study. The study was conducted only in Kampala City Capital. The inclusion of just one rural health centre, which is still part of the KCC is insufficient to be used to drawn conclusions regarding rural setting. The geographic spread and the socio-economic characteristics of the locations of almost all the health centres in the study therefore suggest that the conclusion should be drawn to Kampala City Council rather than to the entire country.\n\nFinally, the authors are commended for the effort in implementing this important research project.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4941", "date": "10 Oct 2019", "name": "Yan Lin", "role": "Author Response", "response": "Reviewer 2:General Comment:The manuscript is on a very important public health issue, which is at the intersection between communicable and non-communicable diseases control. It is especially relevant because of the contextual issues of high TB and HIV burden in Uganda, the increasing burden of DM as a result of changing lifestyles and nutritional patterns, and limited resources in the country.Response: Thank you.Abstract: The abstract is well written and captures the essence of the paper.Response: Thank you.Introduction: The Introduction section is well written, supported with key epidemiological facts and figures on TB and DM, globally, in the Sub-Saharan African region and in Uganda. The authors concisely made a strong case for the rationale of the study, from the epidemiological and clinical perspectives and aptly noted the absence of policy on routine screening for TB and DM in health care settings as well as the paucity of evidence to guide the formulation of such a policy. Having created gaps in knowledge and policy that their paper will contribute toward addressing, the authors stated the objectives of their study.Response: Many thanks.Methods: This section is, on the whole, well written, although it would have been useful to see a justification for the choice of only Kampala Capital City, especially because the research aimed to produce evidence for policy formulation for programmatic screening for TB and DM in the whole country, not just in Kampala Capital City.Response: Point well taken! We have restructured some relevant parts in the method as highlighted in yellow colour to give justification on selection of the health centers. In addition, we have added a sentence in the limitation as “Finally, the selected HCs were only located in Kampala due to financial limitations and transportation barriers. This might have limited the representative value.”Results: This section is also well written. However, there are two issues of concern here. First, since this study was not just an academic exercise but a public health project, the authors should state measures they took, if any, to ensure that patients newly diagnosed with DM and referred to DM clinics actually reached and accessed care at DM clinics. Initial default is a major challenge in such referrals because some of the referred patients fail to go for the needed care.Response: This is excellent advice, thank you. We have rephrased the last sentences in the first paragraph of the Results as “Ninety-two TB patients were diagnosed with DM, and of them, 66 (71.7%) were newly diagnosed DM and were referred to DM services. All DM patients arrived at the DM clinics and received DM care except four patients who died shortly after TB registration. Second, it would have been useful to show the socio-demographic and clinical characteristics of all the patients versus those diagnosed with DM. This would have made this paper (the analysis, results and discussions) much stronger.Response: Thank you for the valuable advice. We agree. To well accept this point, we have rephrased the sentence in the limitation as “We did not collect individual patient data, including socio-demographic data, and perform multivariate analysis and were unable to identify and control potential confounding factors.” Discussion: This is well written and acknowledged the limitation pointed out above about the non-collection and analysis of patient-level data.Response: Thanks.Conclusion: One key conclusion of the study is “Screening TB patients for DM in a primary health care setting in a resource-limited country with high HIV-associated TB epidemic appeared feasible.” This conclusion is apparently not supported by the geographical coverage of the study. The study was conducted only in Kampala City Capital. The inclusion of just one rural health centre, which is still part of the KCC is insufficient to be used to drawn conclusions regarding rural setting. The geographic spread and the socio-economic characteristics of the locations of almost all the health centres in the study therefore suggest that the conclusion should be drawn to Kampala City Council rather than to the entire country. Finally, the authors are commended for the effort in implementing this important research project.Response: Thank you for pointing this out. We have revised the sentence in the conclusion as “Screening TB patients for DM in a primary health care setting in Kampala with high HIV-associated TB epidemic appeared feasible.”" } ] } ]	1	https://f1000research.com/articles/8-872
https://f1000research.com/articles/8-1733/v1	09 Oct 19	{ "type": "Research Article", "title": "Patterns of lymphoma in Misan city, Iraq: A retrospective observational study", "authors": [ "Haider Saadoon Qasim Alhilfi", "Omer Mansib Kassid", "Husam Jihad Imran Jihad", "Ahmed Salih Hussien Alshewered", "Haider Saadoon Qasim Alhilfi", "Omer Mansib Kassid", "Husam Jihad Imran Jihad" ], "abstract": "Background: Lymphomas represent a biologically and clinically heterogeneous group of neoplasms. They have historically and clinically been divided into two groups, Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). This study aimed to identify patterns in lymphomas in Misan city, Iraq, and evaluate the characteristics of this disease. Methods: A retrospective, observational, single-center study was conducted at Al-Shifaa Oncology Center, Al-Sadder Teaching Hospital, Misan city, Iraq, between 1 April 2016 and 30 April 2018. A total of 80 Misanian participants (48 (60%) men and 32 (40%) women) who had lymphoma were involved in this study. The sources of information were patient files, histopathology reports, and patients’ oncologist reports. Results: The mean age (±SD) of participants was 36 ±12.8 years. The male to female ratio was 1.5:1. NHL cases were three times more prevalent than HL. The most frequent stage at presentation was stage IV, in 34 (42.5%) participants. The classical subtypes of HL were present in 14 (70%) of HL cases. The diffuse large B-cell lymphoma (DLBCL) subtype was the most common NHL subtype, being recorded for 44 (73.3%) of participants. Conclusion: Lymphomas were more frequent in men. NHL was more common than HL; one HL case was diagnosed for every three NHL cases. The most common histopathology of HL was mixed cellularity, while DLBCL was the most common subtype of NHL. Most cases presented at an advanced stage, resulting in a late diagnosis.", "keywords": [ "Non Hodgkin lymphoma", "Hodgkin lymphoma", "Misan", "Lymphoid cells", "Iraq" ], "content": "Introduction\n\nThe term lymphoma refers to a heterogeneous group of neoplasms that originate from lymphoid cells. The majority (85%) of lymphomas originate from mature B-cells, and 15% derive from the T-cell lineage1. Historically and clinically, lymphomas have been divided into two groups, Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). HL is characterized by the presence of Reed–Sternberg cells, which arise in a single lymph node or chain of lymph nodes and typically spread in a stepwise fashion to anatomically contiguous nodes1. The crude incidence of HL among Europeans is 2.3 per 100,000 individuals2. Young adults aged 20 to 40 years are most commonly affected1. Histologically, classical HL accounts for 95% of all HL cases1. The subtypes of classical lymphomas include nodular sclerosis (the most common subtype, comprising 60–65% of cases), mixed cellularity (15–30%), lymphocyte-rich (5%), and lymphocyte-depleted (1%)1,3. HL patients present with peripheral lymphadenopathy, with nodes that are not tender, and with no overlying skin changes1,2. NHL are neoplastic transformations of mature B-, T-, or natural killer cells1. In children, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma, and lymphoblastic lymphoma are most common2–4. DLBCL is the most common histological subtype in adults4. The incidence increases with age, while a family history of lymphoma, autoimmune disease, human immunodeficiency virus infection, hepatitis C virus seropositivity, and a high body-mass as a young adult have all been identified as risk factors of DLBCL4,5.\n\nHere, we describe a study designed to show patterns of lymphoma among patients in Misan who presented at our center. Since this group of neoplasms are curable diseases, we need to obtain more information about them to get a significant and timely picture about the current lymphoma situation in Iraq, and in Misan in particular.\n\n\nMethods\n\nThis was a retrospective, observational, single-center study carried out in Misan city, Iraq to identify any patterns in lymphoma prevalence in this governmental administrative area.\n\nThe study was conducted at Al-Shifaa Oncology Center, Al-Sadder Teaching Hospital, Faculty of Medicine, Misan University, Misan city, Iraq, from 1 April 2016 to 30 April, 2018 (Figure 1). The recruitment dates began on the tenth day of each month and continued to the thirtieth day of that month. The period from the first day of the month until the ninth day was the time of patient follow-up. Data were collected on the last day of each month of the study.\n\nImagery ©2019 Maxar Technologies.\n\nA total of 80 participants (48 men and 32 women) from Misan who were diagnosed with lymphoma were included in this study. The age of participants ranged from 10 years to 80 years. Each patient had been previously diagnosed with lymphoma and attended our center for chemotherapy. We included all patients diagnosed with lymphoma without any selectivity (the patients must live in Misan city). Sources of information included patient files, histopathology reports, and patient oncologist reports (these contained all of a patient’s data written by his/her oncologist). Participant follow-up was performed on any day of nine days following a chemotherapy cycle.\n\nHistory and investigation results were documented and recorded for each participant in his/her file, including age, type of residency, occupation, sex, diagnosis, stage of lymphoma, subtype of lymphoma, and class of lymphoma.\n\nThe main sources of data were patient files and histopathology reports. Data written in the files included a patient’s baseline characteristics, lymphoma baseline characteristics, and all investigations done.\n\nWe obtained this number of participants because we included all individuals with lymphoma without any selection methods.\n\nWritten informed consent was obtained from all participants, or the parents of those aged less than 18 years, to participate in this study. The Medical Ethical Committee at Al-Shifaa Oncology Center, Al-Sadder Teaching Hospital, Faculty of Medicine, Misan University approved this study (code: 1000552).\n\nWe used Microsoft Excel (v. 2010) to calculate frequencies and percentages of variables, and also to calculate means and standard deviations (SDs). This study did not have any missing data.\n\n\nResults\n\nAll 80 participants included in this study were examined for eligibility and completion of follow-up. The mean age (±SD) of participants was 36±12.8 years. Half of the participants were in the 31–60 year age group, 24 (30%) of participants were aged >60 years, and just 16 (20%) belonged to the 10–30 year age group. Most participants (53; 67.5%) lived in rural areas, while 27 (32.5%) lived in urban regions. The majority of patients were employed (58; 72.5%). The male to female ratio was 1.5:1 (48 males and 32 females).\n\nNHL was three-times more prevalent than HL, with 60 (75%) and 20 (25%) cases, respectively. Participants most frequently presented with stage IV, in 34 (42.5%) of cases, followed by stage III in 24 (32.5%), stage II in 12 (15%), and stage I in 8 (10%) cases. The classical subtypes of HL were most common, occurring in 14 (70%) cases compared with 6 (30%) cases who had nodular subtypes. The DLBCL subtype was most common among NHL subtypes, being recorded in 44 (73.3%) of cases. Classical HL was subdivided into nodular sclerosis (2; 14.3%), lymphatic-rich subtype (4; 28.6%), and mixed cellularity (8; 57.1%) (Table 1).\n\n\nDiscussion\n\nIn our study, HL and NHL were recorded in 25% and 75% of cases, respectively, which is consistent with the results of a study conducted in Erbil city, in the north of Iraq6. The majority of HL cases presented as the classical subtype (70%), while 30% of cases encountered were of the nodular subtype (30%). The subtypes of classical HL included the nodular sclerosis (NS) subtype (14.3%) and the lymphocyte-rich subtype (28.6%), although the majority of cases comprised the mixed cellularity subtype (57.1%). The most frequent histological subtype of HL was mixed cellularity, which differs from earlier reports from the north of Iraq5,6, but is consistent with earlier reports from nearby countries and India7–17. This changing pattern differs from more recent reports from Saudi Arabia, Jordan, United Arab Emirates (UAE), and Kuwait, where higher relative rates of NS HL were reported, approaching levels seen in the USA and in European countries12–17.\n\nWith regard to NHL subtypes, we found that the majority comprised DLBCL, followed by mantle cell, follicular cell, peripheral T-cell, and Burkitt’s lymphoma. According to WHO classifications, DLBCL was also the most common diagnosis worldwide, at 52.2%, followed by T-cell rich lymphoma12. The relative proportion of DLBCL seen in the current study is almost twice the proportion seen in the USA and Europe, and is much higher than the proportion reported in India2,4,17,18, but is closer to figures reported from UAE14, Kuwait15, Jordan13, and Turkey19.\n\nWe found that the majority of lymphoma cases occurred in those aged 31 to 60 years, which was the same as findings in the USA and other countries15,18–20.\n\nThe majority of cases in our study presented at stage III or stage IV. Stage IV comprised 42.5% of cases and stage III 32.5%, while the remainder were stage II (15%) and stage I (10%). These results are in agreement with the results of a study by Robert et al.21.\n\nLimitations of this study include that it was a single-centre study, with a small number of participants, and the participants represent residents of the Misan government administrative area only.\n\n\nConclusions\n\nThis study showed that in Misan city, Iraq, lymphoma occurs most frequently among males. NHL is more common than HL, and the most common histopathology of HL is mixed cellularity, while DLBCL is the most common histopathology for NHL. Most cases presented with stage III or IV, which reflects delays in diagnosis, and decreases the chance of complete recovery, reflecting a more aggressive course and behavior of the disease.\n\n\nData availability\n\nZenodo: Lymphoma diseases patients patterns in Misan Government, http://doi.org/10.5281/zenodo.345880622.", "appendix": "References\n\nCheson BD, Fisher RI, Barrington SF, et al.: Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014; 32(27): 3059–3068. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSant M, Allemani C, Tereanu C, et al.: Incidence of hematologic malignancies in Europe by morphologic subtype: results of the HAEMACARE project. Blood. 2010; 116(19): 3724–3734. PubMed Abstract \| Publisher Full Text\n\nMorton LM, Slager SL, Cerhan JR, et al.: Etiologic heterogeneity among non-Hodgkin lymphoma subtypes: the InterLymph Non-Hodgkin Lymphoma Subtypes Project. J Natl Cancer Inst Monogr. 2014; 2014(48): 130–144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSant M, Minicozzi P, Mounier M, et al.: Survival for haematological malignancies in Europe between 1997 and 2008 by region and age: results of EUROCARE-5, a population-based study. Lancet Oncol. 2014; 15(9): 931–942. PubMed Abstract \| Publisher Full Text\n\nYahya HI, Al-Saleem T, Tikriti F, et al.: Hodgkin's disease in Iraq: a clinico-pathologic study of 85 cases. Clin Oncol. 1979; 5(1): 69–71. PubMed Abstract\n\nAlsabti EA: Histopathological subtypes of Hodgkin's disease in childhood in Iraq. Jpn J Exp Med. 1979; 49(5): 319–24. PubMed Abstract\n\nAlmasri N, Thunold S, Devi KR, et al.: Hodgkins lymphoma in North Jordan. Does it have a different pattern? Saudi Med J. 2004; 25(12): 1917–21. PubMed Abstract\n\nBamanikar S, Thunold S, Devi KR, et al.: The pattern of malignant lymphoma in Oman. J Trop Med Hyg. 1995; 98(5): 351–4. PubMed Abstract\n\nShome DK, George SM, Al-Hilli F, et al.: Spectrum of malignant lymphomas in Bahrain. Leitmotif of a regional pattern. Saudi Med J. 2004; 25(2): 164–7. PubMed Abstract\n\nAlsabti EA: Histopathological subtypes of Hodgkin's disease in childhood in Iraq. Jpn J Exp Med. 1979; 49(5): 319–24. PubMed Abstract\n\nAl-Bahrani ZR, Al-Mondhiry H, Bakir F, et al.: Clinical and pathologic subtypes of primary intestinal lymphoma. Experience with 132 patients over a 14-year period. Cancer. 1983; 52(9): 1666–72. PubMed Abstract \| Publisher Full Text\n\nAl-Diab AI, Siddiqui N, Sogiawalla FF, et al.: The changing trends of adult Hodgkin's disease in Saudi Arabia. Saudi Med J. 2003; 24(6): 617–22. PubMed Abstract\n\nHaddadin WJ: Malignant lymphoma in Jordan: a retrospective analysis of 347 cases according to the World Health Organization classification. Ann Saudi Med. 2005; 25(5): 398–403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCastella A, Joshi S, Raaaschou T, et al.: The Pattern of malignant lymphoma in the United Arab Emirates--a histopathologic and immunologic study in 208 native patients. Acta Oncol. 2002; 40(5): 660–4. PubMed Abstract \| Publisher Full Text\n\nal-Bahar S, Pandita R, al-Bahar E, et al.: Recent trends in the incidence of lymphomas in Kuwait. Neoplasma. 1996; 43(4): 253–7. PubMed Abstract\n\nAnderson JR, Armitage JO, Weisenburger DD: Epidemiology of the non-Hodgkin's lymphomas: distributions of the major subtypes differ by geographic locations. Non-Hodgkin's Lymphoma Classification Project. Ann Oncol. 1998; 9(7): 717–20. PubMed Abstract \| Publisher Full Text\n\nNaresh KN, Srinivas V, Soman CS: Distribution of various subtypes of non-Hodgkin's lymphoma in India: a study of 2773 lymphomas using R.E.A.L. and WHO Classifications. Ann Oncol. 2000; 11 Suppl 1: 63–7. PubMed Abstract \| Publisher Full Text\n\nNaresh KN, Agarwal B, Nathwani BN, et al.: Use of the World Health Organization (WHO) classification of non-Hodgkin's lymphoma in Mumbai, India: a review of 200 consecutive cases by a panel of five expert hematopathologists. Leuk Lymphoma. 2004; 45(8): 1569–77. PubMed Abstract \| Publisher Full Text\n\nIsikdogan A, Ayyildiz O, Buyukcelik A, et al.: Non-Hodgkin's lymphoma in southeast Turkey: clinicopathologic features of 490 cases. Ann Hematol. 2004; 83(5): 265–9. PubMed Abstract \| Publisher Full Text\n\nNaresh KN, Advani S, Adde M, et al.: Report of an International Network of Cancer Treatment and Research workshop on non-Hodgkin's lymphoma in developing countries. Blood Cells Mol Dis. 2004; 33(3): 330–7. PubMed Abstract \| Publisher Full Text\n\nNewton R, Ferlay J, Beral V, et al.: The epidemiology of non-Hodgkin's lymphoma: comparison of nodal and extra-nodal sites. Int J Cancer. 1997; 72(6): 923–30. PubMed Abstract \| Publisher Full Text\n\nAlhelfi HQ: Lymphoma diseases patients patterns in Misan Government. 2019. http://www.doi.org/10.5281/zenodo.3458806" }	[ { "id": "67869", "date": "26 Aug 2020", "name": "Maria Luisa Moleti", "expertise": [ "Reviewer Expertise Pediatric and Young adult hematological malignancies", "particularly Hodgkin and non Hodgkin lymphomas" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report the results of a retrospective study conducted at Al-Shifaa Oncology Center, Al-Sadder Teaching Hospital, Misan city, Iraq, on 80 lymphoma patients diagnosed and treated from April 2016 to April 2018. The aim of the study was to describe the histopathological and clinical patterns of lymphoma patients from the Misan area. The important issue of the spectrum of lymphomas in the different geographic areas is the object of several reports and this paper could add new data regarding the Middle East area. However, some comments need to be made.\nIntroduction\nThe Introduction should focus primarily on the topic of different histological lymphoma subtypes in different geographic areas.\n\nMethods\nInformation concerning the Histopathologic Classification system, Stadiation system and stadiation modality, and also the inclusion criteria are not described.\n\nThe methods description is repetitive and unclear and is not completely congruent with the objectives and results of the study. For example: 1)“The recruitment dates began on the tenth day of each month and continued to the thirtieth day of that month. The period from the first day of the month until the ninth day was the time of patient follow-up. Data were collected on the last day of each month of the study.” What do the authors mean? It is not clear. Why do the authors mention the follow-up? It is not relevant to this study. 2) “We included all patients diagnosed with lymphoma without any selectivity (the patients must live in Misan city)”. What do the authors mean? It is not clear. 3) ”Study size” Can be omitted. It does not make sense; moreover, the authors already mentioned that all diagnosed patients were included.\n\nResults\nThe authors could describe the results in a more detailed table, distinguishing the characteristics of the patients with HL and NHL, both for the entire population and also according to the three different age ranges.\n\nMedian age instead of mean age may give more information on the age distribution.\n\nHow many patients are in the pediatric age (<15)? This is relevant because of the different histological distribution of pediatric lymphomas.\n\nThe description of the clinical presentation is missing; it could add interest to the study and could be included in the table.\n\nDiscussion\nThe authors compare their results to the literature in detail. Other more recent papers on the same topic may be included in the discussion. A table comparing the results of papers reporting the Middle East lymphoma distribution might be interesting.\n\nSome comments on the low incidence of low-grade lymphomas might be interesting.\n\nReferences\nIn the literature, there are other papers on this topic, for example, Perry, Hematologica 2016, (NHL in the developing world)1; Gross, Br J. Hematol 2016, (pediatric population in LIC)2; Monabati, Ann. Hematol. 2016 and Mozaheb, Cancer Epidemiology 2011 (data from Iran)3,4; Saglam, Turkish Journal of Medical Sciences 2018 (data from Turkey).5\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "58788", "date": "26 Aug 2020", "name": "Wendy Cozen", "expertise": [ "Reviewer Expertise Epidemiology and pathology of hematologic neoplasms." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study describes characteristics of lymphoma patients at a teaching hospital in Misan, Iraq. The subject matter is interesting because the distribution of histologic subtype of lymphoma varies internationally. There is not much known about the distribution of lymphoma types in Iraq so this study will contribute knowledge.\nHowever, the authors miss an opportunity to present more useful data and there are a few problems with the way the paper is written. Comments are below:\nThe representativeness of the lymphoma patients diagnosed at Misan University hospital is unclear. What % of all patients in the region are diagnosed at this hospital? Does the hospital preferentially treat refractory or difficult cases? More details are needed to understand the generalizability of the patients to the Iraqi population.\n\nFigure 1 is not necessary.\n\nTable 1 - frequencies should be presented by sex.\n\nAdditional details would be helpful, for example, what is the distribution of germinal center vs. activated B-cell among DLBCL cases? How many of the Hodgkin lymphoma cases are EBV+?\n\nThe characteristics in Table 1 should be stratified by Hodgkin and non-Hodgkin lymphoma and not lumped together.\n\nA pie chart might be a better way to show the histological distributions, perhaps comparing to other countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1733
https://f1000research.com/articles/8-1729/v1	09 Oct 19	{ "type": "Case Report", "title": "Case Report: Facial injury due to a firearm projectile in a Brazilian adolescent", "authors": [ "Alessandro Leite Cavalcanti", "Lunna Farias", "Isla Camilla Carvalho Laureano", "Damião Edgleys Porto", "Josuel Raimundo Cavalcante", "Davi da Silva Barbirato", "Alidianne Fábia Cabral Cavalcanti", "Lunna Farias", "Isla Camilla Carvalho Laureano", "Damião Edgleys Porto", "Josuel Raimundo Cavalcante", "Davi da Silva Barbirato", "Alidianne Fábia Cabral Cavalcanti" ], "abstract": "Background: Deaths and injuries from firearms are significant public health problems. This article presents a case of face injury caused by a firearm projectile with atrial involvement. Case report: A 13-year-old male Black patient was admitted as an emergency victim of an accident caused by a firearm projectile. On physical examination, a hemorrhage was diagnosed in the right ear pinna region from the wound, and an increase of volume, of hardened consistency, in the right genic region, suggestive of local infection. On radiographic examination, a radiopaque, dense, foreign body was identified in the right zygomatic process region. The patient underwent surgery to remove the projectile. Conclusion: The care provided to the victim of a firearm injury depends on the systemic conditions, the available professional staff, the resources and the infrastructure of the environment. Prior to initiating therapy, it is important to stabilize the patient to ensure survival.", "keywords": [ "violence", "firearms", "facial injuries", "maxillofacial injuries." ], "content": "Introduction\n\nFirearms are one of the leading causes of injury and death in children and adolescents in the world1,2, constituting a public health problem that affects mainly large urban centers3. Costly treatment, long length of hospital stay, and considerable decreased productivity are significant factors related to this health issue4,5.\n\nThe lesions can range from small lesions to large tissue avulsions, depending on the amount of energy released at the moment of impact3. The characteristics and severity of the lesions are determined by several factors such as load power, number and shape of the projectiles, distance and path of the shot, as well as elasticity and vascularization of the affected tissue3. Reduced distances, high-velocity projectile wounds, and gunshot wounds can result in devastating aesthetic and functional consequences6,7.\n\nTraumas produce irreversible damages, which can incapacitate individuals, leaving them unable to work, and generates demands for care to the health sector in services of various levels of complexity, from prehospital to physical and mental rehabilitation8,9. Thus, they increase costs for the Brazilian Unified Health System and other sectors, the national social security system and for the families themselves8,9. This article presents a case of face injury caused by a firearm projectile with atrial involvement.\n\n\nCase report\n\nA 13-year-old male Black patient, was taken to emergency department in April 2014 to a local hospital of Picuí, Paraíba, Brazil. He was victim of an intentional firearm injury and suffered one gunshot wound. At admission, he was cooperative and fully conscious. His vital signs and neurological examination were normal when he arrived at the hospital.\n\nOn physical examination, a hemorrhage was diagnosed in the right ear pinna region from the wound (Figure 1), and an increase of volume, of hardened consistency, in the right genic region. On radiographic examination (Figure 2), a radiopaque, dense, foreign body image was identified in the right zygomatic process region. The patient was diagnosed with a face lesion caused by penetration of a firearm projectile through the skin requiring urgent surgical intervention.\n\nThe surgical procedure was performed under general anesthesia with orotracheal intubation and local infiltration of 2% lidocaine (Xylestesin 2.0% with vasoconstrictor, (Cristália Prod. Quím. Farm. Ltda., São Paulo, Brazil). Then, a linear incision was made immediately at the entry wound of the projectile, followed by divulsion of the fascial planes, exposure and subsequent removal of the projectile. Surgical cleaning of the area was performed using irrigation with 0.9% sodium chloride. The wounds were sutured (EthiconTM, Johnson & Johnson, São José dos Campos, Brazil) a penrose drain no.1 installed and skin sutured (ShalonTM, Goiânia, Brazil) with single interrupted stitches.\n\nAfter the surgery (immediately postoperative), the patient was treated with anti-tetanus serum 1000 UI/mL (Butantan Institute, São Paulo, Brazil).\n\nCephalothin (Keflin) 1 gram intravenously every 6 hours and after discharge 500 mg every 6 hours orally for 10 (ten) days) and an analgesic (Tylenol, 500 mg orally every 6 hours for three days) were prescribed. On the second postoperative day, the patient was discharged from the hospital and was advised about the medication prescription and the care related to hygiene and antiseptic treatment of the wounds, as well as when to return for follow-up (after seven days) for removal of the sutures and drain. The region around the wound’s appearance has significantly improved at follow up.\n\n\nDiscussion\n\nThis work is important because it provides guidance on how to proceed with emergency care in patients with firearms, as there are no specific treatment protocols. For the treatment of gunshot wounds, it is essential to know the ballistics of the wound in order to establish the prognosis and the treatment. Ballistics studies the movement of firearm projectiles and the trauma caused by the impact such projectiles on the human body. This knowledge results in better treatment and reduces the occurrence of complications related to the trajectory of the projectile9.\n\nThe first objective of the trauma team is to stabilize the patient maintaining free airways through intraoral aspiration, positioning and tongue trapping10. When necessary, orotracheal or nasotracheal intubation should be performed and, when indicated, a cricothyrotomy or tracheostomy performed11.\n\nIn the case reported, accurate medical care was given to the victim at admission to the hospital, which reduced potential risks to life and enabled immediate medical treatment. The availability of qualified and experienced professionals in this setting and, the access to imaging equipment, allowed the detection of the agent firearm projectile quickly. The patient was immediately referred for surgery after diagnosis.\n\nSurgical removal of the projectile should be considered when it is superficial or compromises the function of affected structures. However, when it is close to vital structures or is difficult access, the surgeon can choose to maintain it and follow-up (using radiographs, CT scans or digital arteriography’s)7. Computed tomography (CT) is the gold standard for determining the nature of complex head and neck injury12.\n\nIn this case, complete removal of the firearm projectile was performed because it was superficially housed in the auricular region. A drain was placed due to the wound being infected and the presence of edema.\n\nPostoperative antibiotic therapy was imperative to assist in the elimination of the existing infection and to prevent further infection. The use of anti-tetanus serum was necessary due to the contaminated nature of the wound due to the projectile and its secondary remnants, such as lead, gunpowder, tissue fragments and cartridge7. Prophylactic antibiotics with broad coverage specific for oral and skin flora should be administered at this time12.\n\nInjuries to the face should not be seen only as a medical problem, but also social and economic13,14. The costs incurred in providing care to the victims by the health teams and the losses to the patient (loss of wages, permanent or transitory disabilities) can limit the social reintegration of the victims and their return to the labor market13. In addition, psychological issues should also be considered.\n\nThe limitations of the study are that although we have been following the patient for a short time, and have employed this treatment protocol on all firearm patients who reach the maxillofacial complex, this is only a report of a clinical experience in which this protocol was used. Therefore, further study is required to evaluate the effectiveness of this protocol.\n\n\nConclusion\n\nThe care provided to the victim of a firearm depends on the systemic condition of the patient, available professional staff, resources and infrastructure of the healthcare center. Prior to initiating therapy, it is important to stabilize the patient to ensure survival. In cases involving head and neck injuries, it is important that a oral and maxillofacial surgeon be part of the team. In addition, surgical cleansing, antibiotic therapy and anti-tetanus serum protocols should be followed in order to prevent postoperative complications.\n\n\nConsent\n\nWritten informed consent for publication of the patients’ clinical details and clinical images was obtained from the parents of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nBrewer JW Jr, Cox CS, Fletcher SA, et al.: Analysis of pediatric gunshot wounds in Houston, Texas: A social perspective. J Pediatr Surg. 2019; 54(4): 783–91. PubMed Abstract \| Publisher Full Text\n\nLeite Cavalcanti A, Barros De Alencar CY, Sant'Anna Araujo Rodrigues I, et al.: Injuries to the head and face in Brazilian adolescents and teenagers victims of non-natural deaths. J Forensic Odontostomatol. 2012; 30(1): 13–21. PubMed Abstract \| Free Full Text\n\nLeite Segundo AV, Zimmermman RD, Nogueira EFC, et al.: Inclusion of ballistic study on management of facial wounds caused by gunshot. Rev Cir Traumatol Buco-Maxilo-Fac. 2013; 13(4): 63–70.\n\nMiguens SAQ Jr, Borges TS, Dietrich LAB, et al.: A retrospective study of oral and maxillofacial injuries in an emergency hospital in Southern Brazil. Braz Res Pediatr Dent Integr Clin. 2016; 16(1): 339–50. Publisher Full Text\n\nCosta RC, Nóbrega JBM, Dantas ELA, et al.: Profile of Hospitalizations and Deaths from Craniofacial Fractures in Brazilian Children and Adolescents: An Ecological Study. Braz Res Pediatr Dent Integr Clin. 2016; 16(1): 99–111. Publisher Full Text\n\nHollier L, Grantcharova EP, Kattash M: Facial gunshot wounds: a 4-year experience. J Oral Maxillofac Surg. 2001; 59(3): 277–82. PubMed Abstract \| Publisher Full Text\n\nBermejo PR, Coléte JZ, Momesso GAC, et al.: Surgical treatment of mandibular fracture after gunshot injury: case report. Arch Health Invest. 2016; 5(6): 330–5.\n\nSanches S, Duarte SJH, Pontes ERJC: Characterization of victims injured by firearms assisted by the mobile emergency care service in Campo Grande-MS. Saúde Soc. 2009; 18(1): 95–102. Publisher Full Text\n\nRibeiro AP, Souza ER, Sousa CAM: Injuries caused by firearms treated at Brazilian urgent and emergency healthcare services. Cien Saúde Colet. 2017; 22(9): 2851–60. PubMed Abstract \| Publisher Full Text\n\nNeupert EA, 3rd, Boyd SB: Retrospective analysis of low-velocity gunshot wounds to the mandible. Oral Surg Oral Med Oral Pathol. 1991; 72(4): 383–7. PubMed Abstract \| Publisher Full Text\n\nBehnia H, Motamedi MH: Reconstruction and rehabilitation of short-range, high-velocity gunshot injury to the lower face: a case report. J Craniomaxillofac Surg. 1997; 25(4): 220–7. PubMed Abstract \| Publisher Full Text\n\nKaufman Y, Cole P, Hollier LH Jr: Facial gunshot wounds: trends in management. Craniomaxillofac Trauma Reconstr. 2009; 2(2): 85–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZamboni RA, Wagner JCB, Volkweis MR, et al.: Epidemiological study of facial fractures at the Oral and Maxillofacial Surgery Service, Santa Casa de Misericordia Hospital Complex, Porto Alegre - RS - Brazil. Rev Col Bras Cir. 2017; 44(5): 491–7. PubMed Abstract \| Publisher Full Text\n\nSousa RIM, Bernardino IM, Castro RD, et al.: Maxillofacial Trauma Resulting from Physical Violence against Older Adults: A 4-year Study in a Brazilian Forensic Service. Braz Res Pediatr Dent Integr Clin. 2016; 16(1): 313–22. Publisher Full Text" }	[ { "id": "59072", "date": "06 Feb 2020", "name": "Diah Ayu Maharani", "expertise": [ "Reviewer Expertise Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for your efforts in preparing the manuscript. The paper is elegantly written on an important topic it should be indexed. However, there are some remarks the authors should consider to improve the manuscript:\nPlease make sure to present the case using the CARE (CAse REport) as guidelines.\n\n\"case report\" should be added in keywords.\n\nPatient information should be added such as possible primary concerns and symptoms of the patients or from the parents. As well as medical, family and psycho social history.\n\nWere patient asked to share their perspective on the treatment received? Please address in the text.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "85191", "date": "28 Jun 2021", "name": "Carisa Parrish", "expertise": [ "Reviewer Expertise child and parent distress and coping after pediatric physical injuries (psychologist)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provided a good background to introduce the specific case of a 13yo patient with a single gunshot wound. This introduction very concisely summarized multiple clinical variables to consider in medical treatment of gunshot wounds, as well as contextualizing the medical, functional, cosmetic, and economic factors that make effective medical intervention so crucial. Given the location of the injury and the quantity of discussion appropriately dedicated to proactive management of potential infections, perhaps the authors could consider adding 1-2 citations regarding infection control strategy used, likelihood of infections after GSWs. I provided 2 example articles; I defer to the authors regarding whether these versus other articles would be most appropriate based on similarity to the specific GSW under discussion:\nSimpson et al. (20031).\n\nvon See et al. (20112).\n\nThe authors stated in the discussion that there are no specific treatment protocols regarding how to proceed with emergency care in patients with firearm injuries. Could the authors provide any citation of studies regarding common elements in effective management of GSWs based on other case report(s) or small series of case studies? Likewise, could the authors reference general trauma management protocols that should be referenced in effective intervention, including aspects that may vary in the case of firearm injuries?\n\nAs a psychologist, I sincerely appreciate the authors referencing the burden of mental rehabilitation and risk for developing psychological issues. I would respectfully request that the authors use this forum to briefly highlight importance of proactive emotional \"injury\" risk, just as they do with the proactive infection risk. Clearly medical stabilization is of top priority to establish; however, once patients are stable, trauma teams would do well to consider the long-term implications of GSW to mental health. In the discussion, the authors stated, \"psychological issues should be considered.\" I would strongly urge the authors to briefly elaborate here. Trauma surgery and emergency medicine advocates have the opportunity to highlight the critical need for effective longer term emotional/behavioral supports to complement the establishment of effective medical interventions. The long term negative impact on quality of life warrants more discussion, particularly given the age of the patient under discussion. Adolescents are at increased risk for development of a number of negative mental health outcomes, and suffering a firearm injury is not a neutral variable in that risk accumulation. Here are a few articles for authors to consider citing to assist in the suggested elaboration of psychological issues to consider:\nGreenspan et al. (20023).\n\nJoseph et al. (20194).\n\nVella et al. (20205).\n\nThank you for the opportunity to review this manuscript! Best wishes to the authors for highlighting the case of an adolescent victim of a firearm injury.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1729
https://f1000research.com/articles/8-1726/v1	07 Oct 19	{ "type": "Case Report", "title": "Case Report: Facial mixed fungal infection in a poorly responding non-Hodgkin’s lymphoma patient with suspected immunodeficiency", "authors": [ "Ali Mostafa", "Mervat Gaber", "Hany Abdelrahman", "Noha ElAnwar", "Ahmed Galal", "Ali Mostafa", "Mervat Gaber", "Hany Abdelrahman", "Noha ElAnwar" ], "abstract": "A male child presented with left lacrimal gland and right submandibular masses that were diagnosed as high grade B-cell non-Hodgkin's Lymphoma. Immunodeficiency was suspected, mostly due to secondary to Epstein Barr virus infection. Chemotherapy was started and gradual regression was observed in the size of the lesions forming a central necrosis and facial wound. Cultures from this wound showed mixed gram negative growth of Pseudomonas aeruginosa, and Escherichia coli. Two days later, a follow up swab of the wound was taken and showed fungal growth of mixed Candida tropicalis and Cryptococcus laurentii which was treated with two antifungals (fluconazole and liposomal amphotericin B). After a second chemotherapy cycle, the facial necrotic lesion increased in size and the patient's general condition markedly deteriorated with multi-organ system failure secondary to septic shock by candidemia. Candida glabrata, which is non-Candida albicans fungus was the fungus that appeared in the patient's blood culture. Caspofungin was prescribed in addition to liposomal amphotericin B. two days later, the follow up blood culture revealed growth of methicillin resistant Staphylococcus aureus and the pulmonary condition of the patient then deteriorated gradually. Six days later, the patient developed multi-organ dysfunction syndrome secondary to sepsis. We conclude that we mixed fungal infection in not uncommon among immunocompromised patients, candidemia is fatal even if treated with the correct antifungals.", "keywords": [ "Non-Hodgkins lymphoma", "immunodeficiency", "candida", "disseminated fungal infection", "septic shock" ], "content": "Introduction\n\nImmunocompromised patients, those with hematological malignancies, solid organ or bone marrow transplantation, are highly vulnerable to invasive fungal infection, even with administration of systemic antifungal prophylaxis1. Skin damage, which is a natural barrier and considered as one components of innate immunity, may rarely lead to fungal invasion2. Blood stream infection by Candida is very serious and has a mortality rate of 30–60%3. Here, we present a case of fatal mixed fungal infection that started by cutaneous mixed fungal infection that progressed to fatal candidemia.\n\n\nCase presentation\n\nA male patient 22 months old, 2nd offspring of a consanguineous marriage (1st degree relatives), presented with left lacrimal gland mass that was assumed to be an abscess (a timeline of care is given in Table 1). Empirical antibiotics (amoxicillin/calvulinic: 25 mg/kg/12 hours; for 7 days) were started but with no improvement. A week later another mass appeared at the right submandibular region, firm in consistency, adherent to the underlying tissue, with no skin infiltration. A positron emission tomography (PET)/CT scan was performed, which showed multiple nodal and soft tissue lesions at the left inner canthus infiltrating left nasal bone and lamina propria, and extending to the left orbit, right submandibular lymph nodes, bilateral scattered lung nodules, abdominal, hilar and mediastinal lymph nodes. Biopsy from the lacrimal lesion was consistent with high grade B-cell non-Hodgkin’s Lymphoma.\n\nFull immunological assessment was done as the parents reported a history of recurrent chest infections since birth. Virology PCR for cytomegalovirus, Epstein Barr virus (EBV), herpes simplex virus and HIV was performed (only EBV was positive). The serum level of IgG (226 mg/dl; normal range 330–1260) and CD4 were low (4%; normal range at this age is 32– 51%); however other immunoglobulin levels were normal. Initial bone marrow aspirate and cerebrospinal fluid were normal with no infiltration with atypical cells.\n\nFirst cycle of chemotherapy (rituximab at a dose of 12.5 mg/kg/d, cyclophosphamide at a dose of 10 mg/kg/d, methylprednisolone at a dose of 12mg/m2/d and vincristine at a dose of 0.33mg/kg/d for one day only) was started; gradual regression was observed in the size of the lesions especially the lacrimal lesion over the next 12 days. Empirical antibiotics (cefepim at a dose of 50 mg/kg/8 hours for 3 days) was added when fever neutropenia occurs at day 4 after chemotherapy.\n\nCentral necrosis started to appear at the lacrimal and submandibular lesions. Necrosis increased gradually causing exposure keratitis. Initial culture from the facial lesions showed mixed gram negative growth of Pseudomonas aeruginosa and Escherichia coli. Anti-gram negative antibiotics (meropenem at a dose of 20mg/kg/day for 32 days and amikacin at a dose of 7.5 mg/kg/12 hours for 14 days) were started. Empirical antifungal liposomal amphotericin B at a dose of 3mg/kg/day was stated at the fifth day of fever neutropenia and continued for 32 days. CT paranasal sinus and chest were done as fungal screening at day 6 of chemotherapy and showed stationary pulmonary nodules and other facial and neck lesions. Serum galactomannan was negative. Three days later, a follow-up swab of the wound was taken and showed the first fungal growth of mixed Candida tropicalis and Cryptococcus laurentii. Fluconazole at a dose of 10mg/kg/day; was added to the current liposomal amphotericin B and continued for 7 days.\n\nAfter 14 days of the first cycle, patient neutrophils were recovered and all culture specimen became negative with no isolated microorganism. So the second cycle chemotherapy (doxorubicin at a dose 0.66 mg/kg/day for 1 day, cyclophosphamide at a dose of 25 mg/kg/day for 2 days, Rituximab at a dose of 12.5 mg/kg/d for 1 day, methylprednisolone at a dose 1.2 mg/kg/day for 5 days, and vincristine at a dose of 0.046 mg/kg/d for only one day) was given.\n\nAt day 10 of the second cycle of chemotherapy; the patient became neutropenic again and the facial necrotic lesion had increased in size and a combined surgical consultation (ophthalmology and otorhinolaryngology) decision was made to perform ethmoidectomy and enucleation to remove the source of infection whenever the general condition of the patient permitted.\n\nTwo days later, the patient’s general condition markedly deteriorated. The patient was highly febrile, tachycardiac, tachypneic, hypotensive, and shock developed. The patient was transferred immediately to the ICU; initial resuscitative measures were done including IV fluids, mechanical ventilation. Vancomycin at a dose of 15 mg/kg/6 hours was added to broaden the spectrum of antibiotics and continued for 8 days. After 24 hours of ICU admission, when the general condition permitted, follow-up CT maxillofacial and chest showed stationary course regarding the nodal and pulmonary lesions (Figure 1A and B, respectively). Initial blood and tracheal aspirate culture showed heavy growth of Candida glabrata; facial swab wound culture showed Aspergillus flavus (Figure 2A and B, respectively). At this point, caspofungin at a dose of 70mg/m2/day was added to liposomal amphotericin B instead of fluconazole for 8 days. After 2 days, follow-up blood culture gave growth to methicillin resistant Staphylococcus aureus. The patient deteriorated gradually regarding pulmonary condition, ventilator demands increased and follow-up chest imaging revealed newly developed bilateral apical and basal consolidative patches. After 8 days of ICU admission, the patient developed muti-organ dysfunction syndrome secondary to sepsis leading to patient’s cardiac arrest with failed cardio-pulmonary resuscitation.\n\n(A) maxillofacial CT scan showing ulcerating lesion in the left inner canthus with erosion of the left nasal bone. Left intra-orbital upper lateral cystic lesion measuring 14 * 12 * 7 mm in its maximal diameters. (B) Well defined right side pulmonary nodule measuring 19*22 mm in maximal diameter.\n\n(A) Early fungal after 24 hours, where growth is still not identified. (B) Late fungal growth after 90 hours of incubation 30°C in a humidified environment.\n\n\nDiscussion\n\nThis case describes mixed fungal infection in severely immunocompromised patient after chemotherapy. The infection started local and superficial till ended up with candida septic shock. Early diagnosis of fungal infection is critical to effective treatment that improve the outcome4. Diagnosis of fungal infection can be confirmed by either etiologic agent is isolated from clinical sterile specimens by culture or by histopathological diagnosis of a specimen or biopsy5. In our case we found it difficult to take any tissue biopsy due to rapid deterioration of patient’s general condition which is one of the limitations in our work. Mixed fungal infection is a rare condition, it was reported in 2014 in a case with severe hand injury after corrective surgery6. Other study reported only three mixed fungal infections from 178 immunocompromised pneumonia patients7.\n\nEarly and aggressive treatment of invasive fungal infection is very important to improve the outcome. A high index of suspicion is needed, especially with few antifungal agents available and increasing resistance8. Early administration of antifungal combination may salvage the patient’s condition. But in our case the patient died due to his immunocompromised condition.\n\n\nConclusion\n\nNeutropenic patients are greatly vulnerable to life threatening fungal infections. Aggressive, early administration of double antifungal drugs may be needed in mixed fungal infection.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the parents of the patient.", "appendix": "References\n\nNeofytos D, Lu K, Hatfield-Seung A, et al.: Epidemiology, outcomes, and risk factors of invasive fungal infections in adult patients with acute myelogenous leukemia after induction chemotherapy. Diagn Microbiol Infect Dis. 2013; 75(2): 144–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVitrat-Hincky V, Lebeau B, Bozonnet E, et al.: Severe filamentous fungal infections after widespread tissue damage due to traumatic injury: six cases and review of the literature. Scandinavian Journal of Infectious Diseases. 2009; 41(6–7): 491–500. PubMed Abstract \| Publisher Full Text\n\nHirano R, Sakamoto Y, Kudo K, et al.: Retrospective analysis of mortality and Candida isolates of 75 patients with candidemia: a single hospital experience. Infect Drug Resist. 2015; 8: 199–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKozel TR, Wickes B: Fungal diagnostics. Cold Spring Harb Perspect Med. 2014; 4(4): a019299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBadiee P, Hashemizadeh Z: Opportunistic invasive fungal infections: diagnosis & clinical management. Indian J Med Res. 2014; 139(2): 195–204. PubMed Abstract \| Free Full Text\n\nMilana Obradovic-Tomasev M, Popovic A, Vuckovic N, et al.: Mixed Fungal Infection (Aspergillus, Mucor, and Candida) of Severe Hand Injury. Case Rep Infect Dis. 2014; 2014: 954186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVon Eiff M, Roos N, Fegeler W, et al.: Pulmonary fungal infections in immunocompromised patients: incidence and risk factors. Mycoses. 1994; 37(9–10): 329–35. PubMed Abstract \| Publisher Full Text\n\nBadiee P, Alborzi A, Moeini M, et al.: Antifungal susceptibility of the Aspergillus species by Etest and CLSI reference methods. Arch Iran Med. 2012; 15(7): 429–32. PubMed Abstract" }	[ { "id": "54787", "date": "28 Oct 2019", "name": "Hironobu Nakayama", "expertise": [ "Reviewer Expertise Mycology and molecular biology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors isolated fungi and bacteria from the immunocompromised patient, and reported failure of correct antifungal therapy against this patient due to mixed fungal infection. Since there are few case reports of mixed fungal infections, such a report could provide information concerning to fungal therapy. I however believe the report would be more informative when the authors reveal the sensitivity of isolated fungi against used antifungals (fluconazole, amphotericinB, and Caspofungin) and compare them with their type culture strains.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "79406", "date": "15 Feb 2021", "name": "Joanna Zawitkowska", "expertise": [ "Reviewer Expertise Hematologic malignacies", "especially acute lymphoblastic leukemia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a facial mixed fungal infection in a poorly responding non-Hodgkin’s lymphoma patient with suspected immunodeficiency. The fungal infection are still therapeutic problem in children with hematologic malignancies. The manuscript is potentially interesting, but requires a major revision.\nComment 1\nThe manuscript should be edited for proper English language, grammar, punctuation, spelling, and overall style.\nComment 2\nTitle: I suggest shortening the title.\nComment 3\nAbstract: “Candida glabrata, which is non-Candida albicans fungus was the fungus that appeared in the patient's blood culture.” I suggest to remove “…..which is non-Candida albicans fungus”. May be better: “The results of the patient's blood culture was Candida glabrata” or the like.\n\n“two days later, the follow up blood culture revealed growth of methicillin resistant” This sentence should be started from the capital letter.\nComment 4\nIntroduction: the introduction contains too little information about this subject.\nComment 5\nCase reports: Has the patient received antifungal prophylaxis at the time of diagnosis and during the first course of chemotherapy.\n\nWas the patient undergoing a CT scan of chest at the time of diagnosis?\n\nIt is unclear why the patient received Fluconazole only for 7 days?\n\nWas GCS-F used during neutropenia?\nComment 6\n“Initial bone marrow aspirate and cerebrospinal fluid were normal with no infiltration with atypical cells.” I suggest to remove “were normal”, but “…. were no infiltrated by …..”\n\nCT – please, explain abbreviation\nComment 7\nDiscussion: The article does not use the latest knowledge about this subject. In the literature used, all of the items are older than 5 years. This section is too short.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1726
https://f1000research.com/articles/8-401/v1	08 Apr 19	{ "type": "Data Note", "title": "Draft genome assembly and transcriptome sequencing of the golden algae Hydrurus foetidus (Chrysophyceae)", "authors": [ "Jon Bråte", "Janina Fuss", "Kjetill S. Jakobsen", "Dag Klaveness", "Janina Fuss", "Kjetill S. Jakobsen", "Dag Klaveness" ], "abstract": "Hydrurus foetidus is a freshwater alga belonging to the phylum Heterokonta. It thrives in cold rivers in polar and high alpine regions. It has several morphological traits reminiscent of single-celled eukaryotes, but can also form macroscopic thalli. Despite its ability to produce polyunsaturated fatty acids, its life under cold conditions and its variable morphology, very little is known about its genome and transcriptome. Here, we present an extensive set of next-generation sequencing data, including genomic short reads from Illumina sequencing and long reads from Nanopore sequencing, as well as full length cDNAs from PacBio IsoSeq sequencing and a small RNA dataset (smaller than 200 bp) sequenced with Illumina. We combined this data with, to our knowledge, the first draft genome assembly of a chrysophyte algae. The assembly consists of 5069 contigs to a total assembly size of 171 Mb and a 77% BUSCO completeness. The new data generated here may contribute to a better understanding of the evolution and ecological roles of chrysophyte algae, as well as to resolve the branching patterns within the Heterokonta.", "keywords": [ "Hydrurus foetidus", "Chrysophyceae", "golden algae", "genome", "transcriptome", "Nanopore", "PacBio" ], "content": "Introduction\n\nHere, we present extensive genome sequencing data, including a multiapproach assembly, as well as transcriptome data of mRNAs and small RNAs of the golden algae Hydrurus foetidus (Villars) Trevisan.\n\nThere has been considerable interest in the golden algae for many reasons: they are ecologically diverse, important as primary producers (phototrophs) in oligotrophic to dystrophic lakes (Kristiansen, 2005; Nicholls & Wujek, 2015), some are also mixotrophs, phagotrophs or osmotrophs (Kristiansen & Preisig, 2001; Pringsheim, 1963). The chrysophytes span a large range of feeding and nutrient uptake modes (Kristiansen, 2005) and therefore play a significant role in aquatic food webs. Chrysophytes also make up a significant fraction of sequence reads and novel operational taxonomic units in clone libraries from freshwater environmental samples (del Campo & Massana, 2011).\n\nHowever, chrysophytes have also attracted considerable interest from an evolutionary point of view. They belong to the division (phylum) Heterokonta (Cavalier-Smith, 1986), an immensely diverse group of eukaryotes with many basal branches in the phylogeny still not resolved, despite numerous molecular phylogenetic studies, including multigene phylogenomics (e.g. Burki, 2014; Grossmann et al., 2016; Riisberg et al., 2009; Scoble & Cavalier-Smith, 2014). One reason for this is the presence of cryptic species and many groups with extremely similar morphology (Grossmann et al., 2016). Another reason is the complex evolutionary history of the Heterokonta, including an elaborate plastid evolution (e.g. Kim et al., 2019) and heterotrophic lineages which have lost the plastids altogether (Graupner et al., 2018; Pringsheim, 1963). The lack of genomic or transcriptomic data from many taxa, and even whole orders, which limits the power of multigene phylogenies (Beisser et al., 2017), is yet another motivation for genomic and transcriptomic studies. However, recently, there has been a significant addition of transcriptomic data for chrysophyte taxa (e.g. Beisser et al., 2017; Graupner et al., 2018; Keeling et al., 2014; Kraus et al., 2019; Lie et al., 2017).\n\nHydrurus foetidus is not a typical representative of the golden algae. It is macroscopic and benthic (e.g. Klaveness et al., 2011; Rostafinski, 1882: Tab II, Szklarczyk, 1953), whereas most chrysophytes are microscopic single cells or colonial plankton (Sandgren, 1988; Kristiansen, 2005). Furthermore, Hydrurus is native to polar, peri-glacial and alpine rivers in Norway and similar regions around the world (e.g. Klaveness, 2019; Rott et al., 2006; Rott & Schneider, 2014) and can only live in cold waters (2–10 °C) (Bursa, 1934; Kann, 1978). Members of the Hydrurus clade may cause colored snow and ice, and may be found on permanent ice sheets (Klaveness et al., 2011; Lutz et al., 2018; Remias et al., 2013).\n\nHydrurus has a number of peculiar morphological characteristics relevant for understanding chrysophyte and heterokont evolution. Although it is multicellular, the cells in the thalli are not physically connected, and under some growth conditions the cells may slide away from each other in their wall-less polysaccharide tubes, or be released as single-celled swarmers (Klaveness et al., 2011). Other characteristic features, which may be considered primitive for a thallose alga, are contractive vacuoles, often more than one in each cell (Fott, 1959; Klaveness, 2019).\n\nWe have assembled a draft genome of Hydrurus foetidus using a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The assembly consists of 5069 contigs yielding a total size of 171 Mb and a 77% BUSCO completeness. In addition to the deep genomic sequencing, we have also sequenced full-length poly(A) transcripts using PacBio IsoSeq, as well as sequencing the expressed small RNAs. This extensive dataset will be important, not only for studies of heterokont and crysophyte evolution but also for elucidating the genetic mechanisms behind cold water adaptation, like the production of polyunsaturated fatty acids (Klaveness, 2017) and the regulation of a complex multicellular lifestyle.\n\n\nMaterials and methods\n\nThe specimen of Hydrurus foetidus (Villars) Trevisan (strain G070301) used in this study was sampled from the river at the Finse Alpine Research Center (60°36' N. 07°30' E) in March 2007 and is currently kept in culture at University of Oslo. H. foetidus was isolated in an adapted Guillard & Lorensen’s WC (Wright’s Chu) medium (Guillard & Lorenzen, 1972) as described by Klaveness & Lindstrøm (2011). To prepare for DNA isolation, the growth of large thalli was promoted by repeated transfer of individual thalli into fresh culture media. Large thalli (0.5–1.0 g wet frozen weight) were collected by removal from the culture medium and immediate transfer to -80°C and storage until further processing. The culture will be deposited in a special culture collection, at the Fraunhofer Culture Collection of Cryophilic Algae (CCCryo).\n\nSix individual thalli were used for the DNA isolation. DNA isolation was performed using the DNeasy Plant Mini Kit from Qiagen (Qiagen Inc., Valencia, CA, US). To ensure efficient lysis and homogenization of the external polysaccharide sheath, a few titanium beads were added to the frozen samples and the tubes were shaken using TissueLyser II machine (Qiagen Inc., Valencia, CA, US) for four minutes. After the addition of the lysis buffer and the RNase, tubes were placed in a thermomixer set at 65°C and 800 rpm with 20 second intervals for 30 mins. After adding Buffer AP2 to the lysate, the incubation was done on ice for 15 minutes to allow for better precipitation of the polysaccharides. Further, the extraction kit protocol was followed as is, until the second elution step. Here we reused the flow through from the previous step elution to avoid excessive dilution of the samples. Afterwards, the samples were de-salted and concentrated by ethanol precipitation and resuspension in 100 μl of Milli Q water. Finally, the samples were concentrated even further by pooling all the samples and freeze drying with a Leybold-Heraeus Lyovac GT2 (Leybold-Heraeus, Köln, Germany).\n\nThe isolated and freeze-dried genomic DNA was sent to the Norwegian Sequencing Center (NSC) at the University of Oslo for library preparation and sequencing. The library was made with 600-700 bp fragment size and sequenced on two lanes of Illumina HiSeq 2500 with 250 bp paired-end reads (Table 1).\n\nGenomic DNA was isolated from two thalli as described above, except that tissue lysis was done using MagNA Lyser Green Beads (Roche, Penzberg, Germany) and shaken for 15 sec at 4 m/sec and the incubation at 65°C was done for 10 min. In addition, the supernatant (after adding buffer AP2) was run through QiaShredder columns to further homogenize the lysate. The DNA was eluted (twice, but re-using the elution buffer) in 50 µl AE elution buffer. To further clean and concentrate the samples, the samples were pooled and cleaned using the Zymo DNA Clean & Concentrate kit (Zymo Research, CA, US). The sample was double-eluted (as before) in 50 µl kit provided elution buffer.\n\nDNA sequencing was done using the MinION (MIN-101B) sequencer, the R9.5 Flow Cell and following the SQK-LSK108 protocol (version GDE_9002_v108_revT_18Oct2016) (Oxford Nanopore, Oxford, UK). Approximately 1 µg of starting DNA was used and inspection of the DNA on a 0.7% agarose gel run at 30 volts from 18 hours showed that the majority of the DNA was between 20-30 kbp, but with a long tail of shorter fragments. The sequencing was run using the MinKNOW software (Oxford Nanopore, Oxford, UK; downloaded October 2017) on an iMac and stopped after 36 hours. Base-calling of the raw Nanopore sequence data was done using Albacore v.2.1.10 (Linux, Python 3.5 version) with default settings. The process was run on the Abel computing cluster at the University of Oslo (Table 1).\n\nTotal RNA was isolated from one frozen thallus using Qiagen RNeasy Plant kit, including a QiaShredder column and lysis using MagNA lyser beads as described above, otherwise following the kit protocol. Isolated RNA was sent to NSC for library preparation and PacBio sequencing. Three size fractions (1-2 kbp, 2-3 kbp and 3-5 kbp) were prepared using the IsoSeq library preparation protocol and sequenced on RSII SMRT cells (Pacific Biosciences, CA, US) (Table 1 and Table 2).\n\n1-2kb_high – accession ERR2869477; 1-2kb_low - accession ERR2882521; 2-3kb_high - accession ERR2869478; 2-3kb_low - accession ERR2869481; 3-6kb_high - accession ERR2869483; 3-6kb_low - accession ERR2869484.\n\nSmall RNAs (below 200 bp) were isolated from a frozen thallus using the Sigma mirPremier kit (Sigma-Aldrich, MO, US) following the manufacturer’s instructions, but including lysis with MagNA beads as described above. The sample was sent to the NSC for library preparation and sequencing. Sequencing library (up to approx. 40 nt fragment size) was prepared and sequenced with Illumina NextSeq 500 as single-end 75 bp reads (Table 1).\n\nThe basecalled Nanopore reads were processed with Porechop v0.2.2 using default parameters to remove sequencing adapters. Next, the reads were filtered with Nanofilt v2.0.0 (De Coster et al., 2018) to remove reads shorter than 500 bp and average quality below 9. The filtered reads were further error-corrected with LoRDEC v0.7 (Salmela & Rivals, 2014) using the Illumina reads. First the Illumina reads were quality assessed by removing sequencing adapters and bases with an average quality below 20 (average score across 4 bases), in addition to leading and trailing bases with a quality below 20. This was done using Trimmomatic v0.36 (Bolger et al., 2014). Then lordec-correct (options -k 21 -s 3) was run with the trimmed Illumina reads to correct the filtered Nanopore reads. Then the corrected reads were run through Canu v1.6 (Koren et al., 2017) for further correction (canu -correct with genome Size set to 300 m) and trimming (canu -trim) before assembly (canu -assemble). The assembly was done with two different corrected error rates, 0.144 and 0.146. The two assemblies were almost identical, but the results from using the corrected error rate of 0.144 were used further because the total size was slightly larger and also had the largest contig. The Canu assembly was then polished using the trimmed Illumina reads (described above) by running three rounds of Pilon v1.22 (Walker et al., 2014). The final genome assembly consisted of 5069 contigs with a total length of 171 183 409 nt. The N50 was 43,856 nt and the longest contig of 5,118,963 nt (Table 3).\n\naThe genome size estimation was based on k-mer frequencies on the Illumina data.\n\nbBUSCO was run against the Eukaryota dataset.\n\n\nData availability\n\nAll Hydrurus foetidus datasets produced in this study are available, study accession number PRJEB29405: https://identifiers.org/ena.embl/PRJEB29405.", "appendix": "Author contributions\n\n\n\nJB performed DNA and RNA isolation, Nanopore sequencing, genome assembly, prepared and submitted data and wrote the manuscript. JF monitored and performed culturing, isolated DNA and RNA and prepared for Illumina and PacBio sequencing, prepared and submitted data and commented on the manuscript. KSJ planned and monitored the entire project and commented on the manuscript. DK maintained and developed the cultures and isolation protocol and wrote the manuscript.\n\n\nGrant information\n\nJB was funded by the Research Council of Norway (grant numbers: 213703 and 240284).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank the Norwegian Sequencing Centre for all sequence library preparations and sequencing services.\n\n\nReferences\n\nBeisser D, Graupner N, Bock C, et al.: Comprehensive transcriptome analysis provides new insights into nutritional strategies and phylogenetic relationships of chrysophytes. PeerJ. 2017; 5: e2832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. 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Hydrobiol. 2006; 562(1): 195–216. Publisher Full Text\n\nRott E, Schneider SC: A comparison of ecological optima of soft-bodied benthic algae in Norwegian and Austrian rivers and consequences for river monitoring in Europe. Sci Total Environ. 2014; 475: 180–186. PubMed Abstract \| Publisher Full Text\n\nSandgren CD: The Ecology of Chrysophyte Flagellates: their Growth and Perennation Strategies as Freshwater Phytoplankton. In: Sandgern, C.D. (ed.) Growth and reproductive strategies of freshwater phytoplankton (Ch. 2). Cambridge University Press, New York. 1988; 442. Reference Source\n\nSalmela L, Rivals E: LoRDEC: accurate and efficient long read error correction. Bioinformatics. 2014; 30(24): 3506–3514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScoble JM, Cavalier-Smith T: Scale evolution in Paraphysomonadida (Chrysophyceae): Sequence phylogeny and revised taxonomy of Paraphysomonas, new genus Clathromonas, and 25 new species. Eur J Protistol. 2014; 50(5): 551–592. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "47028", "date": "09 May 2019", "name": "Daniela Beisser", "expertise": [ "Reviewer Expertise High-throughput sequencing analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease find below the comments to the article “Draft genome assembly and transcriptome sequencing of the golden algae Hydrurus foetidus (Chrysophyceae)” by Jon Bråte, Janina Fuss, Kjetill S. Jakobsen and Dag Klaveness. The authors present in the article draft genome data for the chrysophyte Hydrurus foetidus. H. foetidus was chosen because it is a special chrysophyte which is, in contrast to most other chrysophytes, macroscopic, but cells can slide apart under some growth conditions, benthic and thrives under cold conditions in polar and high alpine regions. The genome was sequenced using Illumina and Nanopore sequencing, in addition mRNA reads sequenced with PacBio and small RNA reads sequenced with Illumina are provided. The genome was assembled in a hybrid approach, resulting in a length of 171 Mb and 5,069 contigs with a BUSCO completeness of 77%. DNA extraction, sequencing and assembly generation are described in detail, but some information is missing, which is listed below. Overall, the work is technically sound and should be indexed after minor corrections.\nMinor comments:\n\nAbstract: “We combined this data with, to our knowledge, the first draft genome assembly of a chrysophyte algae”. The data is not combined with the draft genome, maybe it should be “We combined this data to create, to our knowledge, the first draft genome assembly of a chrysophyte algae”. Further, there are two draft genomes listed at JGI for Ochromonas and Paraphysomonas species (https://genome.jgi.doe.gov/portal/OchCCMStandDraft_FD/OchCCMStandDraft_FD.info.html, https://genome.jgi.doe.gov/portal/ParimpEvaluation_FD/ParimpEvaluation_FD.info.html). They cannot be accessed without registration, but perhaps they should be cited. Abstract: “...belonging to the phylum Heterokonta.” Recent classification of Eukaryotes (Adl et al 2005, 2012, 2018)1,2,3 place the Chrysophyceae into the Stramenopiles which themselves belong to the SAR supergroup. The current classification should be adapted. Abstract: An assembly of 171 Mb was obtained, is this size expected? Is there other data available which suggests this genome size? On the other hand, the k-mer based genome size estimation suggests a much larger genome. Introduction: “multiapproach assembly”, should better be described as hybrid assembly. Culturing of H. foetidus: The introduction mentions plastid reduction and heterotrophy in chrysophyte species. H. foetidus in called alga, so I assume it is phototrophic, but it would be good to state that somewhere explicitly. Otherwise one would wonder if the cultures were axenic. Concerning the sequencing: Was there quality control performed by the sequencing center (RIN values etc.) before sequencing? Which library preparation protocols were used for the different sequencing data? Was the RNA rRNA-depleted before sequencing? What was the average sequence quality before and after filtering? An overview table of all the sequencing data including quality and number of reads/yield should be added. Nanopore sequencing of genomic DNA: “Albacore v2.1.10 (Linux, Python 3.5 version)” Please provide a reference for Albacore. The information that it was run under Linux with Python 3.5 is probably not necessary. Next line: Table 1 is not the correct reference for the sentence. Table 2: It is not described in the text or caption what “_high” and “_low” means. Table 3: The genome size estimation based on k-mers and BUSCO are not described in the methods section. Maybe the GC content could also be added to the table.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "4862", "date": "09 Oct 2019", "name": "Jon Bråte", "role": "Author Response", "response": "We thank the reviewer for the critical reading. Here are our responses to the minor comments:1. We have rephrased the sentence and it no longer states that we present the first chrysophyte draft genome assembly.2. We have rephrased the first sentence of the abstract. And the first time Heterokonta now is mentioned in the introduction we have also added that Adl et al. uses Stramenopila and added the reference. However, we prefer the name Heterokonta as it has seniority to Stramenopila, and is still used by for instance Cavalier-Smith (e.g. Ruggiero et al. 2015; A Higher Level Classification of All Living Organisms. Plos One: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418965/).3. The k-mer estimation was done on the Illumina data alone. While the assembly was done on the Nanopore reads alone (after error correcting with the Illumina reads). Also, k-mer basted estimations of genome size can be inaccurate, especially since we know nothing about the ploidy level or the amount of genetic variation between chromosome copies. Therefore we don't think that this discrepancy is particularly large or surprising.4. Corrected.5. We have added a sentence explicitly mentioning that H. foetidus is photosynthetic to the methods section under the paragraph describing the culture procedure. 6. The PacBio transcriptome sequencing was performed by selecting for polydenylated transcripts (information now added to the manuscript), and for the smallRNA sequencing the totalRNA was size fractionated to remove ribosomal RNA. Information about concentration, integrity and library preparation protocols have been added for the Illumina DNA sequencing under the \"Illumina sequencing of genomic DNA\" section. Information about concentration and integrity of the DNA for Nanopore sequencing has been added to the section \"Nanopore sequencing of genomic DNA\". In our opinion, there is not much added value in showing the average sequence quality before trimming/filtering of the different datasets because the various quality thresholds used for the DNA sequences used in the assembly is described. For the small RNA and the transcriptome data we have not performed any filtering or assembly and the raw data is provided.7. There are different versions of Albacore for different versions of Python. So, we believe it is more specific to keep this information. Albacore is not published (and it is not developed any more) but we added a reference to Oxford Nanopore who developed the software.8. Corrected.9. The information has been added.10. The information has been added." } ] }, { "id": "49312", "date": "12 Aug 2019", "name": "Blake T. Hovde", "expertise": [ "Reviewer Expertise Algal genomics", "genome editing", "metabolic engineering", "genome sequencing technology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of Draft Genomes: Draft genome assembly and transcriptome sequencing of the golden algae Hydrurus foetidus (Chrysophyceae):\n\nThis data note represents a significant contribution to the algal genomics research community in couple different ways. This project represents the first publicly available draft genome of a chrysophyte (golden algae) to date, making it a critical contribution to the study of this particular clade of algae and will be a much-utilized resource for other chrysophyte genome analyses as they emerge. Because this is the first representative of Chrysophyceae to be sequenced is will be a de facto resource, which leads to the next important contribution of the note – which is the importance of the modern sequencing technologies used in development of this genome and transcriptome assembly. The tools used are likely to be the gold-standard tools of de novo genome and transcriptome sequencing and assembly. The use of Pacific Biosciences “Isoseq” long read, full length transcriptome sequencing approach will allow this team to generate high quality gene annotations, a great advantage for a new reference such as this genome. Additionally, the use of long read technologies (either Nanopore or PacBio) will be used to generate higher quality genome assemblies with fewer, longer contigs and scaffolds.\n\nWhile broad strokes are painted about the contribution of this draft genome to phylogenetic (or phylogenomic) analysis and the importance of the algae as primary producers it is unclear what additional analyses this team will be doing to utilize this genome assembly. Next steps may be to perform biosynthetic pathway analyses and phylogenetic analysis of the Chysophte class or higher level algal/protist analysis. It would be beneficial if the authors could point to some specific follow-ups.\n\nI was unable to locate the genome assembly using accession number ERZ780628as listed in Table 1. Though, I was able to find the assembly under NCBI accession number: GCA_900617105.1\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "4863", "date": "30 Aug 2019", "name": "Jon Bråte", "role": "Author Response", "response": "We thank the reviewer for the critical reading. We have updated the accession number to the genome assembly (now GCA_900617105.1) and this should work both on EBI and NCBI." } ] } ]	1	https://f1000research.com/articles/8-401
https://f1000research.com/articles/8-1722/v1	04 Oct 19	{ "type": "Case Report", "title": "Case Report: Renal impairment and bilateral cataracts in a patient with Maffucci syndrome", "authors": [ "Abid Nawaz", "Aqsa Iqbal", "Nauman Shaukat", "Muhammad Aslam Khan", "FNU Farukhuddin", "Anil A. Kumar", "Vinesh Kumar", "Aqsa Iqbal", "Nauman Shaukat", "Muhammad Aslam Khan", "FNU Farukhuddin", "Anil A. Kumar", "Vinesh Kumar" ], "abstract": "Maffucci syndrome is a sporadic, non-hereditary condition that is characterized by the presence of multiple skeletal and vascular lesions. We present the case of a 37-year-old man who complained of multiple swellings on the hands, face and wrists affecting the quality of his life. Bilateral cataract and renal impairment were two other features identified in our patient. Renal impairment was likely due to renal vascular malformation, as all other causes of renal compromise were ruled out. Radiographic images showed multiple radiolucent areas in both hands. Multiple enchondromas with pathologic confirmation of skin lesions as cavernous haemangiomas confirmed the diagnosis of Maffucci syndrome in our patient. The patient was referred to the orthopaedic department for osteotomy. Currently, the patient is on dialysis and no other complications have been observed in follow-up visits. This is the first report of Maffucci syndrome which discusses renal involvement. Regular screening of renal functions in patients with Maffucci syndrome could prevent renal complications.", "keywords": [ "Maffucci Syndrome", "enchondromas", "hemangiomas", "renal impairment", "bilateral cataract" ], "content": "Introduction\n\nMaffucci syndrome, also called Morbis Ollier, is a rare, nonhereditary, congenital condition characterized by a combination of multiple enchondromas with haemangiomas and less often lymphangiomas1. Angelo Maffucci first described it in 1881 when he reported all the main characteristics of the syndrome in the autopsy of an elderly woman who died following an arm amputation. In 1940, Carleton et al. proposed the eponym Maffucci syndrome2. There is no gender or racial predilection, but the syndrome usually becomes evident by puberty in 78% of patients3. Multiple cases of this syndrome have been reported. The involvement of many organ systems, including the liver, spleen, adrenal cortex and nervous system, have been reported in Maffucci syndrome4–7. Here, we describe a case of Maffucci syndrome, which along with the typical findings of enchondromas and haemangiomas, also presented with renal impairment and bilateral cataracts.\n\n\nCase presentation\n\nA 37-year-old male patient presented with complaints of multiple swellings on both hands that affected his quality of life. He noticed these swellings at the age of 15 and since then the swellings had increased in size. The progressive increase in the size of swellings interfered with the patient’s day to day life and made it difficult for him to perform routine tasks. The patient had not undergone any medical or surgical treatment for this syndrome since diagnosis. The patient had no history of melena, haematochezia or hematemesis. There was no history of diabetes, hypertension, or drug or alcohol use. Physical examination revealed multiple firm nontender swellings on both hands and a cystic swelling present on the face above the right eyelid. Ophthalmologic examination showed bilateral cataracts.\n\nThe patient also had a cystic rubbery, painless, non-tender mass present on the left wrist, which is suggestive of cystic lymphangioma (Figure 1).\n\nWe did not find any stippled calcification within the right humerus, femur, tibia and fibular head, right scapular and pelvic dyschondroplasias, and there was no phlebolith in the soft tissue.\n\nSignificant lab findings included urea of 200 mg/dl (normal range, 7–30 mg) and creatinine 1.9 mg/dl (normal range, 0.6–1.2 mg/dl), which might be due to renal vascular malformation, as the patient was young, and all other causes of renal failure were ruled out, which included diabetes (by assessing HBA1c), hypertension (by assessing blood pressure), use of any nephrotoxic drugs, or urinary tract obstruction (by ultrasound). An x-ray showed irregular radiolucent areas at the proximal end of the metatarsals of both hands (Figure 2).\n\nEndoscopy and colonoscopy were performed to detect the presence of hemangioma. Endoscopy revealed grade 1 esophagitis of moderate intensity and erosive gastritis in the antrum. No vascular malformations were detected on colonoscopy.\n\nBiopsy of the skin lesions was performed, which showed irregularly shaped, convoluted ectatic vascular malformation, lined by epithelial cells diagnosed as a cavernous haemangioma. There was no family history of Maffucci syndrome. He was referred to the Dermatology department for the treatment of face and hand lesions and to the Orthopaedic department for osteotomy and curettage of bone lesions. The patient is currently on dialysis and no other complications were observed on follow-up visits after osteotomy.\n\n\nDiscussion\n\nWe presented a case of a 37-year-old man who noticed multiple swellings on his hands at the age of 15, which is the typical age for the manifestation of Maffucci syndrome; in 78% of patients, symptoms appear at the age of 15 years2. Maffucci syndrome is considered as a progressive form of Ollier’s disease. Enchondromas and haemangiomas are the main diagnostic features of Maffucci syndrome. Enchondromas are benign growth of cartilage that may develop at any site, but in Maffucci syndrome, enchondromas are found on phalanges and long bones. It may also affect ribs or skull bones. Soft tissue tumours usually develop within these bone lesions8,9. Our patient presented with multiple enchondromas on both hands. Haemangiomas are benign tumours of blood vessels, which differentiates Maffucci syndrome from Ollier’s disease. Haemangiomas can occur anywhere around the body, and our patient presented with multiple haemangiomas on the face.\n\nAccording to recent research, Maffucci syndrome occurs due to mutation of isocitrate dehydrogenase (IDH) enzymes 1 and 2 genes. Maffucci syndrome is considered as non-hereditary because mutations occur after fertilization10. Mutations of parathyroid hormone-related protein-1 (PTHrP1) have also been linked to Maffucci syndrome; PTHrP1 mutation causes activation of the signalling pathway, which in turn leads to abnormal proliferation and differentiation of chondrocytes11.\n\nDiagnosis of Maffucci syndrome is made based on clinical, histopathological, and radiographic findings. Clinically, our patient had multiple enchondromas on both hands, a cystic firm mass on the left wrist, and cystic swellings on the face. Radiographically, patients with Maffucci syndrome present with translucency in the bony regions which are enchondromas, and opaque spots, which represent phleboliths12. X-rays of our patient showed multiple well defined, irregular radiolucent areas in all metatarsals of both hands, which are typical radiographic findings in Maffucci syndrome. Histopathology of skin lesions in our patient revealed cavernous haemangiomas, which are characteristic of Maffucci syndrome. Clinical findings in combination with histopathological and radiographic findings confirmed the diagnosis of Maffucci syndrome in our patient.\n\nTwo distinct features of our patient were bilateral cataract and renal impairment. According to a literature search, to the best of our knowledge, this is the first case report of Maffucci syndrome where these findings are observed. The relationship between renal impairment and cavernous haemangiomas has been addressed in the past. Bui et al. published a case report in which they discussed a 23-year-old patient with multiple haemangiomas and end-stage renal disease13. Although the end-stage renal disease has been suggested to be the cause of renal mesenchymal tumours, which include haemangioma and angiomyolipoma, we suggest that it could be the other way around. Mesenchymal renal tumours or vascular malformation could cause renal impairment. Due to financial constraints and patient reluctance to undergo further diagnostic procedures, we could not confirm our findings to be part of Maffucci syndrome, but we firmly believe that renal impairment in our patient is a manifestation of Maffucci syndrome as we ruled out all other causes of renal impairment. We recommend monitoring renal function in patients with Maffucci syndrome. Several case reports discuss the involvement of various organs in patients with Maffucci syndrome. Involvement of the liver, spleen, adrenal gland, and brain have been reported in the literature14,15.\n\nWe also emphasize the importance of diagnosing Maffucci syndrome early in the disease process. Multiple masses on limbs should prompt the diagnosis of Ollier’s disease or Maffucci syndrome. Our patient noticed swellings at the age of 15, but diagnosis did not occur until 37 years of age. Maffucci syndrome increases the risk of developing malignancy in patients. The overall incidence of malignancy in patients is 23-100%16.\n\nEnchondromas and haemangiomas that cause pain, thrombosis, fracture or other complications should be removed. Patients should be monitored regularly for any complications of enchondromas or haemangiomas. Cancer surveillance should also be done. Full body MRI should be done to find any obsolete haemangiomas. Organ functions should also be tested after the confirmation of diagnosis as Maffucci syndrome can affect any organ.\n\n\nStrengths and weaknesses\n\nThe strength of this report is that is shows that regular screening of patients for cataracts and renal impairment can lead to avoidance of these complications. Unfortunately, since the patient had a poor socioeconomic status, we were unable to perform comprehensive diagnostic tests to confirm renal involvement as a part of Maffucci syndrome.\n\n\nConclusions\n\nOur patient exhibited a typical case of Maffucci syndrome with two new features, bilateral cataract and renal impairment, never before reported in the literature, to the best of our knowledge. Cancer surveillance should be done in patients after diagnosis, since Maffucci syndrome carries a high risk of malignancy.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nGao H, Wang B, Zhang X, et al.: Maffucci syndrome with unilateral limb: a case report and review of the literature. Chin J Cancer Res. 2013; 25(2): 254–258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBean WB: Dyschondroplasia and hemangiomata (Maffucci's syndrome). II. AMA Arch Intern Med. 1958; 102(4): 544–550. PubMed Abstract \| Publisher Full Text\n\nLewis RJ, Ketcham AS: Maffucci's syndrome: functional and neoplastic significance. Case report and review of the literature. J Bone Joint Surg Am. 1973; 55(7): 1465–1479. PubMed Abstract\n\nSchnall AM, Genuth SM: Multiple endocrine adenomas in a patient with the Maffucci syndrome. Am J Med. 1976; 61(6): 952–956. PubMed Abstract \| Publisher Full Text\n\nJirarattanaphochai K, Jitpimolmard S, Jirarattanaphochai K: Maffucci's syndrome with frontal lobe astrocytoma. J Med Assoc Thai. 1990; 73(5): 288–293. PubMed Abstract\n\nHuang XD, Jiao HS, Yang Z, et al.: Sclerosing angiomatoid nodular transformation of the spleen in a patient with Maffucci syndrome: a case report and review of literature. Diagn Pathol. 2017; 12(1): 79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPezzilli R, Serra C, Tomassetti P, et al.: Maffucci Syndrome with Hemangioma of the Liver. Case Rep Gastroenterol. 2009; 3(1): 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiber C, Ergun P, Turay UY, et al.: A case of Maffucci 's syndrome with pleural effusion: ten-year follow-up. Ann Acad Med Singapore. 2004; 33(3): 347–350. PubMed Abstract\n\nDuke D, Dvorak A, Harris TJ, et al.: Multiple retiform hemangioendotheliomas. A low-grade angiosarcoma. Am J Dermatopathol. 1996; 18(6): 606–610. PubMed Abstract \| Publisher Full Text\n\nProkopchuk O, Andres S, Becker K, et al.: Maffucci syndrome and neoplasms: a case report and review of the literature. BMC Res Notes. 2016; 9: 126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta N, Kabra M: Maffucci syndrome. Indian Pediatr. 2007; 44(2): 149–150. PubMed Abstract\n\nZwenneke Flach H, Ginai AZ, Wolter Oosterhuis J: Best cases from the AFIP. Maffucci syndrome: radiologic and pathologic findings. Armed Forces Institutes of Pathology. Radiographics. 2001; 21(5): 1311–1316. PubMed Abstract \| Publisher Full Text\n\nBui TL, Glavis-Bloom J, Liu HK: Multiple renal capillary hemangiomas in a patient with end-stage renal disease. Radiol Case Rep. 2019; 14(6): 750–754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBüttner M, Kufer V, Brunner K, et al.: Benign mesenchymal tumours and tumour-like lesions in end-stage renal disease. Histopathology. 2013; 62(2): 229–236. PubMed Abstract \| Publisher Full Text\n\nPuech-Bret N, Bret J, Bennet A, et al.: Maffucci syndrome and adrenal cortex tumor. Joint Bone Spine. 2009; 76(5): 556–558. PubMed Abstract \| Publisher Full Text\n\nAhmed SK, Lee WC, Irving RM, et al.: Is Ollier’s disease an understaging of Maffucci’s syndrome? J Laryngol Otol. 1999; 113(9): 861–864. PubMed Abstract \| Publisher Full Text" }	[ { "id": "81917", "date": "09 Apr 2021", "name": "Dennis Mazingi", "expertise": [ "Reviewer Expertise Surgery", "Global surgery", "congenital malformations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to read this report. It describes a brief case of Maffucci's syndrome with two unique clinical findings not seen before in the literature. The authors describe the case and then offer possible explanations for renal failure. While they failed to substantiate their argument for renal failure, it remains an interesting theory. This report has the potential to assist clinicians in the future to be mindful of the potential issues that patients with Maffucci’s syndrome may face it may be premature to recommend routine screening for cataracts and renal failure since this is the only case that has seen this combination of findings. Because this was a brief report whose aim was to report an interesting clinical finding there are not many details on the treatment or follow-up of this patient as laid out in the SCARE guidelines. However, it may have been interesting to describe the implications of the clinical findings on further management or prognosis. For example, what is the implication of renal failure on operative management, perioperative risk and the decision to operate in someone with end-stage renal impairment when the reasons for the operation were for aesthetic reasons or for mild functional impairment? In this way, they could have made the case more impactful. In addition, the patient’s perspective was not explicitly described as well as their reasons for refusal of potentially helpful investigations. The authors state that they ruled out other causes of hypertension with multiple tests but did not present the results of said tests for the reader to use for themselves. There are also other simple tests that may have helped to narrow down the cause of the renal failure.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "88615", "date": "01 Jul 2021", "name": "Ian Murdoch", "expertise": [ "Reviewer Expertise Ophthalmology", "glaucoma", "epidemiology", "public health", "telemedicine", "health economics", "education" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a case of Maffucci’s syndrome in which two additional findings were noted: renal impairment and cataract. Whilst the literature review seems reasonable given the background to the syndrome and prior reports, the two novel findings are inadequately presented. As my fellow reviewer points out, some more detailed medical history and further simple (not expensive) tests might have helped with the renal impairment. From an ophthalmological point of view the description is totally inadequate. Again, history and examination details are required (FH, past medical history including other potential causes for cataract, present history, refraction, acuity, ocular status, type of cataract etc).\nWhilst I totally agree that with such rare conditions case-reports are completely appropriate; if they are to be of use then some concentration needs to be shown on firstly ensuring the correct diagnosis, and secondly detailing the novel finding as much as possible. I appreciate the difficulties of working in resource poor environments but that does not stop good basic medical examination.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/8-1722
https://f1000research.com/articles/8-1721/v1	04 Oct 19	{ "type": "Review", "title": "Functional genomic approaches to improve crop plant heat stress tolerance", "authors": [ "Baljeet Singh", "Neha Salaria", "Kajal Thakur", "Sarvjeet Kukreja", "Shristy Gautam", "Umesh Goutam", "Baljeet Singh", "Neha Salaria", "Kajal Thakur", "Sarvjeet Kukreja", "Shristy Gautam" ], "abstract": "Heat stress as a yield limiting issue has become a major threat for food security as global warming progresses. Being sessile, plants cannot avoid heat stress. They respond to heat stress by activating complex molecular networks, such as signal transduction, metabolite production and expressions of heat stress-associated genes. Some plants have developed an intricate signalling network to respond and adapt it. Heat stress tolerance is a polygenic trait, which is regulated by various genes, transcriptional factors, proteins and hormones. Therefore, to improve heat stress tolerance, a sound knowledge of various mechanisms involved in the response to heat stress is required. The classical breeding methods employed to enhance heat stress tolerance has had limited success. In this era of genomics, next generation sequencing techniques, availability of genome sequences and advanced biotechnological tools open several windows of opportunities to improve heat stress tolerance in crop plants. This review discusses the potential of various functional genomic approaches, such as genome wide association studies, microarray, and suppression subtractive hybridization, in the process of discovering novel genes related to heat stress, and their functional validation using both reverse and forward genetic approaches. This review also discusses how these functionally validated genes can be used to improve heat stress tolerance through plant breeding, transgenics and genome editing approaches.", "keywords": [ "GWAS", "VIGS", "T-DNA", "CRISPR", "Heat stress", "Functional genomics" ], "content": "Introduction\n\nAbiotic stresses have numerous adverse effects on crop plants, which further lead to yield and quality losses (Figure 1). To feed the whole world in the scenario of the changing climate, new and better heat tolerant varieties of various crops is needed1. The understanding of various physiological, molecular and biochemical pathways can facilitate the development of superior heat tolerant varieties2. However, previous efforts, aimed at improving plant heat stress tolerance, have had limited success3,4 because of the poor understanding of the genetics of heat tolerance. Fortunately, nowadays reference genomes of major food crops and model plant species are available publicly, which provide a solid platform for crop improvement. Moreover, wild species and various landraces of various crops have unknown heat tolerant genes that should be identified and incorporated to high yielding modern cultivars5. The functional genomic approaches such as genome wide association studies (GWAS) and gene expression profiling using microarrays can catalyse the discovery of novel genes associated to heat stress6–8. In addition, suppression subtractive hybridization (SSH) is another effective and productive technique used for the screening and cloning of the genes/ESTs that express differentially under heat stress9,10. Reverse genetic techniques can improve the understanding of their expression patterns under heat stress. The plant breeding strategies and new biotechnological tools including genome editing techniques can use these validated genes to enhance heat stress tolerance in crop plants (Figure 2).\n\n\nMining of stress linked genes\n\nPresent crop varieties have limited heat tolerance because earlier domestication, green revolution and conventional breeding were focused to increase yield and qualitative traits11. However, the knowledge of genes/markers/QTL regions associated to heat tolerance is now required to improve thermo tolerance. Previous studies suggested that vast genetic diversity still exists in the germplasms of various crops12–14. GWAS emerged as a powerful tool to identify the genetic basis behind complex phenotypic traits15,16, and it provides high mapping resolution compared with conventional genetic mapping17,18. So far this approach has been applied to major food crops, including wheat7,19,20, rice21, maize22, sorghum23and Brassica napus L.24, to identify the natural variation associated with heat stress and to understand this genetic basis. Another way to identify and understand the key molecular mechanisms in response to heat stress is a transcriptomic study25,26; plants respond to heat stress by inducing various heat responsive genes, thus transcriptomic studies provide an effective screening of heat responsive candidate genes6. For example, microarray studies allow the screening of genes on the basis of their expression patterns under stressed conditions at a particular plant developmental stage6,26,27. Singh et al. (2015)6 investigated the heat responsive genes for potato tuberization and Ginzberg et al.28 identified the candidate heat responsive genes for potato periderm formation using microarrays. In addition, SSH is an easy and efficient approach for the identification of genes/ESTs with differential expression under heat stress. This technique is preferred when the genome sequence information is not available9. It can identify the tissue specific differentially expressed transcripts. To identify heat responsive ESTs cDNA libraries can be generated from plants grown under heat stressed conditions29. For example, SSH library of potato skin present 108 candidate genes for suberin and periderm formation30. To investigate the genes/ESTs involved in heat tolerance at the stage of grain filling in wheat, SSH library was constructed by using the leaf RNA samples from heat stressed plants9,29. The results of these studies provided many heat responsive genes/ESTs, which can be used to develop thermo tolerant wheat varieties.\n\n\nValidating the stress responsive genes\n\nThe above approaches can identify potential candidate genes linked to heat stress tolerance. However, the functions of the candidate genes must be validated before incorporating them into present cultivars. Both forward and reverse genetic approaches can be employed for functional validation of genes (see examples in Table 1). Forward genetics detect variations in the nucleic acid sequence responsible for a given phenotype31, while reverse genetics detect the gene’s functionality by observing the change in the phenotype due to alterations in known genetic sequence32.\n\nVirus-induced gene silencing (VIGS) is a rapid, efficient and cost effective post-transcriptional gene silencing (PTGS) technique used to study target gene(s) functionality53. It can be used as both the forward and reverse genetic approach48,54. Plants can sense and then respond to heat stress by activating various transcriptional cascades55. Being a PTGS technique, VIGS can be used to knockdown the expression of target genes after transcription. VIGS takes an advantage of a plant’s innate defence mechanism against virus infection. In this technique, a fragment of the target gene is first inserted into a suitable viral vector and then that vector is transformed into the plant, where the viral genome harbouring the fragment of target gene start replicating and produce dsRNA. Then an enzyme DICER cut this dsRNA into multiple siRNA of about 21 nucleotide long. Later these siRNA unwind into two single stranded RNAs, one out of which is degraded and the other one binds to RNA induced silencing complex (RISC), which later degrade the targeted endogenous gene and the effect on gene knockdown can be observed on by phenotypic analysis56–58. Many candidate genes and transcriptional factors associated with heat stress response/tolerance have been validated successfully through VIGS58,59. For example, TRV-VIGS based silencing of CabZIP63 gene lowered the tolerance to heat stress in pepper plants, suggesting that CabZIP63 is a positive regulator for thermos tolerance44. The functionality of ATG5, ATG7,FAD7 and NtEDS1genes in response to heat stress have been successfully studied in tomato using tobacco rattle virus (TRV) based VIGS technique48,60. Recently, VIGS has also been employed to investigate the involvement of small heat shock proteins (CaHSP16.4 and CaHsp25. 9) in heat stress tolerance61,62.\n\nThe role of candidate genes in heat stress tolerance can also be verified through the generation of transfer (T)-DNA mutants63. Like VIGS, insertion of T-DNA in the target gene’s sequence disrupt its functionality, which results in the change of phenotype. This approach is widely accepted because of the genome wide distribution of transposable elements with superior insertions in the gene sequences, resulting in the direct gene knockout64. In addition, T-DNA can also be used as a gain of function approach to study the target gene’s functionality, called as activation tagging65. For example, T-DNA having a tetramer of cauliflower mosaic virus 35S promoter can cause gene activation mutations65. Since plant responses to environmental stresses are polygenic and complex traits, model plants, such as Arabidopsis, are used to first study adaptive responses66. T-DNA mutant lines of many heat tolerant ecotypes of Arabidopsis have been discovered and are available at Nottingham Arabidopsis Stock Centre (NASC) or The Arabidopsis Information Resource (TAIR).\n\nTargeting induced local lesions in genome (TILLING) is another non-transgenic approach that allows the PCR based identification of directed mutations in the target gene sequence and the function of target gene can be analysed from the modified phenotype due to that mutation67,68. It is a fast and cost efficient technique for the screening of point mutations and for the functional validation gene of interest69. It take advantage of conventional insertional mutagenesis and availability of genomic sequences70. These point mutations can be generated with the help of chemical mutagens such as ethyl methane sulfonate (EMS)71. Nowadays, the genome sequences of many crop plant species are available, which make this technique more effective. The plants under heat stress exhibit different phenotypes associated with allelic variations in their genomic sequence. The TILLING approach used to study these natural variations or SNP mutations in individuals is called as EcoTILLING. The next generation sequencing techniques allow inexpensive TILLING by sequencing method to screen SNP variations72. Recently, the functionality of heat shock binding protein 1 (HSBP1) was examined with TILLING. The chemically induced mutations disrupted the functionality of HSBP1 partially and the mutant plants exhibited increased heat stress tolerance. These findings confirmed that HSBP1 is a negative regulator of heat stress response in tomato73.\n\nIn addition, the genome-editing techniques such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) and clustered regularly interspace short palindromic repeat (CRISPR) can also be used as reverse genetic approaches to study the target gene function. TALENs using sequence specific nucleases (SSNs) became a powerful genome editing technique, which can also be applied as a reverse genetic approach to understand the function of a target gene. It consists of one customizable DNA-binding domain and a nuclease, which generate double stranded DNA breaks (DSBs) at the target gene sequence74. Similar to CRISPR, these DSB are repaired either via NHEJ pathway or via homologous recombination. Both these recovery pathways allow insertion, deletion and intentional replacements in the target gene sequence. These modifications in the target gene’s sequence may cause a variation in the phenotype, which suggests the function of that gene74,75. The ZFNs are the synthetic proteins having a DNA binding domain that consists two finger modules and a DNA cleaving domain. ZFNs causes DSBs in the targeted DNA sequence and facilitate site-specific mutagenesis, or base substation, which alter or may knockout the gene expression76. The ZFNs have revealed the function of various genes in model plants as well as in crop species77.\n\nAmong all genome-editing techniques, CRISPR-Cas9 has emerged as a powerful tool for precise genome editing to study the molecular pathways linked to heat stress and to enhance thermo tolerance in crop plants78,79. It is comparatively simpler, more accurate and faster than other genome editing techniques. In brief, CRISPR involves designing of a guide RNA of ~20 nucleotides complementary to the gene of interest and a Cas9 nuclease enzyme that cut 3–4 bases next to the protospacer adjacent motif, which is later repaired either by homology directed repair pathway or via error prone non-homologous end joining80,81. Therefore, this technique can be used to generate gene knockout mutant lines to study the function of targeted gene(s). For example, annexin gene OsAnn3 knockout mutant lines developed via CRISPR-Cas9 technique revealed the role of OsAnn3 gene in cold stress tolerance in rice82.\n\n\nApproaches to enhance heat tolerance\n\nPlants have inherent mechanisms to survive under heat stressed conditions but the heat tolerance capacity of plants varies species-to-species and even within the species. If heat tolerant genes are present in sexually compatible species, then marker-assisted selection (MAS), new generation molecular breeding, precision breeding and genome editing techniques can be used.\n\nThermo tolerance is a complex multigenic trait, which is influenced by genotype X environment interactions83. Development of heat tolerant crop varieties through traditional breeding is very labours and time consuming. However, precision breeding with the help of MAS can accelerate the plant breeding programs with high efficiency84. SNPs and simple sequence repeats (SSR) are being used widely in plant breeding experiments aimed to enhance abiotic stress tolerance. Presently the use of SNPs become more common in plant breeding than SSR markers85,86. Garg et al.87 found one SNP in the sequence of heat shock protein (HSP16.9) between a heat tolerant and heat susceptible wheat genotypes. This SNP contribute 29.89% phenotypic variation for grain weight per spike. Recently, many SNPs associated to heat stress tolerance have been identified in major food crops7,88–90. However, heat stress tolerance is a polygenic trait and a single molecular marker contribute little to improve it. Therefore, it is important to incorporate several SNPs associated to various QTLs that are controlling the heat stress tolerance mechanisms89,91. However, the accessibility of genome editing techniques opened various new windows to introduce targeted editing of plant genomes to understand the molecular aspects involved in heat stress tolerance92,93. For example, ethylene response factors (ERFs) are the stress induced transcriptional factors that take part in abiotic stress tolerance. The CRISPR-Cas9 based genome editing of one such ethylene response factor from AP2/ERF superfamily enhanced abiotic stress tolerance in crop plants94.\n\nIn cases when heat tolerant genes do not exist in sexually compatible species, these methods cannot be applied. Advanced biotechnological tools can increase the limited heat stress tolerance in crop plants. The transfer of heat tolerant genes through recombinant DNA technology can generate heat tolerant transgenic lines in a short amount of time. This method also allows utilisation of potential genes from other species to enhance thermo tolerance in target crops, e.g. AmDREB2C, from Ammopiptanthus mongolicus has been used to increase heat stress tolerance in transgenic Arabidopsis plants95. In addition, many genes responsible for heat stress tolerance have been identified and validated in model plants and also in major food crops that can be introduced to heat susceptible cultivars or their expression levels can be increased by generating their stable overexpression lines. For instance, the overexpression TaPEPKR2 gene enhanced heat stress tolerance in wheat and Arabidopsis plants96.\n\n\nConclusion\n\nHeat stress affects crop production significantly. Plants respond to heat stress by activating complex molecular networks, such as signal transduction, metabolite production and expressions of heat stress-associated genes. With the developments in plant functional genomics techniques, many novel genes related to heat stress tolerance have been identified and are being used to improve stress tolerance with the help of advanced biotechnological approaches. Next generation sequencing and genome-editing techniques will play crucial roles in crop improvement. In the near future, the scientists will have a better understanding of plant heat tolerant mechanisms and farmers will be able to grow better high yielding heat tolerant crop varieties in the fields.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nLesk C, Rowhani P, Ramankutty N: Influence of extreme weather disasters on global crop production. Nature. 2016; 529(7584): 84–7. 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PubMed Abstract \| Publisher Full Text\n\nJaganathan D, Ramasamy K, Sellamuthu G, et al.: CRISPR for Crop Improvement: An Update Review. Front Plant Sci. 2018; 9: 985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang F, Doudna JA: CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017; 46: 505–529. PubMed Abstract \| Publisher Full Text\n\nShen C, Que Z, Xia Y, et al.: Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice. J Plant Biol. 2017; 60(6): 539–547. Publisher Full Text\n\nTayade R, Nguyen T, Oh SA, et al.: Effective Strategies for Enhancing Tolerance to High-Temperature Stress in Rice during the Reproductive and Ripening Stages. Plant Breed Biotechnol. 2018; 6(1): 1–18. Publisher Full Text\n\nZafar SA, Hameed A, Nawaz MA, et al.: Mechanisms and molecular approaches for heat tolerance in rice (Oryza sativa L.) under climate change scenario. J Integr Agr. 2018; 17(4): 726–38. Publisher Full Text\n\nMammadov J, Aggarwal R, Buyyarapu R, et al.: SNP markers and their impact on plant breeding. In: The Role of Bioinformatics in Agriculture. 2014; 387–413. Reference Source\n\nSingh N, Choudhury DR, Singh AK, et al.: Comparison of SSR and SNP markers in estimation of genetic diversity and population structure of Indian rice varieties. PLoS One. 2013; 8(12): e84136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarg D, Sareen S, Dalal S, et al.: Heat shock protein based snp marker for terminal heat stress in wheat (Triticum aestivum L.). Aust J Crop Sci. 2012; 6(11): 1516–1521. Reference Source\n\nRuggieri V, Calafiore R, Schettini C, et al.: Exploiting Genetic and Genomic Resources to Enhance Heat-Tolerance in Tomatoes. Agronomy. 2019; 9(1): 22. Publisher Full Text\n\nKilasi NL, Singh J, Vallejos CE, et al.: Heat Stress Tolerance in Rice (Oryza sativa L.): Identification of Quantitative Trait Loci and Candidate Genes for Seedling Growth Under Heat Stress. Front Plant Sci. 2018; 9: 1578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuan Y, Cairns JE, Babu R, et al.: Genome-Wide Association Mapping and Genomic Prediction Analyses Reveal the Genetic Architecture of Grain Yield and Flowering Time Under Drought and Heat Stress Conditions in Maize. Front Plant Sci. 2019; 9: 1919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaul PJ, Samineni S, Thudi M, et al.: Molecular Mapping of QTLs for Heat Tolerance in Chickpea. Int J Mol Sci. 2018; 19(8): pii: E2166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParmar N, Singh KH, Sharma D, et al.: Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review. 3 Biotech. 2017; 7(4): 239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaque E, Taniguchi H, Hassan MM, et al.: Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges. Front Plant Sci. 2018; 9: 617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDebbarma J, Sarki YN, Saikia B, et al.: Ethylene Response Factor (ERF) Family Proteins in Abiotic Stresses and CRISPR-Cas9 Genome Editing of ERFs for Multiple Abiotic Stress Tolerance in Crop Plants: A Review. Mol Biotechnol. 2019; 61(2): 153–172. PubMed Abstract \| Publisher Full Text\n\nYin Y, Jiang X, Ren M, et al.: AmDREB2C, from Ammopiptanthus mongolicus, enhances abiotic stress tolerance and regulates fatty acid composition in transgenic Arabidopsis. Plant Physiol Biochem. 2018; 130: 517–528. PubMed Abstract \| Publisher Full Text\n\nZang X, Geng X, He K, et al.: Overexpression of the Wheat (Triticum aestivum L.) TaPEPKR2 Gene Enhances Heat and Dehydration Tolerance in Both Wheat and Arabidopsis. Front Plant Sci. 2018; 9: 1710. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54724", "date": "28 Oct 2019", "name": "Himanshu Sharma", "expertise": [ "Reviewer Expertise Plant Genetics and Genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review entitled “Functional genomic approaches to improve crop plant heat stress tolerance” is relevant considering the present climate change scenario and global food security. The review highlighted the negative effects of heat stress on crop plants. Heat stress is a major yield-limiting factor in this climate change scenario. This review presents recent achievements in understanding the molecular basis of heat tolerance in crop plants. Further, it discussed the usage of appropriate approaches from the identification of heat tolerant genes to their incorporation into cultivated crop varieties. The article is written and organised nicely but still some minor improvements can be done.\nSpecific comments Title The title of the article is appropriate and accurately reflect the content of the paper.\nAbstract It represent the article nicely.\nIntroduction Last two lines of introduction can be improved. Authors written reverse genetic techniques can improve the understanding of their expression patterns under heat stress. I suggest that instead of writing reverse genetic techniques they should mention the names of these techniques.\nMining of stress linked genes Authors mentioned: Singh et al. (2015) investigated the heat responsive genes for potato tuberization and Ginzberg et al. identified the candidate heat responsive genes for potato periderm formation using microarrays. I suggest, either write the year with Ginzberg et al. or remove year from Singh et al. (2015) to keep the same formatting.\nValidating the stress responsive genes I found this section is very interesting.\nApproaches to enhance heat tolerance Do not write the gene name; AmDREB2C in Italics as rest of the gene names in text are not written in Italics.\nConclusion Summarized the article in a coherent way.\nOverall, the article is logically structured and well-organised, and provides a useful compilation of subject matter related to addressed topic. It is an important contribution and I highly recommend it.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "56551", "date": "05 Dec 2019", "name": "Neila Abdi", "expertise": [ "Reviewer Expertise legume-rhizobia symbiosis under abiotic constraints" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDuring review process, I appreciated this study. Authors discuss new approach that is well written. However, I found following points to be check for further improvement in manuscript:\nAbstract: Please correct \"in this area\" instead of \"in this era\".\n\nIntroduction: Please replace \"tolerant varieties\" instead of \"new and better heat tolerant varieties\".\n\nThe conclusion need to be ameliorate because it is very brief.\nThe review discussed very well and comprehensively the context of current literature.\nAll statements are correct and author can refer to others citations to make the paper more supported.\nThe review is written with good English and it did not need any improvement.\nAuthor can refer the following paper to explain more:\nZafar S.A., Zaidi S.S.A., Gaba Y., Singla-Pareek S.L., Dhankher O.P., Li X., Mansoor S., Pareek A (2019). Engineering abiotic stress tolerance via CRISPR/ Cas-mediated genome editing. Journal of Experimental Botany, doi:10.1093/jxb/erz476, pp 1-101. Cushman J.C., Bohnert H.J. 2000. Genomic approaches to plant stress tolerance. Genome studies and molecular genetics, 3:117–1242.\nIn general, this review can be accepted with minor revision.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "56662", "date": "09 Jan 2020", "name": "Ajaya Biswal", "expertise": [ "Reviewer Expertise Plant Biotechnology", "Genomics", "Plant cell wall", "Polysaccharide and Bioenergy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlants are constantly exposed to different abiotic stresses. They have developed a wide variety of adaptation mechanisms through molecular, physiological, and biochemical alterations to cope with different stress conditions. Heat stress is still considered as one of the major limiting factors for crop yields. Simultaneously, heat stress significantly contributes to climate change. This review well describes the wide potential of various functional genomics strategies from gene selection and identification of heat tolerance genes to genetic manipulation by genetic engineering and genome editing. However, I have a few comments that need to be addressed:\nThe authors should have discussed the functional role of heat shock protein (HSPs) and molecular chaperones under extreme temperatures. I believe a potential discussion about the possible role of HSP/chaperones towards heat stress adaptation to plants will strengthen this review.\n\nI do not find a specific reason for selecting tolerant genes from a few selected crops/plants (Table I). I encourage to refer a number of heat-tolerant gene lists from other major crops.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1721
https://f1000research.com/articles/8-1720/v1	04 Oct 19	{ "type": "Case Report", "title": "Case Report: Cardiac arrest due to traumatic coronary artery dissection treated by extracorporeal membrane resuscitation", "authors": [ "Kumiko Tanaka", "Taka-aki Nakada", "Tadayuki Kadohira", "Shigeto Oda", "Taka-aki Nakada", "Tadayuki Kadohira", "Shigeto Oda" ], "abstract": "Traumatic coronary artery dissection, which is rare in blunt trauma, has high risk of acute myocardial infarction and cardiac arrest. A 44-year-old man who had a traffic accident was transferred to the emergency department with refractory ventricular fibrillation (VF). After conventional cardiopulmonary resuscitation, we introduced extracorporeal cardiopulmonary resuscitation (ECPR) and obtained return of spontaneous circulation with ST-elevation electrocardiogram at V4-6. Subsequent coronary angiography and intravascular ultrasound supported by extracorporeal membrane oxygenation (ECMO) revealed complete occlusions of left anterior descending and left circumflex artery due to dissections. Drug-eluting stents were placed with restorations of TIMI 2 flows. After ICU admission, his left ventricular function gradually recovered; he was successfully weaned from VA-ECMO on day 9. ECPR may be a valuable option to allow time and stable hemodynamic condition to treat the cause of cardiac arrest.", "keywords": [ "cardiac arrest", "traumatic coronary artery dissection", "refractory ventricular fibrillation", "extracorporeal cardiopulmonary resuscitation", "hemostasis" ], "content": "Introduction\n\nTraumatic coronary artery dissection is rare but delayed diagnosis of this condition can lead to life-threatening1. We report a case of cardiac arrest in emergency department (ED) due to traumatic coronary artery dissection, which is treated with extracorporeal membrane oxygenation (ECMO) and extracorporeal cardiopulmonary resuscitation (ECPR).\n\n\nCase presentation\n\nA 44-year-old man with no previous history of heart disease had a traffic accident, his vehicle ramming into a wall. There were no witness and no information about the speed and mechanism of accident. On arrival of emergency medical service personnel at the scene, he had an open airway, a respiratory rate of 24 breaths/min, a blood pressure of 68/32 mmHg, a pulse rate of 120 beats/min and a Glasgow coma scale of E4V2M4. After 30 minutes, he was transferred to the ED of our hospital. Just before his arrival to the ED, he had a sudden ventricular fibrillation (VF). The VF was refractory to defibrillations, 5 mg epinephrine and 150 mg amiodaron; no abnormal findings was detected in focused assessment with sonography for trauma. After 11 minutes, we initiated to switch conventional CPR to ECPR. Two minutes after introducing ECPR, his electrocardiogram recovered from VF to sinus rhythm with ST-elevation at V4–6. Echocardiography revealed hypokinesis of the basal and anterior septal segment with an ejection fraction of 20% without pericardial effusion. He also had right scapula fracture, left chest wall hematoma, spleen laceration and open left femoral shaft fracture, which needs external fixation (Injury Severity Score38, probability of survival by Trauma Injury Severity Score=3.9%). We prioritized coronary angiography due to a suspicion of acute coronary syndrome. The coronary angiography and intravascular ultrasound (IVUS) supported with ECMO revealed complete occlusions of left anterior descending (LAD) and left circumflex (LCX) artery due to dissections of distal left main coronary artery (LMA) without atheroma; normal right coronary artery was normal (Figure 1, Figure 2). Drug-eluting stents were placed 3 hours after admission: 1 in #6–7 and #11–13 with restorations of TIMI 2 flows of LAD and LCX. Subsequently, he was treated for the open femoral fracture with external fixation and then admitted to the intensive care unit (ICU). After ICU admission, his left ventricular function gradually recovered; he was successfully weaned from VA-ECMO on day 9. Despite recovery from traumatic injuries, he had acquired cytomegalovirus and candida infections (diagnosed on day 23 of his hospital stay by bronchoalveolar lavage) in hospital due to his compromised status, he had no infections on admission. Medical therapy with 1.25 mg/kg/day ganciclovir for 19 days and 150 mg/day micafangin for 13 days had been introduced, but he died on day 51 because of advanced multiple organ failure due to resistant virus infection.\n\nArrows indicates the site of coronary artery dissection.\n\n\nDiscussion\n\nAcceleration and deceleration forces of blunt trauma potentially causes vascular spasm, intimal tears, dissection or a rupture of an existing plaque within the thrombus formation in coronary arteries, which can result in acute coronary syndrome after trauma2,3. In the present case, intimal tears caused non-atherosclerotic myocardial infarction. In a previous report including 76 cases of traumatic acute myocardial infarction4, which included 12 cases with coronary artery dissection, LAD was most frequently affected (LMA, 6.4%; LAD, 71.4%; RCA, 19%; LCX 3.2%); in the present case, the distal LMA were dissected. Spontaneous coronary artery dissection is an increasingly recognized cause of non-atherosclerotic myocardial infarction5. However, the right scapula fracture, left chest wall hematoma and spleen laceration, which was located on the same coronary plane may be evidence of traumatic dissection. Since reported incidence of cardiac injury in blunt thoracic trauma was 0.3% in the U.S. National Trauma Data Bank, cardiac injury is relatively rare6.\n\nBleeding is the most common complication during ECMO; the use of ECMO in patients with trauma of bleeding shock is uncommon7. The literature search of ECPR in trauma during 1974–2018 identified 67 trauma patients who had cardiac arrest treated with ECPR (bleeding shock=14, severe pulmonary contusion=6, hypothermia=3, hypoxia=1, unknown=24)8–17, but not refractory VF due to AMI or coronary dissection. Recent guidelines from the American Heart Association concerning cardiac arrest described the following: “ECPR may be considered for select patients for whom the suspected etiology of the cardiac arrest is potentially reversible during a limited period of mechanical cardiorespiratory support”18. In the present case, percutaneous intervention to the coronary dissection was successfully performed in the hemodynamically stable condition by ECMO.\n\nTo conclude, traumatic coronary artery dissection potentially leads to development of refractory VF on ED. ECPR may be a valuable option to allow time and a stable hemodynamic condition to treat the cause of cardiac arrest.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from the patient and his parent.", "appendix": "References\n\nLobay KW, MacGougan CK: Traumatic coronary artery dissection: a case report and literature review. J Emerg Med. 2012; 43(4): e239–43. PubMed Abstract \| Publisher Full Text\n\nColombo F, Zuffi A, Lupi A: Left main dissection complicating blunt chest trauma: case report and review of literature. Cardiovasc Revasc Med. 2014; 15(6–7): 354–6. PubMed Abstract \| Publisher Full Text\n\nRadojevic N, Radunovic M: Traumatic acute myocardial ischaemia involving two vessels. J Forensic Leg Med. 2014; 23: 9–11. PubMed Abstract \| Publisher Full Text\n\nChristensen MD, Nielsen PE, Sleight P: Prior blunt chest trauma may be a cause of single vessel coronary disease; hypothesis and review. Int J Cardiol. 2006; 108(1): 1–5. PubMed Abstract \| Publisher Full Text\n\nIngrassia J, Diver D, Vashist A: Update in Spontaneous Coronary Artery Dissection. J Clin Med. 2018; 7(9): pii: E228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrigorian A, Milliken J, Livingston JK, et al.: National risk factors for blunt cardiac injury: Hemopneumothorax is the strongest predictor. Am J Surg. 2019; 217(4): 639–642. PubMed Abstract \| Publisher Full Text\n\nCordell-Smith JA, Roberts N, Peek GJ, et al.: Traumatic lung injury treated by extracorporeal membrane oxygenation (ECMO). Injury. 2006; 37(1): 29–32. PubMed Abstract \| Publisher Full Text\n\nBonacchi M, Spina R, Torracchi L, et al.: Extracorporeal life support in patients with severe trauma: an advanced treatment strategy for refractory clinical settings. J Thorac Cardiovasc Surg. 2013; 145(6): 1617–26. PubMed Abstract \| Publisher Full Text\n\nTseng YH, Wu TI, Liu YC, et al.: Venoarterial extracorporeal life support in post-traumatic shock and cardiac arrest: lessons learned. Scand J Trauma Resusc Emerg Med. 2014; 22: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKudo S, Tanaka K, Okada K, et al.: Extracorporeal cardiopulmonary resuscitation for blunt cardiac rupture. Am J Emerg Med. 2017; 35(11): 1789 e1–e2. PubMed Abstract \| Publisher Full Text\n\nDarocha T, Kosiński S, Jarosz A, et al.: Extracorporeal Rewarming From Accidental Hypothermia of Patient With Suspected Trauma. Medicine (Baltimore). 2015; 94(27): e1086. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArlt M, Philipp A, Voelkel S, et al.: Extracorporeal membrane oxygenation in severe trauma patients with bleeding shock. Resuscitation. 2010; 81(7): 804–9. PubMed Abstract \| Publisher Full Text\n\nHuh U, Song S, Chung SW, et al.: Is extracorporeal cardiopulmonary resuscitation practical in severe chest trauma? A systematic review in single center of developing country. J Trauma Acute Care Surg. 2017; 83(5): 903–7. PubMed Abstract \| Publisher Full Text\n\nLin CY, Tsai FC, Lee HA, et al.: Extracorporeal membrane oxygenation support in post-traumatic cardiopulmonary failure: A 10-year single institutional experience. Medicine (Baltimore). 2017; 96(6): e6067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerchinsky MJ, Long WB, Hill JG, et al.: Extracorporeal cardiopulmonary life support with heparin-bonded circuitry in the resuscitation of massively injured trauma patients. Am J Surg. 1995; 169(5): 488–91. PubMed Abstract \| Publisher Full Text\n\nYen TS, Liau CC, Chen YS, et al.: Extracorporeal membrane oxygenation resuscitation for traumatic brain injury after decompressive craniotomy. Clin Neurol Neurosurg. 2008; 110(3): 295–7. PubMed Abstract \| Publisher Full Text\n\nJones DR, Hill RC, Vasilakis A, et al.: The successful resuscitation of a hypothermic multitrauma patient. W V Med J. 1991; 87(7): 298–301. PubMed Abstract\n\nNeumar RW, Shuster M, Callaway CW, et al.: Part 1: Executive Summary: 2015 American Heart Association Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation. 2015; 132(18 Suppl 2): S315–67. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56838", "date": "20 Nov 2019", "name": "Gerald Chi", "expertise": [ "Reviewer Expertise cardiology", "coronary artery disease", "coronary angiography" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTanaka and colleagues report a case of a 44-year-old man with traumatic coronary artery dissection in the distal left main coronary artery after a traffic accident, which was successfully managed with drug-eluting stent implantation. The background, case presentation, and discussion are well-rounded.\nSeveral comments to be considered:\nCase presentation, Page 2: Imaging from intravascular ultrasound may be supplemented to confirm the absence of underlying atherosclerosis or other coronary artery diseases.\n\nDiscussion, Page 2: “In a previous report including 76 cases of traumatic acute myocardial infarction, which included 12 cases with coronary artery dissection, LAD was most frequently affected (LMA, 6.4%; LAD, 71.4%; RCA, 19%; LCX 3.2%).” Regarding the distribution of coronary arteries affected by traumatic dissection, it is advisable to reference the more recent and comprehensive literature review by Lobay and colleagues (J Emerg Med. 2012 Oct;43(4):e239-431).\n\nTreatment options for traumatic coronary artery dissection include coronary artery bypass grafting, percutaneous coronary intervention with stent placement, and conservative management. The decision rationale for this case may be further discussed.\n\nThe caption for Figure 1 and Figure 2 appears to be in reverse.\n\nCase presentation, Page 2: “…normal right coronary artery was normal.” The sentence should read: “…right coronary artery was normal.”\n\nTypographical errors to be corrected: amiodarone (page 2); micafungin (page 2).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "68133", "date": "11 Aug 2020", "name": "L Christian Napp", "expertise": [ "Reviewer Expertise Interventional Cardiology", "Intensive Care Medicine", "Heart Failure", "Mechanical Circulatory Support" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors submit a case report on a rather young man with cardiac arrest likely due to coronary dissection, which was successfully treated with ECPR. In general the report is well written but left me with some major concerns.\n\nGeneral\nIs this really a case of traumatic dissection, or a car accident due to spontaneous dissection?\n\nPlease discuss the rationale of ECMO vs. ECPELLA, in a patient with acute myocardial injury.\n\nWas there some underlying condition, as CMV and candida infection are unusual causes of death in previously healthy people.\n\nThe manuscript might benefit from language revision.\nIntroduction\nThere seems to be a word missing (‘life-threatening …’).\n\nECMO during resuscitation is the same as ECPR, but in this sentence (‘and’) it looks like it is two different measures.\nResults\nPlease provide more details regarding LV function over time.\nFigures\nLegends of Figures are mixed up.\n\nFrom Figure 2 I am not really sure whether LAD was recanalized, maybe the authors want to submit another angulation after PCI.\n\nMaybe the authors can submit an ECG from the ED.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1720
https://f1000research.com/articles/8-1718/v1	04 Oct 19	{ "type": "Research Article", "title": "Randomized controlled trials in ophthalmology: a bibliometric study", "authors": [ "Saif Aldeen AlRyalat", "Areen Abukahel", "Khaled Ali Elubous", "Areen Abukahel", "Khaled Ali Elubous" ], "abstract": "Background: Randomized controlled trials (RCTs) are situated at the top of hierarchy of evidence-based medicine, where its number and quality are important in the assessment of quality of evidence in a medical field. In this study, we aim to assess the status of RCTs in Ophthalmology. Methods: On 15th of May 2019, we performed a PubMed search for randomized controlled trials published in the field of ophthalmology using relevant filters and search terms. We categorized the results into specific topics in ophthalmology according to Medical Subject Heading (MeSH) database classification system. We used Altmetric explorer to identify journals and articles with the highest number of RCTs and highest citations. Results: We found a total of 540,427 publications in the field of ophthalmology, of which only 11,634 (2.15%) of them were RCTs. ‘Retinal diseases’ was the topic with the highest number of RCTs, followed by ‘glaucoma’ and ‘conjunctival diseases’. The trial with highest number of citations was on retinal diseases. Only around 18% of all ophthalmology RCTs are published in the top 10 ophthalmology journals, with a maximum percentage of RCTs was (5.53%) published in Ophthalmology. Conclusion: RCTs in ophthalmology primarily concern the retina, glaucoma, and a few other sub-topics, with little focus on sclera, orbit, and the eyelids. Most of the high impact RCTs are published in non-ophthalmology journals.", "keywords": [ "Ophthalmology", "Randomized Controlled Trials", "PubMed", "Retina", "Journals", "Bibliometrics." ], "content": "Introduction\n\nSince the conception of the term “evidence-based medicine” in clinical practice in 19921, where well-conducted randomized controlled trials (RCTs) are situated at the top of hierarchy of evidence, there has been an emphasis on accepting high quality evidence in terms of RCTs in clinical practice. Moreover, previous reports showed that RCTs have generally higher methodological rigor than observational studies2. However, despite the rapid growth in ophthalmology literature in the recent years, this growth has not been paralleled by a growth in the quality of evidence3. This is evident by the number of Cochrane reviews that don’t include any RCTs (i.e. empty review), which were estimated to be half of the total reviews on Cochrane Eyes and Vision in 20134. In this study, we aim to assess the status of RCTs in ophthalmology, and will focus on publishing trends for RCTs in ophthalmology in the recent years with regards to different ophthalmology topics.\n\n\nMethods\n\nOn 15th of May 2019, we performed a PubMed search for randomized controlled trials published in the field of ophthalmology. We used the following search filters:\n\nOphthalmology studies: eye diseases [MeSH Terms]\n\nRCT: Randomized Controlled Trial [Publication Type]\n\nTo categorize the results into specific topics in ophthalmology, we used the Medical Subject Heading (MeSH) database to identify the topics within ophthalmology, where the following were included:\n\nOrbital Diseases\n\nConjunctival Diseases\n\nCorneal Diseases\n\nEyelid Diseases\n\nLacrimal Apparatus Diseases\n\nLens Diseases\n\nGlaucoma\n\nRefractive Errors\n\nScleral Diseases\n\nUveal Diseases\n\nRetinal Diseases\n\nFor each topic, we added the query as a MeSH term to the search to identify relevant articles (e.g. Orbital diseases[Mesh Terms]. It is worth noting that trials might be categorized in more than one topic.\n\nTo identify journals with the highest number of RCTs and top articles with highest citations, we used Altmetric database, where we inputted the PubMed query we used in the PubMed search in the search field; the database yielded citation information about searched articles along with information about the journals these articles published previously5.\n\nFor each RCT, we extracted data regarding the topic of the study and categorized them into the following: RCTs per year, percentage of each sub-specialty, Articles per sub-specialty per year, Top 10 journals with their respective data, Top 10 articles with highest dimensions citations\n\n\nResults\n\nA total of 540,427 publications in the field of ophthalmology were identified, of which only 11,634 (2.15%) of them were RCTs. There was a total of 482,791 RCT identified in all disciplines, of which only 2.4% are in the field of ophthalmology. Of these trials, 124 were phase 1 trials, 270 were phase 2 trials, 380 were phase 3 trials, and 42 phase 4 trials; all others did not have phases. Number of RCTs peaked in 2015 with a total of 583 trials. Figure 1 shows the trend in number of RCTs in the field of ophthalmology.\n\n‘Retinal diseases’ is the topic with the highest number of RCTs, with a total of 2915 trials, followed by ‘glaucoma’, with 2118 trials, and ‘conjunctival diseases’, with 1230 trials. Figure 2 details the number of trials for each topic.\n\nThe trial with highest number of citations discussed retinal complications of diabetes mellitus entitled “The Effect of Intensive Treatment of Diabetes on the Development and Progression of Long-Term Complications in Insulin-Dependent Diabetes Mellitus”, published in The New England Journal of Medicine6. Table 1 details the top 10 RCTs with highest citations. A total of 2090 (18%) of the RCTs were published in 10 journals, with “Ophthalmology” being the top journal with highest number of RCT published in it (643 RCTs). Table 2 details the top 10 journals with highest number of RCTs published in them.\n\nOA, open access.\n\n\nDiscussion\n\nIn the current study, we observed a peak in the annual number of RCTs on 2015, after which a steady decrease observed till 2018. Retinal diseases is the topic with the highest number of RCTs, followed by glaucoma and conjunctival diseases. The trial with highest citation was on retinal diseases and was published in The New England Journal of Medicine, where also other top cited trials were published in general non-ophthalmology journals. The total RCTs published in top 100 ophthalmology journals was only 2090 (17.96%).\n\nIn general, there has been an increase in the number of RCTs in ophthalmology since the late 1990s. In a study assessing the frequency of prospective studies published in the American Journal of Ophthalmology and British Journal of Ophthalmology, they found an increase from 1% to 12% during the years 1980 to 19997. We observed a low number of RCTs among the ophthalmology literature, a percentage that didn’t exceed 2.5% of the overall ophthalmology literature. In a previous study assessing the frequency of RCTs published in the major four ophthalmology journals, they found that only around 3.5% of their annual publications are RCTS8. Moreover, we found that only around 18% of all ophthalmology RCTs are published in the top 10 ophthalmology journals, with the most RCTs (5.53%) published in Ophthalmology. In a study that reviewed risk of bias in RCTs published in major ophthalmology journals found that a risk of bias was observed in 29.4% of published RCTs9. In another study that assessed fragility of RCT’s that included the comparison between two groups found a high proportion of fragile results in ophthalmology RCTs10. In a study that assessed types of articles published in core pediatric journals, they found that only 0.3% were RCTs11, which supports our findings that a large proportion of RCTs were published in high-impact general medical journals.\n\nOne of the main limitations in this study is that it didn’t assess the quality of RCTs, so we included RCTs from our PubMed search regardless of their quality. Recent studies have stated that ophthalmology literature is of questionable methodological robustness, where RCTs become the center of the scope when methodological robustness is assessed, as they are the source of the highest level of evidence10,21. Future studies should focus on assessing quality of RCTs rather than the quantity (which was the scope of this study), where the Cochrane Eyes and Vision library criteria for RCT robustness can be utilized22.\n\n\nData availability\n\nHarvard Dataverse: Ophthalmology randomized controlled trials. https://doi.org/10.7910/DVN/TXEYDX23.\n\nThis project contains the articles identified during this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nEvidence-Based Medicine Working Group: Evidence-based medicine. A new approach to teaching the practice of medicine. JAMA. 1992; 268(17): 2420–5. PubMed Abstract \| Publisher Full Text\n\nLai TY, Leung GM, Wong VW, et al.: How evidence-based are publications in clinical ophthalmic journals?. Invest Ophthalmol Vis Sci. 2006; 47(5): 1831–8. PubMed Abstract \| Publisher Full Text\n\nBoudry C, Baudouin C, Mouriaux F: International publication trends in dry eye disease research: A bibliometric analysis. Ocul Surf. 2018; 16(1): 173–9. PubMed Abstract \| Publisher Full Text\n\nWormald R, Dickersin K, Cochrane E, et al.: Evidence-Based Ophthalmology. Ophthalmology. 2013; 120(12): 2361–2363.e1. PubMed Abstract \| Publisher Full Text\n\nPriem J, Groth P, Taraborelli D: The altmetrics collection. PLoS One. 2012; 7(11): e48753. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiabetes Control and Complications Trial Research Group, Nathan DM, Genuth S, et al.: Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med. 1993; 329(14): 977–86. PubMed Abstract \| Publisher Full Text\n\nAng A, Tong L, Bhan A: Analysis of publication trends in two internationally renowned ophthalmology journals. Br J Ophthalmol. 2001; 85(12): 1497–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBojikian KD, Gupta D, Dettori JM, et al.: Evidence in ophthalmology: Are we doing better? Ophthalmology. 2015; 122(12): 2584–6. PubMed Abstract \| Publisher Full Text\n\nJoksimovic L, Koucheki R, Popovic M, et al.: Risk of bias assessment of randomised controlled trials in high-impact ophthalmology journals and general medical journals: a systematic review. Br J Ophthalmol. 2017; 101(10): 1309–14. PubMed Abstract \| Publisher Full Text\n\nShen C, Shamsudeen I, Farrokhyar F, et al.: Fragility of results in ophthalmology randomized controlled trials: a systematic review. Ophthalmology. 2018; 125(5): 642–8. PubMed Abstract \| Publisher Full Text\n\nHardin WD Jr, Stylianos S, Lally KP: Evidence-based practice in pediatric surgery. J Pediatr Surg. 1999; 34(5): 908–13; discussion 912-3. PubMed Abstract \| Publisher Full Text\n\nUK Prospective Diabetes Study Group: Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. UK Prospective Diabetes Study Group. BMJ 1998; 317(7160): 703–13. PubMed Abstract \| Free Full Text\n\nRosenfeld PJ, Brown DM, Heier JS, et al.: Ranibizumab for neovascular age-related macular degeneration. N Engl J Med. 2006; 355(14): 1419–31. PubMed Abstract \| Publisher Full Text\n\nBrown DM, Kaiser PK, Michels M, et al.: Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N Engl J Med. 2006; 355(14): 1432–44. PubMed Abstract \| Publisher Full Text\n\nKass MA, Heuer DK, Higginbotham EJ, et al.: The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch Ophthalmol. 2002; 120(6): 701–13. PubMed Abstract \| Publisher Full Text\n\nGragoudas ES, Adamis AP, Cunningham ET Jr, et al.: Pegaptanib for neovascular age-related macular degeneration. N Engl J Med. 2004; 351(27): 2805–16. PubMed Abstract \| Publisher Full Text\n\nAgis Investigators: The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration.The AGIS Investigators. Am J Ophthalmol. 2000; 130(4): 429–40. PubMed Abstract \| Publisher Full Text\n\nGordon MO, Beiser JA, Brandt JD, et al.: The Ocular Hypertension Treatment Study: baseline factors that predict the onset of primary open-angle glaucoma. Arch Ophthalmol. 2002; 120(6): 714–20. PubMed Abstract \| Publisher Full Text\n\nShankaran S, Laptook AR, Ehrenkranz RA, et al.: Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med. 2005; 353(15): 1574–84. PubMed Abstract \| Publisher Full Text\n\nEarly Treatment Diabetic Retinopathy Study Research Group: Grading diabetic retinopathy from stereoscopic color fundus photographs--an extension of the modified Airlie House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. Ophthalmology. 1991; 98(5 Suppl): 786–806. PubMed Abstract \| Publisher Full Text\n\nSanfilippo PG, Casson RJ, Yazar S, et al.: Review of null hypothesis significance testing in the ophthalmic literature: are most 'significant' P values false positives? Clin Exp Ophthalmol. 2016; 44(1): 52–61. PubMed Abstract \| Publisher Full Text\n\nHiggins JP, Green S, editors: Cochrane handbook for systematic reviews of interventions. John Wiley & Sons; 2011. Reference Source\n\nAlRyalat SA: \"Ophthalmology randomized controlled trials\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/TXEYDX" }	[ { "id": "54690", "date": "21 Oct 2019", "name": "Peter Y Chang", "expertise": [ "Reviewer Expertise Ocular inflammatory diseases", "vitreoretinal surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI applaud the authors' efforts in sifting through the large volume of data in determining the proportion of ophthlamology publications that are cateogorized as RCTs. The authors also found that most of the RCTs concern retinal diseases. This makes sense, as one of the most dramatic therapeutic advancements since early 2000s- not just in Ophthalmology but all of medicine - is the employment of anti-VEGF therapy in the treatment of age-related macular degeneration and diabetic retinopathy. These two conditions, along with glaucoma, are perhaps the 3 most common ophthalmic conditions for which many newer therapeutics are being developed. It is also encouraging to see that the number of RCT published annually in Ophthalmology has generally trended upward, and that they are published in well-regarded peer-reviewed journals. Overall, though, there remains a lack of RCTs in Ophthalmology, with majority of published works being presumably retrospective in nature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "61423", "date": "08 Apr 2020", "name": "Sa'ed H. Zyoud", "expertise": [ "Reviewer Expertise Toxicology", "bibliometric" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to congratulate you on the work done. This helps to analyze the most active researchers in the subject, facilitating future collaborations. I would like to ask some questions that were not very clear for me in the paper.\nWhy did you only use PubMed to survey data?\n\nHow did you consider collaborations?\n\nYou could have done more interesting things, such as a heat map (VOSViewer has a great one and is free to use - but have used it in a previous bibliometric analysis).\n\nBibliometric is an aid in the characterization of a field, but scholarly communication is too complex to explain the history, status, and development of any field of science. Bibliometric has a lot of limitations that should be recognized in order to avoid a simplistic misuse of the indicators and methodologies.\nBorgman, C. L., and Furner, J. (2002). Scholarly communication and bibliometrics. Annu. Rev. Inform. Sci. Technol. 36, 3-72. doi:10.1002/aris.14403601021\nHicks, D., Wouters, P., Waltman, L., Rijcke, S. D., and Rafols, I. (2015). Bibliometrics: the Leiden Manifesto for research metrics. Nature 520, 429-431.2\n\nWithout further comments or questions, I congratulate again the initiative.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1718
https://f1000research.com/articles/8-1717/v1	04 Oct 19	{ "type": "Case Report", "title": "Case Report: Bladder adenocarcinoma: primary or urachal?", "authors": [ "J. Eduardo Tejeda-Mariaca", "Marco Ordoñez-Alcantara", "Aldo Bello-Sedano", "Victor Perez-Cornejo", "J. Antonio Grandez-Urbina", "J. Eduardo Tejeda-Mariaca", "Marco Ordoñez-Alcantara", "Aldo Bello-Sedano", "Victor Perez-Cornejo" ], "abstract": "Background: Bladder adenocarcinoma (AC) is a scarce histological variant and there are few studies on its proper management. No previous case reports present the management of a urachal tumor and the incidental finding of bladder adenocarcinoma. Clinical case: We present the case of a young woman with nonspecific symptoms, who presented with a prior history of dysuria, bladder tenesmus, suprapubic pain and urinary urgency for one year, which had been treated as recurrent urinary tract infection. A partial cystectomy plus extended lymphadenectomy was scheduled. We found a bladder tumor with characteristics of a urachal tumor and the pathological report indicated a primary bladder AC. The patient had a complete recovery at one year of follow-up. Conclusions: A patient can present with a tumor with urachal characteristics; however, the pathology report can show primary AC. The decision to perform partial cystectomy was an appropriate option for the location of this tumor, with optimal surgical results. Still, a long-term follow-up is necessary. More specific management guidelines are required for the treatment of AC.", "keywords": [ "Urinary Bladder Neoplasms", "Adenocarcinoma", "Surgical Pathology" ], "content": "Background\n\nWithin bladder tumors, adenocarcinoma (AC) is a histological variant that represents only 0.5–2% of cases. Its prognosis is the poorest, given that it is diagnosed in more advanced stages due to its rarity. There is little literature about its management and there are no standard treatment guidelines.\n\nIts association with a history of bladder exstrophy, schistosomiasis and chronic bladder irritation has been described. Due to intramural growth, the symptoms occur later in disease progression and AC is diagnosed in more advanced stages, so the prognosis is worse. Only 5% of cases are diagnosed during the initial stages. Hematuria is the most frequent symptom (60–100% of cases), and irritative symptoms and mucosuria are also common (25–80% of cases)1.\n\nBladder AC can be classified as primary or secondary, the latter occurring by direct extension or by metastasis from a distant site like the colon, prostate, endometrium, cervix or breast. Strictly, the urachus is not an intrinsic component of the bladder. However, urachal AC is usually described with bladder tumors because they share pathological and clinical features. Therefore, bladder AC can be classified as urachal AC (10–30%) and non-urachal AC (70–90%).\n\nPrimary bladder AC shows a pure glandular phenotype. It usually arises from the trigone and the posterior wall but can be found anywhere in the bladder. It usually presents as a solitary lesion2. Histologically, it shows several growth patterns: enteric, morphologically identical to its colonic counterpart; or mucinous, with abundant extravasated mucin, including signet ring cells.\n\nUrachal AC develops from the remnant of urachus. It presents as a solitary polypoid mass in the dome of the bladder, although it can be seen anywhere along the anterior midline, and it can affect the Retzius space and the anterior abdominal wall. Microscopically, it is very similar to primary AC, the mucinous variant being the most frequent.\n\nHere, we report an incidental case of a patient with bladder AC treated as urachal AC who presented good oncological results at one year of follow-up. What is unique about this case is that urachal AC tumor management was proposed because of the clinical findings; however, the urachus was ultimately found to be tumor-free.\n\n\nClinical case\n\nThe patient was a 35-year-old mestizo woman, who works as a junior manager, with no clinical history of hematuria, bladder tumors or prior surgical interventions and no family history of bladder tumors. The patient presented to the urology practice with a prior history of dysuria, bladder tenesmus, suprapubic pain and urinary urgency for one year, which had been treated as recurrent urinary tract infection with broad spectrum antibiotics. The patient presented negative cultures; however, the symptoms did not disappear. A timeline of the major timepoints in the patient’s history, diagnosis and treatment is provided (Figure 1).\n\nA physical abdominal and genitourinary exploration was carried out. There were no positive findings upon physical examination and no painful trigger points were found. There were no signs of vulvar irritation or palpable abdominal mass.\n\nA bladder screening ultrasound was performed in order to identify any abnormal structure or urinary retention. During the exam the bladder was full, and a bladder dome mass was noted. A unique, polypoid mass with mucoid characteristics of 4.0cm was found using urethrocytoscopy. A lower abdomen contrasted CT scan was performed and a collection/mass was located on the anterior and superior edge of the bladder of 60 by 40mm, which was cystic and solid (density of 30UH) and had peripheral calcifications (Figure 2). Following this, a transurethral resection was performed. In the transurethral resection pathological report, moderately differentiated muscle invasive mucinous AC was reported. Taking into account these findings, an endoscopy, colonoscopy and mammography were performed, but there was no evidence of tumor in the exams. A solid or cystic mass in the midline with calcifications is considered a major finding indicative of urachal AC and so the diagnosis of urachal AC was proposed.\n\nA bladder dome mass of approximately 5cm was resected.\n\nMobile solitary tumors that are away from the base may potentially benefit from partial cystectomy, so a partial cystectomy plus extended lymphadenectomy was scheduled (Figure 3). There were no pre-intervention considerations. The patient was placed in dorsal decubitus position and spinal anesthesia plus epidural catheter, with bupivacaine hydrochloride at 0.5%, without adrenaline and without preservatives, was administered without any complications.\n\na) Primary adenocarcinoma (AC) and infiltrated muscle layer (M) (blue arrows). b) Tumor-free urachus.\n\nThe surgical intervention was performed by an experienced surgeon without complications. A bladder dome mass of approximately 5cm was resected. In the partial cystectomy pathology report, an invasion of the proximal third of muscle layer was described. Clear surgical margins were reported, and no positive lymphatic nodules were found. There was no evidence of infiltration in the area corresponding to the remnant of urachus. Immunohistochemical analysis showed the tumor tested positive for Cytokeratin 20 (CK20) and Cytokeratin 7 (CK7) that are distributed in epithelia and their neoplasms. However, the test for carcinoembryonic antigen (CEA), which is a marker of colon carcinoma cells, was negative.\n\nThe urachus was tumor-free (Figure 4). However, the bladder layer presented a tumor in its dome without any evidence of secondary AC. Therefore, the final diagnosis was primary bladder AC.\n\nThe patient was discharged 10 days after the surgical intervention. Cephalexin 500mg three times a day was prescribed for five days after discharge. The Foley catheter was removed 14 days after surgery. No complications and no urinary fistula were reported. No chemotherapy was administered. No signs of recurrence were observed during a CT scan and urethrocystoscopy performed after a follow-up period of one year.\n\n\nDiscussion\n\nAlthough the bladder is not a common site of metastasis, secondary AC is more frequent than primary AC. Cancer cells can spread by direct extension or by the hematogenous/lymphatic route. During diagnosis, ultrasound is useful as an initial imaging test; however, CT scanning and MRI provide solid information to determine the extent of the disease, rule out metastases and evaluate if it is potentially resectable. In our case report there was evidence in the CT scan that a collection/mass was located on the anterior and superior edge of the bladder; however, it may have been interesting perform an MRI in order to precisely identify any urachal involvement.\n\nA mass in the midline, solid or cystic, with calcifications is considered a major finding indicative of urachal AC. Cystoscopy and transurethral resection of the tumor confirms the diagnosis. Peritoneal carcinomatosis, as well as peritoneal pseudomyxoma, can be a finding in patients with metastatic disease. The analysis of CEA, CA125 and CA19-9 antigen levels should be carried out, which may be elevated in 40%–60% of these patients. The diagnosis of primary bladder AC should be made only after the exclusion of a secondary AC. Therefore, it is necessary to perform colonoscopy, endoscopy, mammography and colposcopy. The histopathological findings are difficult to use to differentiate between the types of AC and immunohistochemistry has limited utility for the differential diagnosis. The diversity of AC means that cytological preparations are a challenge because immunohistochemistry has limited utility.\n\nThe low frequency of AC and the absence of large studies explain the absence of clearly established therapeutic guidelines. In primary AC, radical cystectomy and dissection of pelvic lymph nodes are the first option. However, mobile solitary tumors that are away from the base may potentially benefit from partial cystectomy1. In urachal AC, partial cystectomy is the standard procedure, with block resection of the bladder dome, urachal ligament, and umbilicus3,4. Lymphadenectomy (LD) is necessary when the incidence of lymph node metastasis in AC is high at the time of diagnosis. LD improves survival, time before recurrence and staging. Therefore, performing extended LD would be the most appropriate option in these patients5.\n\nThe role of chemotherapy is not yet clear. However, some cohort studies have shown benefit in high-risk patients (advanced stage, positive margins, positive nodes). This is based on cisplatin and 5-fluorouracil4. The use of radiotherapy is also not clear in bladder AC. Some studies showed better oncological results with positive nodes and recurrence. Despite this, its advantage in terms of oncological results has not been established with adequate studies. It can be recommended for local control only6.\n\n\nConclusions\n\nA patient can present with a tumor with urachal characteristics, however, the pathology report can show primary AC.\n\nThe decision to perform partial cystectomy was an appropriate option for the location of this tumor, with optimal surgical results. Still, a long-term follow-up is necessary.\n\nMore specific management guidelines are required for AC.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nOtero JR, Ojeda JMD, Betriu GC, et al.: Adenocarcinoma vesical primario: nuestra experiencia. Actas Urol Españolas. 2005; 29(3): 257–60. Publisher Full Text\n\nDadhania V, Czerniak B, Guo CC: Adenocarcinoma of the urinary bladder. Am J Clin Exp Urol. 2015; 3(2): 51–63. PubMed Abstract \| Free Full Text\n\nSiefker-Radtke A: Urachal adenocarcinoma: a clinician's guide for treatment. Semin Oncol. Elsevier Inc.; 2012; 39(5): 619–24. PubMed Abstract \| Publisher Full Text\n\nSzarvas T, Módos O, Niedworok C, et al.: Clinical, prognostic, and therapeutic aspects of urachal carcinoma-A comprehensive review with meta-analysis of 1,010 cases. Urol Oncol. Elsevier; 2016; 34(9): 388–98. PubMed Abstract \| Publisher Full Text\n\nCrozier J, Demkiw S, Lawrentschuk N: Utility of lymphadenectomy following cystectomy for non-urothelial bladder cancer: a systematic review. Minerva Urol Nefrol. 2016; 68(2): 185–91. PubMed Abstract\n\nFlaig TW, Spiess PE, Agarwal N, et al.: NCCN Guidelines Insights: Bladder Cancer, Version 5.2018. J Natl Compr Canc Netw. 2018; 16(9): 1041–53. PubMed Abstract \| Publisher Full Text" }	[ { "id": "59590", "date": "21 Feb 2020", "name": "Orsolya Módos", "expertise": [ "Reviewer Expertise urachal cancer", "urological cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report presents a case of a young woman with adenocarcinoma presented in the bladder. With endoscopic procedures, secondary involvement of the bladder was excluded. Based on imaging findings the presence of urachal carcinoma was supposed, therefore partial cystectomy with extended lymphaedenctomy and the removal of the umbilical remnant and the umbilicus was performed. The pathological examination of the tumor found primer bladder adenocarcinoma.\nThe case report is well written, the train of thought is traceable, but there are some diagnostic tools which could suggest the presence of urachal cancer before open surgical treatment. Therefore I have some minor point to discuss:\n\nSerum tumor markers as CEA, CA-19-9, CA125 and CA-724 can be elevated in urachal cancer.1,2 What was the level of these serum tumor markers before and after partial cystectomy?\n\nHowever, there is no reliable immunohistochemical marker which can distinguish between urachal and primary bladder adenocarcinoma, some of them can suspect the origin of the examined adenocarcinoma. Were immunohistochemical examinations performed of the TURB tumor sample (e.g. AMACR, CK34bE12, GATA3)? This should be mentioned in the manuscript.\n\nHistological examination of partial cystectomy resulted primary bladder adenocarcinoma. What was the TNM-stage of this?\n\nIn the case of primary bladder adenocarcinoma, radical cystectomy is suggested. In this case, urachal cancer was supposed before surgical treatment, therefore partial cystectomy was performed, however the final diagnosis was primary adenocarcinoma. In the future, is radical cystectomy proposed and if so, when?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "65012", "date": "06 Jul 2020", "name": "Marlon Perera", "expertise": [ "Reviewer Expertise Urology", "urooncology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a case report on the management of muscle-invasive adenocarcinoma of the bladder with partial cystectomy. As discussed by the authors, this is not standard or recommended treatment for this disease. This treatment strategy has been well addressed in the literature.\nBackground:\nSchistosomiasis and chronic bladder irritation are associated with squamous cell carcinoma of the bladder (not adenocarcinoma).\n\nCase report:\n\nIt is unclear the duration of followup. It would be useful to know longer-term oncologic outcomes. Specifically, the authors are advocating for partial cystectomy in T2 adenocarcinoma of the bladder without a clear report of the oncologic outcome.\n\nI'm not entirely sure of the novelty of this case and its' suitability for publication.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "67953", "date": "07 Aug 2020", "name": "Oriana Rivera", "expertise": [ "Reviewer Expertise EPIDEMIOLOGY AND PUBLIC HEALTH" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well structured and fulfills one of the educational functions.\n\nThe introduction expresses the reason why it is intended to publish, there is a review of the topic and explains the relevance of the topic.\n\nThe case description has important antecedents, the interrogation, the physical examination, the diagnostic support studies, and the results.\n\nIn the discussion, the authors emphasize why the case is remarkable and explains or clarifies the important aspects and compares themselves with other students.\n\nIn conclusion, the document is suitable for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "67951", "date": "12 Aug 2020", "name": "Alejandro Carvajal Obando", "expertise": [ "Reviewer Expertise General Urology", "Andrology", "MAle Infertility" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is an interesting work and an unusual find in that geography. It is valuable in keeping urologists aware of the possibility of such a differential diagnosis. However, I make several clarifications:\nThe abstract is not clear, it is not understood why the surgical treatment is noted (partial cystectomy) and the full text must be read to understand it\n\nDuring the discussion, they focused little on the pathophysiology and epidemiology of the type of tumor, I think it should be expanded further because they discussed the diagnosis and treatment options.\n\nThe conclusions of the work should not justify the findings of the case, but rather contextualize the analysis of the case\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1717
https://f1000research.com/articles/8-1593/v1	05 Sep 19	{ "type": "Case Report", "title": "Case Report: Massive epistaxis from juvenile angiofibroma in an adolescent with severe haemophilia A", "authors": [ "Jose Florencio F. Lapeña", "Olivia Agnes D. Mejia", "Olivia Agnes D. Mejia" ], "abstract": "Epistaxis may be profuse in individuals with normal bleeding parameters, but in an individual with haemophilia, it may be life-threatening. It is even more dangerous when epistaxis is caused by an undetected concomitant juvenile angiofibroma, and only one such case has been reported in the English literature. We report another case, of an 18-year-old Filipino adolescent with severe haemophilia A who was referred for repeated massive epistaxis. The epistaxis had been attributed to his haemophilia and managed with nasal packing, multiple blood transfusions and Factor VIII administration. After two years of unsuccessful management, nasal endoscopy was performed for the first time, revealing an intranasal mass. Imaging showed a right intranasal vascular tumour supplied mainly by the right sphenopalatine artery. He subsequently underwent preoperative embolization and endoscopic excision of the tumour with Factor VIII transfused pre-, intra-, and post-operatively, and recombinant Factor VII added post-operatively. Final histopathology was consistent with juvenile angiofibroma. There has been no nasal obstruction or recurrence of epistaxis seven years since the surgery. Clinicians should be more meticulous in assessing epistaxis in any patient with a bleeding disorder and investigate more subtle symptoms such as nasal obstruction. Verification of the source by direct visualization and ancillary diagnostic techniques (such as imaging) when indicated should be the standard of care for all patients presenting with epistaxis, whether or not a concomitant bleeding disorder exists. A high index of suspicion for juvenile angiofibroma should be maintained in adolescent males with epistaxis and nasal obstruction.", "keywords": [ "epistaxis", "juvenile angiofibroma", "haemophilia a", "male adolescents", "nasal endoscopy", "nasal surgical procedures", "computed tomography angiography" ], "content": "Introduction\n\nJuvenile angiofibroma (JA) is a benign vascular tumour accounting for 0.5% of all head and neck neoplasms1. It occurs almost exclusively in adolescent males nine to 19-years-old, with a mean age at diagnosis of 15 years2. The clinical presentation involves unilateral epistaxis, nasal obstruction, and an intranasal mass. Epistaxis may be profuse and require nasal packing, vasopressors, antifibrinolytics and transfusions, even in individuals with normal bleeding parameters. However, with haemophilia, such epistaxis is more difficult to control and can be life-threatening. To our knowledge, only one case of JA in a haemophiliac has been reported in the English literature3. We report another case here.\n\n\nCase presentation\n\nAn 18-year-old male Filipino adolescent was referred to the Department of Otorhinolaryngology of the Philippine General Hospital for recurrent epistaxis. Previously diagnosed with severe haemophilia A at age 16, he initially presented with recurrent right nasal congestion and an episode of predominantly right-sided epistaxis described as sudden and profuse, amounting to 1500 ml. At that time, he was admitted to a provincial hospital and received blood and cryoprecipitate transfusions. Following discharge, epistaxis of 100 ml recurred almost daily, requiring nasal packs, repeated hospitalizations of one to two weeks in duration, and transfusions. Cryoprecipitate was often used to control the bleeding since plasma-derived Factor VIII (pFVIII) was seldom available due to shortage of supply and cost. His past history also included hemarthroses and gum bleeding since early childhood, but his symptoms were initially ignored and later only attributed to haemophilia although nasal congestion gradually progressed to obstruction.\n\nAfter two years of such management, nasal endoscopy performed for the first time by a visiting otorhinolaryngologist revealed a right intranasal mass. He was referred to our institution and admitted with an impression of JA (Radkowski IA) and severe haemophilia A. Following admission, he suffered from hypovolemic shock several times due to difficulty in acquiring blood, cryoprecipitate and Factor VIII. With previous Factor VIII Assay levels less than 1%, 1900 units of Factor VIII concentrate were empirically administered (calculated by weight) to raise levels to normal. His condition was compounded by development of Factor VIII antibodies because of previous, repeated cryoprecipitate transfusions in a suboptimal health-care setting. Although his baseline inhibitor titre had been negative, the preoperative inhibitor titre following multiple transfusions with various blood products was positive 3.5 Bethesda units (BU), necessitating pre-, intra- and post-operative transfusion with recombinant Factor VII (rFVIIa) in addition to higher doses of Factor VIII. Unfortunately, rFVIIa only became available post-operatively.\n\nContrast-enhanced computed tomography (CT) scans showed a hyperdense right intranasal mass corroborated by preoperative embolization angiography as an intranasal vascular tumour supplied by the right sphenopalatine artery and internal maxillary artery (IMA) (Figure 1A and 1B). The vast majority (90%) of the blood supply arose from distal sphenopalatine branches of the right IMA, while the remaining 10% came from both ascending pharyngeal arteries (Figure 1B).\n\nA. CECT Scan showing enhancing nasopharyngeal mass (asterisk) and B. Angiography showing vascular tumour (asterisk) supplied by sphenopalatine (black arrowhead) and internal maxillary (white arrowhead) arteries. Adobe Photoshop CC 20.01 release was used to erase identifying patient details, remove pixelated areas from black background, and enhance contrast to sharpen image (applied to entire image).\n\nWithin 24 hours post-embolization, the patient underwent endoscopic surgery under general endotracheal anaesthesia with Sevoflurane. Factor VIII was given before, during, and after surgery, with recombinant Factor VII added post-operatively. Intraoperatively, a fleshy, vascular 4.7 × 3.2 × 2.7 cm mass was seen arising from the right sphenopalatine foramen. The sphenopalatine artery was cauterized and ligated, and the mass was delivered trans-orally (Figure 2A and 2B). Intraoperative blood loss was 300cc and post-operative bleeding was negligible. In total, the patient received 39,500 units of commercially available pFVIII, 24 mg of rFVIIa, 22 units of packed red blood cells (PRBC), 301 units of cryoprecipitate, 1 unit of whole blood and 3 units of fresh frozen plasma (FFP). Final haematoxylin-eosin stained histopathology findings showed endothelium-lined capillaries with absent smooth muscle cells in a fibrous stroma, consistent with JA. The patient was discharged after two months in hospital and has followed up regularly, with no evidence of tumour on nasal endoscopy and no recurrence of nasal obstruction or epistaxis reported by the patient for seven years. He has completed a vocational course at college and is well. Figure 3 summarizes the timeline.\n\nA. Intraoperative endoscopic view of the sphenopalatine artery (black arrowhead) supplying the mass (black asterisk) and B. Gross specimen measuring 4.7 × 3.2 × 2.7 cm. Adobe Photoshop CC 20.01 release was used to erase identifying patient details and sharpen the image (applied to entire image 2B).\n\n\nDiscussion\n\nTo our knowledge, there is only one previous case of JA and concomitant haemophilia in the English literature, twice reported by Ozturk et al. in 19993 and by Celiker et al. in 20044. In their case, the preliminary diagnosis of JA was confirmed by biopsy at a different medical centre, where massive haemorrhage jeopardized the patient’s life. On referral to their institution, preoperative embolization, surgical excision, and adequate Factor VIII replacement saved the patient4.\n\nSimilarly, significant risk to our patient’s life was posed by delayed diagnosis from hasty attribution of epistaxis to haemophilia alone, and not the possibility of a vascular tumour such as JA. Per haemophilia management guidelines, the long history of “spontaneous bleeding into joints or muscles” in our patient corresponded to the baseline Factor VIII assay clotting factor level of “<1 IU/dL or <1% of normal” seen in severe haemophilia5. While recent-onset of bleeding from “mucous membranes in the mouth, gums, nose, and genitourinary tract” was serious, massive bleeding with “neck/throat” involvement was “life-threatening.” This degree of epistaxis should not have been expected in patients with haemophilia A alone, where major bleeding from these areas only occurs 5–10% of the time5. Moreover, the symptom of nasal obstruction was long-overlooked. Unfortunately, two full years passed before the underlying tumour was discovered.\n\nCurrent guidelines5 advise otolaryngologist referral only for “persistent or recurrent” epistaxis, but the emphasis in this recommendation is for control of bleeding only and not to investigate a different underlying cause such as JA. Our experience demonstrates that vascular lesions causing epistaxis may remain undetected when presumptively attributed to pre-existing bleeding disorders and are likely to remain undetected unless sought.\n\nIn conclusion, although guidelines do not mention vascular lesions such as JA, a high index of suspicion should be maintained in adolescent males with epistaxis and nasal obstruction. Clinicians should carefully assess the cause of epistaxis in any patient with a bleeding disorder, and direct visualization of the source should be attempted (and verified by ancillary diagnostic techniques such as imaging when indicated) in all patients with epistaxis, regardless of the presence of a concomitant bleeding disorder.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional data are required.\n\n\nConsent\n\nWritten informed consent for publication of his clinical details and clinical images was obtained from the patient.", "appendix": "Acknowledgements\n\nWe acknowledge Dr. Arsenio Claro A. Cabungcal and Dr. Alzhes R. Buelva for their surgical contributions to patient care, Dr. Cheryl Lyn A. Diez for her expertise in haematology that made the surgery possible, and Mary Angeline R. Bagabaldo for her expert assistance with the deidentification, contrast-improvement, sharpening, labelling, and layout of the Figures.\n\n\nReferences\n\nLund VJ, Stammberger H, Nicolai P, et al.: European position paper on endoscopic management of tumours of the nose, paranasal sinuses and skull base. Rhinol Suppl. 2010; 22: 1–143. PubMed Abstract\n\nNicolai P, Schreiber A, Bolzoni Villaret A: Juvenile angiofibroma: Evolution of management. Int J Pediatr. 2012; 2012: 412545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOztürk MA, Haznedaroğlu IC, Oztürk MH, et al.: Nasopharyngeal angiofibroma in a patient with haemophilia A: a bleeding tumour in a bleeding-prone patient. Haemophilia. 1999; 5(3): 207–8. PubMed Abstract \| Publisher Full Text\n\nCeliker V, Basgul E, Karagoz AH, et al.: Anesthesia in a patient with nasopharyngeal angiofibroma and hemophilia A. J Cardiothorac Vasc Anesth. 2004; 18(6): 819. PubMed Abstract \| Publisher Full Text\n\nSrivastava A, Brewer AK, Mauser-Bunschoten EP, et al.: Guidelines for the management of hemophilia. Haemophilia. 2013; 19(1): e1–47. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53496", "date": "09 Sep 2019", "name": "Alberto Maria Saibene", "expertise": [ "Reviewer Expertise Otolaryngology", "rhinology", "head and neck surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors present a compelling case report where the concomitance of a rare sinonasal vascular tumour, i.e. a juvenile angiofibroma, and haemophilia A delayed diagnosis and complicated clinical management. As the authors correctly demonstrate in the report, a sub-optimal healthcare setting led to haemophilia first diagnosis delay and, foremost, delayed the identification of the neoplasm despite the recurrence of profuse bleeding. The article is well written, both by a grammar and literary standpoint, and present all clinical information on diagnosis and management in a complete fashion. The clinical management is sound and clinical decisions are consistent with current guidelines and good clinical practice. The article could be publishable in this present form, but I'd like to point out a couple of ideas that might add some teaching relevance to the article:\nFirst of all, it might be worth mentioning that in good rhinologic practice, performing sinonasal tumors biopsies without adequate imaging can result to harmful or fatal incidents. Had the clinicians decided to perform a biopsy in an outpatient setting, a massive epistaxis could have led to serious complications for this patient, without helping further clinical decisions. This is the case of the - correctly cited - case report already published by Ozturk.\n\nSecondly, while it is true that haemophilia guidelines do not advise routine evaluation for epistaxis, on the other hand epistaxis guidelines1 do recommend for a thorough evaluation of the patient in order to identify the bleeding source in all case, starting with anterior rhinoscopy and escalating to nasal endoscopy whenever the source of bleeding cannot be easily identified. Therefore, it is worth mentioning that correct management of all epistaxis cases required identification of the bleeding source.\n\nLast, while the CT scan the authors provided allows a good depiction of the clinical picture, it would be interesting to see whether the CT scans showed enlargement of the sphenopalatine foramen. While such enlargement is not constant in all JA patients, an enlargement >3mm in presence of a unilateral sinonasal mass can point the diagnosis towards JA.\nIn conclusion, this is an extremely interesting article well deserving publication, with an interesting teaching value that could be further increased with some more information as above stated.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "4944", "date": "02 Oct 2019", "name": "Jose Florencio Lapeña", "role": "Author Response", "response": "We thank the reviewer for his excellent review and valuable comments and recommendations: 1. \"First of all, it might be worth mentioning that in good rhinologic practice, performing sinonasal tumors biopsies without adequate imaging can result to harmful or fatal incidents. Had the clinicians decided to perform a biopsy in an outpatient setting, a massive epistaxis could have led to serious complications for this patient, without helping further clinical decisions. This is the case of the - correctly cited - case report already published by Ozturk.\"Yes, indeed. A biopsy in the case of our patient would have been catastrophic (and we have witnessed this happen to patients of other unsuspecting physicians). Any nasal mass suspicious for angiofibroma should not be manipulated (unless in the operating theatre under double set-up). However, we felt that mentioning this important management caveat would detract from the main message of initial diagnosis necessitating visualising the bleeding source (directed toward general practitioners, primary care providers, family physicians, and paediatricians), and opted to reiterate the point in our response to the review instead.2. \"Secondly, while it is true that haemophilia guidelines do not advise routine evaluation for epistaxis, on the other hand epistaxis guidelines (1) do recommend for a thorough evaluation of the patient in order to identify the bleeding source in all case, starting with anterior rhinoscopy and escalating to nasal endoscopy whenever the source of bleeding cannot be easily identified. Therefore, it is worth mentioning that correct management of all epistaxis cases required identification of the bleeding source.\"We have added the statement \"On the other hand, epistaxis guidelines(6) do recommend 'anterior rhinoscopy with headlight following nasal decongestion' escalating to 'rigid endoscopy or microscopy … where anterior rhinoscopy fails to identify a bleeding point.' \" to the Discussion, for which the additional reference (6) provided by the reviewer was cited:National ENT Trainee Research Network. The British Rhinological Society multidisciplinary consensus recommendations on the hospital management of epistaxis. J Laryngol Otol. 2017;131(12):1142-1156. 10.1017/S0022215117002018.3. \"Last, while the CT scan the authors provided allows a good depiction of the clinical picture, it would be interesting to see whether the CT scans showed enlargement of the sphenopalatine foramen. While such enlargement is not constant in all JA patients, an enlargement >3mm in presence of a unilateral sinonasal mass can point the diagnosis towards JA.\"We totally agree that the educational value of the article can be enhanced by mentioning that \"in the presence of a unilateral sinonasal mass,\" such an enlargement of the sphenopalatine foramen \"can point to the diagnosis of JA.\" However (and in relation to the first point above), we opted to maintain the primary focus of our discussion and perhaps encourage our non-otolaryngologist colleagues to consider urgent referrals to ENT surgeons following initial diagnosis in such cases." } ] }, { "id": "53498", "date": "23 Sep 2019", "name": "J. Paul Moxham", "expertise": [ "Reviewer Expertise I am an Academic Pediatric Otolaryngologist at BC Children's Hospital and an Associate Clinical Professor of Surgery at the University of British Columbia in Vancouver", "Canada. I am a member of the Triological Society and the American Society of Pediatric Otolaryngology. My main areas of interest are bone grow factors in craniofacial models." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent and well written case report about a young adult with a hematologic disorder and a coexisting angiofibroma. It delves into the difficulties this case presents to the treating surgeon, reviews the relevant literature (of which there is only one previous report), and reminds us that just because someone has a bleeding disorder does not mean they cannot also have a rare vascular tumour.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "4945", "date": "02 Oct 2019", "name": "Jose Florencio Lapeña", "role": "Author Response", "response": "We thank the reviewer for his clear and concise review, recapitulating the main theme and \"take-home\" message of our case report \"that just because someone has a bleeding disorder does not mean they cannot also have a rare vascular tumour.\"" } ] }, { "id": "53497", "date": "24 Sep 2019", "name": "Robert G. Berkowitz", "expertise": [ "Reviewer Expertise paediatric otolaryngology and airway disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a case of juvenile angiofibroma (JA) occurring in a patient with haemophilia, where severe epistaxes were ascribed to the bleeding disorder and no underlying cause was sought for a period of two years. Following transfer to the author's institution, the JA was managed successfully by surgery with pre-operative embolisation and optimisation of haemophila therapy. There were no surgical complications and the patient has remained symptom free after long term follow-up.\n\nThis represents only the second reported case of JA occurring in a patient with haemophilia. The report underscores the importance of considering a separate explanation for epistaxis in a patient with an underlying coagulopathy, and particularly where there are other symptoms suggestive of intra-nasal pathology.\nThe article is well written and carries a clear message. Minor points for the authors to consider providing further information are:\n\nIt is implied, but not actually stated, that during the two year period where the JA was overlooked, the patient's haemophilia was poorly controlled (which presumably contributed to overlooking the diagnosis of JA). Is this the case?\n\nIf treatment proceeded uneventfully, why did the patient require hospitalisation for a period of 2 months?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "4943", "date": "02 Oct 2019", "name": "Jose Florencio Lapeña", "role": "Author Response", "response": "We thank the reviewer for his kind review and for raising minor (but important) points for consideration:1. \"It is implied, but not actually stated, that during the two year period where the JA was overlooked, the patient's haemophilia was poorly controlled (which presumably contributed to overlooking the diagnosis of JA). Is this the case?\"Yes, \"during the two year period where the JA was overlooked, the patient's haemophilia was poorly controlled (which presumably contributed to overlooking the diagnosis of JA).\" We were hoping the first paragraph conveyed a sense of this poor control without being overly dramatic. As mentioned in the Case presentation, this can be attributed to inadequate factor VIII replacement therapy (\"seldom available due to shortage of supply and cost\"). Indeed, \"this makes the case even more interesting, and highlights that not only when there is a known coagulopathy present, and even when it is not being adequately treated, a possible intra-nasal cause should still be considered.\" 2. \"If treatment proceeded uneventfully, why did the patient require hospitalisation for a period of 2 months?\"I hope we did not give the impression that \"treatment proceeded uneventfully.\" Our \"patient require(d) hospitalisation for a period of 2 months\" because of a very stormy preoperative course wherein \"he suffered from hypovolemic shock several times due to difficulty in acquiring blood\" and blood products, and \"his condition was compounded by development of Factor VIII antibodies.\" He also \"received 39,500 units of commercially available pFVIII, 24 mg of rFVIIa, 22 units of packed red blood cells (PRBC), 301 units of cryoprecipitate, 1 unit of whole blood and 3 units of fresh frozen plasma (FFP).\" We have revised the Case presentation by adding the statement \"Post-operative recovery was uneventful and the patient was discharged within a week of surgery (after two months in hospital).\" to clarify that the prolonged hospitalisation was due to the stormy pre-operative course." } ] } ]	1	https://f1000research.com/articles/8-1593
https://f1000research.com/articles/8-417/v1	09 Apr 19	{ "type": "Research Article", "title": "Evaluation of the geriatric curriculum implemented at Shiraz University of Medical Sciences, Iran, since 2017: A qualitative study", "authors": [ "Faezeh Jaafari", "Somayeh Delavari", "Leila Bazrafkan", "Faezeh Jaafari", "Somayeh Delavari" ], "abstract": "Background: Recently, there has been an increase in life expectancy due to improvements in nutrition, health, and sanitation. The aim of this study was to evaluate the geriatric curriculum in the field of general medicine at Shiraz University of Medical Sciences (SUMS), Iran to improve the quality of services provided to this population in the community. Methods: This was a qualitative study. Six educational hospitals and ambulatory centers of Shiraz University of Medical Sciences participated in this study. Within these centers, 15 medical education faculty members and educational experts, 6 medical students, 6 elderly patients and 6 nurses working in the university related to the geriatric field were selected using purposive sampling. Data were gathered through semi-structured interviews, focus group discussion and field observations in the teaching hospital and ambulatory setting of SUMS from June 2017 to May 2018. Based on the qualitative research, the data underwent conventional content analysis and the main themes were developed from this. Results: Three main themes were extracted from the data: effective clinical education, geriatrics curriculum challenges and promotion strategies for geriatric medicine. Subcategories that emerged were a competent curriculum teacher, a challenging program, management of resources, promotion of the program, and the revision required in the curriculum, which were related to other concepts and described in the real-world situation of the geriatric curriculum in the university, as observed in field observations. Conclusions: This study identified three concepts as main themes that can be used to explain how to implement a geriatric curriculum in a medical university. The main contributing factor to different views of the participants was identified as the revision required to the curriculum for integrative care in a geriatric patient. This should be taken into consideration while planning any programs and decisions aimed at education of medical students on this topic.", "keywords": [ "Geriatric Curriculum", "Evaluation", "Qualitative Research", "General Medicine" ], "content": "Introduction\n\nOne of the issues that the medical profession has to face is prioritizing elderly care, as the number of older people suffering chronic illness and multiple comorbidities is on the rise1. For example, in Ireland research shows that individuals aged 65 years and over make up 11.6% of the population in 2011, which is believed to reach 22% by 20412, while in Australia, it is predicted that the number of individuals aged 65 years and over will increase from 13% in 2007 to 23% in 20611. Aging population is on increase both in developed and developing countries3. According to evidence4, individuals over 65 years old make up nearly two thirds (65%) of those admitted to hospital in the UK4,5; also, studies show that 34–59% of individuals over 75 years old take five or more drugs according to their medical backgrounds6. Most hospital beds are occupied by older patients and they have a lengthy stay (more than 30 days)3, functional decline, high re-admission rates, falls, and institutionalization2.\n\nFollowing the rise in life expectancy and medical advances, multi-morbidity and poly-pharmacy has increased recently; this trend has been very significant in the elderly6. Hence, the medical profession needs to handle these patients effectively1,7,8. However, research conducted in the UK shows that medical undergraduate students have not been trained for treatment of these patients sufficiently, and it was also reported that less than two weeks of a five-year educational program had been dedicated to elderly health care1,9. In Australia, medical students spend 78% of their clinical placement time in hospitals; however, only 0.5% this time is dedicated to residential aged care facilities1. The number of geriatric fellowship-trained physicians (geriatricians), who provide appropriate care for older adults, is not expected to meet the needs of a growing aging population. As a result, many older adults will rely on general physicians for their care10.\n\nThe main challenge to which researchers and planners of community and health services in the twenty-first century face is a better quality of life. To reach this end, geriatric medicine is recommended to be included in the curriculum of medicine.\n\nA specific geriatrics curriculum has been designed and implemented in Shiraz University of Medical Sciences, Iran since June 2017. The purpose of this study is to evaluate this curriculum. Educational evaluation aims to determine the effectiveness, quality of a program, processes, goals and the curriculum itself11. Today, due to the significant changes that have emerged in educational systems, evaluation is one of the most widespread and important components of curriculums through which we can find out the achievement levels of specific goals of the curriculum and also deficiencies in its design. If necessary, the educational activities can then be improved or revised12,13. Using curriculum evaluation as a determinant of economic, cultural, social and educational development is very common12. However, in evaluation of education, quantitative approaches which contribute to understanding the educational process are usually used; however, the main goal of educational evaluation is improvement of the conditions of educational contexts, and this cannot be assessed using quantitative research14.\n\nSince Shiraz University of Medical Sciences designed and implemented the geriatrics curriculum from 2017 the aim of this study was qualitative evaluation of this curriculum using a content analysis method. Furthermore, dissatisfaction of graduates, faculty members and students to the curriculum and concerns of educational staff about general medicine educators were also assessed in this study.\n\n\nMethods\n\nSince the main objective of this study was to evaluate the geriatrics curriculum, a content analysis approach was applied for data collection. Therefore, the present study is a qualitative research using content analysis which focuses on systematic human experiences and human science paradigms15. In fact, this method attempts to clarify the structure or nature of an experience in order to describe a phenomenon correctly16. Therefore, in this study we used the participants’ experiences, perceptions and interpretations of geriatric issues and the associated problems, aiming to detect new perspectives to be used in educating medical students in this regard.\n\nIn the present study, the settings for the delivery of medical services related to geriatric medicine, including clinics, hospitals and public and teaching centers, in Shiraz were selected as the research environment.\n\nIn qualitative research, the participants are selected based on purposive sampling so that the individuals selected have experience of the topic being assessed17 and their experiences serve as the data18.\n\nFor this reason, in this study the participants were selected from the medical students of various levels in clinics, clinical professors involved in the geriatrics curriculum, elderly patients, clinical nurses and key informants who were involved in curriculum planning and needs analysis.\n\nThese participants were introduced to the education development center of the university. At first, they were selected by purposive sampling to encompass a maximum variation in work experience, and field of experience. Sampling was continued to data saturation, resulting in selection of six medical students undergoing training courses in hospitals and teaching clinics of Shiraz University of Medical Science, six elderly patients (four admitted patients, and two outpatients), 15 faculty members and health education experts, and five employed nurses.\n\nThree observation methods were used in the study for data collection: observation with no participants (field observations), and semi-structured interviews and focus groups with participants.\n\nSemi-structured interviews. At first, the interviews started with a general question; however, in the process of the interviews more specific questions were asked based on initial interviews and the development of the main themes alongside the research purposes. After the general question, exploratory and more in-depth questions were asked based on the participants’ responses. The interviews were audio recorded and downloaded verbatim, and key points were noted. Each interview lasted for 50 to 75 minutes. Any ambiguous responses during interviews were followed up in order to be clarified, or if clarification was required after the interviews were finished, the participants were asked to be interviewed again in order to clarify the point and delve further into the issue.\n\nField observations. The observations were also made in the field determined and the evidence of curriculum and individuals and training staff’s interaction with each other and the elderly was considered.\n\nFocus group discussions. A focus group session was held after interviews by participation of representatives from educational groups in two meetings of educational centers. The participants’ statements were completely recorded and noted. All of the interviews and observations and the results of the focus group were typed and analyzed.\n\nA written invitation was sent to all selected participants. A conference room of a study center with a U-shape table was considered for the meeting and the participants were reminded to take part in the session the day before. In the first meeting, a central group discussion began with presentation of a 5 minute movie of clinical subjects, then the subjects were discussed by the participants. In the second session, the previous discussion was continued after a summary of the first day discussion was provided. The discussions were recorded. In order to prevent data bias, the data were reviewed several times and noted by two researchers (L.B & F.J). Focus group questions can be found in Supplementary File 1.\n\nConventional content analysis was used to analyze the data collected. Only the data gathered from the focus group and interviews through meaning association were analyzed and we didn’t used the data from the direct observations. Data analysis began reading transcripts repeatedly until a complete overview was achieved19. One of the researchers (FJ) based on his perception and understanding of the text, wrote an initial analysis in order to create preconditions for emerging codes and themes. The themes that emerged were categorized based on their similarities and differences. This categorization was performed by organization and categorization of codes in meaningful clusters. Subsequently, considering the quality of the relationship between subcategories, the researchers could reduce the categories by combining and organizing subcategories.\n\nIn order to increase the reliability, revision of the themes was done in two stages, one after completion of 10–50% of categorization and the other at the end of categorization. Then the researcher (FJ) prepared a report according to the themes identified for the requirements, problems and strategies needed to help elderly health care, which was arranged as a table and discussed and reviewed by the participants in the focus group meeting.\n\nThis study was approved by the Ethics Committee of Shiraz University of Medical Sciences (approval number IR.sums.med.rec.1396.s316). In addition, verbal and written information about the study (the purpose of study, how to cooperate, advantages and disadvantages of participating in research and data records, the role of researchers and participants, and optional participation in the study were presented to the participants, and participants provided written consent to participate.\n\nInformation including name, interview tapes and texts were kept confidential and a specific code was used instead of the participants’ names. Participants were ensured about the confidentiality of their conversations. The researcher provided an opportunity for participants to inform the researcher about withdrawal of their participation at any of the research stages by giving the phone number and e-mail of the lead investigator. Participants were ensured that if they requested, their results would be presented to them in a group.\n\n\nResults\n\nThe results include key terms that were categorized into different levels. From the analysis, 428 initial codes were extracted. The codes were then categorized into six categorizes and three themes based on similarity and analogy. The main themes of the curriculum include: effective clinical education, geriatrics curriculum challenges and promotion strategies for elderly medicine (Table 1).\n\nHaving a curriculum and the authority’s supervision on its implementation is the first stage of effective education. Meanwhile, flexible planning in performance, concept-oriented evaluation, continuous emphasis by professors, student preparation after entering the department, appropriate relations between the student and professor, content coverage of elderly headings, support and supervision at higher levels for the description of effective teaching have been used.\n\n“Training at the patient’s bedside for all cases such as medical care for the elderly requires planning. However, the quality of the program and whether the professor is acquainted with the issue is very important. Having an elderly program reminds me, the professor, to have more emphasis on the round or training.” (Contributor #3)\n\nThe research findings indicate that all contributors of the study somehow experienced model training and a range of appropriate models for training. The professional behavior of the professor are among contributors’ experiences in this realm. A student contributor stated:\n\n“When I see professor …. behavior, and how respective he/she is towards the elderly, always calling them mother, father…. we have a duty to fulfill for you, in fact, I as a student am reminded that I should have the same attitude towards the elderly in the future” (Contributor #11)\n\nIn expressing the participants’ experience, effective clinical education is one of the most important and extensive issues that emerged. Most of the participants had experienced one or more points of geriatrics curriculum practically and emphasized the importance of paying attention to the geriatric population. This theme was composed of two subthemes: effective curriculum and competent training staff.\n\n- Effective curriculum: flexible practical curriculum, problem-center evaluation, repeated emphasis on professors, preparation of students at the time of department admission, appropriate ratio of professors to students, coverage of the topics, support and supervision at high levels\n\n- Competent teaching staff: high potential of professors in education, appropriate patterns for education, professional behavior of professors.\n\nAccording to the contributors’ experiences, a structured program and accurate explanations of students’ duties are effective in education at all levels. Problems arise due to lack of related authorities’ awareness regarding new programs and their goals. Based on the experience of contributors, programs remain neglected because they are not evaluated and adequately presented and some people’s standpoint in this regard is due to their lack of information about the philosophy and goals of the program. One of the administrators states in this regard:\n\n“I was not aware of the medical care for elderly program and this was because there was no opportunity to think about the curriculum of the general medicine course. It is not right to have all types of expectations from the professor and expect everything to go well…” (contributor #1)\n\nLack of facilities and problems of the building in training hospitals are among common problems stated by all contributors of this study. Problems of the building and management structure of the training hospital along with problems of numerous students in addition to lack of educational facilities, such as hospital capacity, hinders the administration of educational programs. Thus, this program is carried out such that students come to the patient’s bedside in big groups, and this not only improves services and elderly’s satisfaction, but also is effective in the student’s educational quality. Regarding the facilities, one of the professors stated:\n\n“… the hospital is very non-standard, I myself slipped a few times on the stairs and fell down, now if this happens for an elderly what will come upon them? What will remain of them… first you should think of doing something about this devastating hospital…” (Contributing professor #15)\n\nor another professor on a similar note:\n\n“… the atmosphere and environment should be prepared for training, if the attitude of everyone with the elderly is degrading, what the professor says alone is not sufficient in order for the students’ perception to change in regard to the elderly patients….” (Contributor #5)\n\nChallenges of the geriatrics curriculum were also composed of two subthemes: curriculum challenges and management and facility challenges. Each of these subthemes consisted of subcategories (Table 2).\n\nDeveloping a guideline will facilitate a number of educational problems. However, developing a guideline included a range of requests, from developing a guide for taking care of the elderly to physicians and also insurance.\n\n“Insurances need guidelines themselves, all of our rehabilitation and healthcare demands will be facing problems. Thus, the only solution to carrying out guidelines accurately is to have more powerful insurances” (Contributing professor #2)\n\nEducating society, families and the elderly are among facilitating programs in carrying out medical training for the elderly and modifying the structure of elderly healthcare.\n\n“You must educate them regarding how to react to our problems. It’s not only related to medicine. All individuals should become aware of laws and science in order to have appropriate behavior and adequately care for the elderly” (Contributor # 9)\n\nIn regards to modifying the structure, contributors suggested issues such as modifying processes of providing healthcare services, determining service priorities given to the elderly, enhancing nursing services, elderly welfare facilities, designing software to help the elderly, adequate facilities in clinics and setting up screening for the elderly. In this regard, one of the contributors stated that:\n\n“Society should be prepared to deal with problems, we are not ready, what services exist to provide healthcare to the elderly? If the nurses are not trained about this technology, how can they provide adequate services in today’s world?” (Contributor #12)\n\nAnother of the contributors says in this regard:\n\n“In the coming years we will be facing an outburst of elderly people; we have insufficient hospitals and healthcare centers to respond to these future needs. If so, how are we to take care of the elderly who cannot benefit from family care? Care should be given in a place where their dignity is maintained.” (Contributor #6)\n\nConsequently, the participants consider the challenges of geriatrics able to be reformed and provided some approaches to remedy this situation. According to the results of this study, these approaches are divided into two categories: facilitating geriatrics curriculum and structural modification (Table 3).\n\n\nDiscussion\n\nIn recent decades, with the growth in the elderly population, those aged 65 and older, constituting a larger portion of the population, geriatrics fellowship programs have been developed and physicians are trained so that they can provide specialized care for the elderly. The medical education system requires accredited geriatrics programs to evaluate the efficacy of the trainings provided20.\n\nThis study evaluated the geriatrics curriculum implemented at the Shiraz University of Medical Sciences qualitatively using content analysis.\n\nThe themes that emerged from participants’ input in this study included “effective clinical teaching”, “challenges of geriatrics curriculum” and the approaches for improving geriatrics, which explained implementation of geriatrics in general medicine, such as strength points (opportunities, challenges) and practical approaches and improving geriatrics at university levels. Almost all participants in the study expressed and experienced one or more indicators of clinical teaching. This means that at present, geriatrics instruction at Shiraz University of Medical Sciences is implementing the above-mentioned points. In this regard, there are several studies that improved geriatrics curriculum by emphasizing the mentioned items21,22.\n\nSapieno et al 2007 showed that the vertical integration program can improve knowledge and attitudes of medical students towards geriatrics23. Also Denson et al. (2016) used a group teaching method for 15 courses of residency and fellowship and caused more effective teaching24. According to the experiences of participants in the present study, the role of teaching staff and professors’ competency are also critical in implementing geriatrics teachings in Shiraz University of Medical Sciences. Clinical professors are one of the three sides of the clinical teaching triangle and their input is essential in clinical teaching. Various studies have emphasized the importance of using the professors as role models in clinical teaching, especially in geriatrics25,26. Despite the fact that the importance of physician-patient communication and attention to teaching it is known for everybody, in the present study most of the participants mentioned some weaknesses in the communication skills.\n\nThe study of Bazrafkan et al. is compatible with these results and shows that the main reason of complaint from physicians is lack of communication skills and showed the effect of teaching communication skills on patient’s satisfaction27.\n\nThe quality of the educational environment, unsafe and non-standard care environment, weakness of welfare in departments, lack of examination room and classroom in clinical departments, facing complications of work, special conditions of older patient, unprofessional behaviors of hospital staff, lack of resources and facilities and management of human relations were among the issues that were mentioned as the challenges of geriatrics curriculum and causing dissatisfaction of students, professors and elderly patients. In this regard Baker et al. used patient communication to evaluate teaching hospital care in Ankara, Turkey. The results showed that none of the hospitals evaluated could satisfy patients’ expectations28.\n\nInter-sectional collaboration in different health sections can provide a basis for proper care of elderly patients. Therefore an inter-professional curriculum can increase this interaction and increase student’s interest in geriatrics. However, participating students in the present study didn’t separate geriatrics from general medicine. Unfortunately, in some cases they consider addressing this issue a waste of time and explained that elderly patients have stereotypical problems and no specific teaching point exists in geriatrics. These findings are similar to those found by Maybom et al. (2015). In their study, about the effect of hidden curriculum in elderly care, the authors came to the conclusion that the students don’t consider geriatrics and its problems as a challenging and instructive issue; they consider elderly behaviors stereotypical and unbearable29. Therefore, the clarity of curriculum and attention to its aspects and components, such as the goals and methods of students’ evaluation, affects the outcome of instruction; accordingly, if the students are not provided with the compiled curriculum and they are not informed about the expectations, they are unlikely to reach the teaching goals. Furthermore, student-centered and problem-centered strategies are successful in medical education. Based on the current findings and other studies confirm this result that problem-centered education can satisfy the student and cause motivation and reinforcement of their perspectives30,31.\n\nUnderstanding elderly patient’s problems and considering these clinical problems in different discipline curricula will lead students to gain enough competence in order to care for the elderly in the future. The present study shows that the medical students don’t have enough competency in geriatrics. Most of the participants’ responses related to the second theme that is challenges of implementing geriatrics, which are specialist and fellowship views that can’t meet the needs of all elderly patients. In addition, the findings confirm that the geriatrics curriculum is required in order to integrate elderly care services. This finding confirms other studies that emphasize inter-professional and integrated care32.\n\nThe third theme of this study was to find improvement strategies for the geriatrics curriculum. For example, planning and revision of existing curriculum and reforming the proposed structure. Along with the results of this study, the study of Tian et al shows the importance of considering prospects and planning in order to provide elderly welfare and reduce future problems of this community33.\n\nDespite different cultures, most of the obtained data from present study overlaps with other studies in this context; other studies referred to the evolution of education to adapt with the needs of elderly patients to realities of telemedicine, telenursing and online teaching33,34. Therefore, this shows that the view of health care providers of elderly patients expressed their readiness for reform in geriatrics teaching despite existing problems. In the study of Castel et al the importance of awareness and teaching was highlighted for elderly patients. The findings of that study reveal that families’ responsibility for providing elderly patients with health care is a well-established concept in the health care system and it is essential that the authorities have a plan for this service and help families35.\n\n\nConclusion\n\nThis study evaluated the geriatric curriculum that was implemented at Shiraz University of Medical Sciences in 2016. According to the findings of this study, the geriatrics curriculum has been successful practically, which was expressed by the experiences of the participants. The challenges of this curriculum that emerged were planning and management of resources, which resulted in incompetent teaching and consequently incompetent graduates. Proposing reform strategies can improve teaching in this field and improve students’ competency for general health of elderly patients. Considering common diseases of old age and taking them into account in the curriculum as integrated and comprehensive health care and ease of access to services is necessary for treatment of elderly patients. Using professors, students and elderly patients’ experiences in curriculum reform are of particular importance and can provide comprehensive information to providers and decision makers of health care systems for future planning.\n\n\nEthical statement\n\nThis study was approved by the Ethics committee of Shiraz University of Medical Sciences (approval number, IR.sums.med.rec.1396.s316). Informed written consent to participate was obtained from all participants. The participants took part in the study voluntarily and their information remained confidential.\n\n\nData availability\n\nTranscripts of the focus group discussions (in Persian) are available on request from the corresponding author (bazrafcan@gmail.com).", "appendix": "Grant information\n\nAll of the funds of the present study were provided by the Vice Chancellor of Research of Shiraz University of Medical Sciences.\n\nThe funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis study is obtained from the thesis of Faezeh Jaafari with thesis no 8409 approved by Shiraz University of Medical Sciences. The authors appreciate the Shiraz University of Medical Sciences for financial support of the project, and who helped in the implementation of this project. Also the authors really appreciate those who have cooperated in translating and editing the article.\n\n\nSupplementary material\n\nSupplementary File 1: Focus group protocol.\n\nClick here to access the data\n\n\nReferences\n\nAnnear MJ, Lea E, Lo A, et al.: Encountering aged care: a mixed methods investigation of medical students’ clinical placement experiences. BMC Geriatr. 2016; 16(1): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCullagh R, O’Connell E, O’Meara S, et al.: A study protocol of a randomised controlled trial to measure the effects of an augmented prescribed exercise programme (APEP) for frail older medical patients in the acute setting. BMC Geriatr. 2016; 16(1): 79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarsden E, Taylor A, Wallis M, et al.: A structure, process and outcome evaluation of the Geriatric Emergency Department Intervention model of care: a study protocol. BMC Geriatr. 2017; 17(1): 76. 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Simulation and Modeling Methodologies, Technologies and Applications (SIMULTECH), 2014 International Conference on. 2014; IEEE. Publisher Full Text\n\nDarch J, Baillie L, Gillison F: Nurses as role models in health promotion: a concept analysis. Br J Nurs. 2017; 26(17): 982–8. PubMed Abstract \| Publisher Full Text\n\nBazrafkan L, Shokrpour N, Tabeie S: A survey of patients’ complaints against physicians in a five year period in Fars province: implication for medical education. J Med Educ. 2008; 12(1,2). Reference Source\n\nBakar C, Seval Akgün HS, Al Assaf AF: The role of expectations in patient assessments of hospital care: an example from a university hospital network, Turkey. Int J Health Care Qual Assur. 2008; 21(4): 343–55. PubMed Abstract \| Publisher Full Text\n\nMeiboom A, Diedrich C, Vries HD, et al.: The hidden curriculum of the medical care for elderly patients in medical education: a qualitative study. Gerontol Geriatr Educ. 2015; 36(1): 30–44. PubMed Abstract \| Publisher Full Text\n\nRamnanan CJ, Pound LD: Advances in medical education and practice: student perceptions of the flipped classroom. Adv Med Educ Pract. 2017; 8: 63–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnider T, Melady D, Costa AP: A national survey of Canadian emergency medicine residents’ comfort with geriatric emergency medicine. CJEM. 2017; 19(1): 9–17. PubMed Abstract \| Publisher Full Text\n\nShield RR, Farrell TW, Campbell SE, et al.: Professional development and exposure to geriatrics: Medical student perspectives from narrative journals. Gerontol Geriatr Educ. 2015; 36(2): 144–60. PubMed Abstract \| Publisher Full Text\n\nTian Q: Intergeneration social support affects the subjective well-being of the elderly: Mediator roles of self-esteem and loneliness. J Health Psychol. 2016; 21(6): 1137–44. PubMed Abstract \| Publisher Full Text\n\nMcCrystle SW, Murray LM, Pinheiro SO: Designing a learner‐centered geriatrics curriculum for multilevel medical learners. J Am Geriatr Soc. 2010; 58(1): 142–51. PubMed Abstract \| Publisher Full Text\n\nCastel A, Lluch C, Ribas J, et al.: Effects of a cognitive stimulation program on psychological well-being in a sample of elderly long-term care hospital inpatients. Aging Ment Health. 2017; 21(1): 88–94. PubMed Abstract \| Publisher Full Text" }	[ { "id": "46950", "date": "17 Jun 2019", "name": "Syed Jaffar Abbas Zaidi", "expertise": [ "Reviewer Expertise Qualitative research. Basic sciences research. Clnical sciences research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article has discussed the evaluation and feedback of geriatrics curriculum at Shiraz University of Medical Sciences. This article is based on thesis work and has been translated from Persian.\n\nThe authors have not cited a landmark article by on the evaluation of geriatrics curriculum at 40 US Medical schools and its citation has been added for reference purposes (Anderson et al., 20041). Furthermore, regional articles relating to qualitative evaluation of geriatric curriculum have not been referenced.\n\nThe article suffers from verbosity, unnecessary repetition of words and inherent problems associated with literal translation. It needs to be edited preferably by a native English speaker so that its writing is more coherent and cohesive. There are many grammar mistakes in this article and I suggest the authors to use software to check grammar such as Grammarly to review this article. Please see my annotated copy of the article here, where I have highlighted and commented on some of the sentences of the article that I have paraphrased.\n\nThe authors have not discussed how they have categorized the data into themes. Whether they have software such as NVivo or have they done it manually?\n\nThe authors have not mentioned the five steps of analysis of themes that include familiarization, identification of a thematic framework, indexing, charting, followed by mapping and interpretation.\n\nThe authors have not suggested how curricular reforms can address the lack of integration of geriatric curriculum into the current general medicine curriculum. No details of field observations and their findings have been included in this article.\n\nOne of the key features of curricular evaluation is social responsibility and social accountability and the authors have not mentioned these terminologies anywhere in this article.\n\nThe words “problems of the building & hospital capacity” are not appropriate and should be replaced by “infrastructure problems”.\n\nThe words “Intersection collaboration in different health sections” should be replaced with “Interprofessional education & collaboration”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4857", "date": "01 Oct 2019", "name": "somayeh delavari", "role": "Author Response", "response": "Response to comment 1: Thank you for your comment. In this article, we needed to consult a bilingual native speaker to help us in using the real concepts in the manuscript. Certainly, we consulted a native speaker in Iran, Prof. Nasrin Shokrpour, the chief editor and consultant of English articles in Shiraz University of Medical University and finally check grammar such as Grammarly.Response to comment 2: Data analysis was done manually.Response to comment 3: Using your valuable comment, we revised the methodological issues and the data analysis sections. Conventional content analysis was used to analyze the data collected.Response to comment 4: Your suggestion was completely right and done and add table 2.Response to comment 5: The social responsibility and social accountability were added to the article, based on your valuable comment.Response to comment 6: Your suggestion was completely right and done.Response to comment 7: Your suggestion was completely right.Thank you for your attention to this manuscript and valuable comments. We hope the new version meets your expectations." }, { "c_id": "4903", "date": "01 Oct 2019", "name": "somayeh delavari", "role": "Author Response", "response": "In this new version of the manuscript (Version 2), we needed to consult a bilingual native speaker to help us in using the real concepts in the manuscript. Certainly, we consulted a native speaker in Iran, Prof. Nasrin Shokrpour, the chief editor and consultant of English articles in Shiraz University of Medical University and finally check grammar such as Grammarly. In addition, data analysis was done manually and we revised the methodological issues and the data analysis sections. Conventional content analysis was used to analyze the data collected. In addition, details of field observations and their findings have been included in this article and add new table (table 2). In addition to that, the social responsibility and social accountability were added to the article, based on your valuable comment. In addition, the words “problems of the building & hospital capacity” and “Intersection collaboration in different health sections” are to be replaced by “infrastructure problems” and “Interprofessional education & collaboration” respectively." } ] } ]	1	https://f1000research.com/articles/8-417
https://f1000research.com/articles/8-1000/v1	02 Jul 19	{ "type": "Research Article", "title": "A unique insert in the genomes of high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk", "authors": [ "Noam Auslander", "Yuri I. Wolf", "Svetlana A. Shabalina", "Eugene V. Koonin", "Yuri I. Wolf", "Svetlana A. Shabalina" ], "abstract": "The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6 as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.", "keywords": [ "Papillomaviruses", "cervical cancer", "oncogenic risk", "translation terminatio-reinitiation", "machine learning" ], "content": "Introduction\n\nPersistent infections with carcinogenic human papillomaviruses (HPVs) are the main cause of cervical neoplasia and cancer, with over 99% of the cervical lesions containing HPV sequences1–3. More than 160 HPV types have been characterized4, of which approximately a third are predominantly detected in the cervical epithelium and belong to the Alphapapillomavirus genus5,6. The viruses of this genus are further grouped into high-risk (HR) and low-risk (LR) HPV types based on their association with cervical cancer and pre-cancerous lesions7,8. Most of the HR-HPV variants belong to the Human papillomavirus 16 (alpha-9) or Human papillomavirus 18 (alpha-7) species groups9.\n\nPhylogenetic trees constructed from alignments of complete HPV genomes cluster all oncogenic types together, suggesting a common ancestor for the HR-HPVs. However, in separate trees built from different regions of the genome, the carcinogenic potential co-segregates with the early but not with the late genes10,11. The early HPV proteins E6 and E7 have transforming properties12–14 and are required for the malignant conversion. The involvement of these proteins in tumorigenesis is thought to stem from their interactions with the tumor suppressors p53 and pRB, respectively, as well as other proteins involved in tumorigenesis14–17. Variations in E6 and E7 proteins have been suggested to determine the oncogenic potential of HPV18,19 but the potential discriminating features of the oncogenic variants are frequently observed in LR-HPVs as well20–24. Currently, the most notable molecular feature that distinguishes HR from LR-HPV is the presence of a PDZ-domain recognition motif at the extreme C terminus of the HR-E6 oncoprotein, as opposed to LR-E625–27, enabling interactions with numerous proteins that contain the PDZ domain28–30.\n\nIdentification of signatures of the HR-HPV genotypes that differentiate them from the majority of alphapapillomaviruses that lack oncogenic potential could help elucidate the genetic basis of the carcinogenic properties of HPVs, thus contributing to a better understanding of the biological mechanisms exploited by the virus to trigger neoplasia. However, at present, genomic determinants of the HPV oncogenic risk are not well understood, and the exact nature of the genetic changes that led to the emergence of the HR-HPV oncogenicity remains unknown.\n\nTo better understand HPV carcinogenesis, we revisited the search for specific genomic determinants of HR-HPV types and identified a previously unreported insert of 30 to 60 basepairs (bp) at the 3’-end of the E6 oncoprotein coding region that is present in all HR-HPV types but not in LR-HPV. This insert introduces a new stop codon, separating the nucleotide sequence coding E6 from that coding for E7, eliminating the overlap between E6 and E7 that is characteristic of the LR-HPV types. The insert confers a PDZ binding motif at the end of E6 oncoprotein which is likely important for the oncogenic potentiation. Additionally, it locates the E6 termination codon upstream of and in close proximity to the E7 initiation codon. Furthermore, the insert contains sequences complementary to human 18S rRNA in the regions of hairpins 26 and 27 that are known interactors of 40S ribosomal subunits and viral RNAs, specifically involved in IRES binding. The folding of these regions of rRNA complementarity in E6-E7 mRNAs is typically relaxed in the predicted optimal and sub-optimal secondary structures of HR-HPV strains. We hypothesize that the insert into the E6 coding sequence identified here was the primary cause of the emergence of high oncogenic potential alpha-HPV.\n\n\nResults\n\nThe complete nucleotide sequences and the amino acid sequences of HPV E1, E2, E4, E5, E6, E7, L1 and L2 proteins were collected for all sequenced alpha-HPV strains (Extended data: Table S131). We then constructed a global multiple sequence alignment of the whole genome nucleotide sequences and the amino acid sequences alignments for each protein. Maximum likelihood phylogenetic analysis of these alpha-HPVs, based on the complete nucleotide sequence, as well as the amino acid sequences of most early proteins, identified HR-HPV as a clade, whereas phylogenies of E4, E5, L1 and L2 did not support the monophyly of the HR-HPV (Figure 1; Extended data: Figure S131), in agreement with previous findings9,10. These observations are compatible with a major role of recombination in the HPV evolution.\n\n(A) Maximum likelihood tree obtained with the whole genome nucleotide sequences alignment of alpha-HPV, colored by the different alpha-class categories. (B) Maximum likelihood trees obtained with alignments of E6, E7, E1, E2, E4, E5, L1 and L2 amino acid sequences of alpha-HPV, colored by the oncogenic risk groups.\n\nWe next sought to identify genomic features that might partition alpha-HPV species, in accord with their oncogenic risk, focusing on the E6 and E7 oncogenes.\n\nFirst, we searched for regions of insertions and deletions within the genome nucleotide sequences of E6 and E7 that might differentiate between the risk groups. Specifically, we identified sequences with high frequency of deletions or insertions that are located within high confidence alignment regions (See Methods for details). We then applied Support Vector Machine (SVM) with a leave-one-out cross-validation, aiming to identify regions that classify HPV strains based on their oncogenic risk. This approach resulted in the identification of genomic regions that separated HR-HPV from LR-HPV with high accuracy (over 0.75, with statistical significance; see Methods). Among these, we found one prominent insert (approximately 30-60 nucleotides) located within the E6 gene (Figure 2A). We also performed a similar search for regions separating HR-HPV from LR-HPV, using the amino acid sequence of E6 and E7 oncoproteins. For the purpose of classification, we coded the amino acids with numbers based on their frequencies and the BLOSUM6232 matrix (see Methods). As expected, the divergent region in E6 was identified from the amino acid sequences as well (Figure 2B). In contrast, we did not find any significant differences in E7 protein sequences between the high risk and low risk HPV strains (Extended data: Figure S231).\n\n(A) Nucleotide sequence alignment of alpha-HPVs (upper panel are LR-HPV and lower panel are HR-HPV sequences). Boxed sequences: pink box, E7 start codon for most HPV types; blue boxes, E6 stop codons that are distinct between HR-HPV and LR-HPV. (B) Amino acid sequence alignment of alpha-HPVs (upper panel are LR-HPV and lower panel are HR-HPV sequences). The orange box shows the PDZ-binding motifs in E6 of HR-HPV. (C) Schematic representation of the E6/E7 separation through the insertion in E6 and the proximity of E6 stop and E7 starts in HR-HPV.\n\nThe discriminating region identified in the E6 gene contains an insert with a conserved sequence in all HR-HPV strains. The insert contains an in-frame stop codon for the E6 coding sequence, which eliminates the overlap between the coding sequences of E6 and E7 that is characteristic of the LR-HPV genomes, but results in nearly identical lengths of the E6 proteins in HR and LR-HPV strains albeit with unrelated C-terminal sequences of 8-19 amino acids (Figure 2B). The unique C-terminal sequence of HR-HPV E6 that originates from the insert contains a PDZ domain-binding motif X-T-X-V/L at the very C-terminus of E6 in almost all HR-HPVs. Indeed, several PDZ domain-containing proteins have been identified as binding partners of HR-E6, including hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP126,29,30,33–35. These interactions that are unique for HR-HPV are likely to be a key contributing factor to HR-HPV induced oncogenesis36.\n\nWe observed that the sequence similarity between the insert sequences among HR-HPV strains is more pronounced at the nucleotide level than at the amino acid level (See METHODS for details and Extended data: Figure S331). Combined with the separation between the coding regions of E6 and E7 resulting from the insertion, and the proximity of E6 stop codon to the E7 start codon, this finding led us to hypothesize that the insert has an additional role as a regulatory region. Furthermore, as E7 has been previously identified as the dominant oncogene37, the lack of genomic determinants of HR-HPV within the E7 gene is compatible with the possibility that the insert contains regulatory elements enhancing E7 expression in HR-HPV strains.\n\nIn HR-HPV strains, E6 and E7 proteins are translated from a polycistronic pre-mRNA38, where translation reinitiation has been suggested as mechanism39–41. However, the close proximity of the E6 stop codon to the E7 start codon in HR-HPV (only a few nucleotides separating these codons) could inhibit re-initiation39,40. Therefore, it has been suggested that ribosomal reinitiation is enabled through the E6I splice variant in which the intercistronic distance between the translation termination codon of E6I and the E7 initiation codon is increased41. However, several studies have reported that E7 translation is independent of splicing within the E6 open reading frame40,42,43, undermining this interpretation.\n\nSeveral cases of coupled termination-reinitiation for polycistronic mRNA with proximate stop and start codons are evident for translation of eukaryotic virus genes44–51. The efficiency of this process depends on the close proximity of the termination and reinitiation sites51,52, and the presence of motifs complementary to the 18S rRNA in the mRNA sequence45,46,51,53. Given the proximity of the E6 termination site to the E7 initiation site codon that results from the insertion in the HR-HPV strains conferred by the insert, we investigated the possibility of coupled termination-reinitiaion of E6 and E7 in these strains. Notably, within the inserted sequence in the vicinity of the E7 start codon, we identified two conserved regions that are complementary to the sequences in 18S rRNA hairpins 26 and 27 which are commonly involved in the interactions between ribosomes and virus IRES54 (Figure 3A). The first region of complementarity consistently forms an internal loop and a relaxed, unpaired structure in the predicted optimal and sub-optimal E6-E7 mRNA foldings of HR-HPV strains54 (Extended data: Figure S431, see Methods). The second region of 18S RNA complementarity overlaps with the E7 start site, and hence might function independently in the re-initiation of E7 translation (Figure 3B). These findings suggest that the insertion could enable coupled termination-reinitiation of E6 and E7 proteins, enhancing their combined expression in HR-HPV, and thus promoting the oncogenic transformation induced by these viruses.\n\n(A) Comparison of 18S rRNA complementary sequences for different HR-HPV strains. The distal and proximal to the E7 start motifs (motifs 1 and 2) to the E7 start codon are shown in red and blue, respectively. The E6 stop codons (TAA) and E7 start codons are marked in bold. (B) An illustration of the potential interactions between folded HPV16 pre-mRNA (grey, red and blue) and the human 18S rRNA (brown). The two motifs are marked on the HPV-structure in red (motif 1) and blue (motif 2).\n\n\nDiscussion\n\nThe genus Alphapapillomavirus includes HPV types that are uniquely pathogenic. However, the events in the HPV genome evolution that led to the carcinogenic potential of some alpha-HPV types remain poorly understood. Here, we revisited this problem by performing a search for genomic determinants of the oncogenic risk of alpha-HPV types and identified a unique insert in the 3’-terminal region of the E6 oncoprotein gene in all HR HPV strains. To the best of our knowledge, this insert in HR-HPV genomes has not been reported previously. The insertion maintains closely similar lengths of E6 proteins in HR-HPV and LR-HPV types, which could explain why it has been overlooked in previous HPV genome analyses.\n\nWe hypothesize that the insertion makes a dual contribution to the oncogenicity of the HR-HPV types. First, the inserted sequence changes the C-terminal amino acid sequence of E6 and creates a PDZ domain-binding motif that is unique to HR-HPV types. The experimentally demonstrated interaction between the E6 proteins of HR-HPV and several PDZ domain-containing proteins is thought to make a substantial contribution to HPV-induced tumorigenesis27,29,55. Interestingly, PDZ-binding motifs have been identified also in several other oncogenic viruses, such as HTLV-1, adenovirus RhPV1 and beta-HPV856,57. Second, the insert eliminates the overlap between the E6 and E7 coding regions, implying a possible role as a regulatory region. We find that almost all HR-HPV genomes contain the sequence T-A/G-T-A-A-T/A in the insert near the end of the E6 coding sequence, a closely similar sequence to that functioning as the early promoter at the 5’ end of E6 and is employed for producing the mRNAs for both E6 and E758,59. However, in HR-HPV strains, unlike the case of the LR-HPV39, there are no reports of an independent E7 promoter, and E6 and E7 are both translated from a polycistronic mRNA.\n\nFurther investigating the potential regulatory role of the inserted sequence, we identified two conserved motifs that are complementary to the human 18S rRNA; interaction of such motifs with the rRNA has been shown to play a role in coupled termination-reinitiation for several viral genes45,46,51,53. The first motif forms an internal loop in the predicted mRNA secondary structure of E6E7, whereas the second one overlaps with the E7 initiation codon. Given the evolutionary conservation of these motifs and the close proximity of E6 termination site to the E7 initiation site, it appears plausible that coupled termination-reinitiation promoted by the insert sequence is a central mechanism for E7 translation in HR-HPV strains. Given the lack of additional major genomic determinants that would consistently differentiate between HR-HPV and LR-HPV, it seems likely that the insert in E6 is the primary cause behind the emergence of oncogenic HPV.\n\n\nMethods\n\nMultiple alignments of nucleotide and amino acid sequences that were obtained from GenBank (Extended data: Table S131) were generated using MAFFT v7.40760 with default parameters. Maximum likelihood phylogenetic analysis was performed using the resulting alignments and PhyML 3.1 software61, with the Bayesian information criterion, NNI tree improvement and an LRT SH-like likelihood method for support estimation.\n\nTo apply Support Vector Machines (SVM62, using Matlab 2018b fitsvm function) to the nucleotide sequences, we first encoded the data with numbers, where each nucleotide is coded as ‘1’ and each gap as ‘0’. We searched for alignment regions with deletions or insertions in multiple HPV strains that are surrounded by high confidence alignment regions (alignment regions of length > 15 bp containing less than 5% of gaps in each position) because these are most likely to contain relevant differences within conserved genomic regions. Within these regions, we then trained the SVM to classify alpha-HPV strains based on their oncogenic potential. The performance of the SVM was evaluated by leave-one-out cross validation, and regions with the overall balanced accuracy >0.75 (the average of the accuracy for positive and negative classes) were selected for further analysis.\n\nTo apply SVM (using Matlab 2018b fitsvm function) to the amino acid sequences of E6 and E7 proteins, we first encoded the amino acids with numbers using the frequencies of each amino acid in each protein and the BLOSUM62 matrix32. For each position, the most frequent amino acid was identified, and the amino acids in each protein were encoded by their BLOSUM62 distances from the most frequent amino acid in the respective position. We then trained the SVM to classify alpha-HPV strains based on their oncogenic potential using the coded protein sequences. Positions surrounded by high-confidence alignment regions (length > 5 amino acids and containing less than 5% of gaps in each position) were selected for further analysis. For these positions, we evaluated the performance by leave-one-out cross validation, and regions with the overall balanced accuracy >0.75 were selected for further estimation of significance using a permutation test.\n\nTo estimate the significance of the identified regions, i.e. to determine whether similar differences could be observed by chance, we performed a permutation test (Extended data63), controlling for the topology of the reconstructed phylogenetic trees. To this end, the labels were randomly permuted while maintaining unified labels for clades with high similarity. For each identified region, the likelihood of obtaining an equivalent or higher performance for the length of the region within the respective protein was evaluated by calculating an empirical P-value. We consider regions with permutation P-value <0.05.\n\nThe most stable secondary structures were predicted for all HR-HPV strains and their free energy values were calculated using the Vienna package54. Afold and Mfold were applied for prediction of optimal and sub-optimal mRNA structures64,65. Target opening free energy was estimated for motifs 1 and 2 using optimal and sub-optimal RNA structures, as described previously66. The sequence fold variants with the lowest secondary-structure free energy are presented in the Extended data: Figure S431. The formation of intermolecular mRNA-rRNA duplexes and hybridization affinity of the E6-E7 inserts to ribosomal RNA were evaluated using the Hybrid software with default parameters, with a ΔG threshold of ≤−10 kcal/mol67. The human 18S rRNA 2D structure was extracted from the structures of X-ray structure of eukaryotic ribosome (http://apollo.chemistry.gatech.edu/RibosomeGallery H%20sapiens/SSU/index.html; 68,69).\n\n\nData availability\n\nNucleotide sequences of HPV and amino acid sequences of HPV E6 and E7 proteins are available as extended data.\n\nHarvard Dataverse: A unique insert in the genomes of in high-risk human papillomaviruses with a predicted dual role in conferring oncogenic risk, https://doi.org/10.7910/DVN/FUGEUB31.\n\nThis project contains the following extended data:\n\nTable 1: Complete nucleotide sequences and the amino acid sequences of HPV E1, E2, E4, E5, E6, E7, L1 and L2 proteins. This is the only source data that was required and employed for the analysis reported in this work.\n\nFigure S1: Maximum likelihood trees obtained with alignments of E6, E7, E1, E2, E4, E5, L1 and L2 amino acid sequences of alpha-HPV strains.\n\nFigure S2: The balanced accuracy (y-axis) obtained from a leave-one-out cross validation for predicting risk category (HR vs LR) of alpha-HPV strains using BLOSUM62 coding of amino acid sequences, of different positions (x-axis) E6 (A) and E7 (B). Zero-accuracy was assigned to regions surrounded with low confidence alignment.\n\nFigure S3: Boxplots showing the distributions of the identity fraction of each nucleotide (NN) and amino acid (AA) in the genome and protein sequences of the insertion (not considering gaps for both NN and AA). The individual identity fractions of each position are overlaid.\n\nFigure S4: RNA fold secondary structure prediction of HR HPV strains 16 (A), 18 (B), 45 (C) and 31 (D). The nucleotides of the first motif are marked in red, and of the second motif in purple. E7 AUG is noted in red font.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nZenodo: Permutation test controlling for HPV strains, http://doi.org/10.5281/zenodo.324223163. Apache License, Version 2.0.", "appendix": "Grant information\n\nThis work was supported by U.S. Department of Health and Human Services (Intramural funds).\n\nThe funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWalboomers JM, Jacobs MV, Manos MM, et al.: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999; 189(1): 12–9. PubMed Abstract \| Publisher Full Text\n\nBosch FX, de Sanjosé S: Chapter 1: Human papillomavirus and cervical cancer--burden and assessment of causality. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCortes C, Vapnik V: Support-Vector Networks. Mach Learn. 1995; 20(3): 273–97. Publisher Full Text\n\nAuslander N, Wolf IY, Shabalina AS, et al.: Permutation test controlling for HPV strains. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3242231\n\nOgurtsov AY, Shabalina SA, Kondrashov AS, et al.: Analysis of internal loops within the RNA secondary structure in almost quadratic time. Bioinformatics. 2006; 22(11): 1317–24. PubMed Abstract \| Publisher Full Text\n\nMathews DH, Turner DH, Zuker M: RNA secondary structure prediction. Curr Protoc Nucleic Acid Chem. 2007; Chapter 11: Unit 11.2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaure G, Ogurtsov AY, Shabalina SA, et al.: Role of mRNA structure in the control of protein folding. Nucleic Acids Res. 2016; 44(22): 10898–10911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatveeva OV, Shabalina SA: Intermolecular mRNA-rRNA hybridization and the distribution of potential interaction regions in murine 18S rRNA. Nucleic Acids Res. 1993; 21(4): 1007–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgurtsov AY, Mariño-Ramírez L, Johnson GR, et al.: Expression patterns of protein kinases correlate with gene architecture and evolutionary rates. PLoS One. 2008; 3(10): e3599. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMelnikov S, Ben-Shem A, Garreau de Loubresse N, et al.: One core, two shells: bacterial and eukaryotic ribosomes. Nat Struct Mol Biol. 2012; 19(6): 560–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "51173", "date": "31 Jul 2019", "name": "Alison McBride", "expertise": [ "Reviewer Expertise HPV replication", "genomics", "epigenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuslander and colleagues present a comparative sequence analysis of alpha-papillomavirus genomes. They identify and analyze a short 30-60 nucleotide sequence insert between the E6 and E7 open-reading frames that encodes the PDZ domain in the high-oncogenic risk types and propose that this region also contains sequences that enable coupled termination and reinitiation to facilitate translation of the E6 and E7 proteins. This is an intriguing observation as HPVs are associated with ~5% human cancers and understanding how the viral oncoproteins are expressed is crucial. The proposed hypothesis should be testable and indicates that experiments in which the E6 PDZ domain is mutated in the background of the viral genome should be interpreted carefully.\nA weakness of the manuscript is that current literature and data sources are not used/and or cited and the data-set used seems incomplete. For example, there are currently 198 officially numbered HPV types, and 442 HPV types in total. A curated data-set of genome sequences for all papillomavirus types sequenced to date is available at PaVE. There are also many recent publications that describe analyses of PV evolution, oncogenicity and E6 PDZ domains that should be cited.\nThere are sequences available for 64 alpha HPV types that have been officially named by the HPV Reference Centre, yet only the genomic sequences of 44 types are listed in Figure S1. The data-set contains many isolates for some HPV types (e.g. 299 isolates of HPV16) yet almost no representatives of the species alpha 2, 4 and 8. The E6 protein of HPV40 (alpha-8) has been proposed to contain an ancestral alpha PDZ domain and so the genomes of the alpha-8 species should be closely examined/discussed. Nevertheless, a nucleotide alignment of all 64 alpha-PV nucleotide sequences from PaVE does support the authors’ conclusions that only HR species-5, 6 7, 9 and 11 contain an insert separating the E6 and E7 ORFs.\nMinor issues:\nIn Figure 1A, alpha-11 is listed in the key for both LR and HR.\n\nIn Figure 2, the resolution of the sequence images should be improved. The legend for 2B should make it clear that these are just the C-terminal sequences of the E6 polypeptides. In 2C, the symbols and colored blocks should be described.\n\nIn Figure 3A, the color/bold identification of the motifs and TAA/AUG are not easily visualized in the pdf. Perhaps highlight TAA/AUG by underlines or boxes. Labelling the groups of different HPV species along the left would also be helpful. In 3B, the labelling of the 18S rRNA hairpin (orange text) is confusing. What is 26es7?\n\nIn Figure S4: clarify in the legend that this is the E6-E7 RNA sequence for each HPV type shown.\n\nThe reference for IRES (54), I think should be 53.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "4923", "date": "01 Oct 2019", "name": "Eugene Koonin", "role": "Author Response F1000Research Advisory Board Member", "response": "We appreciate the constructive and very helpful review in response to which the following changes have been made to the manuscript: We now mention the most recent number of HPV types that have been formally recognized (198), and the reference supporting this has been updated. We added numerous references to recent publications that describe analyses of HPV evolution, oncogenicity, oncoprotein interactions and E6 interactions with PDZ domains. We now include all 64 alpha HPV types from PaVE, and all analyses and figures 1-3 include those HPV strains. Resolution of the new figures has been improved. The legend to Figure 2B has been modified as suggested. The legend to Figure 2C describes all symbols and colored blocks. Figure 3A has been updated to show the motifs clearly and arrange the strains by the order in the phylogenetic tree. Figure 3B has been updated, confusing labelling of 18S rRNA removed." }, { "c_id": "4925", "date": "01 Oct 2019", "name": "Eugene Koonin", "role": "Author Response F1000Research Advisory Board Member", "response": "Although the referee has suggested a statistical review of the permutation test that was used in this work to assess the significance of the results, we are confident that this is unnecessary because the statistical technique we used is simple and standard." } ] }, { "id": "51570", "date": "06 Aug 2019", "name": "Elizabeth A. White", "expertise": [ "Reviewer Expertise HPV E6 and E7", "protein-protein interactions", "transformation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Auslander and colleagues compare nucleotide and amino acid sequences for a subset of genus alpha HPV genotypes. They observe that high-risk HPV genomes share a short sequence insert at the 3’ end of the E6 ORF. The insertion adds a PDZ binding motif to the C-terminus of high-risk HPV E6 proteins and alters the location of the E6 termination codon relative to the E7 initiation codon.\nThis observation is consistent with previous findings. It has been appreciated for some time that high-risk HPV E6 and E7 are transcribed from a polycistronic mRNA whereas low-risk E6 and E7 are transcribed from separate promoters. In addition, a frequent splicing event occurs within the E6 gene in the bicistronic high-risk HPV early mRNA. This report adds to those observations. It proposes that the 3’ high-risk specific insert is another feature that differs between high- and low-risk HPV and that it might drive differences in expression of high-risk versus low-risk HPV oncoproteins. Understanding the differences between oncogenic and non-oncogenic HPV is an area of intensive research and new contributions in this area are potentially significant. This report makes a useful contribution to the literature.\n\nWeaknesses of the manuscript are that the current literature is not cited and that other features of high-risk HPV E6 that might also account for their oncogenic activities are not discussed. Although comparing nucleotide sequence differences is informative, for HPV E6 this comparison does not completely reflect the biology of the proteins. There are high-risk HPV E6-specific protein binding partners other than PDZ domain proteins. Several of these are listed in a useful 2012 review1; others were identified by proteomic analyses from several groups23. For example, the authors do not mention that TP53 binding and degradation is a feature of high-risk HPV E6 not shared by low-risk HPV E6. It also appears that not all of the high-risk HPV E6 interact with the same subset of cellular PDZ proteins. It is unclear how these diverse HPV E6-PDZ protein interactions might account for the shared oncogenic features of high-risk HPV E6, and this point is not discussed in the manuscript.\nIt is established in the HPV literature that small differences in HPV oncoprotein amino acid sequence result in significant differences in interactions with host cellular proteins. This is beautifully illustrated by the structural studies of Gilles Trave and colleagues, who have determined that subtle differences in E6 enable a range of specific interactions with cellular LxxLL-containing proteins. Other recent studies highlight high-risk HPV-specific activities of the E7 oncoprotein. In light of findings like these, this manuscript seems to overstate the claim that the PDZ binding motif is the ‘most notable molecular feature’ distinguishing high- from low-risk HPV. The potential importance of the insert sequence to E6/E7 translation regulation is high and should be tested; the discussion of the importance of the PDZ binding motif should be tempered and put in context with other recent findings.\n\nAdditional points:\nMirabello and colleagues recently reported an analysis of sequence variants from >5000 HPV16-positive cervical samples4. HPV16 E6 sequences exhibited variation across the length of the ORF that was similar in high-grade versus control lesions. A possible interpretation of this finding is that the protein sequence in the C-terminus of E6 is not important for oncogenic transformation. How does this fit into the authors’ model? It would be useful to include a discussion of these data.\n\nThe phylogenetic trees in Figure 1B might be easier to interpret if they were presented in a linear format. It is difficult to determine where the boundaries between the high-risk/low-risk groupings overlap with the major branch points of the tree.\n\nHPV diversity is much greater that that reflected solely in genus alpha. It would be interesting to know whether HPV from other genera have also acquired sequences in this region that might provide some or all of the activities suggested by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4924", "date": "01 Oct 2019", "name": "Eugene Koonin", "role": "Author Response F1000Research Advisory Board Member", "response": "We appreciate this highly constructive and helpful review in response to which the following modifications have been made too the article: Many references to the current literature were added, and several other features of HR-HPV E6 are now discussed. The suggested references to HR-HPV E6 specific binding partners have been included. The discussion on the importance of the PDZ binding motif in distinguishing HR from LR HPV has been tempered, and the context for its potential relevance has been clarified. We now discuss the recent analysis of 5570 cervical HPV16 genomes (Mirabello and colleagues) and have substantially expanded the discussion of the effect of the insert on E7 production. Figure 1B has been updated, phylogenetic trees are now presented in a linear format. In addition, although the referee have suggested a statistical review of the permutation test that was used in this work to assess the significance of the results, we are confident that this is unnecessary because the statistical technique we used is simple and standard." } ] } ]	1	https://f1000research.com/articles/8-1000
https://f1000research.com/articles/8-1705/v1	30 Sep 19	{ "type": "Opinion Article", "title": "Rethinking heritability", "authors": [ "Alex Gamma", "Michael Liebrenz", "Michael Liebrenz" ], "abstract": "Two markedly different concepts of heritability co-exist in the social and life sciences. Behavioral genetics has popularized a highly technical, quantitative concept: heritability as the proportion of genetic variance relative to the total phenotypic variance of a trait in a population. At the same time, a more common biological notion simply refers to the transmission of phenotypic traits across generations via the transmission of genes. It is argued here that the behavioral-genetic concept is of little use overall, while the common biological concept is overly narrow and implies a false view of the significance of genes in development. By appropriately expanding heritability into a general causal concept based on its role in evolution, we will arrive at a new view of development, heritability, and evolution that recognizes the importance of non-genetic inheritance and the causal parity of all determinants of phenotypic traits.", "keywords": [ "heritability", "behavioral genetics", "genes", "development", "evolution", "environment" ], "content": "Introduction\n\nBehavioral genetics shares with many other scientific disciplines the goal of learning about the various factors that shape human beings and about what such knowledge implies for the possibility of positive change. However, the particular way in which behavioral genetics has traditionally raised and answered those questions is the subject of continuing controversy (Barnes et al., 2014; Burt & Simons, 2014; Burt & Simons, 2015; Moffitt & Beckely, 2015; Wright et al., 2017).\n\nThe problems noted by critics are diverse: they range from statistical assumptions made in estimating heritability, the meaning of those estimates and their relationship to the causes of human traits, to the conceptual distinction between nature (genes) and nurture (environment) underlying behavioral genetics’ core methodology. On a more abstract level, these issues translate into doubts about whether the questions asked by behavioral geneticists make sense, and, if so, whether the methods used to answer them are valid.\n\nThis paper has two aims: to diagnose the situation and to offer a way out. The diagnosis will involve showing why the concept of heritability in behavioral genetics lacks utility. The way out leads from recognizing the relationship of heritability in the behavioral genetic sense to its common meaning in biology and then to generalizing this biological notion into a principled concept of heritability based on its causal role in evolution. In the process, a modern view of heritability, development and evolution, united by causal principles, will emerge, and will complete the diagnosis of why, in our view, the traditional behavioral genetic approach has become obsolete.\n\nThis paper will focus exclusively on the classical methods of behavioral genetics - variance partitioning into a genetic and one or two environmental components - and will ignore more modern approaches involving the actual measurement of specific genes or environmental factors (i.e. it focusses on G vs E, and ACE models). The reason for this is twofold: first, even though some behavioral geneticists may now disavow the classical methods, these have produced the main body of literature in the field, and understanding the problems with this body is a prerequisite to understanding whether or not modern extensions of behavioral genetics have successfully overcome the problems. Second, even if \"only\" the classic results were shown to be invalid, it would force the abandonment of a great many core findings about heritability which still inform modern thinking about the genetic nature of traits such as intelligence and diseases such as schizophrenia.\n\nIn what follows, we assume the falsity of genetic determinism. This means we reject the idea that genes have more power than other developmental causes to determine the phenotype. We thus exclude a spectrum of views, from strong genetic determinism, the claim that genes fully determine a phenotype no matter what other developmental causes do, to the so-called \"information metaphor\", the claim that genes, and only genes, carry information in the sense of instructions or a program to control development. The information metaphor is the most widespread form of genetic determinism today, but it has been shown to lack any solid theoretical or empirical basis (Godfrey-Smith, 1999; Godfrey-Smith, 2008; Griffiths, 2001; Griffiths & Gray, 1994; Johnston, 1987; Keller, 1995; Neumann-Held, 2006; Nijhout, 1990; Moss, 2004; Oyama, 1985; Sarkar, 1996; Šustar, 2007).\n\n\nThe origins of nature and nurture\n\nCharles Darwin shattered the complacent view of his predecessors that God was taking care of guaranteeing the similarity of parents and offspring. After Darwin, the fact of parent-offspring resemblance found itself in urgent need of a non-theistic explanation. Theories of biological inheritance sprang up, and the role of environment and upbringing called for clarification. Darwin’s cousin Francis Galton was perhaps the first to explicitly bring biology (the innate qualities of a person, which he called “nature”) into opposition with the environmental influences that impinge on a person after birth (calling the acquired qualities “nurture”). As is well known, the biologically inherited part later became identified with genes. The view that emerged saw human development as the battleground of two fundamental forces: genes and environment. The question became: which of the two was stronger, and how could their relative strengths be measured?\n\nGalton had already suggested that observing identical twins would be useful in addressing these questions, as their virtual indistinguishability from birth indicated that their biological inheritances (genes) were the same. The degree to which they turned out different should then be a measure of the influence of environment.\n\nBy the 1930s, the statistical tools necessary to decompose the effects of nature and nurture had become available in the form of the analysis of variance. It allowed quantification of how much of the variance in an organismic trait was “due to” the variance in any number of explanatory variables. For example, the height to which a plant would grow was expected to depend both on its genotype and on the environment, such as the amount of water in the soil. In a simple experimental design, an agriculturist could plant seeds of different genotypes and expose each genotype to different amounts of water. After the plants had grown to their full height, she would measure how much height varied among the different genotypes (averaged across all water conditions), obtaining the amount of genetic variance. Similarly, she would measure the variation in height across the different levels of exposure to water (averaged across all genotypes) and obtain the environmental variance. If the effects of genotype and water were independent of each other, summing the two variances would yield the total variance in height across all the plants. The ratio of the genetic variance to the total height variance (a number between 0 and 1) could be taken as a measure of the relative strength of the effect of differences in genes on differences in plant height. This number became known as the “heritability” of a trait (Lush, 1937). The ratio of environmental variance to height variance was the complement of heritability and was taken to indicate the strength of environmental differences on trait differences. Comparing these two ratios would supply the answer to Galton’s question as to which was stronger, nature or nurture?\n\n\nVariance partitioning in humans\n\nThe way to obtain these numbers in humans is, however, importantly different from that in the example above. The facts that monozygotic (“identical”) twins share all of their genes, and that dizygotic (“fraternal”) twins share half of their genes can be used as a shortcut to estimating genetic and environmental variances (how this is done mathematically will not concern us here). The simplest partitioning only comprises a term for genes (G) and one for everything else, called environment (E). The total variance in some phenotypic trait (P) is then simply the sum of the genetic and environmental variance:\n\nP = G + E\n\nA slightly more complex approach is to split the environmental variance in two: one term for environmental influences that make twins more similar to each other (called \"shared environmental effects\") and one for environmental influences that do not make twins more similar (called \"non-shared environmental effects\"). This is called the ACE model, where A stands for (additive) genetic effects, C for shared and E for non-shared environmental effects1.\n\nWhat is important to understand is that apart from the trait of interest (e.g. intelligence, antisocial behavior, political preference), nothing is measured in a classic behavioral genetic study. The subjects’ genotype is not quantified or identified in absolute terms. All that enters the calculations is the assumption that all the genetic variance of monozygotic (MZ) twins contributes to their phenotypic similarity (since they share all of their genes), while only half of the genetic variance of dizygotic (DZ) twins contributes to their phenotypic similarity (since they share only half of their genes). Environmental influences are not measured at all. Instead, the term for the environment (E) is simply the remainder after the phenotypic (P) and genetic (G) variances have been calculated (E = P - G).\n\nThis has an important corollary: since the environment is treated as a leftover category, any environmental differences that produce the same pattern of effects as do genetic differences will be counted as genetic effects, not environmental ones. This is true even if the environmental differences actually cause the differences in phenotype.\n\nPerhaps the most striking example of this is the fact that the phenotypic effects of the common egg cell that MZ twins develop from is entirely neglected in twin study designs. MZ twins are created when one single fertilized egg splits in two and the two resulting daughter cells each grow to become a separate individual, while DZ twins stem from two different egg cells fertilized by two different sperm cells. Twin studies make use of the fact that in MZ twins, the two daughter cells will have the same genome - copies of the parent cell’s genome - but ignore that the whole non-genetic part of the two cells (called the “cytoplasm”) also stems from the common parent cell and will therefore also be the same or very similar, whereas the two cells from which DZ twins develop are likely to have less similar cytoplasms. The implication is that in addition to (and in conjunction with) their common DNA, their common cytoplasm will also make MZ twins more similar to each other than DZ twins. To the extent that this effect operates in the 2:1 fashion specified by the mathematical formalism of the ACE model, it is subsumed under the genetic component A, while in reality it is a shared environmental effect belonging to component C. The result is high values for A and low values for C, as is repeatedly found in behavioral genetics studies.\n\nOne context in which this matters is the continued debate about the causes of individual or racial IQ differences (see, e.g., recent debate in Vox [Haider, 2017a; Haider, 2017b; Turkheimer et al., 2017a; Turkheimer et al., 2017b]). If twin-based methods of identifying sources of variation confound genetic with cytoplasmic effects, the importance of genes for IQ will have to be re-evaluated. The same is true in another important context: the debate about the relative importance of parents vs. peers, where behavioral genetic findings have given rise to dramatic claims about the global inefficacy of parenting (Boutwell, 2015a; Boutwell, 2015b; Boutwell, 2017a; Boutwell, 2017b; Boutwell & Khan, 2016; Harris, 1998; Rowe, 1994). In fact, what makes cytoplasmic confounding particularly nasty is that there is no known way of disentangling genetic from cytoplasmic effects. The problem affects the entire body of research on twins and heritability. Everything, from antisocial personality to political preferences, that has been claimed to be heritable could potentially be an effect of cytoplasmic similarity, or, more realistically, a joint effect of genetic and cytoplasmic similarity.\n\n\nThe interactionist challenge\n\nA more fundamental charge is sometimes leveled at variance partitioning in the context of developmental causes, a charge that has been considered by a number of critics to be the fatal flaw of behavioral genetics (Burt & Simons, 2014; Burt & Simons, 2015; Moore, 2003). This “interactionist challenge” states that the development of organismic traits is a process of interacting causes that are inextricably entangled, such that their individual causal contributions cannot be separated, let alone quantified. Therefore, attempting to attach numbers to the relative importance of genes and environment in a trait is doomed to fail. Genes and environmental causes are both necessary (and none by itself sufficient) to create a phenotype, it is said, and if that is the case, it is impossible to quantify their relative contributions. Variance partitioning with regard to the causes of development is fatally flawed.\n\nFor example, in their recent challenge to behavioral genetics, Burt and Simons (Burt & Simons, 2015) put the case as follows:\n\n“[G]enes and environments are involved in an interpenetrating and interdependent dynamic relationship that renders the attempt to demarcate separate influences— the goal of heritability studies—illogical at both the individual and population levels” (p. 105).\n\nThe argument is often illustrated by analogy. One such comes from Richard Lewontin (Lewontin, 1974), who has us imagine two men building a brick wall, where one is laying the bricks and the other is applying the mortar. The two men are analogous to two different developmental causes, and the brick wall represents a phenotypic trait. After their work is done, does it make sense to ask which of the two men has contributed more to the finished wall? The answer is no, because both their contributions are necessary, but not by themselves sufficient, to build the wall.\n\nAnother popular analogy is the area of a rectangle (analogous to a trait) and its two causes, length and width. Does it make sense to ask which of them, its length or its width, contributes more to the rectangle’s area? Obviously not. Both length and width are necessary for the area to even exist, so neither can be considered more important. Trying to quantify their relative contributions makes no sense.\n\nHow can behavioral geneticists respond to this challenge? First, there is some common ground: the defender of behavioral genetics agrees with the critic’s characterization of how causes operate in development (Barnes et al., 2014; Rutter, 2006)2. Second, however, she correctly notes that this characterization pertains to how individual traits arise in an individual organism, i.e. to the etiology of traits. It says nothing about what causes differences in traits between different individuals. Third, she points out that the latter is what behavioral geneticists are interested in and what they use variance partitioning for. Fourth, she claims that variance partitioning is still possible, even in the case of inextricably interacting developmental causes.\n\nThis last claim is correct. In their behavioral genetics textbook, Plomin et al. (2008, p. 85) use the example of the rectangle to show that, although both its length and width are necessary for any individual rectangle’s area to exist, a population of rectangles may not only show variation in length and width, but these two variations may well contribute unequally to the variation in area. In other words, in a particular population of rectangles, the variation in length might be much greater than that in width, and therefore contribute much more to the variation in surface area. In the context of the causes of differences (variation) in a population, length and width are not inseparable; either one of them can exhibit larger variation than the other one and therefore contribute more to differences in the trait of interest, in this case surface area.\n\nHaving established the feasibility of variance partitioning for interacting causes, the crucial question becomes: can it actually tell us anything useful about such causes? Critics of behavioral genetics may think not; they may think that variance partitioning is irrelevant, or even illogical when it comes to questions of trait etiology (Burt & Simons, 2014; Burt & Simons, 2015; Moore, 2003). However, far from being irrelevant, variance partitioning is actually one of the major scientific tools used to identify causes in general, including the causes of organismic traits. It is the core of two of the most widespread statistical methods in all of the natural sciences: ANOVA and linear regression, both employed nearly universally in experimental and observational studies to provide clues as to the causes of human and animal traits. Crucially, variance partitioning provides these clues not only when causes are operating independently, but also in the case of interacting developmental causes. This is true simply because if something is a cause of some outcome, varying its level or its presence will change the outcome. Co-variation with an outcome is therefore an indicator of causation. If, for example, some genes are causes of a trait, changing or removing those genes will typically change the trait. (The 'typically' qualifier is necessary because there might be other causal mechanisms preventing the co-variation.) It does not matter whether a cause interacts with or is independent of other causes. One can begin to see this by setting up a simple statistical simulation that embodies the constraints of two interacting causal variables and running a regression analysis, as has been done by Gamma & Liebrenz (2017). Results show positive relationships between the two variables and the outcome, correctly identifying them as possible causes.\n\nThe upshot is that the interactionist charge that variance partitioning is useless in the case of interacting developmental causes is invalid. Analysis of variance on the population level can, in principle, identify the causes of traits in individuals, even if these causes are \"non-separable\" in the sense of being jointly necessary, but singly insufficient, to cause a trait. There is nothing illogical or categorically wrong about this endeavor. Employing variance partitioning per se is not what makes heritability estimates useless. They are useless for quite another reason.\n\n\nHeritability in behavioral genetics: relevance to causes of traits\n\nIt is not uncommon for technical terms used in science to have a pre-existing meaning. “Heritability” is an example. Its meaning in behavioral genetics is very different from that in common language, including its common meaning in biology. Behavioral genetic heritability (which we will henceforth call “heritabilityBG“) is a single number estimated from twin studies that denotes the proportion of total variance in an outcome variable that is genetic variance. In other words, it is simply the ratio of genetic to total phenotypic variance. As such, its value lies between 0 (no heritabilityBG) and 1 (complete heritabilityBG). In the context of variance partitioning as discussed above, heritabilityBG is the relative size of the variance component due to genes, where “genes” are one of two (or three) causes being considered (the other being “environment”).\n\nBefore discussing the implications of the different meanings of “heritability”, we need to assess the utility or lack thereof of heritabilityBG for identifying the developmental causes of traits. We have shown that variance partitioning per se can be useful for identifying such causes, but that is not at all the same as showing that estimating heritabilityBG is useful for this. In fact, the very reason why heritabilityBG estimates lack utility is because they are a particularly unhelpful way of partitioning variance.\n\nThe main problem is that the two (or three) sources of variance in behavioral genetic studies are such broad, unspecific and unsystematic categories as to make it virtually impossible to draw any useful causal inferences from them. The ACE model seems to make a potentially useful distinction between two kinds of environmental effects (shared vs. non-shared), but these effects are identified in terms of whether they make twins more similar to each other or not. They do not identify any actual environmental conditions3. Even assuming that heritabilityBG precisely estimates the proportion of genetic variance, all a non-zero value tells us is that genes make a difference to a trait in a population. It leaves us entirely in the dark as to which specific genes are involved in the formation of that trait (Sober, 2001).\n\nIt may be objected that the simple statement that genes contribute to a trait (that a trait “is genetic”) already constitutes significant knowledge. Thus, the behavioral geneticist Michael Rutter (Rutter, 2006) writes\n\n\"…the overall pattern demands acceptance of the importance of genetic influences. That is, the findings are incompatible with a zero genetic effect\". (p. 60)\n\nFrankly, however, that genes are involved universally in the formation of traits is one of the fundamental facts of developmental biology. There was never a reason to assume that a ubiquitous cellular component whose contributions to most aspects of cell life have been extensively documented would somehow magically fail to affect some classes of traits. Another behavioral geneticist, Eric Turkheimer, calls this a “pointless null hypothesis” (Turkheimer, 2011).\n\nLooking at variance partitioning in any field other than behavioral genetics will show how differently it is used. For example, a study of obesity may measure intake of fat, carbohydrates and proteins and find a statistical effect of carbs. This result gives a useful pointer to possible causes, and a follow-up study might then look at different types of sugary foods to narrow down the causes of the effect. Studies all over the natural sciences use experimental manipulations or observational variables specific enough to give potentially useful hints for further investigation. In contrast, knowing that some variance in a trait is genetic, even knowing the amount of that variance, provides little useful knowledge and little idea how to proceed further.\n\nAs far as the identification of causes is concerned, heritabilityBG tells us little that is useful. Nevertheless, the brute fact of genetic involvement as indicated by a positive heritabilityBG estimate might be regarded as carrying further significance: it might tell us something about the malleability of a trait.\n\n\nHeritability in behavioral genetics: relevance to malleability\n\nAccording to a still-common view, genetic involvement means that a trait, being inherited and therefore “inborn”, is more hard-wired, more fixed, and less amenable to change than a trait that is environmentally caused. Therefore, the fact of genetic involvement is taken to be much more consequential than, for example, the involvement of diet, in causing a trait.\n\nThis is not the case, however. Whether genes are or are not involved in the causation of a trait says nothing about how difficult it is to change that trait. In other words, genetically influenced traits are not a priori more difficult to change. A given trait’s malleability is always an empirical question, and the answer may not only be different for different traits, but even for different individuals carrying the same trait.\n\nPerhaps, if a genetically influenced trait could only be altered by changing the relevant genes, an argument could be made that due to the relative inaccessibility as well as the structural and functional complexity of the genome, changing such a trait would always be difficult compared to changing other traits whose etiology does not involve genes. But changing a genetically influenced trait does not necessarily require changing the relevant genes. Traits are always multi-causal, and each of the causes involved, whether genetic or environmental, is, in principle, an access point for causal manipulation.\n\nA famous example is that of the disease phenylketonuria (PKU). It involves a faulty gene that fails to produce an enzyme required to break down a certain amino acid. If left untreated, the amino acid accumulates in the body, leading to early mental retardation. There is a treatment that essentially cures the disease, but it has nothing to do with genes. It consists of a special diet that is free of the offending amino acid, so that a toxic build-up is prevented. A causally simple environmental intervention prevents all symptoms of a genetic disease. Many other such examples could be found, all illustrating the same underlying principle: if multiple causes co-determine an outcome, manipulating any of them can potentially change the outcome. This is true regardless of whether any one of the causes is genetic or not.\n\nIt is important to realize that heritabilityBG estimates are not informative about contexts such as this, in which a treatment for a disease trait, or an intervention to improve a beneficial trait (e.g. intelligence) is sought. In particular, the question of whether there could be an environmental intervention to change the trait in a desired direction is not addressed by heritabilityBG, since such an as-yet-unknown intervention cannot be part of the environment of the population from which the heritabilityBG estimate was derived. For example, before a dietary treatment became available, the heritabilityBG of PKU was high, as environmental variation in the disease phenotype was low. This meant nothing, however, about the prospects of finding a successful environmental intervention.\n\nHeritabilityBG does not indicate a trait’s malleability. Genetic involvement per se does not determine malleability, and even the fact that a trait such as PKU is “genetic”, i.e. originates from a gene defect, does not allow any a priori conclusions about how difficult it is to change, and where best to intervene to change it.\n\n\nHeritability in behavioral genetics: relevance to biological heritability\n\nIn biology, heritability is most commonly understood as a trait's potential to recur across generations due to genetic inheritance. We will henceforth call this notion “heritabilityBio“. To say that a trait is heritableBio means that if it exists in parents, it can recur in offspring because they receive from their parents the genes that give rise to the trait. HeritabilityBio therefore incorporates not only the aspect of \"passing on\" something to someone, but also a particular causal mechanism for doing so. That mechanism is genetic inheritance, because in the standard view of biology, genes are seen as the only causal substrate for trait inheritance. In \"The Selfish Gene\", Richard Dawkins puts this bluntly by stating that \"genetic factors replicate themselves, blemishes and all, but non-genetic factors do not\" (Dawkins, 1976, pp. 98/99).\n\nWhat is the relationship between heritabilityBG and heritabilityBio? Why do behavioral geneticists call a statistical measure of the proportion of genetic variance in a trait \"heritability\"?\n\nThe answer is that, given the view that heritabilityBio is based exclusively on the transmission of genes, measuring the influence of genetic differences on trait differences in a population would simultaneously capture the extent to which such trait differences are biologically inherited. While superficially plausible, it is clear that the procedure by which heritabilityBG is estimated does not directly probe any aspect of the process of genetic transmission from parents to offspring. It is not surprising, then, that the behavioral genetic measure of heritability sometimes diverges sharply from the biological concept.\n\nFor example, consider traits such as bipedality (having two legs) or pentadactyly (having five fingers) in humans. These are paradigmatic features of the \"design\" of Homo sapiens and universally considered to be highly heritable. However, almost all variation in these traits is environmental, stemming from accidents, and therefore heritabilityBG is basically zero.\n\nThe inverse case is possible, too: consider the trait of wearing skirts, which, as most would agree, is primarily culturally determined and not heritableBio. Wearing skirts, however, is almost perfectly correlated with sex, which itself is usually considered genetically determined. Giving precedence to genetic over environmental variation, even when the two correlate, the heritabilityBG of wearing skirts would turn out to be very high. Analogous considerations would show that racism and sexism are also highly heritable, even if in reality they were entirely sociocultural phenomena.\n\nHow do these discrepancies occur? In the case of bipedality, what goes wrong is that there is genetic causation, but no genetic variation. And without genetic variation, heritabilityBG is zero. In the other cases, there is environmental causation and therefore correlation, but it is congruent with a genetic correlation, so the effect is ascribed to genes and heritabilityBG is high.\n\nThese examples illustrate that behavioral genetic heritability does not track biological heritability as commonly understood.\n\nTo summarize the previous three sections, heritabilityBG is of little use in determining the specific causes of a trait, in indicating the malleability of a trait, and in tracking heritability as commonly understood in biology.\n\nTruth is not decided by authority, but it should at least be known that authorities in (the philosophy of) biology share this verdict (Bateson, 2001; Feldman & Lewontin, 1975; Keller, 2010; Lewontin, 2006; Sober, 2001), even if they are perhaps more diplomatic about it than we are here. We specifically mention these scholars because it is philosophers of biology who have done most of the work in developing the conceptual basis of biology, particularly addressing issues such as the role of genes in evolution and development (see for example the collection of essays in Neumann-Held & Rehmann-Sutter, 2006). But prominent behavioral geneticists also acknowledge that estimating heritabilityBG in itself is not very useful. According to Michael Rutter (Rutter, 2006), \"It is not that, on its own, it matters very much whether the heritability is 20 percent or 80 percent” (p. 89), while Eric Turkheimer finds variance partitioning as practiced by behavioral genetics to be without “scientific content” entirely (Turkheimer, 2011, p. 598).\n\n\nExtending standard biological heritability\n\nUnfortunately, it turns out that the common biological concept of heritability (heritabilityBio) is itself seriously wanting. By fixing it, however, we can arrive at an elegant, unified view of evolution based on a principled definition of heritability. This definition will abstract from contingent, particular mechanisms of inheritance and instead focus on the causal role of heritability in Darwinian evolution. It will be called heritabilityX (the “X” stands for “extended”).\n\nWhy was Darwin able to formulate his theory of evolution by natural selection without knowing anything about the actual mechanisms of biological inheritance? Because the actual mechanisms do not matter for the theory, only the fact of heritability itself, understood as the fact that parental traits tend to recur in the offspring generation or, simply put, that offspring tend to resemble their parents.\n\nThis basic observation is one part of the “holy trinity” of Darwinism, the three criteria that enable evolution by natural selection: phenotypic variation, differential fitness, and heritability (Sterelny & Griffiths, 1999, p. 32). Together they specify a causal recipe for evolution by natural selection. Importantly, this recipe is abstract enough to leave its physical implementation open. It is defined, rather, by the causal roles it specifies. As long as these roles are implemented in any way in any system, the system will exhibit Darwinian evolution.\n\nIn the world of organisms, the recipe specifies that there must be variation in a trait among a population of individuals. Some of the variants cause their carriers to have higher fitness, i.e. to produce more offspring than carriers of other variants. Finally, the trait in question must be heritable, i.e. offspring of carriers of the “successful” trait must be more likely to have the trait themselves. As a result, the trait increases in frequency in the offspring generation.\n\nThis three-step process is one iteration of the cycle of Darwinian evolution, and it will repeat as long as there is a fitness differential and no countervailing forces. The successful trait will continue to increase in the population across many generations, which is another way of saying that it evolves by natural selection.\n\n\nThe causal role of heritability in Darwinian evolution\n\nNothing in this account depends on the particularities of the mechanism by which offspring come to be similar to parents. Rather, the heritability requirement is defined by a particular causal role. This role consists of causing parental traits to recur in offspring. This can happen in potentially many ways, but, following Oyama (1985), we suggest that these ways can be subsumed under three broad categories, using one simple background assumption.\n\nThe assumption is that, since traits are multiply caused, the presence of the same or similar causes (in different organisms of the same population) will tend to result in the same or similar traits. Put somewhat informally, the idea is simply “same input - same output”. This is certainly plausible.\n\nA parental trait will therefore recur in offspring to the extent that the same causes that gave rise to the trait in parents are also present in offspring. For a trait to be inherited, therefore, the same or very similar “trait-forming” causes need to be present in both parents and offspring. Oyama (1985) identifies three broad ways in which this can happen: first, the trait-forming causes simply persist across generations; second, the trait-forming causes are physically copied and transported from parents to offspring; and, third, the trait-forming causes are reconstructed in each generation anew. These categories should cover most, if not all, of the possibilities of trait inheritance.\n\nTo make the idea more concrete, we will give examples of each category. Persistent trait-forming causes include fixed features of the environment such as sunlight, oxygen, gravity, and humidity. These are always there, and will exert their effect in every generation. Copied trait-forming causes include the familiar case of DNA, which is physically replicated before being passed on to offspring. DNA methylation patterns, if reliably copied across generations, are another example. Reconstructed trait-forming causes are probably the major category. They include all trait-forming causes that recur in every generation without having been directly physically copied or having been always there. Examples are diverse and include the womb (present in every generation, but reconstructed anew from “parts” in every generation), nutrition, social interaction with parents and conspecifics, behavioral habits, cultural norms and practices, and language.\n\nAs these examples show, heritability in the evolutionarily relevant sense is more accurately described as “repeated presence across generations”, because this removes the narrow focus on something physical having to be passed on from parents to offspring. Here, however, we will stick to the shorter term “heritabilityX”.\n\n\nNon-genetic inheritance in nature\n\nAt this point, it is natural to ask what the empirical evidence for non-genetic inheritance in humans is. The answer is that it is all around. This is less a case of having to discover novel non-genetic inheritance mechanisms (although that is part of it, as for example with epigenetic inheritance), but one of many basic and well-known observations that fall into place as soon as they are put in a new conceptual framework. That oxygen and sunlight are reliably present in every generation of our species is a simple fact. That every child starts out from a cell containing its mother’s cytoplasm and begins growing in the rich environment of a womb is another one. That diets can remain stable for long periods of time in local populations is also well-known. That offspring in each generation experience very similar maternal behavior, soak up local cultural norms and practices and learn the local language is no secret, either.\n\nVastly more than just DNA is stable across generations (Griffiths & Gray, 2001; Griffiths & Gray, 1994; Jablonka & Lamb, 2005; Jablonka & Lamb, 2007; Jablonka & Raz, 2009; Oyama, 1985). This should be uncontroversial. All that is needed then is to recognize that these other trans-generationally stable trait-forming causes meet the heritabilityX requirement for Darwinian evolution just as genes do. They all fulfill the causal role of being reliably present in repeated generations, poised to contribute to the building of the phenotype. They are thus heritable in the one sense that matters, the sense that enables evolution by natural selection.\n\nIn human beings, culture will be the major source of non-genetic inheritance (Acerbi & Mesoudi, 2015; Richerson & Boyd, 2008). Factors such as how we raise our children, how we school them, what norms and values we teach, what habits and lifestyles we have, how and where we live, how and where we work, what societies we build, what political and legal systems we have, what technologies we use, what we eat, whether and how we exercise, all this and more is part of a large package of culture children in every generation are shaped by. These packages will be different across different parts of the globe, and different parts of the package will have different “time constants”, i.e. will change faster or more slowly than others. But all will to some extent contribute to the stability or similarity of the phenotypes related across generations by descent.\n\nNon-genetic inheritance is also found in animals with little or no culture. First, persistent trait-forming causes belonging to the physical, geological or biological environment affect most animals just as they do humans. Second, so do reconstructed trait-forming causes like wombs and eggs, a population- or species-specific diet, social interaction with parents and conspecifics, specific forms of housing, etc. Third, there are other, more particular non-genetic inheritance systems in animals that are described in detail in the work of geneticists Jablonka & Lamb (2005); Jablonka & Lamb (2007). The subject matter, particularly in humans, far transcends the current fashionability of “epigenetics” (here, narrowly understood as chemical and structural modifications of DNA).\n\n\nHeritability, extended\n\nAn extended view of heritability also resolves a contradiction inherent in popular notions that deny genetic determinism, while also finding nothing wrong with the standard biological concept of heritability based exclusively on genetic inheritance. The problem becomes evident in a phrase we used earlier to characterize heritabilityBio: “To say that a trait is heritableBio means that if it exists in parents, it can recur in offspring because they receive from their parents the genes that give rise to the trait.”\n\nThis mechanism of inheritance can only work as described if having the right genes more often than not causes the trait in question, regardless of the configuration of other, non-genetic trait-forming causes. In other words, it can only work with a liberal measure of genetic determinism. When genetic determinism is rejected, however, a “paradox” arises: how could trans-generationally similar genes “give rise to” trans-generationally similar traits without other trait-forming causes also being trans-generationally similar?\n\nIf genes do not have special deterministic or instructive powers, how could the mere presence of certain genes guarantee the formation of a certain trait, irrespective of the status of other, non-genetic co-determinants of the trait?\n\nThe answer is that it could not. Development is paradigmatically a matter of multiple causes interacting to create a phenotype. If only one type of cause (genes) were stable across generations, and all other causes were free to vary arbitrarily, there would be no way of making sure that offspring develop the same traits as their parents4. It is here that the interactionist challenge really bites!\n\nAs soon as this is accepted, the pieces fall into place: trait heritability in the evolutionarily relevant sense can only be achieved if all or most or many trait-forming causes are stable across generations - certainly not if only one kind of cause is stable. The rest is re-description: we call all trans-generationally stable trait-forming causes inherited, because that is what they are in the sense relevant to Darwinian evolution (Griffiths, 2001; Griffiths & Gray, 1994; Griffiths & Gray, 1997; Griffiths & Gray, 2001)5. And we call all trans-generationally stable traits heritable, because that is what they are in the sense that is relevant to Darwinian evolution. What we end up with is a view in which many or most traits are heritable, and many or most causes of traits are inherited, at least to some extent. In her book-length treatment of the nature-nurture question, philosopher of science Evelyn Fox Keller (2010) summarizes this new view of heritability:\n\n\"…let us acknowledge that… almost all human traits are transmitted from one generation to the next … [and] let us also accept the fact that the mechanisms of transmission are very varied. They may be genetic, epigenetic, cultural, or even linguistic.\" (p. 80)\n\n\nHeritability, development, evolution: a broader view\n\nIt can now be seen that both the behavioral genetic and the standard biological concept of heritability are based on the false view that only genes are inherited, or at least that it is only genetic inheritance that matters. That view does not reflect the reality of non-genetic, in particular cultural, inheritance. A deeper understanding of heritability, development, and evolution can only follow from an extended notion of heritability which has a principled causal basis in evolutionary theory.\n\nTo elaborate: first, the concept of heritabilityX deepens the understanding of heritability qua parent-offspring resemblance. Far from being a phenomenon with a narrow causal basis limited to genetic inheritance, it is actually supported by a broad range of causal mechanisms. Everything that contributes to the stability of traits across generations is part of an inheritance mechanism. If, for example, offspring reliably get their dietary habits from their parents, then the underlying process of social learning is part of an inheritance mechanism, and diet becomes an inherited (and heritable) trait in the full, evolutionarily licensed, sense of the word.\n\nBroadening the notion of heritability makes sense of fundamental observations about the world, in particular the human world: that a vast array of conditions and stimuli that impinge on us are present in every generation, from the obvious, such as sunlight and oxygen, to the more subtle, such as cultural norms of behavior. Although it is certainly impressive that the DNA molecule is faithfully copied from parents to offspring, it is not only genes that provide stable inputs to the developmental process, and DNA-grade fidelity of replication is required neither for trait heritability nor for biological or cultural evolution (Henrich et al., 2008; Lewis & Laland, 2012; Richerson & Boyd, 2008, p. 83 ff). Rather, there are gradients of similarity of inherited trait-forming causes that lead to gradients of similarity in inherited traits.\n\nSecond, if non-genetic inheritance leads to stable changes in the phenotype of a population, there is no reason to deny this process the status of evolution. One might object that the time frame of many non-genetic inheritance phenomena is much shorter than the millions of years traditionally associated with genetic evolution, and that this should militate against them being counted as truly evolutionary. However, we do not see this as a principled objection. For one thing, research in recent decades has shown many examples of genetically driven evolution occurring at time scales as small as months (Gibbs & Grant, 1987; Herrel et al., 2008; Losos et al., 2006; Reznick et al., 1997). Further, it would be unprincipled just to draw a line somewhere and sort all instances of phenotypic change into evolutionary and non-evolutionary based on an arbitrary length threshold.\n\nTo take an example: adult body height has increased in Western populations for over a century. On average, current Westerners are over 10 centimeters taller than their ancestors 150 years ago (Danubio & Sanna, 2008). The main reasons for this secular trend are thought to be improvements in diet and hygiene, while genetic effects are deemed unlikely (Silventoinen, 2003). If this is correct, then, since diet and standards of hygiene are certainly (at least partly) culturally inherited, there is no reason not to view this increase in height as true evolutionary change6.\n\nThird, an extended notion of heritability connects with the nature of development in various ways. The most important connection has already been mentioned: it is the fact that rejecting genetic determinism actually implies the notion of heritabilityX. If genes alone cannot and do not determine traits, how could it ever be sufficient for a parental trait to be \"rebuilt\" in offspring to just inherit the \"right\" genes? It could not, and therefore non-genetic trait-forming causes must also be inherited from parents7.\n\nTraits are always the product of multiple, interacting causes, none of which is a priori more determinative of the outcome than any other. Heritable traits do not remain stable across generations because there is one special trait-forming cause that instructs the developmental process, but because a sufficient number of different trait-forming causes are inherited so that the process results in a stable phenotype. The principle “same input - same output” applies.\n\nFinally, the view of evolution that emerges is one in which an interacting network of internal and external trait-forming causes envelops and permeates the developing organism, spanning the generations and leading to the reliable recurrence - the heritability - of traits, or to trait variation, depending on the stability or variability of the network components involved (see Figure 1).\n\n\nConclusions\n\nThe behavioral genetic concept of heritability - what we have called “heritabilityBG“ - has three major flaws: first, it does not track common expectations about biological heritability; second, it lacks utility for human research since it does not identify specific causes of traits, is silent on the potential success of new therapeutic interventions, and has no bearing on the malleability of traits. Third, what heritabilityBG shares with the common understanding of biological heritability (“heritabilityBio”) is the popular misconception that heritability is based exclusively on genetic inheritance. In other words, both versions confuse “heritable” with “genetic”. This is both theoretically and empirically false; considering the origin and causal role of heritability in Darwin’s scheme of evolution by natural selection shows that, conceptually, any mechanism that guarantees that traits reliably recur across generations will fulfill the heritability requirement of evolution. In human beings, cultural inheritance is the most varied and important such mechanism, but others are possible, and many have in fact been described in various species of animal (Jablonka & Lamb, 2005). A number of fundamental observations about the stability of cultural and physical environments can be re-interpreted as phenomena of inheritance in the Darwinian sense. This leads to an extended causally defined concept of heritability, which we call “heritabilityX“.\n\nHeritabilityX incorporates the insight that most trait-forming causes - genetic and non-genetic - recur in every generation and are therefore heritable in the relevant sense, which is as trans-generational enablers of Darwinian evolution. Standard neo-Darwinism takes inheritance to be exclusively genetic and therefore implicitly requires a form of genetic determinism, typically the so called \"information metaphor\", the idea that genes embody instructions or even a program to direct the formation of traits. However, there was never any solid theoretical or empirical reason to believe in the information metaphor, as close scrutiny by philosophers of science has demonstrated (Godfrey-Smith, 1999; Godfrey-Smith, 2008; Griffiths, 2001; Griffiths & Gray, 1994; Johnston, 1987; Keller, 1995; Moss, 2004; Neumann-Held, 2006; Nijhout, 1990; Oyama, 1985; Sarkar, 1996; Šustar, 2007).\n\nIn a new, updated view of life, traits from the parental generation are successfully re-created in offspring because a sufficient number of parental trait-forming causes - genetic and non-genetic - are inherited and serve as “inputs” to offspring development. Same or similar inputs tend to produce same or similar outputs.\n\nThis perspective does justice to the most fundamental observation about development: that it is paradigmatically a process of different kinds of causes interacting constantly to produce traits or phenotypes, and that, in general, these causes are all jointly necessary, but not by themselves sufficient, to generate a certain outcome. There is a “causal parity” (Griffiths & Knight, 1998) of different developmental causes, with none of them playing an a priori more significant or more determinative role than any other.\n\nThe causal parity of genes and other developmental factors also implies that genes cannot constitute sufficient causal routes to traits, let alone provide complete explanations of traits. Full-blown explanations will integrate various kinds of causes across different levels of organizational hierarchy, and across the divide between the internal and the external. The impossibly broad categories of nature vs. nurture that captured the imagination of our intellectual ancestors a century ago are no longer fit for the science of today.\n\n\nData availability\n\nNo data are associated with this study.", "appendix": "Acknowledgments\n\nAn earlier version of this article can be found on SocArXiv: https://doi.org/10.31235/osf.io/h4sjy.\n\n\nFootnotes\n\n1We will not deal with the assumption of additivity and the concomitant ommission of an interaction term (G x E) from the basic variance partitioning scheme in classical behavioral genetics. We believe the assumption of additivity is a significant flaw that might in itself render heritability estimates useless. However, our intention here is to show the problems with heritability estimates, even granting the unimportance of G x E interactions.\n\n2From Barnes et al., 2014: ”What this necessarily means is that for a single individual, his or her genes and environment in interaction contribute to his or her phenotypic score…” (p. 615).\n\n3The shared environmental effect C is often understood as those influences that are shared by twins (notably the family environment, see e.g. Rowe [1994], p. 53), whereas the non-shared environmental effect E is taken to be those influences and experiences that are unique to each twin (e.g. peers). Behavioral genetics studies have repeatedly found that C is small, while E is quite substantial. This has been taken to show that the whole family environment - including everything that parents do - has very little effect on children’s development, while it is those experiences outside the home, e.g. peer groups, that mostly shape their personalities (Rowe, 1994; Harris, 1998). If this were true, it would have dramatic implications for parents, schools and any kind of intervention targeted at improving children’s abilities and behavior.\n\nHowever, what these variance components really stand for are environmental influences that make twins more similar (C) to each other or not (E). Better names for these effects would be something like “similar-making” and “not similar-making”. Although it may seem self-evident to some that common experiences, including much of what happens in the home, will have similar effects, making the twins more alike, and that unique experiences (such as with peers) will make them different, this is not at all a given (Rutter, 2012). While plausible-sounding, it is pure speculation, and as such, nothing that could bear the weight of far-reaching conclusions about the effectiveness of parenting. Finally, it should be obvious on reflection that without measuring anything about the actual environments encountered by the study subjects, it is hard to conclude anything about their effects. A mere mathematical formalism cannot achieve this.\n\n4It follows that to equate “heritable” or “inherited” with “genetic” is to tacitly accept some form of genetic determinism, because it is granted to genes alone to ensure the development of the traits that are inherited.\n\n5”…we should define “inheritance” so that something is inherited just if it passes from generation to generation in such a way that evolution can act on its variant forms. Hence, every element of the developmental matrix which is reliably replicated in each generation and which plays a role in the production of the evolved life cycle of the organism counts as something which is inherited.” (Griffiths, 2001, p. 402)\n\n6Cultural modes of inheritance necessitate a re-conceptualization of Darwinian fitness. In standard evolutionary theory, traits are passed only from parents to offpsring. Cultural inheritance, however, cannot only be “vertical”, it can also be “diagonal” and “horizontal”. Consider the trait of sharing. Children will learn to share from their parents (vertical transmission), but also from siblings and peers (horizontal transmission within the offspring generation) and from teachers and other adults (diagonal transmission from non-parental members of the parent generation to offspring generation). 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Reference Source\n\nStotz K, Griffiths PE: Dancing in the Dark: Evolutionary Psychology and the Argument from Design. In D. S. Scher & F. Rauscher, eds. Evolutionary Psychology: Alternative Approaches. Dordrecht: Kluwer. 2001. Reference Source\n\nŠustar P: Crick's notion of genetic information and the ‘central dogma’of molecular biology. Br J Philos Sci. 2007; 58(1): 13–24. Publisher Full Text\n\nTurkheimer E: Commentary: variation and causation in the environment and genome. Int J Epidemiol. 2011; 40(3): 598–601. PubMed Abstract \| Publisher Full Text\n\nTurkheimer E, Harden KP, Nisbett RE: Charles Murray is once again peddling junk science about race and IQ. Vox. 2017a; [Accessed July 29, 2017]. Reference Source\n\nTurkheimer E, Harden KP, Nisbett RE: There’s still no good reason to believe black-white IQ differences are due to genes. Vox. 2017b; [Accessed July 29, 2017]. Reference Source\n\nWright JP, Barnes JC, Boutwell B, et al.: Mathematical Proof Is Not Minutiae and Irreducible Complexity Is Not a Theory: a Final Response to Burt and Simons and a Call to Criminologists. Criminology. 2017; 53(1): 113–120. Publisher Full Text" }	[ { "id": "54497", "date": "18 Oct 2019", "name": "Eric Turkheimer", "expertise": [ "Reviewer Expertise Behavior genetics", "quantitative methods", "philosophy of science." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reframes the concept of heritability in a developmentalist and evolutionary framework. Although the article makes several interesting points and advances the discussion of the heritability of behavior, I think it could be improved in two complementary ways: better attention to the past, and better attention to the future.\nStarting with the latter, my strongest point on the level of peer review is that the decision to limit the BG portion of the article to twin studies is a serious limitation on its overall relevance. Twin studies are getting rarer and rarer, as they are replaced by GWAS and related methods that are based on measured DNA obtained from samples of unrelated participants. These new methods have changed the field in many profound ways. They have, for example, reduced most estimated heritabilities considerably. They have changed the way analysis of variance is used to compute heritability, and in some ways found ways to estimate heritability without analysis of variance (e.g. polygenic risk scores). They have also changed the way we think about questions of the relation between ANOVA and causation, as we are now able to at least consider the causal properties of some DNA-based effects on behavior.\nI do not mean to say that modern genomics refutes the argument that is presented here. In some ways it supports it, but in general it complicates the picture, and this paper would be much more relevant to the current state of affairs if it grappled with the field as it is now rather than what it was like 20 years ago, in the heyday of big twin studies and Richard Lewontin.\nEven as a reflection of older models of behavior genetics, the limitation of heritability to twin studies is problematic. It omits, for example, adoption studies, which are in many ways more directly related to issues of parent-child transmission that interest these authors. Even more generally, twin and adoption studies are actually special cases of a larger paradigm called quantitative genetics, which involves estimating variance components from analysis of familial relationships, which includes twins and adoptees but potentially many other relations as well. More complicated quantitative genetic models incorporate much more sophisticated results than the simple ACE or AE models the authors present here. Again, I don’t necessarily think consideration of such models would invalidate the authors’ arguments, but as it stands they run the risk of being accused of arguing with a very dated straw man.\nMy final point along these lines is that I don’t really think it is correct to characterize what this model of heritability as heritabilityBG. Quantitative genetics as applied to humans doesn’t actually have anything to do with behavior per se, and as the authors demonstrate the methods can be applied just as easily to height. There is a very large domain of twin studies of medical conditions. The authors are correct that the deep issues are about variance and causation, not behavior per se. I would call it heritability QG.\nAs for the inclusion of the past, there are several places along the way that the authors don’t recognize previous versions of very similar discussions. It’s not a matter of citing this or that study, it is a question of recognizing that this discussion has been going on for a very long time. I will include those issues as I proceed to enumerate more specific concerns below.\nThe discussion of cytoplasmic effects, while potentially interesting, is introduced without evidence that it is actually relevant to analyses of human behavior. It is also an instance of a broader issue - the equal environments assumption - that has a very long history in the theory of twin studies.\n\nThe discussion of anova and causation is incomplete. The classic article on the subject, Lewontin’s Analysis of Variance and Analysis of Causes, is mentioned but not considered in any detail. Much of what is said here is a recapitulation of that article. The authors might also want to look at the critiques of behavior genetics made by the developmental biologist Gilbert Gottlieb in the nineties, along with replies from myself and Irving Gottesman.\n\nI’m not sure I see the point of the first full paragraph in the right column of Page 5. If a correlational study of food intake is sufficient to establish causal effects of dietary intake on BMI, why wouldn’t an adoption study showing that BMI of adopted away children is correlated with that of their biological parents (or a polygenic score computed from their genome) do the same for genes?\n\nIt seems to me that two issues are being conflated here. The first is Lewontin’s concern with variation and cause. Down’s syndrome is a cause of reduced IQ, but it is not a source of important IQ variance in the population. The other is correlation and causation. Observing that environmental or genetic variables are correlated with BMI is not the same as showing that they cause BMI because of all the complex developmental issues the authors discuss.\n\nThe statement, “It leaves us entirely in the dark as to which specific genes are involved in the formation of that trait (Sober, 2001)” is no longer true in the DNA era. We are hardly enlightened about such things nowadays, but we are not as in the dark as we used to be.\n\nIn the discussion of both variance and causation, and heritability and malleability, the authors might want to have a look at recent philosophical work on causation, focusing on the idea of actual and potential difference makers. Heritability is about actual difference makers; malleability is about potential difference makers. These issues also come up in the Turkheimer and Gottesman responses to Gottlieb.\nThat brings me to heritabilityX and the authors conclusion, so I will stop numbering and consider the conclusion broadly. I don’t object in principle to their characterization of heritability as arising from causal consistency of any kind across generations. My objection to such developmentalist views are more practical than principled. It seems to me that they encourage a view of development that says all cats are gray in the dark, whereas in fact we can make real distinctions about cats, at least out on the edges of the forest where a little light gets in. Whether those principles apply to more complex problems deeper in the dark forest of human behavior is another question, but I don’t think it helps to ignore the distinctions that can be made.\nSo start with single gene disorders. Huntington’s disease is transmitted across generations because the gene is transmitted, and because the environment that supports the expression of the gene (which, as far as we know, is all environments) is also transmitted. We wouldn’t want to conclude, however, that distinctions between genetic and environmental transmission of HD are pointless, because we understand how it works. It could in principle turn out that there is an environmental treatment for HD, and if that happens the story will change, but for the time being it is meaningful to talk about HD as genetic.\nThe case for PKU is more complex because there is a therapeutic environment. But it still doesn’t create a major problem, once again because we generally understand the mechanism. In fact, no one actually bothers to compute the heritability of PKU, because in the presence of biological understanding it is pretty much irrelevant: the etiology of PKU is what it is, regardless of how various components of that etiology happen to vary at a particular time and place.\nNow do height. Why is height transmitted across generations? OK, it is because both genetic and environmental causes of height are preserved. But if you are taller than me, and we are both well-fed modern Americans, we are willing to presume that the cause of that difference is genetic, on the reasonable assumption that relevant environmental causes are not operating. If we observe that Americans are taller now than they were in 1900 we conclude the opposite, because we presume that relevant genetic causes are not operating. On this planet, we don’t have to conclude that no distinctions can be made between genetic and environmental transmission of height.\nBut for IQ, it is more problematic. We know that there are both genetic and environmental causes, and that both of them operate among individuals in the contemporary world. In addition we know that these causes are correlated (rGE would be a useful concept throughout) and that they interact (so would GxE). If you are smarter than me, is that because of genetic or environmental difference between us, or some complex combination of the two? We have no mechanistic knowledge, and no reasonable way of finding out, so the problem becomes intractable. Polygenic human behavior is by and large a gray cat.\nMy conclusion from all this is that the deep problem in understanding the heritability of human behavior is more practical than theoretical. The problem is not so much with the notion of heritability that is applied, but more with the limitations of our knowledge about how G and E work together to produce human behavioral phenotypes. I think this article does an excellent (albeit, as I have said, somewhat incomplete) job of laying out these issues, even if I don’t fully agree with their conclusion.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "56084", "date": "06 Nov 2019", "name": "Pierrick Bourrat", "expertise": [ "Reviewer Expertise Philosophy of science (biology) and evolutionary biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary In this article, the authors argue for an extension of the concept of heritability. They make three main points which I reconstruct as follows. First, they argue that the notion of heritability used in behavioral genetic (heritabilityBG), in spite of tracking causation, is problematic for a number of reasons. In fact, among other things, it does not permit to locate genes causally involved in the production of a phenotype preventing us from effectively being able to intervene on deleterious phenotypes. It also elicits the erroneous interpretation that phenotypes with a high heritability are immutable. Furthermore, this notion, the authors claim, does not correspond to the notion of heritability used in biology (heritabilityBio) which they define as the capacity for a trait to be genetically transmitted from one generation to the other. Second, the authors claim that heritabilityBio is also problematic because it focuses solely on genetic transmission which is too restrictive. Finally, they propose to extend the concept of heritability within an evolutionary framework to account for non-genetic inheritance (heritabilityX).\n\nAssessment Barring a few reservations, I am overall in agreement with the authors’ reasoning and arguments. However, I believe that a few distinctions should be drawn and incorporated in the revision of the manuscript or response, in order to help the authors position themselves in relation to the existing literature and prevent them from being misunderstood.\nThe first important distinction I would like to draw is between theoretical heritability and a heritability estimate. In principle, obtaining the theoretical heritability of a trait is pretty straightforward. One just takes the ratio of genotypic variance to phenotypic variance. One problem, however, is that genotypic variance cannot be measured directly. Instead, one must use an algorithm (e.g., apply a parent-offspring regression, Falconer’s formula, or any of the methods based on genome-wide association studies (GWAS)) to obtain an estimate of it. It is helpful when criticizing the usefulness of “heritability”, to know whether a criticism is addressed to the method for estimating heritability or to the concept itself. If the former, there is scope for improvement; if the latter, the prospects are much bleaker: a perfect estimate of the theoretical value would be unhelpful. Of course, whether one can obtain an accurate estimate in a given context is an important question, but it should nevertheless be distinguished from a criticism of the theoretical underpinnings of heritability itself.\nGoing back to the different claims made by the authors, when they mention that, following some of the classical models used in behavioral genetics, the heritability values obtained are prey to different confounding variables, this represents a criticism of the estimates (and the underlying methods to obtain them), not the concept. I take it that some of the authors’ criticisms are directed at some estimates (in particular those obtained from twin studies), while other criticisms are directed at the concept itself. It would be welcome to see this spelled out.\nSecond, it would be useful to see the distinction between broad-sense and narrow-sense heritability clearly drawn in the context of the article. This is a classical distinction used in the literature (Downes, 20171). When one says that the causes for a phenotype are genetic, this can be understood in two different ways. They refer either to an ontogenic timescale or to a phylogenetic timescale. In the former case, the effects of genes and of their interactions are taken into account for the production of the phenotype—this corresponds to broad-sense heritability. In the latter case, only the additive component of genotypic variance is taken into account—this corresponds to narrow-sense heritability since particular gene-gene interactions (e.g., dominance, epistasis) are, in the long run, eliminated during sexual reproduction.\nMirroring the two notions of heritability are two different scientific projects in the study of the interactions between genes and the environment for the production of a phenotype. One project aims at understanding these interactions during development (ontogenic timescale), while the second project aims at understanding them in the context of evolution (phylogenic timescale). Narrow-sense heritability is clearly relevant for the latter project, while the relevance of heritability (whether in the narrow or the broad sense) for the former project is much less obvious. It would be useful that the authors tell us whether, in their view, many of the criticisms they make apply to both narrow- and broad-sense heritability and to clarify which of the two research projects I outlined they are targeting. It looks to me as if the authors’ distinction between heritabilityBG and heritabilityBio is, at least in part, captured by the narrow/broad-sense heritability distinction, though not fully since they claim that heritabilityBio is a notion used vernacularly in biology (see Minor point 2 below).\nThird, I think it is important to note, as the authors do, that the traditional methods for estimating heritability have been devised while the support for genetic information was unknown. Why does it matter? Simply because what is meant by “genetic” (or more accurately genotypic) in the context of a twin-study or more generally in quantitative genetics, does not perfectly overlap with what is meant with the same word in molecular biology (Griffiths & Stotz, 20132). The difference has important implications when one calls for the extension of an idea in which the words “gene” or “genetic” are used (see for instance Lu & Bourrat, 20183 in the context of the extended evolutionary synthesis). A gene in quantitative genetics, or more generally in evolutionary theory, is a substrate-neutral entity of which one physical realizer is a molecular gene. “Genetic variance” in this context refers to anything physical that behaves like a molecular gene including some epigenetic factors. Thus, I believe that the extension the authors call for is, for part, already accounted for by the classical conceptual apparatus. Two things should be noted, however:\nEstimation methods of heritability relying on genomic data (e.g., Visscher, Yang, & Goddard, 20104) refer to a very narrow notion of the gene, namely one in which a gene is a given DNA-sequence variant with at least one non-silent substitution. Other types of mutations, such as additions and deletions, as well as other DNA changes such as chromosomic changes are not accounted for by these methods (Bourrat, accepted5) and they are one of the reasons a large discrepancy exists between traditional methods of estimation and those based on GWAS (Bourrat & Lu, 20176; Danchin, 20137). All these “genetic” differences are relevant evolutionarily and should be included for obtaining an accurate estimate of heritability relevant for evolution. In this respect a call for extending heritability (estimates) to non-genetic factors (where “non-genetic” here is in reference to the molecular concept of the gene) is justified.\n\nNon-genetic factors (where “non-genetic” here is in reference to the evolutionary concept of the gene) can be transmitted over time. This calls for a substrate neutral concept of heritability, which can be defined abstractly as a relative population-level measure of parent-offspring resemblance. When defined as such, no particular mechanism of inheritance is considered. Although this notion of heritability is sometimes preferred by some (see for instance the exchange between Downes, 20098 and Okasha, 20109) because it is even more abstract than the gene-centered concept, it can lead to a number of epistemic difficulties, such as for instance assessing the causal structure underlying the inheritance of different factors from one generation to the other. For instance, as shown by Lynch and Bourrat (2017)10, when there exists a genotype-environment correlation, the causal origin of the correlation can be different. One implication of this difference is that in cases where the environment can be considered as part of the extended phenotype of a genotype, one should include any gene-environment correlation resulting from this causal relationship as part of the heritability estimate. On the contrary, when environment and genotype are causally independent, the correlation should not be included in the estimate. One danger of calling for an extension of the heritability concept to diverse types of physical substrates is to neglect that many of them might be extended phenotypes. Thus, calling for an extension of the concept of heritability to include non-genetic factors (including environmental ones) can be wrongheaded in some situations. To be clear, I am not defending here a naïve version of genetic determinism, just hedging against a strong version of the causal parity thesis.[1] For these reasons, I found the Figure 1 slightly misleading as each factor is represented by a line which does not cross with any other. This tends to suggest that these factors are independent from one another. In reality, many of these factors interact (non-additively) with one another, and some will be causally involved in the determination of other transmitted factors. I think it would be useful to visually represent this on the figure or write it somewhere in the caption and/or text. Additionally, if heritability is invoked with the aim of separating the effects of individual properties from those of the environment in their contribution to parent-offspring phenotypic resemblance, it must be defined in reference to an environment even when the abstract substrate-neutral concept is used. This implies that the environment (or at least part of it) must be considered independently from the properties of the individuals forming a population. Calling for an extension of heritability to include the environment (or part of it), without being very clear what is meant by the word “environment” could lead to the view that any factors of the environment correlated with individual properties should be included in a heritability measure. This should be resisted. This last point is to be related to the problematic use of the notion recurrence by the authors when they refer to heritability. Recurrence does not necessarily imply transmission, while a trait being heritable implies that it has been transmitted. I am not saying that the authors have fallen prey to the different problems outlined above with respect to extending the concept of heritability. Rather, I am saying that while an extension of the notion of heritability is welcome, the authors might want to ponder upon this type of considerations to guard themselves against these potential difficulties.\n\nMore minor points:\nI think I agree with Eric Turkheimer (Reviewer 1) that the authors might focus too narrowly on behavioral genetics. I also recommend using the term “heritabilityQG” instead of “heritabilityBG”.\n\nI was unconvinced by the claim on page 6 that bipedality is universally considered has heritable while its heritabilityBG is nil. Most evolutionary biologists would say that bipedality has a low heritability but would recognize that the heredity of this trait is high. I am thus wondering whether the authors’ intention is not contrast heredity with a population measure of it, namely heritability, the latter of which is a very imperfect measure of the former.\n\nRelated to the previous point, I would argue that the word heritability is most often used in a technical sense while the words inheritance and heredity are used more casually. In their proposal of extending heritability to non-genetic factors, do the authors have in mind a technical or vernacular notion? Since they used “heritability”, and they want their extension to be framed evolutionarily, I took it they had in mind a technical notion. In fact, in evolutionary biology heritability overwhelmingly refers to the technical meaning of quantitative genetics. But if they don’t then they should be very clear about it since this could be an important way in which their article is misunderstood.\n\nI would cite Lewontin (1970)11 when mentioning the three conditions for evolution by natural selection since this paper was very influential in making the recipe approach to natural selection a popular one in the last 50 years.\n\nI think the authors should recognize previous calls to extend the concept of heritability. One such call in evolutionary theory has been made by Danchin (2013)7.\n\n[1] Note furthermore that another reason it is not a defense of naïve determinism is that this reasoning could be applied whether genes or any other inherited factors is transmitted from parent to offspring.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-1705

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