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https://f1000research.com/articles/4-131/v1	28 May 15	{ "type": "Review", "title": "Sepsis breakthroughs in 2014", "authors": [ "James A. Russell", "Keith R. Walley", "Keith R. Walley" ], "abstract": "The mortality of sepsis may be decreasing and, because there are more survivors, it is increasingly important to understand the epidemiology, pathogenesis, genetics, prevention, and treatment of the impaired long-term outcomes of sepsis. Recent insights on the clearance of bacterial products during sepsis suggest new strategies for early intervention. Immune suppression/immune reprogramming to decrease later secondary infections is a novel strategy now in clinical trials. The Protocolized Care for the Early Septic Shock (ProCESS), Australasian Resuscitation in Sepsis Evaluation (ARISE) and ProMISe randomized controlled trials (RCTs) of early goal-directed therapy (EGDT) versus usual care found no differences between groups in mortality. Fluid therapies may not require full-on EGDT, but rather emphasize the importance of early recognition and resuscitation of sepsis. The Albumin Italian Outcome Sepsis (ALBIOS) RCT did not find a difference between albumin (titrated to serum albumin >30 g/L) and crystalloid in severe sepsis. However, in a subgroup analysis, mortality was lower in the albumin group in patients who had septic shock. Therapeutic use of albumin may be beneficial in septic shock, but requires further evaluation in RCTs. A recent RCT of conservative versus liberal transfusion strategies (70 versus 90 g/L, respectively) found no difference in mortality in septic shock. The transfusion threshold in septic shock is now 70–90 g/L. Although there was no difference in mortality between a usual or a high target mean arterial pressure (MAP) for septic shock resuscitation, a higher MAP target may be beneficial in patients who have pre-existing hypertension, because higher MAP may decrease the incidence of acute kidney injury (AKI) and need for renal replacement therapy (RRT). Nutrition practice can continue with enteral nutrition started on days 2–3 (i.e., early but there is no indication for very early parenteral nutrition). Acute respiratory distress syndrome (ARDS) is the commonest complication of sepsis. Two recent RCTs of simvastatin and rosuvastatin in ARDS were not positive. Early statins at appropriate doses and plasma levels deserve a trial in sepsis. In future, perhaps three changes could improve the chances of having positive trials in sepsis: the use of biomarkers to stratify patients; adaptive trial design to enhance dose selection and reject compounds that are unlikely to be suitable at Phase 2; and the use of composite organ dysfunction as the primary outcome.", "keywords": [ "Sepsis", "Trials", "Septic Shock" ], "content": "The global burden of critical care and decreasing sepsis mortality\n\nThe global burden of critical care1 is increasing rapidly for many reasons, one of which is the increasing incidence and prevalence of sepsis. Sepsis is a common cause of admission to an Intensive Care Unit (ICU) and the commonest cause of death in ICU. Interestingly, despite no new drug approvals for sepsis since activated protein C (APC) (and the withdrawal of APC from the world market), sepsis mortality appears to be decreasing over the last decade2 from about 40% to about 20%. Most attribute this success to the improved process of care, including the earlier identification of sepsis in the Emergency Department and earlier initiation of appropriate antibiotics, fluid and vasopressor resuscitation, and low tidal volume during mechanical ventilation3–8. The decreasing mortality, by definition, leads to increasing numbers of survivors of severe sepsis and septic shock. Thus, the long-term outcome and morbidity of survivors is an emerging, centrally important research and clinical care concern in sepsis.\n\nA very recent, very large (n = 1,171,197!) cohort study evaluated whether the numbers of systemic inflammatory response syndrome (SIRS) criteria a patient has when presenting with sepsis is associated with increased mortality9. The vast majority (88%) had SIRS-positive severe sepsis whereas only about 12% had SIRS-negative severe sepsis. Interestingly, the declines in mortality rates described above were similar in these two groups, SISR positive and SIRS negative. The authors therefore conclude that SIRS criteria to define severe sepsis may not be helpful or useful because the usual definition of needing two or more SIRS criteria to define severe sepsis excluded about 12% of patients with severe sepsis. However, in the SIRS-positive group, mortality increased as the number of SIRS criteria was positive, suggesting that the sum of SIRS criteria does have prognostic value. This will create more controversy because a growing number of authors have questioned the value and need for SIRS criteria to define sepsis and severe sepsis.\n\nIt is important to highlight that the parallel increase in the incidence of severe sepsis (and septic shock) worldwide and the reduction in its mortality rate may have another “administrative” explanation and that the changes may not be as real initially considered. Several investigators10,11 have argued that the higher incidence of reported cases of “mild sepsis” is due to greater awareness of the problem (and the sepsis definition) as well as enhanced reimbursement for a sepsis diagnosis, thus incentivizing the diagnosis of sepsis definition). Ironically, while the incidence of organ-specific infections remains fairly constant over the last decade, the incidence of severe sepsis continues to increase10. And perhaps even more important, the greater inclusion of mild cases under the umbrella of “severe sepsis”, which were less often reported in the past, may artificially decrease the pooled mortality rate of sepsis in cohorts and administrative databases. These observations challenge partially the statement that the mortality of severe sepsis has decreased, although there could also be a true decrease of mortality too.\n\nWhat about the mortality rates of randomized controlled trials of different eras? Why have those mortality rates also been decreasing? Perhaps the patient populations of severe sepsis included in “old” RCTs differ from more recent RCTs. Even in the more recent RCTs, there is a marked discrepancy between mortality rates of European trials compared to North American and Australian RCTs. One possible explanation is that baseline characteristics and risks of death of the study populations enrolled differ across continents and jurisdictions. In support of this hypothesis, the incidence of mechanical support (which may be a surrogate of the overall severity and risk of death) at the time of study enrollment is about 85–90% of the study populations in European RCTs, versus only about 40–50% in the American and Australian trials. VASST12 (vasopressin vs. norepinephrine in septic shock) and CORTICUS13 (European RCT of corticosteroids vs. placebo in septic shock) were reported in 2008 and both had an incidence of mechanical ventilation of about 85–90%. Thus, it is difficult to compare RCTs of different continents and of different eras as there are uncontrolled differences and changes over time in processes of care (and that could improve outcomes) as well as changes in populations included (and that could alter risk of death).\n\n\nLong-term outcome of sepsis\n\nAs the mortality of severe sepsis and septic shock continues to decrease, the number of survivors is increasing steadily; however, this is not all good news. Long-term outcome is impaired in 1-year survivors in the ensuing 1–10 year period after septic shock, even after adjusting for underlying co-morbidities14. Furthermore, even mild stage 1 AKI is significantly associated with impaired long-term (10 years) outcome of septic shock15. Cognitive dysfunction, neuromuscular weakness and neuromyopathies, post-traumatic stress disorder (PTSD), and depression are common long-term complications of septic shock16. Cognitive and functional decline, in part, reflect pre-existing declines in those domains that may place such patients at risk for sepsis17.\n\n\nRandomized controlled trials (RCTs) in sepsis\n\nMost RCTs have been negative in sepsis and, in particular, no new therapies for modification of the host response have been successfully introduced into clinical practice. Recent examples include eritoran18, and polyclonal anti-tumor necrosis factor (TNF)19. Eritoran is an inhibitor of the early innate immune response that signals via Toll-Like receptor 4 (TLR4) and its associated molecule, MD2. Eritoran did not alter mortality or secondary outcomes in 1,961 severe sepsis patients. Similarly, anti-TNF did not display an efficacy signal in a Phase 2b multicenter RCT. Talactoferrin actually increased mortality (http://www.genengnews.com/gen-news-highlights/agennix-stops-phase-ii-iii-talactoferrin-study-in-sepsis-due-to-higher-death-rates/81246312), despite a seemingly positive Phase 2 RCT20. Accordingly, new RCT designs are needed and new approaches to sepsis therapy must be considered.\n\n\nNew RCT designs are necessary in sepsis\n\nNew RCT designs are needed in sepsis21. Three changes could increase the chances of having positive RCTs in sepsis: the use of biomarkers, response adaptive trial design, and composite organ dysfunction endpoints.\n\nPredictive biomarkers define subgroups of septic patients who have an enhanced (or adverse) response to a specific treatment. The use of predictive biomarkers (such as Her2neu for Herceptin use) has revolutionized cancer RCTs, and clinical drug protocols leading to improved outcomes of many cancers. Furthermore, biomarkers could improve patient selection through more accurate diagnosis of sepsis. Finally, outcomes of sepsis vary widely depending on the source of sepsis22, so perhaps future sepsis RCTs should stratify by the source of sepsis or only include particular sources of sepsis (e.g., severe community-acquired pneumonia) in RCTs in sepsis.\n\nResponse-adaptive trial design can improve the efficiency of sepsis RCTs by optimizing dose selection and by stopping futile trials earlier because complex adaptive trial design makes use of interim data in nearly real time to adjust randomization and to select optimal dosing prior to completion. While industry is increasingly using adaptive trial design, academia has been slower to adopt this method. It is difficult to design outcomes in sepsis phase 2 trials due to the competing risk of death. Therefore, current phase 2 trials may not be predictive of success in phase 3 trials and the response-adaptive trials could help improve this situation.\n\nThe many failed RCTs of therapeutics for sepsis used 30-day mortality as the primary endpoint. The usual primary sepsis trial endpoint of 28 or 30-day mortality is roughly 6–8% lower than 90 day mortality in recent RCTs of sepsis. This observation, along with the prevalence of impaired long-term outcomes beyond 90 days, suggests that the “standard” 28-day mortality outcome in sepsis trial is an inadequate measure of the burden of sepsis. The recent RCTs (ProCESS23, ARISE24, ProMISe25 and Cohort studies2 show that the mortality rates of sepsis are lower than in RCTs published in 2008 of vasopressin versus norepinephrine12 (mortality rate 35–39%) and corticosteroids versus placebo13 (mortality rate 36–39%) (Table 1 and Table 2).\n\nAccordingly, there are greater numbers of sepsis survivors, many of whom have ongoing morbidities and increased long-term (10-year) mortality rates. Therefore, it is appropriate and timely to adjust to the use of composite endpoints in sepsis RCTs. Examples of composite outcomes could include days alive and free of vasopressors and ventilation (for a drug that modified cardiovascular and pulmonary dysfunction) or days alive and free of vasopressors, ventilation and renal replacement therapy (for a drug that modulated these three organ dysfunctions). Composite cardiovascular endpoints have been the norm in academic and industry cardiovascular RCTs for several decades. As discussed under the section long-term outcomes of sepsis, such outcomes are also of value for therapies that could have such sustained benefits.\n\nWe also note that another critical strategy to improve the likelihood of future RCTs in severe sepsis and septic shock being positive depends upon on a better understanding of the molecular and pathophysiological rationale upon which we may decide to test, or not, a specific drug or device. Furthermore, the field is now widely aware that acute short-term models in healthy animals are poor models of human severe sepsis and septic shock. It is likely that the reason for some examples of negative RCTs in severe sepsis and septic shock is an incompletely clear molecular and pathophysiological rationale based on inadequate pre-clinical models.\n\nEGDT, Early goal-directed therapy; MAP, mean arterial pressure; RBCs, red blood cells.\n\nEGDT, Early goal-directed therapy; MAP, mean arterial pressure; RBCs, red blood cells.\n\n\nMany new randomized controlled trials published in 2014 and early 2015\n\nThere were a number of large multicenter RCTs published in 2014 and early 2015 that are directly or indirectly concerned with severe sepsis and septic shock (Table 1 and Table 2). The eight RCTs highlighted in Table 1 and Table 2 recruited a total of 13,282 critically ill patients, a remarkably large number. Unfortunately, none of these RCTs achieved pre-specified statistically significant results on their respective primary endpoints!\n\n\nMore novel targets are necessary in sepsis\n\nThe majority of failed clinical RCTs of novel biologics for treatment of sepsis share key features. First, most of the trials aimed to modulate the host response to infection. Modulation of the host response to sepsis may have failed, in part, because the host septic inflammatory response is complex, involving many parallel redundant biological pathways that are expressed to a lesser or greater extent at different times, in different tissues, in different patients26. Furthermore, a number of the pathways are detrimental in some patients and therefore could reasonably be inhibited (for example, TNFα detrimentally drives a systemic inflammatory response) but not in other patients (TNFα helps wall off focal infection, such as localized bowel perforation). Second, enrollment in these RCTs was delayed by the process of identifying sepsis, by the processes of obtaining informed consent and delivering the therapeutic to the patient; thus, often, the novel therapeutics were delivered to the patient 12 hours or even more than 72 hours after the onset of sepsis. A key lesson learned from early EGDT studies, and by the more recent ProCESS23 and ARISE24 trials, is that early identification and treatment, notably including early antibiotics, profoundly reduces mortality from sepsis, severe sepsis, and septic shock27.\n\nAccordingly, the search for novel therapeutics to treat sepsis should be cautious regarding the modulation of the late host response and, instead, focus on the earliest aspects of sepsis—particularly the pathogen and clearance of pathogen-derived products—that initially trigger the host inflammatory response.\n\nProprotein convertase subtilisin/kexin type-9 (PCSK9) is an exciting candidate for treatment because anti-PCSK9 strategies target the organism(s), not the host response (a strategy that has almost uniformly failed)28 (Figure 1). The potential benefit of this approach arises from an understanding of the normal clearance pathways of sepsis-initiating pathogen molecules. The innate immune system provides the earliest response to infecting pathogens when pathogen-associated molecular patterns (PAMPs) bind and activate innate immune receptors, such as Toll-Like receptors. Key PAMPs from bacteria are lipid-containing molecules that arise from bacterial cell walls, such as lipopolysaccharide (LPS) from Gram-negative bacteria, or lipotechoic acid from Gram-positive bacteria. To clear these pathogen lipids from the circulation of septic patients, these molecules are first bound by transfer proteins, such as LPS-binding protein (LBP) and bactericidal/permeability-inducing protein (BPI). These transfer proteins are highly homologous to other lipid-carrying transfer proteins, such as phospholipid transfer protein (PLTP) and cholesterol ester transfer protein (CETP), molecules that are much more familiar to cardiologists and others caring for patients with hypercholesterolemia and atherosclerotic cardiovascular disease. Like PLTP and CETP, the pathogen lipid transfer proteins are carried within, and equilibrate between, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL)27. Then, pathogen lipids are cleared by the liver via the LDL receptor expressed on hepatocytes and by other less clearly identified routes.\n\nPathogen lipids, such as endotoxins from Gram-negative bacteria and lipotechoic acid from Gram-positive bacteria, are carried within high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) particles in the blood. Pathogen lipids contained within LDL cholesterol (LDL-C) are then cleared via LDL receptors (LDLR) expressed on hepatocytes. The left hand panel illustrates the role that proprotein convertase subtilisin/kexin type 9 (PCSK9) plays in modulating clearance. PCSK9 binds the LDLR and when LDLR is internalized, as part of hepatocyte uptake of LDL-C, bound PCSK9 targets the LDLR for lysosomal degradation. As a result, LDLR density on the hepatocyte cell surface diminishes. The right hand panel shows that when PCSK9 concentrations are reduced, LDLR density increases so that the clearance of LDL-C is increased. In the setting of sepsis, when LDL-C carries pathogen lipids, increased LDLR density also results in the increased clearance of pathogen lipids, leading to a diminished inflammatory response and improved sepsis outcomes.\n\nThe LDL receptor is thus a key step in the clearance of pathogen lipids from the circulation in sepsis, severe sepsis and septic shock28. LDL particles bind to the LDL receptor and, with the LDL receptor, are internalized within the hepatocyte (Figure 1). When cleared of LDL, the receptor is then recirculated to the hepatocyte cell surface. As recently described in the cardiovascular literature30, the number of LDL receptors expressed on hepatocytes is regulated by PCSK9. PCSK9 in the circulation binds LDL receptors on hepatocytes. When the LDL receptor is internalized, bound PCSK9 targets the LDL receptor for lysosomal degradation. This results in decreased LDL receptor density on hepatocytes that, in turn, reduces LDL receptor-mediated clearance of LDL (of interest to physicians treating atherosclerosis risk) but also reduces the clearance of pathogen lipids (of great interest in sepsis). Conversely, pharmaceuticals or genetic variations that reduce PCSK9 function, increase the expression of the LDL receptor on hepatocytes, and thereby increase the clearance of LDL from the blood28.\n\nAccordingly, knockout of the PCSK9 in mice reduces the adverse systemic inflammatory response when LPS is administered, by increasing the clearance rate of LPS28. Inhibition of PCSK9 in a model of polymicrobial septic peritonitis decreases the inflammatory cytokine response and results in increased survival. The beneficial effect of decreased PCSK9 function also appears to have an important effect in human sepsis. Septic shock patients carrying one or more loss-of-function genetic variants of the PCSK9 gene have significantly improved outcomes: decreased inflammatory cytokine response and decreased 28-day mortality. Conversely, septic shock patients carrying gain-of-function PCSK9 genetic variants have adverse outcomes: increased septic inflammatory cytokine response and increased mortality. This raises the hypothesis that pharmacologic inhibition of PCSK9 may improve outcomes in septic patients by enhancing the clearance of pathogen lipids by the liver, thereby decreasing the cytokine inflammatory response and its physiologic and clinical phenotype consequences28.\n\n\nStatins in the commonest organ dysfunction of sepsis: ARDS\n\nIf PCSK9 inhibition (conceptually the new “super statin”) improves the outcome in sepsis, then does conventional statin therapy also improve outcomes from sepsis and related organ dysfunction, such as ARDS? After a number of days of treatment, statins indirectly increase LDL receptor expression as a consequence of the statin-induced decrease in LDL. This should be beneficial in sepsis. However, statin treatment also indirectly increases PCSK9 concentrations, again as a response to decreased LDL. Thus, the net effect of statins was not known. Simvastatin31 and rosuvastatin32 did not alter outcomes of ARDS, the commonest organ dysfunction associated with sepsis. Perhaps it would be logical to test earlier statins and more adequate plasma statin levels during treatment in future RCTs in sepsis. However, PCSK9 inhibition may also be required to counteract the adverse impact of statin-induced elevations of PCSK9 levels. The Papazian study33 of simvastatin for ventilator-associated pneumonia (VAP) was stopped early for futility with a hazard ratio of 1.45 [95% CI, 0.83 to 2.51].\n\n\nRCTs of fluid resuscitation and management of sepsis\n\nEarly protocolized care of septic patients has been almost universally adopted and has transformed patient outcomes2. One major contributor to improved outcomes appears to be earlier identification of sepsis by physician and non-physician Emergency Department (ED) staff, facilitated by the implementation of manual or computerized algorithms. Earlier identification sepsis algorithms also drive the early administration of appropriate antibiotics and that appears to be a key contributor. Another contributor may be early fluid resuscitation and hemodynamic support. The ProCESS23, ARISE24 and ProMISe25 trials sought to identify the key beneficial aspects of Rivers’ early EGDT34 in the current 21st century ED environment, where early identification, antibiotics, and fluid resuscitation are considered usual care. In view of the current ED environment, these studies did not achieve very substantial clinical differences in resuscitation (processes of care) between treatment groups, and all patients had received many aspects of Rivers’ EGDT and had achieved many of the EGDT targets by the time of enrollment into ProCESS and ARISE (e.g., initial mean central venous oxygen saturation in ProCESS was above the EGDT target of >70%, mean Central venous oxygen saturation [ScvO2] was about 65% in ProMISe25). Enrollment in ProCESS and ARISE sometimes took substantially longer (up to 12 hours) compared to Rivers’ EGDT (mean 1.5 hours) so that these later RCTs may have interrogated patients somewhat later and after more substantial resuscitation, and thus improvement. ProMISe recruited a mean of 2.5 hours after meeting inclusion criteria.\n\nThe ProCESS23 and ARISE24 RCTs had low pooled mortality rates (18–22%) and there was no difference in mortality between EGDT and usual care groups (Table 2). ProMISe used 90-day mortality that was as expected higher (pooled mortality 29%), with no difference between EGDT and usual care groups (29.5 vs. 29.2% respectively; Table 2).\n\nIt is challenging to arrive at firm conclusions for RCTs with minimal differences in processes of care between the treatment groups and with no significant difference in outcomes. Nevertheless, the low mortality rates of the control groups in ProCESS and ARISE suggest that many changes in care (e.g., early identification of sepsis, early administration of antibiotics and early, effective source control) have decreased the mortality rate of severe sepsis. The negative results of ProCESS and ARISE suggest that targeting a central venous saturation greater than 70% is one of several effective approaches to determining whether fluid and vasopressor resuscitation adequately reversed hypoperfusion. For example, ProCESS allowed investigators to rely on clinical judgment which, in this setting, appeared to be as good as EGDT guided care, while Jones et al.35 suggest that lactate clearance is as good a target as central venous oxygen saturation targeting. Indeed, Rivers’ EGDT implies that a low mixed venous oxygen saturation (after adequate fluid and vasopressor resuscitation, in the setting of adequate blood oxygen carrying capacity) raises the possibility of impaired cardiac function. With the recent emergence of routine goal-directed echocardiography in the ED, the diagnosis of impaired cardiac function need not await measurements of central venous oxygen saturation36.\n\nThe optimal mean arterial blood pressure (MAP) for septic shock patients is unknown. Higher arterial pressures may be more effective at perfusing some high-resistance vascular beds in vital organs, such as the brain, kidneys and heart, yet higher MAP targets may require greater use of vasopressors with potential adverse consequences. Asfar and colleagues37 compared a high MAP (80–85 mmHg) with a lower MAP (65–70 mmHg) over the course of 5 days, or until cessation of vasopressor infusions in septic shock patients38. They found no overall difference in mortality rates between high and low target MAP. As expected, the high MAP target was associated with increased vasopressor use and, consequently, with an increased incidence of dysrhythmias (especially atrial fibrillation). However, in the a priori subgroup of patients who had pre-existing hypertension, patients randomized to the high MAP group had significantly lower incidences of AKI and need for RRT (32% versus 42%, unadjusted P=0.046)39.\n\nWhether the choice of fluid for septic shock resuscitation alters outcome continues to be debated. ALBIOS40 showed no difference in mortality between albumin resuscitation and maintenance of a serum albumin >30 G/L while in ICU for patients with severe sepsis/septic shock, compared to usual care with crystalloid resuscitation. In a post hoc analysis, there was a lower mortality in the albumin compared to the crystalloid resuscitation group in the subgroup of patients who had septic shock. Optimal choice of fluids from resuscitation, therefore, remains an open issue.\n\n\nTransfusion in septic shock\n\nThe pivotal Transfusion Requirements in Critical Care (TRICC) RCT41 found that a conservative transfusion strategy (to transfuse when Hg <7 g/dL) was similar in efficacy to a liberal transfusion (Hg <9 g/dL) strategy in the critically ill42. However, it remained uncertain whether this observation also applied to patients who had septic shock. This was especially confusing because the EGDT RCT of Rivers and colleagues34 used a transfusion threshold of Hg <10 g/dL, much higher than the conservative TRICC threshold (Hg <7 g/dL). Fortunately, Holst and colleagues43 performed an RCT and again found no difference in mortality between conservative (43% at 90 days) and liberal transfusion thresholds (45% at 90 days, P=0.44, Table 2), but this time in septic shock. Furthermore, there were no differences between groups in ischemic events, adverse events, or numbers of patients who needed life support. Also, when aligned with the results of ProCESS and ARISE described above, it is very reasonable to use a transfusion threshold of 7 g/dL in septic shock. Thus, a conservative transfusion threshold is safe and effective in patients who have septic shock.\n\nThe age of transfused blood may alter the outcome in that older stored blood was associated with poorer outcomes in non-controlled or non-randomized studies. In a related RCT, Lacroix and colleagues44 sought to determine whether age of transfused erythrocytes (RBCs) alters outcomes in the critically ill, many of whom had sepsis; patients received 1 unit of either fresh RBCs (stored for 6.1±4.9 days) or standard-issue RBCs (stored for 22.0±8.4 days) (Table 1 and Table 2). There was no difference in 90-day mortality rates between groups (37% vs. 35.3% respectively, P=0.38). All of the secondary outcomes and subgroup analyses aligned with the major finding: no difference between groups in outcomes. Thus, standard issue RBCs are as safe and as effective in the critically ill, and presumably in sepsis.\n\n\nNutrition in sepsis\n\nA large RCT of two strategies45 found no difference in 30-day mortality between early enteral (34%) and parenteral (33%) nutrition in the critically ill. Parenteral feeding was associated with less hypoglycemia and vomiting, but there were no differences in any other secondary outcomes (e.g., infectious complications, 90-day mortality).\n\n\nAlbumin infusion in severe sepsis or septic shock\n\nA recent randomized controlled trial showed that 20% albumin plus crystalloids did not change mortality compared to patients who received crystalloids alone in patients who had severe sepsis40. The albumin plus crystalloids group had 20% albumin given daily to maintain serum albumin concentration above 30 g/L. Although the albumin group had higher mean arterial pressure and lower fluid balance over the first 7 days, 28-day mortality rates were very similar (31.8% albumin, 32% crystalloid group).\n\n\nConclusions\n\nMortality of sepsis may be decreasing, and so having more survivors of sepsis convinces clinicians and investigators that it’s critically important to understand epidemiology, pathogenesis, genetics, prevention and treatment of long-term complications of sepsis. Many negative RCTs were reported in 2014 that inform the use of EGDT, target mean arterial pressure, albumin infusion, RBC transfusion, nutrition, and the use of statins in severe sepsis and septic shock. Perhaps three changes could improve the possibility of positive RCTs in this field: the use of biomarkers, response adaptive trial design, and a primary outcome of composite organ dysfunction. PCSK9 is an exciting candidate for treatment because anti-PCSK9 strategies target the organism(s), not the host response (a strategy that has almost uniformly failed). Fluid therapies may not require full-on EGDT, but rather emphasize the importance of early recognition and resuscitation of sepsis. Therapeutic use of albumin may be beneficial in septic shock, but requires further evaluation in RCTs. The transfusion threshold in septic shock is now hemoglobin of 70 g/L. A higher MAP target may be beneficial in patients who have pre-existing hypertension because higher MAP may decrease the incidence of AKI and the need for RRT. Nutrition practice can continue with enteral nutrition started on day 2–3 (i.e., early, but there is no indication for very early parenteral nutrition). Early statins at appropriate doses and plasma levels deserve further investigation in trials in sepsis.", "appendix": "Competing interests\n\n\n\nDrs. James Russell and Keith Walley report patents owned by the University of British Columbia (UBC) that are related to proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor(s) and sepsis and related to the use of vasopressin in septic shock. Drs. Russell and Walley are inventors on these patents. Drs. Russell and Walley are founders, Directors and shareholders in Cyon Therapeutics Inc. (developing a sepsis therapy). Dr. Russell has share options in Leading Biosciences Inc.\n\nDr. Russell reports receiving consulting fees from Cubist Pharmaceuticals, formerly Trius Pharmaceuticals (developing antibiotics), Ferring Pharmaceuticals (manufactures vasopressin and is developing selepressin), Grifols (sells albumin), MedImmune (regarding sepsis), Leading Biosciences (developing a sepsis therapeutic), La Jolla Pharmaceuticals (developing a sepsis therapeutic), and Sirius Genomics Inc. (now closed; had done pharmacogenomics research in sepsis).\n\nDr. Russell reports having received grant support from Sirius Genomics, Ferring Pharmaceuticals, and Astra Zeneca that is provided to and administered by UBC.\n\n\nReferences\n\nVincent JL, Marshall JC, Namendys-Silva SA, et al.: Assessment of the worldwide burden of critical illness: the intensive care over nations (ICON) audit. Lancet Respir Med. 2014; 2(5): 380–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKaukonen KM, Bailey M, Suzuki S, et al.: Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand, 2000–2012. JAMA. 2014; 311(13): 1308–16. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nEisner MD, Thompson T, Hudson LD, et al.: Efficacy of low tidal volume ventilation in patients with different clinical risk factors for acute lung injury and the acute respiratory distress syndrome. 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PubMed Abstract \| Publisher Full Text\n\nRussell JA, Walley KR, Singer J, et al.: Vasopressin versus norepinephrine infusion in patients with septic shock. N Engl J Med. 2008; 358(9): 877–87. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy for patients with septic shock. N Engl J Med. 2008; 358(2): 111–24. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLinder A, Guh D, Boyd JH, et al.: Long-term (10-year) mortality of younger previously healthy patients with severe sepsis/septic shock is worse than that of patients with nonseptic critical illness and of the general population. Crit Care Med. 2014; 42(10): 2211–8. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLinder A, Fjell C, Levin A, et al.: Small acute increases in serum creatinine are associated with decreased long-term survival in the critically ill. Am J Respir Crit Care Med. 2014; 189(9): 1075–81. PubMed Abstract \| Publisher Full Text\n\nJackson JC, Pandharipande PP, Girard TD, et al.: Depression, post-traumatic stress disorder, and functional disability in survivors of critical illness in the BRAIN-ICU study: a longitudinal cohort study. Lancet Respir Med. 2014; 2(5): 369–79. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nIwashyna TJ, Ely EW, Smith DM, et al.: Long-term cognitive impairment and functional disability among survivors of severe sepsis. JAMA. 2010; 304(16): 1787–94. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nOpal SM, Laterre PF, Francois B, et al.: Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA. 2013; 309(11): 1154–62. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBernard GR, Francois B, Mira J, et al.: Evaluating the efficacy and safety of two doses of the polyclonal anti-tumor necrosis factor-α fragment antibody AZD9773 in adult patients with severe sepsis and/or septic shock: randomized, double-blind, placebo-controlled phase IIb study. Crit Care Med. 2014; 42(3): 504–11. PubMed Abstract \| Publisher Full Text\n\nGuntupalli K, Dean N, Morris PE, et al.: A phase 2 randomized, double-blind, placebo-controlled study of the safety and efficacy of talactoferrin in patients with severe sepsis. Crit Care Med. 2013; 41(3): 706–16. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nOpal SM, Dellinger RP, Vincent JL, et al.: The next generation of sepsis clinical trial designs: what is next after the demise of recombinant human activated protein C? Crit Care Med. 2014; 42(7): 1714–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeligdowicz A, Dodek PM, Norena M, et al.: Association between source of infection and hospital mortality in patients who have septic shock. Am J Respir Crit Care Med. 2014; 189(10): 1204–13. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nYealy DM, Kellum JA, Huang DT, et al.: A randomized trial of protocol-based care for early septic shock. N Engl J Med. 2014; 370(18): 1683–93. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nPeake SL, Delaney A, Bailey M, et al.: Goal-directed resuscitation for patients with early septic shock. N Engl J Med. 2014; 371(16): 1496–506. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMouncey PR, Osborn TM, Power GS, et al.: Trial of early, goal-directed resuscitation for septic shock. N Engl J Med. 2015; 372(14): 1301–11. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBarochia AV, Cui X, Natanson C, et al.: Risk of death and the efficacy of eritoran tetrasodium (E5564): design considerations for clinical trials of anti-inflammatory agents in sepsis. Crit Care Med. 2010; 38(1): 306–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBarochia AV, Cui X, Vitberg D, et al.: Bundled care for septic shock: an analysis of clinical trials. Crit Care Med. 2010; 38(2): 668–78. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWalley KR, Thain KR, Russell JA, et al.: PCSK9 is a critical regulator of the innate immune response and septic shock outcome. Sci Transl Med. 2014; 6(258): 258ra143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevels JH, Abraham PR, van den Ende A, et al.: Distribution and kinetics of lipoprotein-bound endotoxin. Infect Immun. 2001; 69(5): 2821–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStein EA, Raal F: Reduction of low-density lipoprotein cholesterol by monoclonal antibody inhibition of PCSK9. Annu Rev Med. 2014; 65: 417–31. PubMed Abstract \| Publisher Full Text\n\nMcAuley DF, Laffey JG, O'Kane CM, et al.: Simvastatin in the acute respiratory distress syndrome. N Engl J Med. 2014; 371(18): 1695–703. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nTruwit JD, Bernard GR, Steingrub J, et al.: Rosuvastatin for sepsis-associated acute respiratory distress syndrome. N Engl J Med. 2014; 370(23): 2191–200. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nPapazian L, Roch A, Charles P, et al.: Effect of statin therapy on mortality in patients with ventilator-associated pneumonia: a randomized clinical trial. JAMA. 2013; 310(16): 1692–700. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRivers E, Nguyen B, Havstad S, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001; 345(19): 1368–77. PubMed Abstract \| Publisher Full Text\n\nJones AE, Shapiro NI, Trzeciak S, et al.: Lactate clearance vs central venous oxygen saturation as goals of early sepsis therapy: a randomized clinical trial. JAMA. 2010; 303(8): 739–46. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWalley PE, Walley KR, Goodgame B, et al.: A practical approach to goal-directed echocardiography in the critical care setting. Crit Care. 2014; 18(6): 681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsfar P, Teboul JL, Radermacher P: High versus low blood-pressure target in septic shock. N Engl J Med. 2014; 371(3): 283–4. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nAsfar P, Meziani F, Hamel JF, et al.: High versus low blood-pressure target in patients with septic shock. N Engl J Med. 2014; 370(17): 1583–93. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRussell JA: Is there a good MAP for septic shock? N Engl J Med. 2014; 370(17): 1649–51. PubMed Abstract \| Publisher Full Text\n\nCaironi P, Tognoni G, Gattinoni L: Albumin replacement in severe sepsis or septic shock. N Engl J Med. 2014; 371(1): 84. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHébert PC, Wells G, Blajchman MA, et al.: A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials Group. N Engl J Med. 1999; 340(6): 409–17. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHébert PC, Carson JL: Transfusion threshold of 7 g per deciliter--the new normal. N Engl J Med. 2014; 371(15): 1459–61. PubMed Abstract \| Publisher Full Text\n\nHolst LB, Haase N, Wetterslev J, et al.: Lower versus higher hemoglobin threshold for transfusion in septic shock. N Engl J Med. 2014; 371(15): 1381–91. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLacroix J, Hébert PC, Fergusson DA, et al.: Age of transfused blood in critically ill adults. N Engl J Med. 2015; 372(15): 1410–8. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHarvey SE, Parrott F, Harrison DA, et al.: Trial of the route of early nutritional support in critically ill adults. N Engl J Med. 2014; 371(18): 1673–84. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nCaironi P, Tognoni G, Masson S, et al.: Albumin replacement in patients with severe sepsis or septic shock. N Engl J Med. 2014; 370(15): 1412–21. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation" }	[ { "id": "8797", "date": "28 May 2015", "name": "Taylor Thompson", "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\nDrs Walley and Russell, two internationally renowned sepsis clinical investigators and translational scientists, concisely review the rapidly changing sepsis landscape. Recently published studies on transfusion practices, immune modulation, and pharmacotherapy are reviewed as well as groundbreaking large comparative effectiveness studies of early resuscitation strategies. Exciting insights from lipid biology and clever strategies for enhancing the clearance of bacterial products are reviewed, suggesting a new therapeutic strategy for early intervention. Given the legacy of failed sepsis trials, the authors suggest new approaches to clinical trials, predictive enrichment strategies and adaptive designs that may, along with improved understanding of sepsis pathogenesis, lead the way to effective therapies.", "responses": [] }, { "id": "8798", "date": "28 May 2015", "name": "Anthony Gordon", "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\nThis opinion piece from two leading clinical academics provides an overview of sepsis research in 2015. As well as discussing the latest thoughts about definitions, incidence and outcomes in sepsis, they present a summary of some of the recent important clinical trials. They then propose ways to both improve sepsis trial design and novel therapeutic strategies to be tested.", "responses": [] }, { "id": "8805", "date": "28 May 2015", "name": "Walter Hasibeder", "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", "responses": [] }, { "id": "8806", "date": "28 May 2015", "name": "Pietro Caironi", "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", "responses": [] } ]	1	https://f1000research.com/articles/4-131
https://f1000research.com/articles/4-130/v1	27 May 15	{ "type": "Research Note", "title": "Transgenic supplementation of SIRT1 fails to alleviate acute loss of nigrostriatal dopamine neurons and gliosis in a mouse model of MPTP-induced parkinsonism", "authors": [ "Yasuko Kitao", "Natsumi Ageta-Ishihara", "Ryosuke Takahashi", "Makoto Kinoshita", "Osamu Hori", "Yasuko Kitao", "Natsumi Ageta-Ishihara", "Ryosuke Takahashi" ], "abstract": "BackgroundDopamine (DA) neuron-selective uptake and toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans. Loss of DA neurons via mitochondrial damage and oxidative stress is reproduced by systemic injection of MPTP in animals, which serves as models of parkinsonism and Parkinson’s disease (PD). This study aimed to test whether pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1, which confers partial tolerance to at least three models of stroke and neurodegeneration, could also alleviate MPTP-induced acute pathological changes in nigrostriatal DA neurons and neighboring glia.ResultsWe employed a line of prion promoter-driven Sirt1-transgenic (Sirt1Tg) mice that chronically overexpress murine SIRT1 in the brain and spinal cord. Sirt1Tg and wild-type (WT) male littermates (3‒4 months old) were subjected to intraperitoneal injection of MPTP. Acute histopathological changes in the midbrain and striatum (caudoputamen) were assessed with serial coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA. In the substantia nigra pars compacta (SNpc) of the midbrain, the number of TH-positive neurons and the reactive gliosis were comparable between the Sirt1Tg and WT littermates. In the striatum, the relative fluorescence intensity of TH-positive nerve terminals and the level of gliosis did not differ by the genotypes.ConclusionsSirt1Tg and WT littermate mice exhibited comparable acute histopathological reactions to the systemic injection of MPTP, loss of TH-positive neurons and reactive gliosis. Thus, the genetic supplementation of SIRT1 does not confer histologically recognizable protection on nigrostriatal DA neurons against acute toxicity of MPTP.", "keywords": [ "Dopamine", "1-methyl-4-phenyl-1", "2", "3", "6-tetrahydropyridine (MPTP)", "resveratrol", "SIRT1", "Sirtuin" ], "content": "Introduction\n\nDopamine (DA) transporter-mediated uptake of 1-methyl-4-phenylpyridine (MPP+), an oxidized metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), damage DA neurons by impairing mitochondrial respiratory chain and generating reactive oxygen species1. The DA neuron-selective toxicity is reproduced in animals by systemic administration of MPTP, which serve as models of Parkinson’s disease (PD)2. The neurotoxicity of MPTP is alleviated by pretreating mice with resveratrol (trans-3,5,4'-trihydroxystilbene) or other phytoalexins3–5. Besides directly suppressing oxidative stress of MPP+ as an antioxidant6, resveratrol modulates cytoprotective signaling molecules and enzymes that include nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT17. Given the pleiotropic cytoprotective potentials of SIRT1 through diverse substrates, resveratrol’s antagonism against MPTP may be mediated at least in part by SIRT17,8. SIRT1 alleviates animal and cellular models of amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, and an α-synuclein model of PD9. On the other hand, transgenic mice that overexpress SIRT1 in a neuronal subset via the neuron-specific enolase (NSE) gene promoter were not resistant to MPTP10.\n\nWe have established a distinct line of transgenic mice that overexpress SIRT1 in wider neuronal lineages and additionally in glial and vascular endothelial cells via the murine prion gene promoter (Prp)11,12. Unlike the NSE-SIRT1 mice, our Prp-SIRT1 mice are resistant to cerebral hypoperfusion by bilateral common carotid artery stenosis, due to vascular dilatation which is potentiated by SIRT1-mediated deacetylation of endothelial nitric oxide synthase (eNOS)12. Further, Prp-SIRT1 mice are resistant to proteotoxic stress by an ALS-linked mutant of superoxide dismutase 1 (SOD1), due partly to SIRT1-mediated deacetylation of the heat shock factor 1 (HSF1) and the resulting upregulation of HSP70i11. On the basis of the SIRT1-HSF1 axis, and the protective effects of HSF1/HSPs against neurodegenerative insults such as MPTP and α-synuclein9,13–16, we assessed whether Prp-SIRT1 mice is resistant to acute loss of DA neurons and gliosis by MPTP.\n\n\nMethods\n\nAll animal procedures were done in accordance with the guidelines of the Animal Use and Care Committees of Kyoto University (MedKyo08097), Nagoya University (#13151), and Kanazawa University (AP-101606). A line of transgenic mice with a C57BL/6J background harboring the PrP-Sirt1cDNA transgene had been generated and deposited at RIKEN Bioresource Center (RBRC06467) as described elsewhere in detail (Watanabe et al., 2014). Mice were reared in a specific pathogen-free environment at 23 ± 2°C, and identified by PCR using a pair of primers, 5′-CAAGAGGTTGTTAATGAAGC-3′ and 5′-TTTCCTGTTGCCTTCAATCAGCTATCG-3′. All comparisons were made between 3–4-month-old, wild-type (WT) and transgenic (Tg) male littermates. Eight mice were subjected to intraperitoneal injection of MPTP (20 μg/g body weight) in saline or saline alone, each 4 times with 2 h-intervals17, followed by histological analysis 4 days later3.\n\nMice were deeply anesthetized with sodium pentobarbital (50 μg/g, i.p.), fixed with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Frozen-sectioned 10 μm-thick coronal brain sections were reacted with antibodies for tyrosine hydroxylase (TH, rabbit IgG, Chemicon) and glial fibrillary acidic protein (GFAP, mouse IgG, Sigma)17,19. The sections were reacted with Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-goat IgG (Jackson ImmunoResearch), and observed with a laser scanning confocal microscope (Eclipse TE2000U, Nikon) with the Nikon EZ-C1 software. We counted TH-positive neurons in the SNpc in three planes (−3.08, −3.16, and −3.40 mm from the bregma), and measured immunofluorescence intensity for TH in the striatum (caudoputamen; CPu) as described previously17,19.\n\n\nResults\n\nWe used the original Prp-SIRT1 Tg mouse line that chronically expresses murine Sirt1cDNA in the central nervous system (CNS) under control of the murine prion gene promoter11,18. The expression levels of SIRT1 in the midbrain and striatum assessed by immunoblot were approximately three times higher in heterozygous Tg mice than in the non-Tg (WT) littermates12. Four days after serial administrations of MPTP (20 μg/g body weight), we assessed acute histopathological changes of the nigrostriatal tract in the two genotypes (n = 8).\n\nIn the midbrain of Tg and WT mice without MPTP administration, the distribution and appearance of TH-positive cells (presumed DA neuronal somata and dendrites), surrounding GFAP-positive astrocytes, and the nuclei of these and other cells (consist mostly of non-DA neurons and microglia) were comparable (Figure 1A, top). MPTP administration induced acute, significant loss of TH-positive cells and reactive gliosis at comparable severity between the genotypes (Figure 1A, bottom). The numbers of TH-positive neuronal somata in the SNpc (identified in three serial sections) did not show statistically significant difference (Figure 1B). These data indicate that the supplementation of SIRT1 does not suppress the loss of DA neurons and reactive gliosis by acute MPTP toxicity.\n\n(A) Representative immunofluorescence images of wild-type and Sirt1Tg mouse midbrain 4 days after intraperitoneal injection of saline with or without MPTP. Serial coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA (DAPI) consistently exhibited no recognizable histopathological differences in the loss of TH-positive cells (presumed DA neuronal somata and dendrites) and in the proliferation of GFAP-positive astrocytes. Scale bars, 100 μm. (B) The number of TH-positive neurons identified in the three serial coronal sections of SNpc was comparable between the WT and Sirt1Tg littermates. The bars denote mean ± standard error of the mean (n = 4 × 4).\n\nIn the striatum/caudoputamen without MPTP administration, the staining patterns for TH (mostly axons and axon terminals of DA neurons), GFAP-positive astrocytes, and the nuclei of these and other cells were comparable between the Tg and WT littermates (Figure 2A, left). Loss of TH-positive neuropil and reactive gliosis after MPTP administration were also comparable between the genotypes (Figure 2A, middle; higher magnifications in the right). Fluorescence intensity for TH in the striatum after MPTP administration did not differ (Figure 2B), indicating that the supplementation of SIRT1 does not alleviate the loss of DA nerve terminals by acute MPTP toxicity.\n\n(A) Representative immunofluorescence images of wild-type and Sirt1Tg mouse striatum/caudoputamen 4 days after intraperitoneal injection of saline with or without MPTP. Coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA (DAPI) exhibited no recognizable histopathological differences in the loss of TH-positive cells (presumed axons and axon terminals of DA neurons) and in the proliferation of GFAP-positive astrocytes. Scale bars, 100 μm. (B) The relative immunofluorescence intensity for TH in the striatum was comparable between WT and Tg littermates. The bars denote mean ± standard error of the mean (n = 4 × 4).\n\nOverall, PrP-SIRT1 and WT male littermates exhibited similar responses to MPTP toxicity in terms of acute damages to nigrostriatal DA neurons, and proliferation/remodeling of neighboring astrocytes. These findings indicate that supplementation of SIRT1 in neurons and glia does not alleviate MPTP-induced DA neuronal damages and reactive gliosis.\n\n\nDiscussion\n\nAcute degeneration of DA neurons in the mouse MPTP model can be rescued by resveratrol pre-administration4,5, or by transgenic supplementation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) which controls mitochondrial biogenesis and oxidative phosphorylation19. The rescue effects had been attributed at least partly to SIRT17,8 on the basis that resveratrol directly or indirectly potentiates SIRT120, and that SIRT1 activates PGC-1α by deacetylation21. However, transgenic supplementation of SIRT1 either with the neuron-specific promoter10 or with the neuron/glia/vascular endothelial promoter (this study) did not confer tolerance to MPTP-induced pathology. The consistent results indicate that the resveratrol-mediated tolerance to MPTP is due to SIRT1-independent mechanisms (e.g., antioxidant activity as a polyphenol. See Introduction.), and that SIRT1-mediated activation of PGC-1α is insufficient to confer tolerance (i.e., the upregulation of PGC-1α is necessary). Thus, this study has made a case against the unproven notions that health benefits of resveratrol are attributed mostly to SIRT1, and that potentiation of SIRT1 in neurons and glia nonselectively suppresses neurodegeneration and gliosis. Nevertheless, it is worth testing whether Prp-SIRT1 mice are resistant to chronic neurotoxin models or genetic models of PD22,23, and whether PGC-1α/SIRT1-double Tg mice are more resistant than the original PGC-1α Tg mice19.\n\nOur recent study with Prp-SIRT1 mice has demonstrated their resistance, albeit limited, to spinal cord degeneration caused by chronic overload of a mutant SOD111. The proteotoxic stress by misfolded SOD1 is alleviated at least in part by SIRT1-mediated deacetylation of a master transcription factor HSF1 and the resulting upregulation of HSP70i and perhaps other molecular chaperones11. Intriguingly, either transgenic supplementation of HSP70 or its heat shock-mediated upregulation (i.e., preconditioning) confers recognizable resistance to MPTP13–15. We therefore hypothesize that, in DA neurons of Prp-SIRT1 mice, the expression levels of the SIRT1 substrate HSF1 and the downstream effectors including HSP70i are insufficient to counter the toxicity of MPTP―as with the aforementioned situation of PGC-1α. Thus, an obvious subject for future studies is to test whether some preconditioning or milder insults (e.g., lower dose of MPTP or rotenone) could differentiate Prp-SIRT1 mice from wild-type mice.\n\n\nData availability\n\nAll the original image data are accessible at Kanazawa University Repository, http://dspace.lib.kanazawa-u.ac.jp/dspace/handle/2297/41475?locale=en.\n\nF1000Research: Dataset 1. Raw data of transgenic supplementation of SIRT1 in a mouse model of MPTP-induced parkinsonism. 10.5256/f1000research.6386.d4684124", "appendix": "Author contributions\n\n\n\nYK and OH conducted the toxicological and histological analyses on mice bred by NA-I under the supervision of RT and MK. MK and OH designed the study and wrote the manuscript. All authors read and approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by CREST (Creation of a novel technology for prevention, diagnosis, and therapy for psychiatric and neurological disorders) from JST, Grant-in-Aid for Scientific Research on Innovative Areas, Comprehensive Brain Science Network, and Grants-in-Aid for Scientific Research from the MEXT of Japan.\n\nWe confirm that the 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 D. Borchelt (University of Florida) for Mo-Prp plasmid DNA, S. Imai for murine Sirt1 cDNA, and A. Tanigaki, and R. Hikawa for technical help in the establishment of the PrP-Sirt1 transgenic line.\n\n\nReferences\n\nBove J, Perier C: Neurotoxin-based models of Parkinson's disease. Neuroscience. 2012; 211: 51–76. 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PubMed Abstract \| Publisher Full Text\n\nPan-Montojo F, Anichtchik O, Dening Y, et al.: Progression of Parkinson's disease pathology is reproduced by intragastric administration of rotenone in mice. PLoS One. 2010; 5(1): e8762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKitao Y, Ageta-Ishihara N, Takahashi R, et al.: Dataset 1 in “Transgenic supplementation of SIRT1 fails to alleviate acute loss of nigrostriatal dopamine neurons and gliosis in a mouse model of MPTP-induced parkinsonism”. F1000Research. 2015. Data Source" }	[ { "id": "8792", "date": "05 Jun 2015", "name": "Masahiko Takada", "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\nThis manuscript deals with the possible alleviative effect of pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1 on a mouse model of MPTP-induced parkinsonism. The present work was totally based on negative data. However, the entire experimental procedures are sound and the anatomical results obtained are well presented. Since the present work has a potential to provide a certain contribution to the progress in Parkinson’s disease research, this reviewer recommends that the manuscript may be accepted as it is.", "responses": [] }, { "id": "8871", "date": "12 Jun 2015", "name": "Ralitsa I. Petrova", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study Kitao and colleagues have performed comprehensive in vivo analysis of the effect of high levels of SIRT1 on dopamine (DA) neuron survival and gliosis following MPTP insult. The authors test the effect of acute MPTP injury in their previously characterized transgenic mouse line Prp-SIRT1 over-expressing the longevity-related gene Sirt1 in both neurons and glia. The study reports no alleviation in MPTP-induced neurodegeneration or astrogliosis in Prp-SIRT1 animals compared to wild-type controls. This data is consistent with previously reported inability of SIRT1 gain-of-function to mitigate the effects of MPTP-induced neural damage in a neuron-specific Sirt1 transgenic line (NSE-SIRT1). Despite the negative connotation of the results, Kitao et. al.’s study is very informative and provides scientifically sound qualitative and quantitative analysis. An important point, also noted by the authors in the discussion, is the possibility that SIRT1 contributes only minimally to DA neuron survival - this effect could be unmasked by using a lower dose of MPTP or alternatively model parkinsonism using the neurotoxin 6-Hydroxydopamine. In either case, further analysis is required to completely negate the neuroprotective effects of SIRT1 in Parkinson's in vivo models.", "responses": [] }, { "id": "8790", "date": "29 Sep 2015", "name": "Hiroshi Ichinose", "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\nThe authors examined the possible protective effect of SIRT1 against MPTP-induced neurotoxicity using Prp-SIRT1 transgenic (Tg) mice, which enabled pan-neural over-expression of the SIRT1 gene. Although the other Tg mice expressing SIRT1 under the promoter of neuron-specific enolase gene had failed to protect MPTP-neurotoxicity previously, this study was worthwhile doing because the Prp-SIRT1 Tg mice had exhibited partial tolerance to some animal models of stroke and neurodegeneration.In this paper, the authors found no significant difference in MPTP-induced neurotoxicity between the Prp-SIRT1 Tg mice and wild-type by immunohistochemical analyses. Whereas the experimental methods used in this study are sound, immunohistochemical analyses are in general semi-quantitative compared with biochemical ones. As the authors mentioned in the Discussion, it would be interesting to test the milder insults on the Prp-SIRT1 mice. Further studies are expected.", "responses": [] } ]	1	https://f1000research.com/articles/4-130
https://f1000research.com/articles/4-129/v1	27 May 15	{ "type": "Opinion Article", "title": "An open ecosystem engagement strategy through the lens of global food safety", "authors": [ "Paul Stacey", "Garin Fons", "Theresa M Bernardo", "Paul Stacey", "Garin Fons" ], "abstract": "The Global Food Safety Partnership (GFSP) is a public/private partnership established through the World Bank to improve food safety systems through a globally coordinated and locally-driven approach. This concept paper aims to establish a framework to help GFSP fully leverage the potential of open models. In preparing this paper the authors spoke to many different GFSP stakeholders who asked questions about open models such as:what is it?what’s in it for me?why use an open rather than a proprietary model?how will open models generate equivalent or greater sustainable revenue streams compared to the current “traditional” approaches? This last question came up many times with assertions that traditional service providers need to see opportunity for equivalent or greater revenue dollars before they will buy-in. This paper identifies open value propositions for GFSP stakeholders and proposes a framework for creating and structuring that value. Open Educational Resources (OER) were the primary open practice GFSP partners spoke to us about, as they provide a logical entry point for collaboration. Going forward, funders should consider requiring that educational resources and concomitant data resulting from their sponsorship should be open, as a public good. There are, however, many other forms of open practice that bring value to the GFSP. Nine different open strategies and tactics (Appendix A) are described, including: open content (including OER and open courseware), open data, open access (research), open government, open source software, open standards, open policy, open licensing and open hardware. It is recommended that all stakeholders proactively pursue \"openness\" as an operating principle. This paper presents an overall GFSP Open Ecosystem Engagement Strategy within which specific local case examples can be situated. Two different case examples, China and Colombia, are presented to show both project-based and crowd-sourced, direct-to-public paths through this ecosystem.", "keywords": [ "food safety", "open educational resources", "OER", "open access", "open data", "open policy", "Creative Commons", "knowledge sharing" ], "content": "Introduction\n\nThe Global Food Safety Partnership (GFSP) is a public/private partnership established through the World Bank. Open models have the potential to significantly enhance the GFSP’s goal of improving food safety systems through a globally coordinated and locally-driven food safety approach. This open models concept paper aimed to establish a framework to help leverage that potential.\n\nWe explored a range of open models that could enhance the scalability and sustainability of food safety. Our primary goal was to show how open models could support GFSP’s efforts to help ensure safe food, increase food supply chain value, accelerate economic growth, alleviate rural poverty, and improve public health outcomes.\n\nIn developing open models the sub-working group considered the many stakeholders involved in global food safety including:\n\ngovernments\n\nregulatory agencies - public regulators, inspectors and managers\n\nprivate sector agri-food processors and manufacturers\n\nfarmers and producers\n\nuniversities, service providers, trainers and certification bodies\n\ninternational organizations\n\nnon-governmental organizations (NGOs)\n\nOpen models increase opportunities for access and participation so our models also identified new stakeholders including the general public.\n\nWe set out to define and design open models that generate impact and benefits from a multi-stakeholder perspective. Open models show how the many forms of openness, including such things as Open Educational Resources (OER), open access (OA), open data, and open policy can be adopted across all stakeholders and at different stages of knowledge production and dissemination. Open models provide a new paradigm for multi-stakeholder collaboration and capacity building.\n\nMulti-stakeholder adoption of open models generates cumulative benefits for all stakeholders. The greater the number of stakeholders that use open models, the larger the impact. Going open also means that new stakeholders have the agency to get involved and participate. Open models in this paper show how both traditional and new stakeholders can collaborate in the use of open resources and practices to enhance global food safety.\n\nNew technologies offer opportunities for information sharing, public participation, and collaboration. A frequently cited benefit of openness is lower costs for funders and usersi,ii. Lower costs are amplified by using digital Information and Communication Technology (ICT) which, in combination with openness, creates opportunities for free, or very low cost, large scale access and participation. Open models harness these technologies to make more food safety information public and actively engage citizens in improving and disseminating food safety knowledge.\n\nThe primary goal of open models in this paper is to scale and disseminate food safety knowledge and practices to generate social and economic benefits. While open models can be adopted alongside status quo operations, preserving existing business models and traditional revenue streams were not the priority. Open models often disrupt traditional practices and business models. The open models outlined in this paper involve alternative business model approaches for all stakeholders.\n\nIn preparing this paper the open models sub-working group spoke to many different GFSP stakeholders. Underlying many of those conversations were questions about open models such as:\n\nwhat is it?\n\nwhat’s in it for me?\n\nwhy use an open model rather than a proprietary model?\n\nhow will open models generate equivalent or greater sustainable revenue streams compared to the current “traditional” approaches?\n\nThis last question came up many times with assertions that traditional service providers need to see opportunity for equivalent or greater revenue dollars in real terms before they will buy-in. This paper identifies open value propositions for food safety stakeholders and proposes a framework for creating and structuring that value. However, we recommend the GFSP not attempt to use open models and at the same time try and preserve the traditional business models of all partners. Open models enable access, scale, massive adoption, lower costs, localization and social networks, but only if existing business models are set aside and new ones adopted. Traditional models are resistant to open innovation. As a result open models are often more aggressively and strategically pursued by providers who adopt new open business models and play by different rules. Open models are not “business as usual”. Open models cannot be adopted and driven by questions like “Who pays?” assuming the same players and beneficiaries as in the traditional model. This is not to say that open models ignore the financial underpinnings of global food safety and the need for stakeholders to pay bills and keep the lights on. That is understood, but the economics of open models are different and preserving traditional business models is secondary to achieving global food safety goals. Open models are better framed by questions like “How much of this can be free?” and “Where can I add value?”.\n\nBalancing calls for “show me the money” were aspirations for open models to improve food safety scalability and sustainability. We heard loud and clear the need for a macro, generic open model that depicts food safety as an ecosystem at the global and local level. This open models concept paper presents an overall GFSP Open Ecosystem Engagement Strategy within which specific local case examples can be situated. Two different case examples, China and Colombia, are presented to show both project-based and crowd-sourced, direct-to-public paths through this ecosystem.\n\nOERs were the primary open practice GFSP partners spoke to us about, as they provide a logical entry point for collaboration. Going forward, funders should consider requiring that educational resources and the concomitant data resulting from their sponsorship should be open, in the same manner that publicly funded research (and more recently data) is available as a public good. There are, however, many other forms of open practice that bring value to the GFSP. This paper names and describes nine different open practices (Appendix A) stakeholders can use to generate food safety value including: open content (including OER and open courseware), open data, open access (research), open government, open source software, open standards, open policy, open licensing and open hardware. It is recommended that the GFSP adopt as many of these open practices as possible, not just OER. Parallel to Metcalfe’s law which states that the value of a network is proportional to the square of the number of users, open model value becomes reciprocal and is magnified when a wide range of open practices are adopted by a large number of stakeholders. Overlaid on both the China and Colombia case examples are suggestions for which open practices can be adopted by which stakeholders.\n\nThis paper presents an overarching framework intended to guide GFSP partners in their thinking and adoption of open models. It does not get down into the specifics of identifying open business models and approaches for each individual stakeholder. However, this is a logical next step and is recommended as a follow-on for both global and local GFSP initiatives. For those interested in understanding the economics of open business models a short list of recommended books and readings is provided in Appendix B.\n\n\nAn open ecosystem engagement strategy\n\nFigure 1 depicts food safety as an ecosystem at the global and local level and represents a macro overarching framework for open models. The open aspects of this work make it possible for GFSP to make use of both the global and local food safety knowledge bases. Global food safety knowledge can be reused, revised, remixed, adapted, translated, and localized for local food safety provision. And the reverse is also true, local food safety knowledge can be reused, revised, remixed, adapted, and translated into global resources.\n\nOn the left are global stakeholders who, driven by social needs and economic opportunities, are making public and private investments and creating a coordinated approach to improving food safety. To generate maximum value through open models all global stakeholders adopt open principles which have everyone participating, co-creating, sharing, pooling, using and improving. Nine different open practices can be adopted as a means of fulfilling those principles. Definitions for each of these nine open practices, examples of success, and sample stakeholder value propositions associated with them are in Appendix A at the end of this paper. Collectively this generates an open global food safety knowledge base made up of knowledge assets (e.g. text, graphics, sound, video, lists of experts and expert networks, etc.) and knowledge creators, curators and providers. Local food safety stakeholders are shown on the far right. While the categories are the same the actual organizations are different. To generate maximum value through open models all local stakeholders adopt open principles which have everyone participating, co-creating, sharing, pooling, using and improving. The same nine different open practices can be adopted as a means of fulfilling those principles. Collectively this generates an open local food safety knowledge base made up of knowledge assets and knowledge creators, curators and providers. Credit: The Blue Marble by NASA, Public Domain; Planeta Loarre by Juandc CC BY.\n\nOpen model implementation of local food safety involves the formation of partnerships between global and local stakeholders who then design and distribute the knowledge in a way that meets local social and economic needs.\n\nImpact, scalability, and sustainability can be thought of as an equation where maximum impact, scale, and sustainability are achieved by having the maximum number of stakeholders adopt the maximum number of open practices.\n\n\nCase Example 1: China\n\nThe global food system has changed dramatically as multinational supermarkets and their procurement channels have rapidly expanded into emerging markets. Consumers are demanding safe, high-quality food. In response, governments and industry are collaborating to assure quality and food safety consistently around the world. One area of focus has been the development of protocols and training for suppliers and people responsible for food safety compliance.\n\nStarting in 2008, the Food Safety Knowledge Network (FSKN) a collaboration between Michigan State University (MSU), the Global Food Safety Initiative (GFSI) of the Consumer Goods Forum, and other food industry and public sector partners began strengthening the food industry’s response to the complex food safety knowledge and training challenges that affect emerging markets by providing free access to high-quality, standardized OERsiii. These OERs, for basic and intermediate levels of food manufacturing, are based on competencies developed by the Consumer Goods Forum. Their Global Markets Working Group has defined company characteristics of suppliers which have been used by MSU to create OER and proprietary pre- and post-tests. These are also based on the global and country-specific standards.\n\nGFSP's 5-year work program of demand-driven food safety capacity building and advisory services for low and middle income countries was preceded by an initial programming and preparatory year (2012) that included implementation of a training program developed in partnership with the Asia Pacific Economic Cooperation (APEC) and other partners, on food safety prerequisites and Hazard Analysis & Critical Control Points (HACCP) delivered in Beijing in June, 2012. This program was comprised of 3–4 weeks of online learning followed by a 6 day intensive face-to-face session (with real-time live translation) focused on skills development. More recently in the summer of 2013 a similar program was conducted in Shanghai.\n\nThese programs are making use of existing OERs, building on those developed by FSKN/MSU, in a range of formats from PowerPoint presentations to more narrative and full curricula developed in partnership with APEC and the World Bank. Content for the Basic Global Markets Training Program (Archived at http://www.webcitation.org/6Y28HOsqN) is now up to version 2 and version 3 China specific translations.\n\nThe cumulative build-out of this work has established a global food safety knowledge base made up of knowledge assets and knowledge experts as depicted in Figure 2.\n\nThis figure provides an overview of collaborators, resources and approaches being implemented in China.\n\nGFSP’s current focus on China as a priority is building on this predecessor work. The main effort will be focused on generating economic growth by building out food safety knowledge and competencies of the estimated four hundred thousand food manufacturers and suppliers in China. The plan is to scale up use of existing open resources and roll out a program using a train-the-trainer approach in the fall of 2014.\n\nThe China work involves the formation of partnerships including:\n\nfunders - such as United Nations Industrial Development Organization (UNIDO), Global Food Safety Partnership (GFSP), World Bank, International Finance Corporation and others\n\nnonprofits - U.S. Pharmacopeial Convention, Grocery Manufacturers Association Foundation\n\nuniversities - Shanghai Jiao Tong University\n\nAdditional China-based partners are still being established.\n\nThe business model for the China train-the-trainer program is to reuse existing open educational resources and make little to no upfront investment in training materials. Public investment is being sought to support the initial train-the-trainer delivery. Downstream delivery to food manufacturers and suppliers would entail participants paying a fee.\n\nScalability & sustainability challenges include:\n\nfinding someone entrepreneurial to run this as a business\n\nestablishing facilities and resources for needs assessment, logistics, registration, Learning Management Systems (LMS), etc.\n\nensuring quality of trainers\n\nkeeping the content up to date\n\nbase OERs are country agnostic so need adaptation to fit country and sector needs including preventive control information and country specific requirements to meet local regulations.\n\nsome partners want to do training using their own proprietary content\n\nassessment components of training are proprietary not OER\n\ndownstream, the suppliers pay a fee for training to cover costs and improve materials, but must be affordable to suppliers\n\nafter initial train-the-trainer in-country, partner must be responsible for continuous roll out and scaling up. Earlier initiatives have not scaled up as expected.\n\nneed to move beyond the training – the model needs to incorporate mentoring and skills development and application beyond the initial training\n\nsome partners need to make revenue from service provision\n\nneed an open platform to coordinate the partnership, organize implementation, and reduce duplication of effort\n\nTable 1 indicates the potential starting points for various stakeholder groups in China in using the nine different types of open practices. For example, government and funders are poised to make use of eight of the nine open practices (open content, open data, open access, open government, open sources software, open policy and open licensing), whereas the use of open hardware, which is a relatively new form of open practice, is most likely to be initiated by universities and colleges.\n\n\nCase example 2: Colombia\n\nIn Colombia the provincial government of Cundinamarca in partnership with Convenio Andres Bello and education partners are interested in open and distance education as a means of rural social development including food safety.\n\nConvenio Andres Bello is an international intergovernmental organization including Bolivia, Chile, Colombia, Cuba, Ecuador, Spain, Mexico, Panama, Paraguay, Peru, Dominican Republic and Venezuela. It is led by the Ministers of Education of the member countries. Convenio Andres Bello promotes consensus building among members and joint action plans for culture, education, science and technology. Convenio Andres Bella's strategic plan focuses implementation of an ordered set of initiatives under four program areas:\n\n1. The educational sector and the construction of citizenship\n\n2. Social appropriation of knowledge and learning and citizenship\n\n3. Sustainable development, climate change and citizenship\n\n4. Policies: educational, cultural, scientific, technological and citizenship\n\nCundinamarca and Convenio Andres Bello have asked the OpenCourseWare Consortium (OCWC) for help in designing a digital learning initiative with a goal of adopting open online education in support of these aims. The OCWC is a worldwide community of hundreds of higher education institutions and associated organizations committed to advancing open education and its impact on global education. The consortium seeks to engender a culture of openness in education to allow everyone, everywhere to access the education they desire, while providing a shared body of knowledge and best practices that can be drawn upon for innovative and effective approaches. In addition the OCWC helps to solve social problems through expansion of access to education.\n\nThe OCWC is responding to this request by assembling a group of experts in open and online education to support a redesign project, and proposes to collaborate with the GFSP’s Learning and Knowledge Working Group to offer a curriculum, on a national scale in Colombia, in food safety leading to certification (Figure 3).\n\nThis figure provides an overview of collaborators, resources and approaches being implemented in Colombia.\n\nThe GFSP may benefit through the development of standardized curricula and courses that are aimed at different target groups including:\n\n1. residents of impacted communities\n\n2. technical institutes that may want to include curriculum in food safety aiming at employment as inspectors and other skilled professions\n\n3. K-11 teachers for inclusion in primary and secondary health curricula, and\n\n4. university departments for inclusion in undergraduate and graduate degree programs\n\nThe aim is to start with a substantial body of food safety knowledge already in OER form available through GFSP and other stakeholders. Combined with the existing local curriculum, the intent is to adapt these core resources to address different levels of education. A curriculum component that provides education on what someone needs to know about cleanliness at any level might also be a curriculum component for someone enrolled in a two year program to become a certified food inspector. OER will be adapted for the Colombian context and used to create:\n\neasy start/stop short sequences that tie to a social need\n\ninformal learning that leads to workforce opportunities and could be basis for employment\n\nladder learning that progressively builds gradually into certification.\n\ndifferent kinds of certificate tracks including employment as inspectors as well as K-11, college, and university tracks\n\nIn addition to food safety pertaining to food manufacturing and supply, the Colombia program seeks to:\n\nsituate food safety deep down in society - on the farm, in the home, in the local community, and use open models to make knowledge community based impacting people on the ground\n\nimprove nutrition, reduce sickness\n\nachieve better learning through improved food nutrition\n\nhave the learning lead to employment opportunities, and so reduce poverty\n\nprovide food safety and security related to growing, harvesting, storing, and shipping food, as well as food safety pertaining to food preparation and cleanup\n\nCurricula will be made open to all, enabling local communities, farmers, food vendors, small business food suppliers and even the general public to become active participants in knowledge creation and dissemination. This open model (Figure 3) blends expert and indigenous knowledge into a knowledge co-creation open model. One ambitious concept is supporting a free path to certification – and employment – for members of communities most affected by food security and problems with food safety.\n\nAs part of its redesign for digital learning a full range of contemporary options are being considered including:\n\nonline learning via LMSs\n\nMassive Open Online Courses (MOOCs)\n\nmobile technology\n\nThere also are opportunities for entrepreneurs to play a role in providing web-based, just-in-time knowledge delivery services (like iCow which provides timely information to small-holder cattle farmers).\n\nCertification tracks will be designed in such a way that participants can take courses online at their own pace with a practicum and assessment at the end. National centers will be used for the face-to-face practicum and assessment with colleges and universities being the certification entities.\n\nThe Colombia case example has a unique business model. The concept is that the avoidance of public health problems that are caused by unsafe foods can more than pay for the costs of employment in the area of training, monitoring and reporting on food safety in poor areas, both rural and urban. In raising the profile of food safety in these communities, related issues of nutrition and food security can be included to support even better social results, including lowered health costs and improved educational results. You can save more money on not providing emergency services than what it would cost you to provide education through this open model. Savings generated through improved nutrition, public health, and reduced days lost to illness, clinic visits and use of medical facilities pay for the food safety education. Using an open model could make the cost of the education very low. Revenue will be generated from those seeking formal certification. However, programs targeted at displaced, impacted communities will be government funded or have a publicly subsidized lower fee.\n\nA matrix of stakeholder groups in Colombia and their potential starting points in using the nine different types of open practices is presented in Table 2. For example, the OCWC makes use of open content, open access, open standards, open policy and open licensing. The community, farmers, entrepreneurs, food vendors and suppliers have the potential to make use of open content, open access, open source software, open standards and open hardware.\n\n\nOpen Policy Recommendations\n\nThe adoption of an open policy represents a major culture change. For some it is a leap into uncharted territory. Although many organizations and businesses have successfully exploited the open ecosystem (see examples of success in Appendix A), in many respects it is easier to start something new in an open paradigm than it is to make the transition from a traditional approach. Historically, people have provided value through their proprietary content, process or service. As content, processes and services become available for free on the internet, basic assumptions and accepted business models are being challenged.\n\nRecognizing that there is some information that should remain secured, there is probably more information and data that could be “freed” for mutual benefit than we currently realize. Providing seamless access to resources that could potentially be open while protecting those that need to be secured would be a breakthrough, forging a path for others to follow (and may lead to the creation of businesses around this new service). The GFSP is positioned to lead in this arena.\n\nThe World Bank adopted an Open Access Policy for Formal Publications as of July 1, 2012 which pertains to work carried out by Bank staff as well as outside research funded by the Bank.\n\nAt the GFSP meeting in Singapore Dec 10 – 11th, 2013 a preliminary proposed GFSP Openness Operating Principle was presented as follows:\n\n“GFSP members intend to leverage existing knowledge, reduce duplicative efforts and speed and scale global solutions. One way GFSP members operationalize this intention is to enable the use, reuse, redistribution and remixing of the knowledge they choose to share as part of the partnership.”\n\nTo operationalize this intention, GFSP members agree to:\n\nuse a standard creative commons copyright license to the extent possible.\n\nimplement standard operating procedures for publication, such as editable file formats, standard file descriptions and publishing in web locations accessible to the public without a fee or registration requirements.”\n\nThe following broader statement is proposed to encompass the various facets of openness discussed in Appendix A. This will facilitate a staged approach as comfort with open concepts grows, without necessitating frequent revision of the policy.\n\nIn the interests of improving global food safety in a cost-effective and scalable manner, GFSP partners agree to proactively pursue “openness” as an operating principle. Partners will give consideration to the adoption and use of open strategies and tactics across all GFSP activities including:\n\nopen content (including OERs and open courseware)\n\nopen data\n\nopen access (research)\n\nopen government\n\nopen source software\n\nopen standards\n\nopen policy\n\nopen licensing\n\nopen hardware\n\nIn so doing it is recommended that GFSP Partners:\n\nopenly license all GFSP publicly funded deliverables with CC BY 4.0 license (or CC BY IGO 3.0 if IGO)\n\ndevelop GFSP deliverables in open file formats that are editable, customizable, and adaptable to local contexts\n\nestablish a GFSP web site that makes GFSP openly licensed deliverables publicly available for free. Engage GFSP global network and public in use, reuse, and continuous improvement of openly licensed deliverables\n\nidentify resources they currently have that could contribute to GFSP goals if openly licensed. Will bring forward those resources to the whole GFSP group and assess return on investment of combining those resources collectively across the partnership and with the new GFSP deliverables being developed.\n\nGFSP partners have differing understanding of what openness is and what it means to global food safety and to their respective organizations. Organization of information sessions, workshops, events, activities and resources about openness would increase awareness, adoption and use both strategically and tactically.\n\nTo stimulate innovation in the adoption of open methods and the creation of new business models that leverage open methods GFSP partners should consider the use of open competitions either within specific projects or in general. Competitions are increasingly being utilized as a method to stimulate innovation and could be used to spur the adoption of open methods and the creation of open business models. Competition for grant funding has long been employed, however, in recent years variations such as the Gates Grand Challenges for Global Health (Archived at http://www.webcitation.org/6Y29b9N4L) and the XPRIZE competitions (Archived at http://www.webcitation.org/6Y2A0SkcW) have come into force. XPRIZE creates incentivized prize competitions “to bring about radical breakthroughs for the benefits of humanity, thereby inspiring the formation of new industries and the revitalization of markets”. A precedent for using competitions to encourage the use of open data has already been set by the HealthDataPalooza (Archived at http://www.webcitation.org/6Y2Ay4GJH) which hosts an annual competition for development of the best app using public health data. The competition is attended by several thousand people, including representatives from major health providers, venture capitalists and entrepreneurs and has led to the creation of new businesses. This could be replicated for food safety stakeholders.\n\nThe commitment to an open operating principle is founded on a belief that open strategies uniquely provide opportunities for the GFSP and all food safety stakeholders to disseminate and scale their work for greatest impact and global public good. Through openness the food safety community can leverage existing knowledge, reduce duplicative efforts, and speed and scale global solutions.", "appendix": "Author contributions\n\n\n\nPS, GF and TB conceived the model and article. PS was the primary author of the manuscript. GF and TB provided expertise in food safety and contributed to the writing. All authors were involved in editing the manuscript and have agreed on the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis article is derived from The Open Models Concept paper written by the authors for the World Bank under contract #7170739.\n\n\nAppendix A - Open practice definitions, examples of success, sample stakeholder value propositions\n\nOpen models utilize a range of open practices. Here are nine open practices, their meaning and their value proposition.\n\nOpen Educational Resources (OER) are teaching, learning, and research resources that reside in the public domain or have been released under an intellectual property license that permits their free use and re-purposing by others. OER include full courses, course materials, modules, textbooks, streaming videos, tests, software, and any other tools, materials, or techniques used to support access to knowledge (source: http://www.hewlett.org/programs/education/open-educational-resources Archived at http://www.webcitation.org/6Y2BJd5yH).\n\nExamples of Success\n\nFormer Wall Street hedge fund analyst, Salman Khan, started creating web-based tutorials to help his cousin with her math which grew into the Khan Academy, reaching about 10 million students per month and delivering over 300 million lessons around the world. The Khan academy aims to provide “a free, world-class education for anyone, anywhere” and is one of the stellar examples of successful scaling using open educational materials.\n\nDigital Green (Archived at http://www.webcitation.org/6Y2Bh9Pjy) is a knowledge platform helps farmers share best practices within their communities through production of digital videos. It allows farmers to see techniques demonstrated by their peers in their own language. The Food Safety Knowledge Network is an example of a University initiative providing free and openly licensed food safety learning resources that help stakeholders in emerging markets navigate the complexities of food safety and training.\n\nSample Stakeholder Value Propositions\n\nGovernment entities, including regulatory agencies, ministries of health and human services, environmental services, etc. can provide a wide variety of training documents, generic food safety models, regulatory guidelines, memos, and other forms of open content that clarify and guide stakeholders in meeting regulatory requirements and food safety standards. There is significant value to all stakeholders in having a common authoritative reference point for all industry participants, minimizing the time and cost associated with acquiring accurate and consistent information regarding regulatory requirements and food safety standards.\n\nNGOs play an important role in helping a wide variety of stakeholders better understand regulatory requirements, relevant food safety standards and policies, industry best practices, etc. Creating and releasing documents and other educational and informative content to the public as open content can extend the impact of the NGO in its mission.\n\nEducational institutions have the potential to further their mission to educate the public and can work with faculty and staff to release course materials and other educational materials related to food processing, manufacturing, food management, food safety, etc. Furthermore, educational institutions and faculty can utilize open content to augment or add to gaps in curriculum, leading to opportunities for cost savings for the institution and students, as well as increased opportunities for knowledge sharing and collaboration.\n\nIt is within the interest of food manufacturers to provide suppliers with the knowledge they need to provide food products and other services that meet both the manufacturer’s standard of quality and the regulatory body’s expectation for food safety. Manufacturers, therefore, can provide a wide variety of food safety training documents, plans and procedures that suppliers can leverage to implement adequate training regimes. Providing open content also demonstrates a willingness to have additional public accountability and showcase a commitment to food safety. Additionally, there is potential for manufacturers and other stakeholders to leverage this content to form added value products and services, such as consulting and training services.\n\nFor new business owners or entrepreneurs, knowing where to begin in order to comply with food safety and regulatory compliance related issues can be a time consuming and costly undertaking. Access to open content, including food safety training documents, generic HACCP models and record keeping templates, regulatory compliance guidelines, etc. provides a valuable starting point for small and very small food manufacturers, vendors, and suppliers. This can result in significant cost savings for small business owners and also increase the likelihood that these entities attempt to abide by food safety standards and practices. Those who are successful in meeting compliance guidelines could gain credibility and possibly expand their business by documenting innovative and cost-effective means of compliance and sharing them with others. This might also be an efficient means of proving compliance to multiple buyers of their product.\n\nOpen data is data that can be freely used, reused and redistributed by anyone, anywhere, for any purpose - subject only, at most, to the requirement to attribute and share-alike (summary of OpenDefinition.org). Historically very little open data has been available in areas such as health, energy, education, public safety, and global development. Today more and more of this data is becoming available and used by entrepreneurs, researchers, tech innovators, and others to create countless new applications, tools, services, and businesses.\n\nExamples of Success\n\nSeveral decades ago, the US government made a decision to make geographic information system (GIS) data publicly available and spawned a multi-billion dollar industry. In 2001 the first draft of the human genome was published in Natureiv as a result of international data sharing by researchers and its public release has led to the creation of hundreds of new drugs and new companies based on that data. In 2009 data.gov was created to make other types of US government data more accessible. In 2012, a national annual competition was created as part of the Health Data Initiative to stimulate the innovative use of health data in apps and products. The “Health DataPalooza” is now a sold out event attended by over 2,000 health providers, technology developers, venture capitalists, entrepreneurs and community advocates and has resulted in the launch of new products and companies. OpenFDA (Archived at http://www.webcitation.org/6Y2FDeT3K), providing easy access to public data of the US Food and Drug Administration (FDA) and highlighting projects using these data, was initiated in September of 2014.\n\nSample Stakeholder Value Propositions\n\nGovernments who collect data related to food safety, food industry trends, food safety research and analysis, can make these data open, leading to more informed decision making and the possibility of new business models that can build product and services offerings. These advances, ultimately, feed back into the economy.\n\nUniversities, colleges, and educational institutions engaged in activities and studies related to teaching or researching food safety, food manufacturing, food processing, management, etc. can release a wide variety of data collected about various fields that may enable stakeholders - particularly small businesses and entrepreneurs - to advance research, improve manufacturing or processing practices, develop added-value products or services.\n\nOpen data - whether historical data or current - can be analyzed and leveraged by small to medium enterprises to potentially design new technologies and software, assess particular market or industry trends, to learn about customer segments, and even develop practices that increase efficiencies in business operation and staffing, manufacturing, food processing, etc.\n\nMany small and very small food businesses lack adequate resources to generate data that can be used as analysis tools to validate processes and substantiate food safety practices. Access to open data could enable these stakeholders to replicate processes and achieve food safety benchmarks. Furthermore, open data can be leveraged by the community and entrepreneurs to develop added value products and services that can have direct benefits to economies and businesses.\n\nBy “open access” [to peer-reviewed research literature], we mean its free availability on the public internet, permitting any users to read, download, copy, distribute, print, search, or link to the full texts of these articles, crawl them for indexing, pass them as data to software, or use them for any other lawful purpose, without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. The only constraint on reproduction and distribution, and the only role for copyright in this domain, should be to give authors control over the integrity of their work and the right to be properly acknowledged and cited (source: http://www.budapestopenaccessinitiative.org/boai-10-recommendations).\n\nExamples of Success:\n\nThere are a number of open access journals and online publications that provide free and open access to scholarly articles specific to food safety, foodborne illness, manufacturing and processing practices, etc. In 2007 the US National Institutes of Health enacted an open access policy requiring the researchers they fund to make their final, peer-reviewed manuscripts publicly available no later than 12 months after official date of publication. The number of open access journals is rapidly increasing - the Directory of Open Access Journals lists over 9,000. The Public Library of Science (PLOS) and BioMed Central are two popular examples relevant to food safety.\n\nSample Stakeholder Value Propositions\n\nGovernment agencies can provide and also promote (or mandate) access to publicly funded research and other food safety, processing, and scientific literature, which a wide variety of stakeholders can leverage in substantiating manufacturing practices. The public and economic value that stems from government agencies providing access to this information is far lower than the cost of correcting the public health problems and food safety risks that can stem from a lack of access.\n\nNGOs involved in carrying out or sponsoring studies, research, and the publication of other scientific or relevant food safety literature can demonstrate a significant commitment to the dissemination of this literature through an open access policy, whereby content is freely available for people to freely access. The value to the organization in adopting this approach is in the potential for an increase in the organization’s reach and impact among key stakeholders and the opportunity to receive increased funding based on demonstrated impact. They can also leverage existing open access resources in the creation of additional knowledge and training resources intended for different audiences.\n\nUniversities and other educational institutions involved in publishing scientific and other literature related to food safety and food manufacturing can provide and promote free and open access this literature. A wide variety of stakeholders can leverage this literature and research to support food processing and manufacturing practices.\n\nOpen government is the governing doctrine which holds that citizens have the right to access the documents and proceedings of the government to allow for effective public oversightv. The Open Government Partnership currently involving 63 countries around the world has endorsed an open government declaration that commits them to, increase the availability of information about governmental activities, support civic participation, implement the highest standards of professional integrity, and increase access to new technologies for openness and accountability– (see more at: http://www.opengovpartnership.org/about/open-government-declaration).\n\nExamples of Success\n\nOpen government initiatives are focused on a wide range of topics including access to information, anti-corruption, citizen participation, open data, and budget transparency. Success stories related to these topics can be found here. IPaidABribe.com (Archived at http://www.webcitation.org/6Y3QjqeNs), started by a non-profit in India, invites citizens to anonymously report the bribes they are asked to pay. It has over 20,000 bribe reports filed from 500 Indian cities and NGOs from 26 countries are interested in replicating its model.\n\nSample Stakeholder Value Propositions\n\nBy providing complete and open access to municipal, state, federal regulation, including guidelines, draft legislation, memos, discussion, generic food safety models, etc. government can demonstrate a commitment to transparency and public accountability. Constituents can subsequently provide input that helps guide legislation and guidelines, helping to preserve cultural or local practices and provide an understanding of how regulatory requirements may benefit or hinder the processes and practices of small and very small businesses.\n\nOpen source software is software that can be freely used, changed, and shared (in modified or unmodified form) by anyone (Open Source Definition: opensource.org).\n\nExamples of Success\n\nBoth new and established businesses have created valuable services around free software. Red Hat Inc. is a multinational software company founded in 1993 that provides open-source software products to the enterprise community. IBM is an example of an established multinational corporation that successfully incorporated the provision of open software support services into its business model and its employees actively contribute to open software development. Google and Apple are additional examples of corporations that have thrived by engaging the open source software developer community to build on their platforms.\n\nSample Stakeholder Value Propositions\n\nThe adoption of open source software can be usefully implemented to cut costs within a government organization and also boost innovation efficiency. A variety of technological product and service offerings can be built upon open source software with open application programming interfaces and open software development kits.\n\nEducational institutions can both utilize and also support the development of open source software, including content and learning management systems, learning and other function based applications for mobile devices, and other computer based programs, that assist in helping both educate students and provide other stakeholders with tools and services directly applicable to managing and monitoring food processing and manufacturing operations.\n\nAlthough there is no single definition for open standard (Archived at http://www.webcitation.org/6Y5L4eTYD), according to the French Government’s Law for Confidence in the Digital Economy it is understood to mean any communication, interconnection or interchange protocol, and any interoperable data format whose specifications are public and without any restriction in their access or implementation.\n\nExamples of Success\n\nOpen standards in the technology space include the specification of open formats. The US Department of Labor published a set of guidelines for grantees in their Trade Adjustment Assistance and Community College Career Training grant program for use of open file formats in the development of OER. This ensures the OER can be easily edited and adapted by others.\n\nSample Stakeholder Value Propositions\n\nAll stakeholder entities have the capacity to establish both technical, communication, and document standards (including open document formats) to facilitate public access and exchange of information. Clear standards lead to greater interoperability and cost savings, which feed directly back into the economy.\n\nOpen policy involves organizations (including funders, government entities, non-profit organizations, education providers, and corporations) setting guidelines and policies that require the output of the research or work they fund or are involved with to be free and open to the public for use and repurposing. The underlying goal of open policy is to ensure that publicly funded resources are openly licensed and available to the public. Open policy can also pertain to resources created without public funding.\n\nExamples of success\n\nThe World Bank supports the free online communication and exchange of knowledge as the most effective way of ensuring that the fruits of research, economic and sector work, and development practice are made widely available, read, and built upon. It is therefore committed to open access, which, for authors, enables the widest possible dissemination of their findings and, for readers, increases their ability to discover pertinent information. Open policy requires use of Creative Commons licenses (see full policy at: http://documents.worldbank.org/curated/en/2012/04/16200740/world-bank-open-access-policy-formal-publications).\n\nThe $2 billion US Department of Labor Trade Adjustment Assistance and Community College Career Training (TAACCCT) grant program has a policy that requires all grantees to license the new resources they create with grant funds using a Creative Commons Attribution license. This policy makes the TAACCCT program the biggest OER initiative in the world. A specific policy statement is found in solicitation for grant applications at: http://www.doleta.gov/taaccct/applicantinfo.cfm.\n\nSample stakeholder value propositions\n\nOpen policies significantly increase the amount and quality of publicly funded education, research, data, and software available to all.\n\nCopyright law by default creates closed resources. Open licensing gives everyone from individual creators to large companies and institutions a simple, standardized way to grant permissions to their creative work while still maintaining copyright. Permissions typically pertain to the right to reuse, revise, remix and redistribute along with stipulations that allow commercial and/or non-commercial use and the requirement to share back adaptations. Open licensing requires downstream users to give attribution to the original creator. Open licenses for software and hardware work in a similar way.\n\nExamples of success\n\nOpen licenses are used for software and for content. Openly licensed software includes, Linux, Moodle, DSpace, Android, and many other applications. In the content arena there are well over 500 million resources licensed with Creative Commons including all of Wikipedia, over 300 million photos on Flickr, and millions of videos on YouTube.\n\nSample stakeholder value propositions\n\nManufacturers and suppliers have the capacity to make significant inroads in building food safety awareness among suppliers, small and medium sized enterprises through the dissemination of training resources and other data, research, and literature related to meeting relevant food safety regulation. Providing these resources under open license such as Creative Commons licenses could provide a significant opportunity for organizations, innovators, and entrepreneurs involved in either food production or food safety education and training to develop product and service offerings for local or industry specific food safety related issues.\n\nOpen hardware are physical goods and technologies designed and offered through open design. Open hardware means that information about the hardware such as mechanical drawings, schematics, bills of material, layout data, and the software that drives the hardware, are all released with open licenses.\n\nExamples of success:\n\nSee Open Source Ecology (http://opensourceecology.org/) and Farm Hack (http://farmhack.net) for examples relevant to food production and food safety. Photosynq (Archived at http://www.webcitation.org/6Y5LzH0vQ) is an open research project whose goal is to create a low cost, hand-held measurement device which researchers, educators and citizen scientists can use to build a global database of plant health. A low cost mobile prototype has been developed to replace the large, expensive and stationary equipment that was previously required to measure photosynthesis.\n\nSample stakeholder value propositions\n\nOpen hardware increases innovation by open collaboration making it possible to produce open source industrial machines and physical goods that can be made for a fraction of commercial costs.\n\nThere is a graduated process in adopting open methods that most people go through, a set of steps or stages as follows:\n\n1. Awareness - for most people openness is a new concept they aren’t even aware of.\n\n2. Responding to and overcoming the initial fear reaction. Almost everyone initially expresses a great deal of fear over openness. Fears include issues such as remuneration, loss of livelihood, degradation of the resource and a negative effect on its initial integrity. An upside or opportunity must counteract and outweigh each fear.\n\n3. Looking at examples. People want to see real examples of openness and hear the stories from those who have taken that path.\n\n4. Trying it out. Once a certain level of comfort has been achieved people begin to see how others’ open work can benefit them personally. They dip their toe in and try using something that is open.\n\n5. Going open yourself. Once you've sampled someone else's open work many then become willing to make their own work open - perhaps initially in a small way but gradually more and more.\n\n6. Adopting open as a cornerstone of practice. Once you get to this stage you’re in all the way and usually become an advocate who won’t go back.\n\nAdopting open as a cornerstone of practice. Once you get to this stage you’re in all the way and usually become an advocate who won’t go back.\n\n7. Spreading open. Someone who adopts open in one area (e.g. OER) then becomes interested in other areas of openness and starts to see the synergistic benefits of adopting more and more open practices. The cumulative benefits of multiple forms of openness are greater than each individually.\n\nUsually one can’t move to stage 5, 6, or 7 without going through stages 1 through 4.\n\nAnother essential component of this is understanding the fundamental economic differences between digital and physical. Digital makes abundance possible. Physical is by its very nature a scarcity. Models based on abundance operate very differently than models based on scarcity. Abundance transfers power, knowledge and action from the few to the many.\n\n\nReferences\n\nSenack E: The Student Public Interest Research Groups (Student PIRGS), 2015. Report: Open Textbooks: The Billion-Dollar Solution. 2015. Reference Source\n\nOER Research Hub (OERRH). OER Impact Map: OER Saves Money. 2013. Reference Source\n\nGeith C, Vignare K, Bourquin L, et al.: Designing Corporate Training in Developing Economies Using Open Educational Resources. Journal of Asynchronous Learning Networks. 2010; 14(3): 3–12. Reference Source\n\nLander ES, Linton LM, Birren B, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract \| Publisher Full Text\n\nLathrop D, Ruma L: Open Government: Transparency, Collaboration and Participation in Practice. 1st edition. United States of America: O’Reilly Media; 2010. Reference Source" }	[ { "id": "8944", "date": "09 Jun 2015", "name": "Ted Hanss Jr", "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\nThis is a concept paper proposing an \"open ecosystem\" approach for the World Bank-led Global Food Safety Partnership (GFSP). The title and abstract accurately and succinctly capture the aims of the paper, in which the authors advocate compellingly for abandoning current business practices and replacing them with open models across multiple strategies and tactics for the GFSP, with the aim of improving the ability of GFSP to meet its goals. Through relevant case examples and a thorough elucidation of open strategies (in Appendix A), the authors build the case for their argument. The authors make their case for content re-use by articulating how the GFSP will build on the efforts in China. Modification and re-use are key attributes of openly licensed content that have been well demonstrated and are documented in Appendix A. However, the case studies would benefit from sharing outcome measures, if available, from previous efforts that are relevant to the GFSP, such as demonstrating how open practices in the China example have magnified the reach of the training programs compared with proprietary approaches. Overall, the authors' recommendations follow logically from their analysis. The recommendations, while targeted at a specific World Bank effort, are generalizable, thus making the paper relevant to a wide audience.", "responses": [] }, { "id": "9188", "date": "06 Jul 2015", "name": "Nikos Manouselis", "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\nThis paper discusses how open models may be adopted by a traditional professional training ecosystem - the one of food safety. It explains the basic concepts around open models, explores examples of relevant open cases, and recommends some open policies that the particular ecosystem should follow in order to become more open. It also examines two specific cases of food safety training stakeholders: one in China and one in Colombia.I found very interesting the fact that different types of open practices have been examined - including open data, content, government, software, hardware etc. This is providing a wide range of tools and interventions that can be adopted by the stakeholders in such an ecosystem, so that new business models may be explored. On the other hand, I believe that putting everything on the table in the case of such a traditional training market may be too radical and in a way intimidate stakeholders.I would find it useful if the authors could reflect on this concern, possibly elaborating a bit more about a potential strategy that someone should adopt to introduce such novel models in a traditional training ecosystem. For instance, elaborating a bit on the Innovators and the Early Adopters that can help introduce such a radical innovation in the ecosystem, using ideas from approaches like the ones presented in the classic book Crossing the Chasm (https://en.wikipedia.org/wiki/Crossing_the_Chasm).Overall, a very solid submission and a novel contribution that provides some insight in the way that openness can be introduced in training markets.", "responses": [] } ]	1	https://f1000research.com/articles/4-129
https://f1000research.com/articles/4-128/v1	27 May 15	{ "type": "Review", "title": "Proliferative nephritis in childhood-onset systemic lupus erythematosus: current therapeutic approaches and future perspectives", "authors": [ "Dawn M. Wahezi" ], "abstract": "Renal involvement occurs in 50-75% of children with childhood-onset systemic lupus erythematosus (cSLE). Proliferative lupus nephritis (LN) represents the most common pattern of renal involvement in cSLE. Despite aggressive treatment, progression to end stage renal disease can occur in up to 5-10% of children. Over the last 2 decades, tremendous advancements have been made in the treatment of pediatric LN. Special considerations in children need to address the impact of disease and therapy on both physical and psychological growth and development. This review will focus on pivotal clinical trials in the treatment of proliferative LN, with a focus on pediatric data when available.", "keywords": [ "systemic lupus erythematosus", "pediatrics", "lupus nephritis", "childhood-onset", "treatment" ], "content": "Introduction\n\nSystemic Lupus Erythematosus (SLE) is a chronic, autoimmune disease characterized by multi-organ system involvement, widespread inflammation and the presence of autoantibodies. Childhood-onset SLE (cSLE), defined as SLE with age of onset prior to 18 years of age, occurs in 15–20% of cases. Despite the similarities between cSLE and adult-onset disease, children have a higher frequency of major organ involvement and are more likely to suffer from damage accrual throughout their lifespan. Renal involvement is no exception and occurs in 50–75% of children with cSLE, with the majority demonstrating evidence of renal involvement within the first 2 years of diagnosis.\n\nThe severity of lupus nephritis (LN) varies from mild subclinical disease to diffuse proliferative nephritis. In 1974, the World Health Organization (WHO) introduced a histologic classification criterion for lupus glomerular disease that was revised in 2004 by the International Society of Nephrology/Renal Pathology Society (ISN/RPS)1. Evaluation of renal biopsy using these criteria is critical in determining the acute management and long-term prognosis of patients affected with LN2. Briefly, Class I and II represent mild mesangial disease and typically do not require intensive treatment. In contrast, Class III and IV represent a more progressive proliferative glomerular involvement that requires aggressive induction and maintenance therapy in an attempt to prevent long-term morbidity and mortality. Membranous LN (Class V) is less common and traditionally thought to be less severe.\n\nProliferative LN (Class III and IV) represents the most common pattern of renal involvement in cSLE. Despite aggressive treatment, progression to ESRD can occur in up to 5–10% of children. Risk factors for progression include delay to treatment, African American race, Hispanic ethnicity, elevated serum creatinine, hypertension, nephrotic range proteinuria, anemia, and biopsy findings (including degree of proliferation and chronic tubulointerstitial changes)3–5. In a recent survival analysis conducted by Mok et al., the life expectancy in patients with SLE with renal disease and renal damage was reduced by 15.1 and 23.7 years respectively, as compared to the general population6. These findings further underscore the importance of appropriate classification and prompt treatment of LN.\n\nOver the last two decades, tremendous advancements have been made in the treatment of pediatric LN. The majority of data has been extrapolated from clinical trials in adult-onset SLE. Data in children is primarily based on few prospective studies, retrospective investigations, cross-sectional analysis and anecdotal evidence. This review will focus on pivotal clinical trials in the treatment of proliferative LN, with a focus on pediatric data when available.\n\n\nGeneral treatment considerations\n\nTreatment of cSLE is targeted toward optimizing efficacy while minimizing drug duration and toxicity. Special considerations in children need to address the impact of disease and therapy on both physical and psychological growth and development. The risk of lupus flare and disease relapse is simultaneously a significant concern in adolescents struggling with understanding the concept of a chronic disease and limited insight regarding consequences of poor adherence to therapy. Similar to adults with LN, treatment is often dictated by histological classification seen, with proliferative forms of LN requiring more aggressive management to prevent progression to ESRD.\n\n\nCorticosteroids\n\nWith the introduction of corticosteroids as the mainstay of pharmacological therapy for SLE, patient outcomes have improved dramatically. Treatment regimens vary significantly between physicians and include corticosteroid administration via primarily an oral route, primarily intravenous methylprednisolone pulses or a mixed approach. Recent evidence suggests that high-dose intravenous methylprednisolone pulses have the potential to eliminate the type I interferon signature (a predominant cytokine in the pathogenesis of SLE) by reducing the number of plasmacytoid dendritic cells7. These findings were not seen with administration of lower-dose oral corticosteroids.\n\nDespite the ubiquitous use of corticosteroids in the treatment of cSLE, prolonged usage results in a significant contribution to patient morbidity. Side effects are numerous and include Cushing syndrome, growth suppression, osteoporosis, behavioral disturbances, cardiovascular effects, ophthalmologic toxicity and myopathy. Among all of these, perhaps the most common and most distressing side effect in adolescents with cSLE is the presence of Cushing syndrome, associated with obesity, acne and hirsuitism. These toxicities may be limited by consolidating daily administration to a single dose given in the morning. Alternate day dosing and intravenous pulse therapy have also been shown to minimize adverse effects.\n\nIn order to prevent toxicity in growing children, it has become standard practice to initiate treatment with oral or intravenous corticosteroids at the onset of LN, usually in combination with an additional steroid sparing agent. As will be demonstrated in several of the clinical trials listed below, monotherapy with oral or intravenous corticosteroids for proliferative LN is typically inferior to combination therapy with an additional immunosuppressant8–10.\n\n\nCyclophosphamide\n\nCyclophosphamide (CYC), an alkylating agent, is a nitrogen mustard derivative that functions by binding to guanine in DNA, destroying the purine ring and preventing cell replication. The first controlled trial reporting short-term efficacy of CYC in adults with LN was published in 197111. Since that time, a series of randomized controlled trials (RCT) sponsored by the National Institutes of Health (NIH) have investigated various treatment regimens including CYC and corticosteroids in the management of adults with proliferative LN. In the initial landmark study in 1986, Austin et al. demonstrated efficacy and preservation of renal function in patients receiving intravenous CYC (every 3 months) plus low dose steroids as compared to high-dose steroids alone8. Subsequently, a RCT by Boumpas et al. defined what is now referred to as the “high-dose” regimen of CYC in the management of LN9. In this study, patients received monthly CYC (0.5–1g/m2) for 6 months followed by quarterly pulse CYC for an additional 2 years and demonstrated superior preservation of renal function as compared to high-dose corticosteroids alone. This study was fundamental in demonstrating the need for long-term maintenance therapy to prevent disease flare in patients with LN9. An additional NIH trial in 1996 further confirmed the benefits of intravenous pulse CYC plus corticosteroids in the treatment of proliferative LN, suggesting benefit of combination therapy over either medication alone10.\n\nData from these early NIH trials supported the role of CYC in the management of proliferative LN, however high rates of short-term and long-term side effects including infection and premature ovarian failure limited adoption. As a result, investigators in the Euro-Lupus Nephritis Trial established what is now known as the “low-dose” regimen of CYC, consisting of a fixed dose of 500mg every 2 weeks for a total of six doses12. There were no significant differences in treatment outcomes, renal remission or disease flare in patient receiving low-dose CYC as compared to the conventional high-dose regimen established in the NIH trials. This study, while important in providing an alternative option for CYC dosing in a specified population of patients with LN, has been criticized for having patients with milder forms of renal disease and limited number of higher risk African American patients. It has been suggested that patients of African American descent generally have a less favorable response to CYC as compared to other ethnic groups; thus treatment with high dose CYC or an alternative immunosuppressant (as discussed below) is generally recommended13–15.\n\nSeveral smaller prospective and retrospective studies have supported the use of these CYC regimens in the treatment of pediatric patients with proliferative LN. In a prospective study in children with proliferative LN, Lehman et al. demonstrated a reduction in proteinuria, improved renal function, and a reduction in corticosteroid dose in children receiving high-dose CYC with an extended course of therapy16,17. Similarly, a retrospective study by Baskin et al. demonstrated efficacy of low-dose CYC in the induction therapy of 20 children with proliferative LN18. In a larger retrospective study of 108 Thai children with severe proliferative LN, a positive renal response was noted after induction therapy with CYC, however long-term results appeared less promising19. A randomized trial comparing low-dose versus high-dose intravenous CYC in children with proliferative LN is currently underway (clinicaltrials.gov: NCT01861561).\n\nThe lack of evidence-based recommendations specific to cSLE led the Childhood Arthritis and Rheumatology Research Alliance (CARRA) to develop consensus treatment plans (CTP) to determine the optimal therapeutic strategy for induction therapy in children with proliferative LN. Based on this CTP, physicians were given an option of choosing monthly doses of intravenous CYC (0.5–1g/m2) for a total of six doses to be used in conjunction with one of three options for oral/intravenous corticosteroids20. The expert panel recommended adjusting the CYC dose for renal insufficiency and for a low white blood cell nadir, which occurs 7–10 days after the infusion of CYC10.\n\nThe long-term safety of CYC in children is not well defined. Leukopenia and thrombocytopenia are the most common adverse reactions, although with close monitoring they are rarely clinically significant. Additional risks include infection, bladder toxicity (such as hemorrhagic cystitis), syndrome of inappropriate antidiuretic hormone, GI toxicity, alopecia, malignancy and effects on fertility. It has been suggested that gonadal failure is greatest in sexually mature males, increases with age and is dependent on cumulative dose of CYC. Despite the reduced risk in children as compared to adults, it has been estimated that premature ovarian failure can develop in 11% of girls treated with CYC for cSLE21,22. As a result, strategies to preserve reproductive potential in girls and boys with SLE have been recommended21.\n\n\nAzathioprine\n\nAzathioprine (AZA) is a purine analog that inhibits purine and nucleotide interconversion, interfering with DNA synthesis. It has been used longer than any other second-line agent in the treatment of cSLE. It has advantages of reduced cost, dosing frequency and improved safety profile that has led to the study of AZA as a less toxic alternative to CYC in the treatment of SLE. In early studies of proliferative LN, AZA plus corticosteroids appeared to reduce the risk of all-cause mortality compared to steroids alone, but had minimal effects on long-term renal outcomes23. In a Dutch Lupus Nephritis Study, patients treated with oral AZA (2 mg/kg/day) and corticosteroids as induction therapy for proliferative LN were more likely to have renal relapse and increasing serum creatinine as compared to intravenous CYC in long-term follow up12,24,25. Furthermore, repeat renal biopsies in this group demonstrated a progression in the chronicity index in patients treated with AZA compared to CYC26. As a result of these studies, AZA is generally not recommended for induction therapy in proliferative forms of LN.\n\nIn contrast to these reports, investigations into the use of AZA as maintenance therapy have been more promising. As mentioned previously, the early NIH trials demonstrated the need for longer treatment courses in the therapy of proliferative LN to prevent renal relapse and improve long-term outcomes. Initially achieved with quarterly doses of CYC, this approach was later changed to AZA as a more efficacious and safer agent for long-term maintenance of renal remission after induction with intravenous CYC27,28. As a result of these studies, the American College of Rheumatology (ACR) recommends AZA (2 mg/kg/day) with low dose corticosteroids as a possible treatment option for the maintenance phase of therapy for proliferative LN. AZA may be preferable in women who are in complete remission and desire to become pregnant or in patients with intolerable side effects from alternate immunosuppressive agents.\n\n\nMycophenolate mofetil\n\nMycophenolate mofetil (MMF), an inhibitor of inosine-monophophatase-dehydrogenase, is a selective, non-competitive inhibitor of purine synthesis that primarily affects T- and B-lymphocytes. MMF was introduced during the late 1990s for the treatment of severe forms of LN with demonstration of favorable renal outcomes, reduced toxicity and ease of administration. Several trials have now justified the use of MMF as an alternative therapeutic option in both the induction phase and maintenance phase in the therapy of proliferative LN.\n\nTo evaluate the utility of MMF in the induction therapy of LN, several randomized trials have been performed comparing MMF to both oral and intravenous forms of CYC. The initial study was performed in 2000 in a Chinese population of adults with LN. This was the first comparative study to demonstrate equivocal rates of renal improvement, complete remission and relapse rates in patients treated with MMF (2 g/day) compared to oral CYC (2.5 mg/kg/day)29,30. Furthermore, patients experienced fewer side effects. Subsequently, two large randomized trials compared intravenous CYC versus MMF in a more diverse adult SLE population. In both studies, MMF (initial dose 1g/day, increased to 3g/day) performed as well (if not superior) to intravenous CYC in the induction of complete renal remission31,32. Side effect profile appeared better in the MMF group; however these results were not statistically significant. In a subgroup analysis of the ASPREVA Lupus Management Study (ALMS), patients of African American or Hispanic decent appeared to have better response to MMF as compared to CYC. In the past decade, several meta-analyses have also been performed confirming the equivalence of MMF to CYC in inducing renal remission in proliferative LN33,34. In these analyses, side effect profiles were better in the MMF group, demonstrating less alopecia, amenorrhea and reduced risk of ovarian failure.\n\nMMF has also been studied as a maintenance therapy for proliferative LN and has demonstrated superior efficacy to quarterly intravenous CYC27. Comparison with AZA has been evaluated with several studies demonstrating either equivocal or superior results of MMF over AZA. In the MAINTAIN Nephritis Trial (an extension of the Euro-Lupus Nephritis trial), patients were randomized to receive MMF (2 g/day) versus AZA (2 mg/kg/day) after induction with low-dose CYC. There was no difference in renal outcomes or time to disease flare in either group35. In the larger, more ethnically diverse ALMS study, MMF (2 g/day) performed superior to AZA (2 mg/kg/day) with significantly fewer treatment failures and improved renal outcomes36. As a result of these investigations, the ACR recommends treatment with MMF (2–3 g/day) as induction therapy in African American and Hispanic patients, and as an option for maintenance therapy after successful induction.\n\nStudies on the use of MMF in childhood onset SLE are limited and primarily based on case series and retrospective reviews. These studies have confirmed the use of MMF as an efficacious option in the treatment of children with LN without any recorded major side effects18,37–40. In a sub-analysis of adolescent patients (12–18 years old) who participated in the ALMS study, results were similar to that of the adults with equivocal renal response rates between MMF and intravenous CYC for induction therapy and comparable efficacy between MMF and AZA for the maintenance phase41. Few studies have demonstrated less promising results with MMF in proliferative LN in children; however patient numbers were small and primarily included children with refractory disease42. Based on these results, the CTP for induction therapy in proliferative LN in cSLE also includes MMF (600 mg/m2/dose twice daily) with corticosteroids as an alternative option to intravenous CYC20.\n\nAlthough generally better tolerated than CYC, studies have demonstrated increased risk of gastrointestinal (GI) symptoms and diarrhea with MMF. GI toxicity may be improved by increasing dosing frequency to three or four times per day. Other side effects include cytopenias, infection, malignancy and teratogenicity. This latter issue may be of particular concern in adolescent females with high-risk behavior. As a result, the mycophenolate pregnancy registry has been established to assure enhanced patient education regarding contraceptive options and monitoring of pregnancy status. A final concern notable in the pediatric population is concern for patient adherence. Expert consensus has not been achieved to determine if therapeutic drug monitoring of MMF should be utilized, however random levels may be obtained to screen for patient compliance20.\n\n\nRituximab\n\nRituximab, a chimeric monoclonal antibody against B-cell CD20 receptor, has recently emerged as an important therapeutic option in patients with resistant or relapsing proliferative nephritis. Its role in the induction therapy of proliferative LN has been evaluated in the Lupus Nephritis Assessment with Rituximab (LUNAR) study43. In this study, patients with proliferative LN were randomized to either rituximab or placebo (with a background of MMF and corticosteroids). There were no significant differences in renal response rates between rituximab and placebo. Despite earlier uncontrolled trials suggesting possible benefit, these results were insufficient to support primary use of rituximab; therefore it is not recommended as a first-line agent in the induction therapy of LN. In contrast, favorable results have been reported in small observational studies and case reports of proliferative LN that is resistant to MMF or CYC44–47.\n\nLimited studies in children have demonstrated refractory LN as the primary indication for the use of rituximab (750–1000mg/m2/dose given 2 weeks apart) in cSLE with significant improvements in disease activity48,49. A report by Lehman et al. additionally suggests benefit in combination therapy with rituximab (750 mg/m2/dose, max 1g/dose) and cyclophosphamide (750 mg/m2/dose) in 12 children with refractory SLE50. These patients were given the combination therapy for two doses given 2 weeks apart at the start of the study, month 6 and month 18. The results suggest improvement in disease activity with reduced need for corticosteroid administration.\n\nIn patients who fail CYC or MMF, a switch to rituximab plus corticosteroids is supported by guidelines from the ACR and the European League against Rheumatism (EULAR). Serious adverse events include infusion reactions and anaphylaxis; thus premedication with antihistamines, acetaminophen and/or corticosteroids is typically recommended. Other side effects include hypogammaglobulinemia, infection and progressive multifocal leukoencephalopathy. Reduced levels of immunoglobulins for prolonged periods appear to be more common and significant in children than adults and thus may require immunoglobulin replacement therapy.\n\n\nTacrolimus and Cyclosporine A\n\nCalcineurin inhibitors, with primary effects on T-cell expansion, have additionally been evaluated for a potential role in the treatment of proliferative LN. Tacrolimus has been compared with CYC and MMF in several short-term studies in the induction therapy of Chinese adults with proliferative LN. In one study, a combination of tacrolimus (4 mg/day), MMF (1 g/day) and corticosteroids were compared with high-dose intravenous CYC and found to result in greater renal response rates; however the regimen was also noted to have an increased number of serious adverse events51. Two smaller studies similarly showed equivocal remission rates in Chinese patients treated with tacrolimus, intravenous CYC or MMF52,53.\n\nA small randomized study demonstrated equivocal renal outcomes in children treated with cyclosporine A and steroids as compared to oral CYC54. This study was limited by the fact that induction therapy with high-dose corticosteroids began prior to randomization. In another study targeted toward the maintenance phase of therapy, cyclosporine A was shown to be as effective as AZA after induction with oral CYC55. This data, although promising, had limited follow up and thus has been insufficient to support the use of tacrolimus or cyclosporine A as initial therapeutic agents in management of proliferative LN. In addition, use has been limited due to concerns for renal toxicity, hypertension and increasing serum creatinine. These medications should be considered in patients who cannot tolerate or have contraindications to the first-line agents described above.\n\n\nAbatacept\n\nAbatacept is a fully human, soluble fusion protein composed of the Fc region of IgG1 and the extracellular domain of CTLA-4. It competitively binds to CD80/86 on antigen presenting cells as an inhibitory signal, preventing T-cell activation. Two recent studies in adults with SLE have reported on the use of abatacept as a potential agent in the management of proliferative LN. In the first study, abatacept was administered in combination with low-dose CYC and corticosteroids56. When compared with placebo, abatacept did not appear to have any added benefit on renal outcomes. In another 12-month RCT, patients were randomized to receive placebo, standard dose abatacept (10mg/kg) or higher dose abatacept (30mg/kg followed by 10mg/kg)57. As a result, patients failed to meet their primary end point of reduction in lupus flares and were also noted to have increase adverse events (ie. infection). Despite these disappointing results, abatacept is occasionally considered as an alternative therapy in the management of refractory LN and has anecdotally resulted in positive renal responses in two adolescents in our center with prolonged, refractory disease.\n\n\nBelimumab\n\nBelimumab is a fully humanized monoclonal antibody that binds soluble B-lymphocyte stimulator (BLyS, also known as BAFF), an immunomodulatory cytokine that promotes B-cell survival and differentiation. Two large RCT have demonstrated improved disease activity in adult patients with SLE treated with belimumab as compared to placebo58,59. The outcomes of these trials resulted in belimumab becoming the first new drug approved for lupus in over 50 years. Additional trials are additionally underway to evaluate the safety and efficacy of belimumab in children and to examine potential use in adult patients with active LN (clinicaltrials.gov: NCT01649765 and NCT01639339).\n\n\nEmerging therapies\n\nDespite the major advances that have occurred in the treatment of proliferative LN over recent decades, investigators continue to seek out more effective and less toxic treatment protocols to improve the long-term morbidity and mortality related to this disease. Many trials are currently underway to investigate various combination therapeutic approaches and newer biologic agents that target more specific inflammatory pathways.\n\nOne group has piloted the “steroid- avoiding rituxilup” protocol consisting of two doses of rituximab (1 g/dose) and intravenous methylprednisolone (500 mg) followed by maintenance therapy with MMF for the treatment of adults with LN60. Early reports have demonstrated that 72% of patients achieved complete response by a median time of 36 weeks, with 11 patients experiencing flare within a median time of 65 weeks. An open label RCT is expected to further address the efficacy of this protocol to treat LN with avoidance of oral steroids (clinicaltrials.gov: NCT01773616).\n\nEpratuzumab, a monoclonal antibody against CD22 antigen on B-cells is a biologic agent that has shown promising results in open-label, phase I and phase II trials in adults with SLE61–63. Epratuzumab has been reported to be well-tolerated and effective in treating numerous SLE disease manifestations. Patients with moderate, stable renal involvement have been included in these studies, suggesting the potential utility of this agent in the treatment of LN. Phase 3 clinical trials are currently ongoing (clinicaltrials.gov: NCT01261793). Tocilizumab, a humanized monoclonal antibody against interleukin-6 receptor has additionally been evaluated for the use in mild to moderate SLE. In a small, open-label, phase I trial, tocilizumab was found to have promising clinical and serologic responses64.\n\n\nLess supported therapies\n\nCurrent data do not support the role of plasma exchange or abetimus (an immunomodulatory agent targeted against antibodies to dsDNA) in the treatment of isolated proliferative forms of LN65–67. Placebo controlled trials of abetimus have shown significant reductions in anti-dsDNA antibodies, however time to renal flare or number of renal flares was not significantly better than placebo66,67. Ocrelizumab, a fully humanized anti-CD20 monoclonal antibody was evaluated in two phase III RCTs and found to have similar results to rituximab but was stopped due to the incidence of serious infectious adverse events68. Atacicept, a soluble fully human recombinant anti-APRIL fusion protein, was also tested in phase II trials in adult patients with SLE, however the study was halted prematurely due to two reported deaths in the treatment group69.\n\n\nAdjunctive treatment\n\nIn integral component of improving renal outcomes in children with LN is the use of supportive therapies in combination with the immunosuppressive agents discussed above. Hydroxychloroquine (HCQ) has been shown to prevent SLE flares and may reduce the risk of renal damage and clotting events70,71. Dose adjustments may be required in patients with impaired renal function to prevent ophthalmologic toxicity. In addition, anti-hypertensive agents, diuretics, anticoagulants and lipid lowering agents are the mainstay of adjunctive therapy. In patients with proteinuria, anti-hypertensive agents that inhibit the renin-angiotensin system (ie. angiotensin converting enzyme inhibitors and angiotensin receptor blockers) play a crucial role in reducing urinary protein excretion, which has been shown to be an independent risk factor for progression to ESRD. Calcium and vitamin D supplementation is also beneficial in improving bone health in children with SLE and a prolonged corticosteroid burden. Lastly, infection remains the most common cause of mortality in cSLE, thus close monitoring is required for children on immunosuppression.\n\n\nConclusion\n\nRenal outcomes in pediatric LN have significantly improved with current induction and maintenance protocols. Despite improved survival rates, long-term outcomes and life expectancy in children with proliferative LN remains unacceptable. Increased efforts toward early diagnosis, targeted therapy with reduced toxicities and special attention to infectious and thrombotic complications are critical in optimizing care for these children. In addition, further investigation into the duration of therapy to prevent disease flares while limiting unnecessary long-term medication toxicity is warranted.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWeening JJ, D'Agati VD, Schwartz MM, et al.: The classification of glomerulonephritis in systemic lupus erythematosus revisited. J Am Soc Nephrol. 2004; 15(2): 241–50. PubMed Abstract \| Publisher Full Text\n\nHiramatsu N, Kuroiwa T, Ikeuchi H, et al.: Revised classification of lupus nephritis is valuable in predicting renal outcome with an indication of the proportion of glomeruli affected by chronic lesions. 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PubMed Abstract \| Publisher Full Text\n\nMoroni G, Doria A, Mosca M, et al.: A randomized pilot trial comparing cyclosporine and azathioprine for maintenance therapy in diffuse lupus nephritis over four years. Clin J Am Soc Nephrol. 2006; 1(5): 925–32. PubMed Abstract \| Publisher Full Text\n\nGroup AT: Treatment of lupus nephritis with abatacept: the Abatacept and Cyclophosphamide Combination Efficacy and Safety Study. Arthritis Rheumatol. 2014; 66(11): 3096–104. PubMed Abstract \| Publisher Full Text\n\nFurie R, Nicholls K, Cheng TT, et al.: Efficacy and safety of abatacept in lupus nephritis: a twelve-month, randomized, double-blind study. Arthritis Rheumatol. 2014; 66(2): 379–89. PubMed Abstract \| Publisher Full Text\n\nNavarra SV, Guzmán RM, Gallacher AE, et al.: Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebo-controlled, phase 3 trial. Lancet. 2011; 377(9767): 721–31. PubMed Abstract \| Publisher Full Text\n\nFurie R, Petri M, Zamani O, et al.: A phase III, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, in patients with systemic lupus erythematosus. Arthritis Rheum. 2011; 63(12): 3918–30. PubMed Abstract \| Publisher Full Text\n\nCondon MB, Ashby D, Pepper RJ, et al.: Prospective observational single-centre cohort study to evaluate the effectiveness of treating lupus nephritis with rituximab and mycophenolate mofetil but no oral steroids. Ann Rheum Dis. 2013; 72(8): 1280–6. PubMed Abstract \| Publisher Full Text\n\nDörner T, Kaufmann J, Wegener WA, et al.: Initial clinical trial of epratuzumab (humanized anti-CD22 antibody) for immunotherapy of systemic lupus erythematosus. Arthritis Res Ther. 2006; 8(3): R74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWallace DJ, Gordon C, Strand V, et al.: Efficacy and safety of epratuzumab in patients with moderate/severe flaring systemic lupus erythematosus: results from two randomized, double-blind, placebo-controlled, multicentre studies (ALLEVIATE) and follow-up. Rheumatology (Oxford). 2013; 52(7): 1313–22. PubMed Abstract \| Publisher Full Text\n\nWallace DJ, Kalunian K, Petri MA, et al.: Efficacy and safety of epratuzumab in patients with moderate/severe active systemic lupus erythematosus: results from EMBLEM, a phase IIb, randomised, double-blind, placebo-controlled, multicentre study. Ann Rheum Dis. 2014; 73(1): 183–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIllei GG, Shirota Y, Yarboro CH, et al.: Tocilizumab in systemic lupus erythematosus: data on safety, preliminary efficacy, and impact on circulating plasma cells from an open-label phase I dosage-escalation study. Arthritis Rheum. 2010; 62(2): 542–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis EJ, Hunsicker LG, Lan SP, et al.: A controlled trial of plasmapheresis therapy in severe lupus nephritis. The Lupus Nephritis Collaborative Study Group. N Engl J Med. 1992; 326(21): 1373–9. PubMed Abstract \| Publisher Full Text\n\nAlarcón-Segovia D, Tumlin JA, Furie RA, et al.: LJP 394 for the prevention of renal flare in patients with systemic lupus erythematosus: results from a randomized, double-blind, placebo-controlled study. Arthritis Rheum. 2003; 48(2): 442–54. PubMed Abstract \| Publisher Full Text\n\nFurie R: Abetimus sodium (riquent) for the prevention of nephritic flares in patients with systemic lupus erythematosus. Rheum Dis Clin North Am. 2006; 32(1): 149–56, x. PubMed Abstract \| Publisher Full Text\n\nReddy V, Jayne D, Close D, et al.: B-cell depletion in SLE: clinical and trial experience with rituximab and ocrelizumab and implications for study design. Arthritis Res Ther. 2013; 15(Suppl 1): S2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIsenberg D, Gordon C, Licu D, et al.: Efficacy and safety of atacicept for prevention of flares in patients with moderate-to-severe systemic lupus erythematosus (SLE): 52-week data (APRIL-SLE randomised trial). Ann Rheum Dis. 2014; pii: annrheumdis-2013-205067. PubMed Abstract \| Publisher Full Text\n\nA randomized study of the effect of withdrawing hydroxychloroquine sulfate in systemic lupus erythematosus. The Canadian Hydroxychloroquine Study Group. N Engl J Med. 1991; 324(3): 150–4. PubMed Abstract \| Publisher Full Text\n\nFessler BJ, Alarcón GS, McGwin G Jr, et al.: Systemic lupus erythematosus in three ethnic groups: XVI. Association of hydroxychloroquine use with reduced risk of damage accrual. Arthritis Rheum. 2005; 52(5): 1473–80. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8912", "date": "05 Jun 2015", "name": "Anne Eberhard", "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 author has provided a review of proliferative nephritis in childhood SLE, the aim of the review was to concentrate on the pediatric data where available. Comments:IntroductionReferences are left out from the introduction; the first 4 paragraphs are making statements essentially without any references. In para 3 the study cited, authored by Mok et al., is primarily in adults and that needs to be clarified. Para 4 line 4: add in \"based on a few…\" CorticosteroidsRemove the 2 'primarily's in lines 4 and 5 of para 1. Reference the statement on alternate day dosing and reduced side effects. As the aim of the paper was to outline studies in children, I would expand the studies referenced regarding pediatrics in this section They have been reduced to a paragraph. Refer back to pediatric vs adult outcomes in these studies and if they differ.AzathioprineThe authors have failed to mention studies done in children with AZA – some referenced below:J Rheumatol. 2014 Oct;41(10):1998-2007Immunosuppressive Therapies for the Induction Treatment of Proliferative Lupus Nephritis: A Systematic Review and Network Metaanalysis.Simon Yu Tian, Brian M. Feldman, Joseph Beyene, Patrick E. Brown, Elizabeth M. Uleryk and Earl D. SilvermanJ Rheumatol. 2002 Dec;29(12):2635-42.Longterm followup of childhood lupus nephritis.Hagelberg S, Lee Y, Bargman J, Mah G, Schneider R, Laskin C, Eddy A, Gladman D, Urowitz M, Hebert D, Silverman E. Please also reference the ACR article on using AZA as maintenance.RituximabMention that children will need to be revaccinated, particularly against pneumococcus.BelimumabSpecifically address the efficacy of belimumab in proliferative nephritis in these studies.Adjunctive TherapyFirst word, first line is In but should be An.ConclusionRegarding the statement in last paragraph (life expectancy in proliferative LN) reference this and compare results between adults and children.", "responses": [] }, { "id": "9571", "date": "23 Sep 2015", "name": "Ginger Janow", "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\nOverall this is a thorough review of the current accepted treatments for childhood/pediatric SLE. Data provided was fair and balanced with a good overview of the current literature. First paragraph of introduction could use supporting references. It would be helpful to clarify which studies included pediatric patients throughout (it is clear in the cytoxan section but notably absent in the azathioprine section). Additionally, the corticosteroid section includes recommendations on dosing schedules but is lacking references. Similarly, the Adjunctive Therapy section could also benefit from additional sources.There was a recent study (published after this paper was submitted) on maintenance therapy that would be appropriate for inclusion: Tian SY, Feldman BM, Beyene J, Brown PE, Uleryk EM, Silverman ED. Immunosuppressive Therapies for the Maintenance Treatment of Proliferative Lupus Nephritis: A Systematic Review and Network Metaanalysis.J Rheumatol. 2015 Aug;42(8):1392-400.In \"Less Supported Therapies\" section, it would be useful to know cause of fatalities in study that was concluded early due to deaths.In conclusion, this is a well-researched review with a good summary of the available data. Additional sources could be added to assist the reader looking to do further research, but the article as it stands will provide a useful resource for those looking to learn more about this topic.", "responses": [] } ]	1	https://f1000research.com/articles/4-128
https://f1000research.com/articles/3-33/v1	31 Jan 14	{ "type": "Case Report", "title": "Atypical Cornelia de Lange Syndrome: a case report", "authors": [ "Vito Leanza", "Gabriella Rubbino", "Gianluca Leanza ", "Gabriella Rubbino", "Gianluca Leanza " ], "abstract": "Cornelia de Lange Syndrome (CdLS) (also called Bushy Syndrome or Amsterdam dwarfism), is a genetic disorder that can lead to several alterations. This disease affects both physical and psychical development. The various abnormalities include facial dysmorphia (arched eyebrows, synophrys, depressed nasal bridge, long philtrum, down-turned angles of the mouth), upper-extremity malformations, hirsutism, cardiac defects, and gastrointestinal alterations. The prevalence of this syndrome is approximately one per 15,000. Ultrasound is not the perfect means to diagnose CdLS, however, many abnormalities can be detected prenatally by scrupulous image observation.We report an atypical CdLS case characterized by increased nuchal translucency in the first trimester, normal kariotype, saddle nose, micrognathia with receding jaw, low set ears, facies senilis, arthrogryposis of the hands, absence of the Aranzio ductus venous, dilatation of gallbladder and bowel, a unique umbilical artery, increased volume of amniotic fluid, and intrauterine growth retardation ending with the interruption of pregnancy.", "keywords": [ "Cornelia de Lange Syndrome (CdLS) is a genetic disorder that can lead to several alterations. It affects both physical and psychical development. The several abnormalities include facial dysmorphia (arched eyebrows", "synophrys", "depressed nasal bridge", "long philtrum", "down-turned angles of the mouth)", "upper-extremity malformations", "hirsutism", "cardiac defects", "and gastrointestinal alterations1. The prevalence of this syndrome is approximately one per 15", "0002. Many markers have to be considered. Nuchal translucency (NT) measurement during the first trimester screening between 11 and 14 weeks gestation has now been clearly identified as a marker for aneuploidies and in particular for trisomy 21. Even in the absence of aneuploidy", "increased fetal nuchal translucency has been shown to be a marker for fetal heart malformations and several other fetal defects linked to genetic syndromes when the measure exceeds the 95th percentile (3–5 mm)3. When conventional karyotyping is normal", "enlarged NT is a strong marker for adverse pregnancy outcome", "associated with miscarriage or intrauterine death). Unspecified genetic syndromes involving developmental delay may only emerge after birth and become evident after the first years of life. Several abnormalities have been reported in fetuses with enlarged NT: the majority are cardiac defects", "diaphragmatic hernia", "exomphalos", "body stalk anomaly", "skeletal defects", "certain genetic syndromes (such as congenital adrenal hyperplasia)", "fetal akinesia", "deformation sequence", "Noonan syndrome", "Smith-lemli-Opitz syndrome and spinal muscular atrophy4. The saddle nose", "characterized by a markedly depressed bridge has been described in AIDS embryopathy", "Christ-Siemans-Touraine syndrome", "various deletion syndromes", "fetal trimethadione syndrome", "Laron-type dwarfism", "leprechaunism", "multiple epiphyseal dysplasia", "otospondylomegaepiphyseal dysplasia (OSMED) syndrome", "relapsing polychondritis", "thanatophoric dwarfism", "Wegener’s granulomatosis", "and various conditions that are further characterized by gargoyle-like facies. Micrognathia (mandible of insufficient size) is a malformation of the fetal face characterized by a small mandible. Micrognathia may be idiopathic but is more commonly associated with many different syndromes. Retrognathia (recession of the chin) is assessed through the measurement of the inferior facial angle", "as defined on a mid-sagittal view. With routine ultrasound", "the receded chin may be seen on a profile of the face. Yet", "this diagnosis is often missed during a routine ultrasound examination. The mandible is known to grow significantly during the third trimester. If mandibular alteration is suspected", "particular attention should be paid to the growth of the chin throughout the remainder of the pregnancy. Conditions associated with micrognathia include chromosomal abnormalities", "neuromuscular abnormalities", "single-gene disorders", "and other syndromes. The prognosis of fetal micrognathia is poor", "even in chromosomally normal fetuses. Frequent malformations associated with micrognathia are: Pierre Robin sequence (micrognathia", "cleft palate", "or both)5", "Cerebrocostomandibular syndrome (diagnosed on the basis of micrognathia", "a posterior cleft palate defect", "and characteristic rib gap abnormalities)", "Cornelia de Lange syndrome", "(underdeveloped chin with tetralogy of Fallot", "intrauterine growth restriction", "and an abnormal right hand)6", "hypochondrogenesis type II and Caffey disease." ], "content": "Introduction\n\nCornelia de Lange Syndrome (CdLS) is a genetic disorder that can lead to several alterations. It affects both physical and psychical development. The several abnormalities include facial dysmorphia (arched eyebrows, synophrys, depressed nasal bridge, long philtrum, down-turned angles of the mouth), upper-extremity malformations, hirsutism, cardiac defects, and gastrointestinal alterations1. The prevalence of this syndrome is approximately one per 15,0002. Many markers have to be considered. Nuchal translucency (NT) measurement during the first trimester screening between 11 and 14 weeks gestation has now been clearly identified as a marker for aneuploidies and in particular for trisomy 21. Even in the absence of aneuploidy, increased fetal nuchal translucency has been shown to be a marker for fetal heart malformations and several other fetal defects linked to genetic syndromes when the measure exceeds the 95th percentile (3–5 mm)3. When conventional karyotyping is normal, enlarged NT is a strong marker for adverse pregnancy outcome, associated with miscarriage or intrauterine death). Unspecified genetic syndromes involving developmental delay may only emerge after birth and become evident after the first years of life. Several abnormalities have been reported in fetuses with enlarged NT: the majority are cardiac defects, diaphragmatic hernia, exomphalos, body stalk anomaly, skeletal defects, certain genetic syndromes (such as congenital adrenal hyperplasia), fetal akinesia, deformation sequence, Noonan syndrome, Smith-lemli-Opitz syndrome and spinal muscular atrophy4. The saddle nose, characterized by a markedly depressed bridge has been described in AIDS embryopathy, Christ-Siemans-Touraine syndrome, various deletion syndromes, fetal trimethadione syndrome, Laron-type dwarfism, leprechaunism, multiple epiphyseal dysplasia, otospondylomegaepiphyseal dysplasia (OSMED) syndrome, relapsing polychondritis, thanatophoric dwarfism, Wegener’s granulomatosis, and various conditions that are further characterized by gargoyle-like facies. Micrognathia (mandible of insufficient size) is a malformation of the fetal face characterized by a small mandible. Micrognathia may be idiopathic but is more commonly associated with many different syndromes. Retrognathia (recession of the chin) is assessed through the measurement of the inferior facial angle, as defined on a mid-sagittal view. With routine ultrasound, the receded chin may be seen on a profile of the face. Yet, this diagnosis is often missed during a routine ultrasound examination. The mandible is known to grow significantly during the third trimester. If mandibular alteration is suspected, particular attention should be paid to the growth of the chin throughout the remainder of the pregnancy. Conditions associated with micrognathia include chromosomal abnormalities, neuromuscular abnormalities, single-gene disorders, and other syndromes. The prognosis of fetal micrognathia is poor, even in chromosomally normal fetuses. Frequent malformations associated with micrognathia are: Pierre Robin sequence (micrognathia, cleft palate, or both)5; Cerebrocostomandibular syndrome (diagnosed on the basis of micrognathia, a posterior cleft palate defect, and characteristic rib gap abnormalities); Cornelia de Lange syndrome, (underdeveloped chin with tetralogy of Fallot, intrauterine growth restriction, and an abnormal right hand)6; hypochondrogenesis type II and Caffey disease.\n\n\nCase report\n\nWe report a case of a healthy 31-year-old, gravida 2, para 1 at 30 weeks of gestation that was admitted to S. Bambino University Hospital in Catania for ultrasound examination.\n\nUltrasounds revealed nuchal edema, saddle nose, micrognathia with receding jaw, low set ears, facies senilis at 3D ultrasounds, arthrogryposis of the hands, absence of Aranzio ductus venous, dilation of gallbladder and bowel, single one umbilical artery, increased volume of amniotic fluid. Intrauterine growth retardation was associated as well. (Figure 1) Micrognathia was evident on midsagittal and 3D scan. The biparietal diameter was 68 mm, femur length 47 mm, suggesting fetal growth restriction. Pulsed Doppler sonography showed normal middle cerebral artery and umbilical artery pulsatility indices. The obstetric history revealed increased nuchal translucency thickness (NT) at 11 weeks (4 mm, > 95 centile). No genetic alterations were found at the amniocentesis carried out during 16th week of pregnancy (normal karyotype of 46, XX). Morphological ultrasound at 22 weeks of pregnancy was not able to detect the syndrome. A further ultrasound examination at 29 weeks (Figure 2) of pregnancy pointed out a fetal dimorphism and the pregnant woman asked for our consultation.\n\nSoon after, interruption of pregnancy occurred. The autopsy showed a foetus with a weight between the 5th and the 10th percentile and a dysmorphic syndrome with malformation features amenable to CdLS (Figure 3). The foetus had a typical dysmorphic face, hirsutism, rhizomelic limb, bilateral camptodactyly, single transverse palmar crease, proximal and very short inches with hypoplasia of 1st metacarpal and single phalanx, II-III membranous syndactyly of feet, right diaphragmatic hernia, bilateral cryptorchidism, microcephaly, numerous nodules of heterotopic cerebellar vermis, single umbilical artery and hypotrophic placenta devoid of inflammatory lesions.\n\n\nDiscussion and conclusion\n\nCdLS is a multisystem malformation syndrome recognized primarily on the basis of the morphological characteristics (malformations of the cranial, cardiac, gastrointestinal, and skeletal systems)7. However, there is wide clinical variability of disorders, with milder phenotypes that may be difficult to ascertain on the basis of physical features. In certain cases the diagnosis may be missed when ultrasound examination is not performed accurately8. Criteria to detect CdLS are not standardized. The main alterations are as follows9:\n\nFacial abnormalities (synophrys, long eyelashes, microcephaly, anteverted nostrils)\n\nCardiac defects (defects of ventricles or atria, aortic or pulmonary stenoses, tetralogy of Fallot, atrioventricular canal, single ventricle, aorto-pulmonary window, truncus arteriosus communis)\n\nAbnormalities of upper limbs (ectrodactylia and monodactylism)\n\nGastrointestinal alterations (diaphragmatic hernia)\n\nMusculoskeletal malformations\n\nIntrauterine growth retardation\n\nUltrasound detection of eyelashes is considered a clue for prenatal diagnosis of CdLS, but it can be missed in clinical practice10. CdLS is considered a cohesinopathy. Mutations in cohesin, or its regulators, cause a spectrum of human developmental syndromes. Cohesinopathy disorders include both CdLS and Roberts Syndrome. The discovery of novel roles for chromatid cohesion proteins in regulating gene expression led to the idea that cohesinopathies are caused by dysregulation of multiple genes downstream of mutations in cohesion proteins. Consistent with this idea, there is an altered expression of developmental genes and an incomplete overlap among dysregulated genes in different components of the cohesion apparatus11. CdLS is considered a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion; it also has a key role in genetic regulation. In CdLS cell lines an altered transcription with either NIPBL or HDAC8 mutations has been found12. The proteins produced from mutated genes interfere in the foetal development. Within cells, these proteins help regulate the structure and organization of chromosomes and are involved in the repair of damaged DNA. They also regulate the activity of certain genes in the developing limbs, face, and other parts of the body. Researchers are looking for additional changes in the NIPBL, SMC1A and SMC3 genes, as well as mutations in other genes, that may be responsible for this condition. The majority of cases result from new gene mutations and occur in people with no history of familiarity. CdLS can be prenatally diagnosed in a family with a known mutation in a CdLS gene. The characteristic ultrasonographic profile may allow for prenatal diagnosis of CdLS in subsequent pregnancies for couples with a prior child with CdLS in whom a mutation has not been identified or when there are unexplained signs of fetal abnormality during pregnancy, such as oligo- or polyhydramnios, a low maternal serum PAPP-A level and/or increased NT, fetal growth retardation, or structural anomalies consistent with CdLS13. In some cases the diagnosis is made prenatally in other cases the syndrome is diagnosed after childbirth.\n\nThe diagnosis is seldom in doubt when there is a major longitudinal deficiency defect of the upper limb, severe prenatal and postnatal growth retardation, and severe mental retardation. Features used to make the correct diagnosis include penciled and arched eyebrows, a high set/short anteverted nose, a long flat philtrum, a thin upper lip, downturned corners of the mouth, and micrognathia. Characteristics that are misleading include full or flat brows, a prominent nasal bridge or bulbous tip, and/or a normal or prominent chin. As genetic and biochemical tests are unreliable at present, antenatal detection depends upon identification of some aspects of the phenotype in the fetus using ultrasound imaging14,15. When the syndrome is not recognized during pregnancy, the newborn may survive with a low quality of life and, thus, medical team could become involved in surgical procedures16. Each malformation causes an impact in the psychological sphere of both the individual and the family17,18. Finally, last but not the least, the failure of an early diagnosis may lead to medical-legal issues19.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nVL: conception and design. GR: performed the ecography and drafted the article. All authors revised the manuscript for intellectual content and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSpecial thanks to Professor Salvatore Sciarretta for his precious help in translating this work.\n\n\nReferences\n\nNoor N, Kazmi Z, Mehnaz A: Cornelia de Lange syndrome. J Coll Physicians Surg Pak. 2012; 22(6): 412–3. PubMed Abstract\n\nMurray JE, Walayat M, Gillett P, et al.: An atypical facial appearance and growth pattern in a child with Cornelia de Lange Syndrome: an intragenic deletion predicting loss of the N-terminal region of NIPBL. Clin Dysmorphol. 2012; 21(1): 22–3. PubMed Abstract \| Publisher Full Text\n\nSenat MV, Frydman R: Increased nuchal translucency with normal karyotype. Gynecol Obstet Fertil. 2007; 35(6): 507–15. PubMed Abstract \| Publisher Full Text\n\nMula R, Goncé A, Bennásar M, et al.: Increased nuchal translucency and normal karyotype: perinatal and pediatric outcomes at 2 years of age. Ultrasound Obstet Gynecol. 2012; 39(1): 34–41. PubMed Abstract \| Publisher Full Text\n\nHsieh YY, Chang CC, Tsai HD, et al.: The prenatal diagnosis of Pierre-Robin sequence. Prenat Diagn. 1999; 19(6): 567–569. PubMed Abstract \| Publisher Full Text\n\nBerney TP, Ireland M, Burn J: Behavioural phenotype of Cornelia de Lange syndrome. Arch Dis Child. 1999; 81(4): 333–336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Lange C: Sur un type nouveau d degenerescence (typus Amestelodamensis). Archives de Médecine des Enfants. 1933; 36: 713–719.\n\nBhuiyan ZA, Klein M, Hammond P, et al.: Genotype-phenotype correlations of 39 patients with Cornelia de Lange syndrome: the Dutch experience. J Med Genet. 2006; 43(7): 568–575. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkahori Y, Masuyama H, Masumoto Y, et al.: Three-Dimensional Ultrasound Findings in Cornelia de Lange Syndrome: A Case Report. Obstet Gynecol. 2012; 2012: 568351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpaggiari E, Vuillard E, Khung-Savatovsky S, et al.: Ultrasound Detection of Eyelashes: A Clue for Prenatal Diagnosis of Cornelia de Lange Syndrome. Ultrasound Obstet Gynecol. 2013; 41(3): 341–2. PubMed Abstract \| Publisher Full Text\n\nHorsfield JA, Print CG, Mönnich M: Diverse developmental disorders from the one ring: distinct molecular pathways underlie the cohesinopathies. Front Genet. 2012; 3: 171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeardorff MA, Bando M, Nakato R, et al.: HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature. 2012; 489(7415): 313–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClark DM, Sherer I, Deardorff MA, et al.: Identification of a prenatal profile of Cornelia de Lange syndrome (CdLS): a review of 53 CdLS pregnancies. Am J Med Genet A. 2012; 158A(8): 1848–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeanza V, Sciarretta S: Fundamental obstetric and gynecologic elements. (Test Book) Editor S.p.e. Catania, (Italy). 2011; ISBN 978-88-96808-05-4.\n\nLeanza V: OBSTETRICS Editor MINERVA MEDICA S.P.A. Torino (Italy). 2009; (ISBN 13 978-88-7711-631-4). Reference Source\n\nZanghì G, Di Stefano G, Leanza V, et al.: Incisional hernia in Day Surgery: our personal experience. G Chir. 2012; 33(6–7): 218–20. PubMed Abstract\n\nLeanza V, Passanisi A, Leanza G: A specific application of two psychological measures on female urinary incontinence: perceived negative affective self-efficacy scale and stress psychological measure. Urogynaecologia International Journal. 2011; 25: N°2, 41–48.\n\nLeanza G, Palumbo M, Marilli I, et al.: Double atresic uterus associated with agenesis of cervix and third upper of vagina. Giornale Italiano di Ostetricia e Ginecologia. 2013; 35(1): 262–264. Reference Source\n\nCoco G, Danzì M, Bellocchi P, et al.: Medicolegal epidemiological observatory of contentious procedures in the Vittorio Emanuele-Ferrarotto- S Bambino Catania in a University-Hospital (Italy): retrospective analysis of one decade (1995–2005). International Academy of Legal Medicine. 2006; 160–161. Reference Source" }	[ { "id": "3450", "date": "14 Feb 2014", "name": "Jinglan Liu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a fetus with clinical features suggestive of Cornelia de Lange Syndrome (CdLS).My comments are as follows:Data from mutational testing on known CdLS genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) would be important for this case. The article published by Clark et al. (2012) which was also cited by the authors, recommends molecular analysis of CdLS genes to prenatal diagnosis of CdLS. The authors should compare prenatal findings in the current case with what described in the article by Clark et al. (2012). Without molecular testing data, I would like to see a table demonstrating the current case presenting prenatal clinical features consistent with what has been documented in the literature by studying a larger cohort of probands.\n\nIn the discussion, in the beginning of first paragraph, the authors claim that CdLS is diagnosed primarily by morphological characteristic and cited a paper published in 1933. At present, the diagnosis criteria of CdLS cannot be the same as what was 90 years ago, particularly with the identification of specific gene mutations in CdLS probands. Please re-phrase this part, and cite updated references. In the end of the discussion, what does it mean “genetic tests are not reliable”? As a matter of fact, molecular testing has become a widely accepted concept and a popular diagnostic approach for individuals with congenital anormalies.\n\nMinor things:OMIM numbers of CdLS and disease genes should be given. Please double check the spelling and eliminate typographic errors, also, some terms are not accurately used. Below is listed some examples of those errors:Abstract – “kariotype” -> “karyotype” Introduction – “psychical” - > I’d like to use “neuropsychiatric” 2nd paragraph in case report – “dimorphism” -> “dysmorphism” 2nd paragraph in discussion – “cohesion” -> “cohesin” 2nd paragraph in discussion – please italicize the gene names “NIPBL” and “HDAC8” 2nd paragraph in discussion – “familiarity” ? What does it mean? Why not change to “ no family history”", "responses": [ { "c_id": "957", "date": "01 Sep 2014", "name": "vito leanza", "role": "Author Response", "response": "“Data from mutational testing on known CdLS genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) would be important for this case. The article published by Clark et al. (2012) which was also cited by the authors, recommends molecular analysis of CdLS genes to prenatal diagnosis of CdLS.”In our case report, data from mutational testing on known CdLS genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) are negative , indeed it was called atypical Cornelia de Lange Syndrome (CdLS).Mutations in NIPBL are not present in all cases and they account for about 60% of CdLS; mutations in SMC1A and SMC3 account for a small percentage (Deardorff et al., 2011). “The authors should compare prenatal findings in the current case with what described in the article by Clark et al. (2012). Without molecular testing data, I would like to see a table demonstrating the current case presenting prenatal clinical features consistent with what has been documented in the literature by studying a larger cohort of probands.”Cornelia de Lange Syndrome (CdLS) is a multisystem developmental disorder characterized by growth retardation, cognitive impairment, external and internal structural malformations, and characteristic facial features. Currently, there are no definitive prenatal screening measures that lead to the diagnosis of CdLS. (Clark et al. 2012).In our case (atypical CdLS) increased nuchal translucency in the first trimester, normal karyotype, saddle nose, micrognathia with receding jaw, low set ears, facies senilis, arthrogryposis of the hands, absence of the Aranzio ductus venous, dilatation of gallbladder and bowel, a unique umbilical artery, increased volume of amniotic fluid, and intrauterine growth retardation were found.Fewer than 1% of individuals diagnosed with Cornelia de Lange syndrome have an affected parent.Recommendations for the evaluation of parents of a proband with an apparent de novo mutation include clinical examination for features of CdLS, complete with plotting of growth parameters and molecular genetic testing if the NIPBL mutation has been identified in the proband. In our atypical Cornelia de Lange syndrome molecular tests of parents were negative. “In the discussion, in the beginning of first paragraph, the authors claim that CdLS is diagnosed primarily by morphological characteristic and cited a paper published in 1933. At present, the diagnosis criteria of CdLS cannot be the same as what was 90 years ago, particularly with the identification of specific gene mutations in CdLS probands. Please re-phrase this part, and cite updated references.”Diagnosis is based on clinical findings. High-resolution ultrasound examination to follow growth and to evaluate the limbs, heart, diaphragm, palate, and other organs or structures affected in CdLS is fundamental. Reported prenatal ultrasound findings include: increased nuchal translucency in the first trimester [Sekimoto et al., 2000; Huang & Porto, 2002]; Growth failure, which typically presents in the second trimester; and the typical in utero facial profile of a fetus with CdLS consisting of micrognathia, a prominent upper lip, and a depressed nasal bridge with somewhat anteverted nares [Ranzini et al., 1997; Boog et al., 1999; Urban & Hartung, 2001]. “In the end of the discussion, what does it mean “genetic tests are not reliable”? As a matter of fact, molecular testing has become a widely accepted concept and a popular diagnostic approach for individuals with congenital anormalies.”Genetic tests are not reliable when negative. On the contrary when positive they are important for syndrome.Indeed, mutations in NIPBL are not present in all cases and they account for about 60% of CdLS; mutations in SMC1A and SMC3 account for a small percentage (Deardorff et al., 2011).Minor things:These minor things are easy to correct. I thank the first referee for his clarifications." } ] }, { "id": "4483", "date": "14 Apr 2014", "name": "Laird Jackson", "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\nI agree with the report and comments of Dr Jinglian Liu, who has also reviewed this article. The case report is interesting in its detail of the prenatal ultrasound examination and observations, but completely fails its responsibility to connect those observations to appropriate use of contemporary molecular diagnostic opportunities. As such, this article fails the medical community by not accepting the opportunity to connect their useful prenatal morphological observations to detectable objective molecular findings.", "responses": [] } ]	1	https://f1000research.com/articles/3-33
https://f1000research.com/articles/3-319/v1	31 Dec 14	{ "type": "Research Note", "title": "Dynamics of Ebola epidemics in West Africa 2014", "authors": [ "Robin J. Evans", "Musa Mammadov", "Robin J. Evans" ], "abstract": "This paper investigates the dynamics of Ebola virus transmission in West Africa during 2014. The reproduction numbers for the total period of epidemic and for different consequent time intervals are estimated based on a simple linear model. It contains one major parameter - the average infectious period that defines the dynamics of epidemics.Numerical implementations are carried out on data collected from three countries Guinea, Sierra Leone and Liberia as well as the total data collected worldwide. Predictions are provided by considering different scenarios involving the average times of infectiousness for the next few months and the end of the current epidemic is estimated according to each scenario.", "keywords": [ "The outbreak of the 2014 Ebola virus epidemic in West Africa", "started in late 2013", "does not seem to be under control and accurate predictions appear to be extremely difficult. The major reason for this might be due to unstable treatment conditions that provide different reproduction numbers at different periods. However", "there are also other challenges related to the mathematical modeling of this epidemic. To address these challenges", "several new models have been suggested that show quite different results", "we note a few of them published recently1–7." ], "content": "Introduction\n\nThe outbreak of the 2014 Ebola virus epidemic in West Africa, started in late 2013, does not seem to be under control and accurate predictions appear to be extremely difficult. The major reason for this might be due to unstable treatment conditions that provide different reproduction numbers at different periods. However, there are also other challenges related to the mathematical modeling of this epidemic. To address these challenges, several new models have been suggested that show quite different results, we note a few of them published recently1–7.\n\nIn this article we introduce a new model to study the dynamics of the current outbreak by considering the average infectious period as a time-dependent parameter. It is derived from the well studied SIR (Susceptible-Infectious-Recovery) model with time delay (e.g. [8,9]), where the decrease in the number of susceptible population in compartment S is the major force stopping epidemics. The susceptible population S is often considered as a whole population. A major drawback of this model, in terms of the current epidemic, is that the population infected constitutes a very small proportion of the total population, a very small decrease in S has almost no effect on compartment I.\n\nWe discuss how this drawback could be tackled and introduce a new model that uses only compartment I. This leads to a linear model having some similarities to those models based only on transmission rates from infectious population at different generations (e.g.5). Our main goal is to fit data by estimating fewer and most influential parameters without considering many other issues like the infectiousness in hospitals and death ceremonies.\n\nThis in addition, allows us to have a more robust model with easily interpreted parameters that can be used for more accurate predictions. The main parameter in this model is the average infectious period τ2 (time from onset to hospitalization) that defines the dynamics of infectious population. This parameter can also be considered as a control parameter in the development of control models dealing with the spread of infection.\n\nWe calculate the basic reproduction numbers R0 for each country (Guinea, Sierra Leone and Liberia) as well as the total Ebola data worldwide. We also provide predictions corresponding to different scenarios by considering different values for τ2 for future time periods.\n\n\nMethods\n\nWe use the notation Ia(t) for the number of “active” infectious population at time t; it mainly represents the total number of infectious population that are not yet hospitalized. C(t) and D(t) are the cumulative number of infected cases and deaths, respectively. The population density of a country is denoted by 𝒟. This is used in the definition of the infection force of the disease with coefficient β. Moreover, μ stands for the natural death rate of the population, α for the death rate due to disease, and τ1 for the average latent period (in days) that infected individuals become infectious and τ2 for the average infectious period (in days).\n\nThe main equations of our model are as follows (see Appendix for details):\n\n\n\n\n\n\n\nHere ω is a gamma (cumulative) distribution function (with p.d.f - ωp) for deaths due to disease7; for the values of the parameters see Appendix. We note that there are only three parameters that need to be estimated to fit data for cumulative number of infected and death cases. These parameters are:\n\nα - the death rate due to disease;\n\nβ - the coefficient of the force of infection;\n\nτ2 - the average infectious period.\n\nHere α and β are continuous variables, τ2 is a discrete variable with integer values (days).\n\nBasic Reproduction Number - R0. We calculate the basic reproduction number by considering the stationary states in (1) as follows:\n\n\n\nSince the natural death rate μ is close to zero; that is, 1– μ ≈ 1, from (2) we have\n\n\n\nMoreover, since α∑i=0τ2−1ω(i)<1, the reproduction number R0 ≈ β𝒟τ2. This means that the reproduction number depends almost linearly on τ2.\n\nThe effective reproduction numbers - Rk, k ≥ 1. The effective reproduction numbers Rk are considered on several consecutive time intervals Δk = [tk, tk+1], k = 1, 2, …, with corresponding values of τ2. They are calculated by the same formula as R0.\n\nHere we make a reasonable assumption that the transmission rate β𝒟 describes the interaction of population (that is, in some sense, related to the local conditions and the life style) and should remain relatively stable for a particular country. Then, the efforts in preventing the spread of infection are mainly observed in the change (decrease) in the value of τ2.\n\nTherefore, to calculate the effective reproduction numbers, we fit data and find the optimal values for α and β, that are constant over the whole period, and optimal values τ2k on each interval Δk. Then Rk is calculated by formula (2) setting τ2k.\n\nThe sequence of optimal values τ21, τ22, …, is considered as a method to describe the effectiveness of measures applied for preventing the spread of infection. This sequence very much defines the reproduction numbers on each consecutive time interval and therefore the dynamics of the infected population. It also allows us to consider future scenarios in terms of possible average infectious periods (i.e. times from onset to hospitalization).\n\n\nResults and discussion\n\nData were retrieved from the WHO website (http://www.who.int/csr/disease/ebola/situation-reports/en/) for the cumulative numbers of clinical cases (confirmed, probable and suspected) collected till 11 November 2014. In all numerical experiments, the second half of the available data for each country is used for fitting the cumulative numbers of infected cases and deaths. The global optimization algorithm DSO in Global And Non-Smooth Optimization (GANSO) library10,11 is applied for finding optimal values of parameters.\n\nFirst we consider the whole period of infection in each country and find the best fit in terms of three variables α, β and τ2 (Problem (DF1) in Appendix). The results are presented in Table 1. Although from Figure 1 it can be observed that the best fit for Guinea is not as good as for the other cases, these results provide some estimate for the reproduction number R0 for a whole period of infection till 11-Nov-2014. In all cases (except Guinea), R0 is around 1.20 and for Guinea - 1.09. We note that the dynamics of infected population is much more complicated (especially in Guinea) which suggests that the reproduction number has been changing since the start of Ebola-2014 in almost all countries. This fact has been studied in7 in terms of the instantaneous reproduction number over a 4-week sliding windows for each country (see also the next section for different values for τ2).\n\nR0 is the reproduction number.\n\nThe lines represent the best fits, red and black circles represent the data.\n\nAccording to (2), the basic reproduction number is mainly determined by β and τ2. Since in our model parameter τ2 takes discrete values (days) it would be interesting to study the change of this parameter over time while keeping β the same for the whole period. This approach makes it possible to consider different scenarios for future developments regarding the change in this parameter and to provide corresponding predictions.\n\nWe consider three consequent time intervals Δk = [tk, tk+1] (k = 1, 2, 3) for each country and find optimal values α, β and τ2k (k = 1, 2, 3) (Problem (DF2) in Appendix). The results are presented in Table 2. The last time point t4 is 11-Nov-2014. The values of t1, t2, t3 are as follows: 22-March, 23-May and 20-July for Guinea; 27-May, 20-June and 20-August for Sierra Leone; 16-June, 20-July and 07-Sept for Liberia; and 22-March, 23-May and 07-Sept for the total data (World). Each interval Δk has its own reproduction number Rk that defines the shape of the best fits presented in Figure 2.\n\nThe optimal values for α and β are also provided; they are constant for a whole period.\n\nThe lines represent the best fits, red and black circles represent the data.\n\nThe starting values of parameters (α, β and τ2k, k = 1, 2, 3) are in Table 2 (World). The first time interval (Δ1) is 12/Nov/2014–31/Dec/2014, followed by each next month and the last interval (Δ5) starts from 1/Apr/2015. The reproduction number for τ2 = 3 is R = 0.778; it is less than 1 which leads to stabilization. For corresponding reproduction numbers for τ2 = 4 and 5 see Table 2 (World).\n\nIn all cases the effective reproduction number is still greater than 1. In Liberia it shows a decrease from 1.45 to 0.99 and this can be seen in quite a noticeable decrease in the number of cumulative infected cases (Figure 2).\n\nWe consider only the cumulative number of infected population worldwide. From Table 2 it can be observed that the number τ2 has changed as 4, 5 and 4 from 22-March to 11-Nov. We keep this initial best fit (the optimal values of parameters (World) are in Table 2) and consider different scenarios for possible changes of this parameter in the future while keeping the values of α and β unchanged.\n\nThe future time intervals are designed as follows: the first interval Δ1 is 12/Nov/2014–31/Dec/2014, followed by each next month Δ2 – Δ4, and the last interval Δ5 starts from 1-Apr-2015. The results are presented in Table 3 The reproduction numbers are 0.778 (for τ2 = 3), 1.035 (for τ2 = 4) and 1.284 (for τ2 = 5).\n\nIn the best scenario in Table 3 it is assumed that the current trend stays stable (τ2 = 4) and a 25 percent decrease in the hospitalization time starts from 1-Jan-2015, then the epidemic may continue till Apr-2015 with the total number of infected cases reaching 31,000.\n\nThe starting values of parameters (α, β and τ2k, k = 1, 2, 3) are in Table 2 (World). The first time interval (Δ1) is 12/Nov/2014–31/Dec/2014, followed by each next month and the last interval (Δ5) starts from 1/Apr/2015. The last column presents the date for the end of Ebola epidemic (see also Figure 3) with corresponding number of cumulative infected population Cmax (-/∞ means no stabilization). The version τ2k = 4 for all k means the current trend remains unchanged. The reproduction number for τ2 = 3 is R = 0.778; it is less than 1 which leads to stabilization. For corresponding reproduction numbers for τ2 = 4 and 5 see Table 2 (World).\n\nThe worst case considered in Table 3 assumes that during the next two months (from 12-Nov-2014 to 31-Jan-2015) the average time to hospitalization increases by 25 percent (that is, from 4 days to 5 days) and then gradually decreases in Feb-Mar-2015 (from 5 days to 4 days), in Apr-2015 (from 4 days to 3 days) and stays at this level afterwards. In this case, the Ebola outbreak could be stopped by July-2015 with the total number of infected cases reaching 166,000.", "appendix": "Author contributions\n\n\n\nAll authors contributed equally to the writing and subsequent refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the Collaborative Research Network (CRN) of the Federation University of Australia and the University of Melbourne.\n\n\nAcknowledgements\n\nThe first version of this paper is available in arXive.org: arXiv:1412.1579 [q-bio.PE].\n\n\nAppendix\n\nModel\n\nThe idea behind the model introduced in this paper is related to the SIR model with time delay. Since we are going to implement it on available daily data, a discrete version of this model is considered with the step-size one day. Moreover, since the “recovered” population is not our focus, we will only consider equations related to susceptible (S) and infectious (I) individuals. The most commonly used SIR model1 in the literature is provided below (see, for example,8):\n\n\n\n\n\nHere λ is the recruitment of the population; μ is the natural death rate of the population; α is the death rate due to disease; γ is the recovery rate; and τ1 is the latent period that infected individual becomes infectious.\n\nThe fraction (1–μ)τ1 represents the survival rate of population over the period of [0, τ1] (in continuous-time case it is equivalent to e–μτ1). Below we examine this model in detail and develop an improved model.\n\nSusceptible individuals. Equation (3) describes the dynamics of susceptible individuals S(t). This equation “keeps” the number of infectious individuals I(t) bounded. For example, when the basic reproduction number is greater than 1, there exists8 an endemic equilibrium (S, I) and S(t) → S, I(t) → I as t → ∞. In the case when the birth rate is zero (λ = 0) the relation I(t) → 0 suggests that S(t) → 0.\n\nThus according to this model the epidemic ends because the number of susceptible individuals S(t) decreases over time and the effective reproduction number (as a function of time) becomes less than 1 at some stage; that is, the number of newly infected population F(S(t), I(t)) decreases thanks to the “enough” decrease in the number of susceptible individuals (while I(t) still increases). This might be applicable to epidemics in early 1900s but it is definitely not applicable to recent ones.\n\nThis issue significantly restricts the application of the SIR model for the study of the current Ebola virus epidemic. Below we consider 3 possibilities to overcome this difficulty.\n\n1. The simplest way would be to use a “relatively small” number S(0) for a possible number of susceptible individuals that may become infected. This approach has been implemented in1 where the total population size in each country (Guinea, Sierra Leone and Liberia) was assumed to be 106 individuals.\n\n2. An interesting (and most reliable in our opinion) approach would be considering “relatively small” number of population S(0) as a variable that needs to be estimated. We have implemented this approach and the results show that currently available curve/data is not “long” enough to uniquely determine S(0); that is, almost the same quality of data fit can be achieved for different numbers S(0) (we have tried 50,000, 100,000 and 200,000) leading to different numbers of “stabilized” cumulative infected cases and infection periods. Taking this factor into account, we do not consider this approach, however we note that it might be quite possible soon with the availability of more data points.\n\n3. In this paper we adopt another approach by neglecting the compartment S completely and leaving just the compartment I. The force of infection F(S, I) in this case is the main factor to be determined. We take this function in the form\n\nF(S, I) = β𝒟I (5)\n\nwhere 𝒟 is the population density of a particular country. In a more general setting, one would involve functions nonlinear in I (like F(S, I) = β𝒟Iξ with ξ ≤ 1). However, since the infectious population I is a very small portion of the total population, function F can be assumed linear at least in early stages of epidemics. In this case equation (4) can be represented in the form\n\nI(t + 1) = (1 – μ)τ1 β𝒟I(t – τ1) + (1– μ – α – γ)I(t). (6)\n\nThe major drawback of this model is that I may growth infinitely if the reproduction number is greater than 1; in this model there is no variable/parameter (like S(t) in SIR) that could force I to decrease. On the other hand we believe that it can better describe the behavior of an infected population in “small” time intervals and provide more accurate reproduction numbers.\n\nActive infectious population. Now we discuss the infectious population and equation (6) in more detail. We call “active infectious populations” at time t the infected population that are infectious at that time but are not hospitalized yet. Denote by Ia(t) the number of active infectious populations at time t. We will rewrite equation (6) in terms of Ia.\n\nDenote by τ2 the average infectious period; that is, time from onset (τ1) to hospitalization. Then, an infected person is assumed to be active infectious during the period [τ1, τ1 + τ2]. Since τ2 is relatively small, we can assume that none is recovering during that period. This means that the rate of recovery γ in (6) is no longer needed for Ia(t).\n\nThus, we transform equation (6) by taking into account the time delay τ2. Accordingly, the equation for Ia(t) can be represented in the form\n\n\n\nHere ω(0) = 0 and ω(i), i ≥ 1, is a gamma cumulative distribution function for onset-to-death that well describes the current Ebola virus in West Africa7. We note that in this equation, for each i ≥ 1, the fraction (1–αω(i)) is applied to the remaining infectious (1–μ)i β𝒟Ia(t–τ1–i); that is, the death rate in (7) is slightly different from (6) (indeed, both μ and ω(i) are quite small and this leads to 1–μ–αω(i) ≈ (1–μ)(1–αω(i))).\n\nCumulative number of infected cases. The first term (1 – μ)τ1 β𝒟 Ia(t – τ1) in (7) describes the number of new cases at time t. The cumulative number of infectious cases at (t+1) will be denoted by C(t +1). It can be calculated as\n\n\n\nCumulative number of deaths. To calculate the cumulative number of deaths at time t, we consider all infectious cases (hospitalized or not) in the interval [t–τ1, t–n] where n is a sufficiently large number. In particular we assume that death may occur after the onset. As mentioned above, the distribution of death is described by a gamma distribution function ω with its p.d.f - ωp. Then, the cumulative number of deaths due to disease can be calculated as\n\n\n\nMain parameters. We have formulated the dynamical system (7),(8),(9). Given the observed cumulative number of infected cases - C0(t) and cumulative number of death cases - D0(t), the parameters of the systems can be estimated by the best fit. Before formulating this problem we discuss the parameters to be estimated.\n\nThe density 𝒟 and the natural death rate of the population - μ is available for each country. We set 𝒟 = 41, 80, 36 and 50 for Guinea, Sierra Leone, Liberia and the worlwide data, respectively. The natural death rate is around 10 deaths for 1000 population per year (1 percent yearly) for all the three countries. Thus in all numerical implementations, the daily rate μ is set to be 0.01/365 = 0.0000274. It is reasonable to have the same average latency period - τ1 for infected individual to become infectious. The previous studies (e.g. [7]) suggest that it is between 2–21 days with the mean of 11.4 days. Our numerical experiments show that the values between 6–8 provide better results; we set τ1 = 6 in all cases.\n\nParameters of the gamma distribution can be taken from7. We set\n\n\n\nwith mean value 7.5. Note that the choice of values a and b within reasonable intervals, by keeping the mean value the same, has almost no effect on the quality of data fitting. Taking into account this fact, the parameters of the gamma distribution are chosen as in (10). In all the calculations, we set n=35 (days) in (9) (note that for large i function values ω(i) are almost zero).\n\nInitial values Ia(t), t ≤ 1, for the equation are chosen in the form\n\nIa(t) = ξC0(1), for all t ≤ 1. (11)\n\nwhere C0(1) is the actual cumulative infectious. Numerical experiments show that the choice of ξ in the interval 0.4–0.7 has very little impact on the quality of data fitting. We set ξ = 0.4 in all cases exept Liberia for which the value 0.7 was better. Accordingly, we do not consider ξ as a variable and set the above mentioned values for each country/data.\n\nTherefore, the main parameters that define the dynamics of Ebola epidemics in different countries are α - the death rate due to disease, β - the coefficient of the force of infection and τ2 - the average infectious period.\n\nData fitting. We consider data collected till 11 November 2014 for the cumulative number of infectious (confirmed, probable and suspected) and death individuals; they will be denoted by C0(t) and D0(t), respectively. We will use the root mean square error. Given time interval [T1, T2] and data points C0(ti) and D0(ti), i ≥ 1, we define\n\n\n\nAccording to this formula, we fit the second half of given data in order to decrease the choice of initial values Ia(t), t ≤ 1, defined by (11).\n\nTo calculate the basic reproduction number, the above model is considered on the whole interval. The corresponding data fitting problem is:\n\nProblem (DF1): Given data C0(ti) and D0(ti), i ≥ 1, and time interval [1, T2]:\n\n\n\nThe reproduction number is mainly determined by β and τ2. Since in our model parameter τ2 takes discrete values (days) it would be interesting to study the change of this parameter over time while keeping β the same for a whole period.\n\nWe consider three consequent time intervals Δk = [tk, tk+1] (k = 1, 2, 3) for each country and find optimal values α, β and τ2k (k = 1, 2, 3). The last time point t4 is T2 = 11-Nov-2014. Corresponding data fitting problem is\n\nProblem (DF2): Given data C0(ti) and D0(ti), i ≥ 1, and time interval [t1, t4]:\n\n\n\n1Continuous time version of this model is\n\n\n\n\n\n\nReferences\n\nAlthaus CL: Estimating the reproduction number of ebola virus (EBOV) during the 2014 outbreak in West Africa. PLoS Currents Outbreaks. 2014. Publisher Full Text\n\nBrowne C, Huo X, Magal P, et al.: A model of the 2014 Ebola epidemic in West Africa with contact tracing. arXiv preprint arXiv:1410.3817. 2014. Reference Source\n\nChowell G, Hengartner NW, Castillo-Chavez C, et al.: The basic reproductive number of Ebola and the effects of public health measures: the cases of Congo and Uganda. J Theor Biol. 2004; 229(1): 119–126. PubMed Abstract \| Publisher Full Text\n\nChowell G, Nishiura H: Transmission dynamics and control of Ebola virus disease (EVDI): a review. BMC Med. 2014; 12(1): 196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNishiura H, Chowell G: Early transmission dynamics of Ebola virus disease (EVD), West Africa, March to August 2014. Euro Surveill. 2014; 19(36). pii: 20894. PubMed Abstract\n\nRivers CM, Lofgren ET, Marathe M, et al.: Modeling the impact of interventions on an epidemic of Ebola in Sierra Leone and Liberia. arXiv preprint arXiv:1409.4607. 2014. Reference Source\n\nWHO Ebola Response Team. Ebola virus disease in West Africa--the first 9 months of the epidemic and forward projections. N Engl J Med. 2014; 371(16): 1481–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi M, Liu X: An SIR epidemic model with time delay and general nonlinear incidence rate. 2014; 2014: 1–7. Publisher Full Text\n\nWang J, Wang J, Liu M, et al.: Global stability analysis of an SIR epidemic model with demographics and time delay on networks Physica A: Statistical Mechanics and its Applications. 2014; 410: 268–275. Publisher Full Text\n\nGlobal and Non-Smooth Optimization library (GANSO). Federation university Australia. Reference Source\n\nMammadov M, Rubinov A, Yearwood J: Dynamical systems described by relational elasticities with applications. Continuous Optimisation: Current Trends and Modern Applications, V. Jeyakumar and A. Rubinov (Eds). 2005; 99: 365–385. Publisher Full Text" }	[ { "id": "7898", "date": "12 Mar 2015", "name": "Gerhard-Wilhelm Weber", "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\nThis is an excellent research work for which the researcher(s) related to the study deserve great thanks and a particular recognition!The paper discloses beauty and rigor of modern Mathematics and Operational Research, and it has the potential to strongly contribute to health and entire living conditions of individuals, communities and nations.Moreover, it can very much stimulate and initiate future research works and practical contributions, e.g., via stochastic aspects and regime switches or paradigm changes.", "responses": [] }, { "id": "8331", "date": "01 May 2015", "name": "Yang Kuang", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript introduced some novel ways to connect the current West Africa Ebola outbreak to the standard SI model. However, the author routinely assumed that the infection rate β is a constant which more or less causes the exponential growth profile even when indications suggest otherwise after the month of October, 2014. The key to better modeling any infectious disease and especially in severe ones like Ebola, is to appropriately model the infection rate which is really a function of individual behavior. Arguably, individual behavior is often strongly correlated to the total cases reported. I suggest that the authors take a look at the paper by Chowell et al. below which implicitly incorporated such observations. One can show by a direct differentiation of the logistic model in the paper by Chowell et al. to find out that I'/I is assumed to be a linearly decreasing function of the total case reported.Chowell G, Simonsen L, Viboud C, Kuang Y. 2014. Is West Africa approaching a catastrophicphase or is the 2014 Ebola epidemic slowing down? Different models yield different answers for Liberia. PLOS Currents Outbreaks. 2014 Nov 20. Edition 1. doi: 10.1371/currents.outbreaks. b4690859d91684da963dc40e00f3da81. PLOS Currents Outbreaks.", "responses": [ { "c_id": "1356", "date": "26 May 2015", "name": "Musa Mammadov", "role": "Author Response", "response": "We thank the reviewer for pointing out the importance of the infection rate which is really a key focus of our paper. Our model mainly depends on two parameters: β and τ2 – time to isolation. Initially we also thought that β should be time dependent (in fact piece-wise constant) and obtained good data fitting in this way. However, later we observed that almost the same quality of data fitting can be achieved if we take β constant and vary only τ2. This is one the major findings of our paper and has important implications. For example:Since τ2 is an integer it is possible to generate future scenarios for the prediction of infectious population.As τ2 is time to isolation it can be linked with time to hospitalization and can lead to optimal control problems.Such an optimal control model has already been implemented in our new study (arXiv:1505.00872) where the problem of optimal distribution of bed capacities is investigated.The paper by “Chowell et al” is cited in a new version regarding the behavior of β, alongside “Lekone et al” where β is considered as a control variable with an exponential decrease." } ] } ]	1	https://f1000research.com/articles/3-319
https://f1000research.com/articles/4-126/v1	26 May 15	{ "type": "Opinion Article", "title": "Developmental biology teaching - the importance of a practical approach", "authors": [ "John F Mulley" ], "abstract": "The huge growth in knowledge in many areas of biological sciences over the past few decades has created a major dilemma for those of us in higher education, for not only must we adequately and efficiently convey these new facts and concepts to our students, we must also ensure that they understand and appreciate them.The field of developmental biology has witnessed such a massive growth in knowledge since the mid-1980s, driven mainly by advances in cell and molecular biology, and the development of new imaging techniques and tools. Ensuring that students fully appreciate the four-dimensional nature of embryonic development and morphogenesis is a particular issue, and one that I argue can only be properly learned via direct exposure to embryos via laboratory practicals.", "keywords": [ "Education", "teaching", "developmental biology", "laboratory practicals", "biological sciences", "active learning" ], "content": "Background\n\nDevelopmental biology is a subject with a long and rich history, and also one in which there have been tremendous and rapid advances over the past few decades. Indeed, it has recently been suggested that these advances, especially those in genetics and genomics and cell (particularly stem cell) biology, have been so great as to require a fundamental shift in developmental biology itself (St Johnstone, 2015). The British Society for Developmental Biology (BSDB) has twice in recent years considered renaming itself the “British Society of Developmental and Stem Cell Biology” (Stem Cells in Developmental Biology: a debate at the BSDB) and the flagship journal Development made a conscious effort to “…become an important player in the stem cell field” (Pourquié, 2012). Regardless of these apparent insecurities and image problems, the undoubted growth in knowledge regarding the embryonic development of an ever-growing number of species and the associated explosion of new facts and concepts, together with an extensive historical body of work, raises the issue of how best to convey these facts and concepts to undergraduate students. Whilst this issue is not necessarily unique to developmental biology (Schwartzbauer, 2003; Tunnicliffe & Ueckert, 2007; Yeong, 2012), it has been argued that the absolute requirement for four-dimensional thinking to truly understand the patterns and process of embryonic development is one not necessarily shared with other disciplines in the biological sciences (Hardin, 2008). It has also been suggested (Wood, 2008) that the issue of teaching concepts rather than facts is more complicated in developmental biology, as much of what is taught can be considered to be both a fact and a concept. Thus it is the way that we ask students to use this knowledge that determines whether they regurgitate it as a fact, or fully appreciate it and apply it as a concept. Given these considerations, how are we to ensure that students on developmental biology courses, and, indeed, general biology, zoology or biomedical courses, not only learn, but understand the principles of embryonic development? I would argue that this is only possible through direct exposure to embryos and developmental biology techniques in laboratory-based practicals.\n\nIt is widely acknowledged that students learn best by doing and by having the opportunity to put what they have learned into practise (Kolb, 1984; Moon, 2013). In addition, the ability to design and carry out experiments is a fundamental requirement of training in the sciences (Hofstein & Lunetta, 2004; Kirschner, 1992; Kirschner & Meester, 1988). However, it must be borne in mind that students by their very nature do not usually practice science per se, but rather are learning to practice science (Kirschner, 1992, see also Adams, 2009 for some exceptions). Science teaching should therefore aim to familiarise students with the way that science works (Allen & Tanner, 2003; Allen & Tanner, 2005; Hmelo-Silver, 2004; Kendler & Grove, 2004), whilst remembering that students lack the theoretical knowledge, sophistication and experience of a researcher. In the same way, purely discovery-based approaches can often fail to properly engage learners with the material, and guided discovery better promotes constructivist learning (Mayer, 2004). In short, behavioural activity does not equal cognitive activity and we must be careful to consider why we are getting students to perform particular tasks.\n\nKirschner (1992) identified three motives for implementing practicals:\n\n1. Service to theory – the practical is used to illustrate or affirm theories taught in another setting. In this way the practical is subservient to other forms of instruction and also subservient to theory, where in fact theory and practice are interdependent.\n\n2. Discovery as the only way to achieve meaningful learning – the practical is used to provide discovery learning and process approaches in the absence of prior theoretical context. However, this relies on an assumption that reception learning cannot be meaningful.\n\n3. As a means to distil insight or understanding from empirical work – the practical is used to provide experience to help students to understand a theory. This approach assumes that meaningful learning can take place (i.e. learners can make sense of their observations) in the absence of a robust conceptual framework.\n\nOthers propose that practical sessions are better suited to the development of specific skills (and to counter the shift towards the teaching of generic “key” skills); to learn the academic approach and to allow students to experience phenomena (Abrahams & Millar, 2008; Collis et al., 2008). In addition, co-operative learning and group work allows students to experience multiple roles and aids in the development of collaborative skills and, together with opportunities for group discussion and personal reflection contributes towards the experiential learning cycle (Kolb, 1984; Kolb & Kolb, 2005). Clearly, the practical approach is a powerful one, and one that can have a great impact not only on the learning process in a particular course but more widely on a student’s whole skill set and entire undergraduate experience.\n\n\nPractical developmental biology\n\nTowards the end of my Postgraduate Certificate in Higher Education (PGCertHE) in 2013, I designed a 20 credit practical-based 3rd year module (‘Practical Developmental Biology’) for undergraduate students in the School of Biological Sciences at Bangor University and this ran for the first time in semester 2 of the 2014/15 academic year. The rationale behind the course (reflected in its intended learning outcomes) was to provide students with an understanding of key models and techniques used to study animal development; to enable them to develop practical skills in embryology and to give them the opportunity to combine background knowledge and independent research to interpret experimental results and solve problems. Interestingly, attempts to involve the students in the design of course content via a brief pre-module survey (“What are you hoping to get out of this module?”; “Are there any particular resources that you’d like to have available, either during a module or before it starts?”; “Is there anything that you have particularly liked in other modules that I can steal for this one?”; “Is there anything you have particularly disliked in other modules that I should avoid in this one?”) met with limited success (16% response rate), although such low response rates are common, possibly as a result of survey fatigue (Porter et al., 2004; van Mol, 2014). The module comprised 21 three-hour practical sessions, ranging from fairly basic single sessions involving direct observation of chicken (Gallus gallus), zebrafish (Danio rerio) and axolotl (Ambystoma mexicanum) embryos and setting up crosses of Drosophila melanogaster, to more complex in situ hybridisation and immunohistochemistry experiments to detect gene expression and protein distribution which ran across multiple sessions over several weeks. In addition to developmental biology skills, students were also given the opportunity to practise key molecular biology techniques, such as DNA/RNA extraction, PCR and RT-PCR, agarose gel electrophoresis, as well as to develop and improve existing skills in general numeracy, pipetting and microscopy, among others. The module was assessed via three pieces of written work (2000–2500 word combination practical write-ups and technique reviews, each worth 25%) and a laboratory notebook (also worth 25%).\n\n\nDiscussion\n\nEmbryonic development provides the link between the genes and gene frequencies learned about in genetics modules and the animals bounding around in fields which students (especially those on Zoology courses) so desire to see on field trips. The formation of the nervous and sensory systems during development dictates behaviour, as all behaviour is ultimately dependent on the ability to sense and respond to the environment and it could also be argued that the embryo is the most important level for selection to act on to change morphology. An understanding of developmental biology is therefore important for students of the biological sciences, and vital for those on Zoology degrees. It is now generally accepted that traditional lectures are poorly suited to teaching and learning in the 21st century, with active learning approaches (such as the “flipped classroom” (Jensen et al., 2015; Lage et al., 2000)) becoming increasingly popular. The ability to carry out practical-based laboratory classes set the sciences apart from many other disciplines, and it is therefore important that these are fully exploited in order to provide the widest possible diversity of active learning approaches.\n\nThe unique requirements of developmental biology (discussed above) make this practical approach all the more important. In ‘Practical Developmental Biology’, students were not only given hands-on experience of several key laboratory model organisms (zebrafish, chickens, fruit flies among others), they were also given the opportunity to observe embryonic development for themselves through the regular monitoring of externally-developing zebrafish embryos and the “windowing” of fertilised chicken eggs. Lectures, workshops, textbooks and even videos are no substitute for such experiences, and the obvious fascination of students confronted for the first time by a tiny, beating embryonic heart or developing limb and the eagerness with which they reach for their smartphones to record the event speaks for itself. More basically, the small class size (30) enabled students to work both individually and in pairs and the overlapping nature of the practicals enhanced time-management and self-organisation and this latter was enhanced via the keeping of a combined lab book and learning journal. The requirement to collect, stage and fix embryos of several different species enhanced microscopy and observational skills, and prompted many discussions regarding the issues with assigning distinct stages to what is essentially a continuous process. The use of antibodies to “mystery proteins” and DIG-labelled antisense riboprobes to “mystery genes” (amplified using “mystery primers” by the students themselves, from RNA they extracted and converted to cDNA) ensured that even when following protocols, students were unsure of the outcome, removing the predictable results common to many “interminable, repetitive and boring” undergraduate practical classes (Adams, 2009). Student feedback showed appreciation for “large variety of practical work”; “gaining more skills in the lab, which a general zoology degree seems to lack”; “learning new techniques” and the “…relaxed, informal atmosphere that encouraged individual work and investigation”.\n\n\nConclusions\n\nWhilst the large class sizes on many undergraduate degrees and the associated implications for physical space and resources (equipment, consumables, technical staff) can often be a barrier to practical classes, there are some subjects where the opportunity to experience size, shape, texture, sights and even smells across days or even weeks cannot be replaced. Developmental biology is one such subject and all the innovative alternatives in the world (including audio-visual resources, flipped classrooms and other active learning approaches) cannot substitute for the sheer wonder of observing embryonic development first hand.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author wishes to thank the various PGCertHE practitioners from Bangor and elsewhere with whom aspects of this work were discussed.\n\n\nReferences\n\nAbrahams I, Millar R: Does practical work really work? A study of the effectiveness of practical work as a teaching and learning method in school science. Int J Sci Educ. 2008; 30(14): 1945–1969. Publisher Full Text\n\nAdams DJ: Current Trends in Laboratory Class Teaching in University Bioscience Programmes. Biosci Educ. 2009; 13: 3. Publisher Full Text\n\nAllen D, Tanner K: Approaches to cell biology teaching: learning content in context--problem-based learning. Cell Biol Educ. 2003; 2(2): 73–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen D, Tanner K: Infusing active learning into the large-enrollment biology class: seven strategies, from the simple to complex. Cell Biol Educ. 2005; 4(4): 262–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollis M, Gibson A, Hughes I, et al.: The Student View of 1st year Laboratory Work in Biosciences - score gamma. Biosci Educ. 2008; 11: 2. Publisher Full Text\n\nHardin J: The missing dimension in developmental biology education. CBE Life Sci Educ. 2008; 7(1): 13–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHmelo-Silver CE: Problem-based learning: What and how do students learn? Educ Psychol Rev. 2004; 16(3): 235–266. Publisher Full Text\n\nHofstein A, Lunetta VN: The laboratory in science education: Foundations for the twenty‐first century. Sci Educ. 2004; 88(1): 28–54. Publisher Full Text\n\nJensen JL, Kummer TA, d M Godoy PD: Improvements from a flipped classroom may simply be the fruits of active learning. CBE Life Sci Educ. 2015; 14(1): ar5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKendler BS, Grove PA: Problem-based learning in the biology curriculum. Am Biol Teach. 2004; 66: 348–354. Reference Source\n\nKirschner PA: Epistemology, practical work and academic skills in science education. Science & Education. 1992; 1(3): 273–299. Publisher Full Text\n\nKirschner PA, Meester MAM: The laboratory in higher science education: Problems, premises and objectives. Higher Education. 1988; 17(1): 81–98. Publisher Full Text\n\nKolb DA: Experiential learning: Experience as the source of learning and development. Prentice-Hall Englewood Cliffs NJ. 1984. Reference Source\n\nKolb AY, Kolb DA: Learning Styles and Learning Spaces: Enhancing Experiential Learning in Higher Education. Acad of Manag Learn Edu. 2005; 4(2): 193–212. Publisher Full Text\n\nLage MJ, Platt GJ, Treglia M: Inverting the Classroom: A Gateway to Creating an Inclusive Learning Environment. J Econ Educ. 2000; 31(1): 30–43. Publisher Full Text\n\nMayer RE: Should there be a three-strikes rule against pure discovery learning? The case for guided methods of instruction. Am Psychol. 2004; 59(1): 14–19. PubMed Abstract \| Publisher Full Text\n\nMoon JA: A handbook of reflective and experiential learning: Theory and practice. Routledge. 2013. Reference Source\n\nPorter SR, Whitcomb ME, Weitzer WH: Multiple surveys of students and survey fatigue. New Directions for Institutional Research Special Issue: Overcoming Survey Research Problems. 2004; 2004(121): 63–73. Publisher Full Text\n\nPourquié O: A niche for stem cell research. Development. 2012; 139: 1–2. Publisher Full Text\n\nSchwartzbauer JE: Book Review: Well Worth the Weight! Cell Biol Educ. 2003; 2(1): 16–17. Publisher Full Text \| Free Full Text\n\nSt Johnston D: The renaissance of developmental biology. PLoS Biol. 2015; 13(5): e1002149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTunnicliffe SD, Ueckert C: Teaching biology - the great dilemma. J Biol Educ. 2007; 41(2): 51–52. Publisher Full Text\n\nvan Mol C: Coping with survey fatigue: The impact of late reminders on web survey response. NIDI Working Paper No. 2014/15. The Hague: NIDI. 2014. Reference Source\n\nWood WB: Innovations in teaching undergraduate biology and why we need them. Annu Rev Cell Dev Biol. 2009; 25: 93–112. PubMed Abstract \| Publisher Full Text\n\nYeong FM: Moving Away from Dogmatic Knowledge Dissemination in a Cell Biology Module: Examples from Singapore. Biosci Educ. 2012; 20: 106–115. Publisher Full Text" }	[ { "id": "8757", "date": "29 May 2015", "name": "Graham Scott", "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\nThis short opinion piece extols the value of a practical approach to learning and presents an interesting question: how can we ensure that students both learn facts and concepts/principles. The question is posed in the context of developmental biology but it applies equally well to all disciplinary areas. The author develops/explains this central question well (if briefly). He then presents information about his own practice and in particular describes (very briefly) a module that he has developed and delivered (once). He presents a discussion in which elements of the module are described, and finally he concludes that his discipline is one in which practical experience is essential.Each of these individual parts is interesting but they are not fully developed and I do not feel that they add up to a discussion of the value of a practical approach to developmental biology, nor do they currently address the question that the author poses.An opinion piece is of course an encapsulation of the opinions of the author and in this case I agree with the opinion expressed. However for a reader to be able to evaluate these opinions and assimilate them with their own experiences it is essential that they are supported by evidence. Unfortunately I don’t think the author of this paper has achieved this. The link between the introductory material and the description of practice is not a fully developed one. In fact it would be possible to remove the description of practice here without really changing the message that is conveyed (ideally of course the case study should enhance the message). I would recommend that the author further develops the explanation and evaluation of the design of the module in the context of both its purpose and outcomes.Furthermore the evaluation presented here is very limited. As a reader I would like to better understand the author’s aims in designing the module? the level to which these aims are achieved? how their achievement is measured/assessed? I also feel that a stronger inclusion of the student voice (through an enhanced evaluation of student perception and engagement) would strengthen the argument that this model is right in this context.Finally it is essential that the conclusion be re-written to make better use of the evidence that the author draws directly upon in formulating his opinion.", "responses": [ { "c_id": "1402", "date": "01 Jun 2015", "name": "John Mulley", "role": "Author Response", "response": "Thanks for the helpful review - I'll get to work on incorporating these comments and making the required changes." } ] }, { "id": "8755", "date": "01 Jun 2015", "name": "Anthony Graham", "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\nThis is an interesting article that makes a solid case for teaching developmental biology via practical classes employing a range of different species and using a number of different technical approaches. It is clear that the students would gain a lot of valuable insights and depth of understanding of developmental biology by this approach. Actually seeing embryos is something that is likely to grab the imagination of most students. I would ask for a bit more information on some aspects. Could we have a clearer idea of the assessments used? What exactly were the three pieces of course work? I would also like more information on how well the course worked. Were there some areas that were more successful than others? Were some aspects of the course more difficult for the students to master than others? What aspects of the course were most engaging for the students?", "responses": [] }, { "id": "8756", "date": "08 Jun 2015", "name": "Thomas Butts", "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\nThis is a thought provoking opinion piece on the advantages, indeed the need, for practical teaching for developmental biology. In general, it is a well written piece that makes a strong case, and one that I whole-heartedly endorse. In the background section, I am a little wary of the approach to thinking about practicals whereby such activities have specific independent purposes that can be expressed in a meaningful way. Are Kirschner's three motives mutually exclusive? I would suggest not, and would avoid discussing them as such. The article describes a practical developmental biology course designed and run by the author that sounds very interesting indeed. If anything, a more thorough description of the course (including its assessment components) and its philosophical underpinnings would be useful and interesting. Also useful would be speculation on how such a course could be 'scaled up', if indeed this is envisaged. Overall, this is a interesting piece that outlines an interesting course that would benefit from more thorough elucidation.", "responses": [] } ]	1	https://f1000research.com/articles/4-126
https://f1000research.com/articles/4-125/v1	22 May 15	{ "type": "Antibody Validation Article", "title": "Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody", "authors": [ "Peter van Endert", "Myriam Lawand", "Myriam Lawand" ], "abstract": "In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.", "keywords": [ "ABCB9", "TAP-L transporter", "dendritic cell", "antigen presentation", "MHC", "peptide", "lysosome" ], "content": "Introduction\n\nTAP-L (TAP-Like), also known as ABCB9, is an ATP-dependent membrane half-transporter. It belongs, like TAP, the transporter associated with antigen processing, to the ABC transporter family, the members of which transport various molecules across membranes. TAP-L can form homodimers and is located primarily in lysosomes, presumably importing peptides from the cytosol. TAP-L has broad specificity for peptides ranging from a length of 6 to 59 amino acids, with an optimal activity for peptides of 23 residues (Wolters et al., 2005). TAP-L can transport two peptides at a time (Herget et al., 2009). Considering its similarity to the heterodimeric TAP transporters (ABCB2/3) importing MHC class I peptide ligands into the endoplasmic reticulum, TAP-L is a potential candidate involved in antigen presentation by MHC molecules (Bangert et al., 2011). Indeed, the length of the peptides transported by TAP-L (6-59 residues) is compatible with loading of both MHC class I and class II molecules. Moreover, TAP-L is highly expressed in lysosomes of professional antigen presenting cell (APC) lysosomes, and upregulated during differentiation of dendritic cells. However, such a function remains hypothetical, and the biological role of TAP-L is presently unknown.\n\nIn this article, we describe experimentation designed to specifically detect the ABCB9 protein in bone marrow-derived dendritic cells (BMDCs) by immunoblot. We purchased a rabbit polyclonal antibody generated by Abcam Company using a synthetic peptide as the immunogen, corresponding to a region between residues 475 and 525 of human ABCB9. This antibody is expected to recognize mouse and human ABCB9 and recommended for immunohistochemistry (IHC), immunoprecipitation (IP) and western blot (WB).\n\n\nMaterials and methods\n\nC57/BL6 TAP-L KO/WT heterozygous mice (ABCB9tm1 (KOMP) Vlcg) were purchased from The Komp Repository at the University of California at Davis, CA 95616 (see the results section for details). Heterozygous mice were bred in our laboratory and inter-crossed to obtain homozygous knock out (KO) mice (TAP-L KO/KO) along with their C57/BL6 wild type (WT) littermates.\n\nBone Marrow-derived Dendritic Cells (BMDCs) were generated from precursors isolated from femur and tibia of C57/BL6 WT and TAP-L KO mice and cultured for 6 days in IMDM (Iscove's Modified Dulbecco's Medium) (Sigma Aldrich, St. Quentin Fallavier, France) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine (PAA, Velizy-Villacoublay, France), 100 U/ml penicillin, 100 μg/ml streptomycin (PAA), and 50 μM 2-mercaptoethanol (GIBCO, Cergy Pontoise, France) in the presence of 3% supernatant of J558 hybridoma cells producing GM-CSF (Granulocyte-macrophage colony-stimulating factor) (Inaba et al., 1992).\n\nOn day 6 of culture, WT and TAP-L KO BMDCs (Table 1) were lysed in a buffer containing 20mM Tris-HCl pH 7.4, 150mM NaCl, 5mM MgCl2, 1% NP40 and protease inhibitors (protease inhibitor cocktail, Roche) for 1 h at 4°C. Protein concentration was determined by Lowry’s method, a biochemical assay for determining the total level of protein in a solution, using DC Protein Assay Reagents Package™ (BioRad).\n\nTwenty to 200μg protein from total cell lysate was mixed at a volume ratio of 1:1 with 2x Laemmli buffer containing 62.5mm Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 100mM DTT and heated for 10 min at 95°C.\n\nReagents are listed in Table 2 and Table 3 and the WB protocol is given in Table 4. The samples were loaded on a 10% acrylamide gel for electrophoresis at 80V. Separated proteins were transferred onto polyvinylidine fluoride (PVDF) membrane (pore size 0.4μm) for 1 h at 75V. The membrane was blocked with 5% BSA (Bovine Serum Albumin) in Tris-Buffered Saline (50mM Tris, 150mM NaCl) containing 0.5% Tween 20 (TBS-T) for 1 h at room temperature, then incubated with the polyclonal rabbit ABCB9 antibody (Abcam, Catalog number 86222, Lot number: GR22408–1) diluted 1/2000 in TBS-T with 5% BSA for 1 h at room temperature. The membrane was washed four times for 5 min with TBS-T then incubated with a goat polyclonal anti-Rabbit-HRP (Jackson ImmunoResearch Laboratory; Suffolk, UK) secondary antibody diluted 1/5000 in TBS-Tween 5% BSA for 1 h at room temperature. An enhanced chemiluminescence (ECL) detection system, Immobilon Western HRP (Millipore, Guyancourt, France) was used for developing the membranes. Images were taken with a CCD camera (Fujifilm, Tokyo, Japan). Three independent experiments were performed.\n\n\nResults\n\nSeeking to detect the ABCB9 protein, we performed a series of WBs on whole-cell lysates obtained from BMDCs, thought to correspond to an inflammatory subtype of DCs. It has previously been shown that ABCB9 expression by monocyte-derived human DCs is increased under inflammatory conditions (Demirel et al., 2007). To validate specificity of antibody staining, we included TAP-L deficient BMDCs as a negative control. TAP-L KO/WT heterozygous mice (ABCB9tm1 (KOMP) Vlcg), in which the region located between nucleotides 5625 and 33216 of the TAP-L gene has been removed for the insertion of a cassette of 6085bp containing the cDNA conferring resistance to neomycin (Neo), were purchased from The Komp Repository at the University of California at Davis, CA 95616 (see construction of the KO gene; Figure 2A). Heterozygous mice were bred in our laboratory and inter-crossed to obtain homozygous KO mice (TAP-L KO/KO). To our surprise, the ABCB9 antibody recognized a band, with an apparent molecular weight (84kDa) corresponding to that of ABCB9 protein, both in WT and TAP-L deficient BMDCs (Figure 1). Three different immunoblots were performed in three independent experiments.\n\n20–200μg of total BMDC cell lysate from WT and TAP-L KO BMDCs was loaded on 10% acrylamide gels. The proteins were transferred onto a PVDF membrane. The rabbit ABCB9 antibody was used to detect the TAP-L protein (84kDa), followed by incubation with an HRP-conjugated goat anti-rabbit secondary antibody. An ECL detection system was used for developing the membranes by chemoluminescence. Three immunoblots from three independent experiments are shown.\n\nStrategy for genomic invalidation of the TAP-L/ABCB9 gene (A) and genotyping of WT and TAP-L deficient mice (B). In B, multiple KO mice were tested in the PCRs amplifying the Neo cassette and introns 5, 6 and 9. L, DNA ladder (See the results section for details).\n\nGiven these surprising results, we verified that the TAP-L KO mice were truly deficient for the target gene. We performed a series of polymerase chain reactions (PCRs). Different fragments of the WT allele (located in exons 2, 4, 8, 11 and introns 5, 6, 9) and the expected genomic region in KO mice (located between the upstream or downstream arm and within the Neo cassette) were amplified by PCR.\n\nThe following forward (F) and reverse (R) primers were used:\n\n- Ex1-F: 5'-GTAGTAGTGACGCTGGCCTT-3' and Ex1-R: 5'CTTCTGTAGTGTGGCTCCCG-3', located in exon 1 of the WT allele and amplifying a product of 498bp in the WT allele\n\n- Ex2-F: 5'-AGACCTTCCTGCCCTACTACA-3' and Ex2-R: 5'-CAGCAGGCAAACGACGACAA-3', located in exon 2 of the WT allele and amplifying a product of 101bp in the WT allele\n\n- Ex4-F: 5'-CGCCTCACCTCTGATACCAC-3' and Ex4-R: 5'-TGCCGTAGATGTTGGACACC-3', located in exon 4 of the WT allele and amplifying a product of 181bp in the WT allele\n\n- Ex8-F: 5'-CAAGGTGACAGCTCTGGTGG-3' and Ex8-R: 5'-GCCATCCAACAATACACGGC-3', located in exon 8 of the WT allele and amplifying a product of 106bp in the WT allele\n\n- Ex11-F: 5'-GAGACACACGGTGCTCATCA-3' and Ex11-R: 5'-TGTGTTCAGTGTTGCTGGGT-3', located in exon 11 of the WT allele and amplifying a product of 214bp in the WT allele\n\n- INT5-F: 5'-TACTCGGGTGCCACTACCTG-3' and INT5-R: 5'-GGCACATGCCACCTTCAAGT-3', located in intron 5 of the WT allele and amplifying a product of 379bp in the WT allele\n\n- INT6-F: 5'-TGCTTAAAGGCACTCGGTGA-3' and INT6-R: 5'-CTTCGGGGATACCACAGAGC-3', located in intron 6 of the WT allele and can amplifying a product of 371bp in the WT allele\n\n- INT9-F: 5'-TGCCAAGTTTAGTGCCAGGATG-3' and INT9-R: 5'-GCCCAGGACAAAAAAAGCAATC-3', located in the intron 9 of the WT allele and amplifying a product of 371bp in the WT allele\n\n- KOFwd1: 5'-TTGCATGGAGAAGACCCTCC-3', located in the arm upstream of the Neo cassette (Neo upstream arm), and KORvs1: 5'-GAGGGGACGACGACAGTATC-3', located in the Neo cassette and amplifying a product of 465bp in the KO allele\n\n- KOFwd2: 5'-GCAGCCTCTGTTCCACATACACTTCA-3', located in the Neo cassette and KORvs2: 5'-GCTTAGTTCTCTCCCAGACATCCTCC-3', located in the arm downstream of the Neo cassette (Neo downstream arm) and amplifying 425bp in the KO allele.\n\nPCRs were performed in a total volume of 25μl containing: 17.3μl H2O (DEPC treated water, pathogen free, DNase/RNase Free-Invitrogen), 5 μl 5x GoTaq Green Reaction Buffer (Promega), 0.5μl dNTP (10mM), 20 μM primers, 0.2μl polymerase (5 U/μl) (GoTaq-Promega polymerase) and 1μl DNA or water (negative control). The amplification reaction was performed as follows: for the WT allele, an initial denaturation at 94°C for 5 min, 10 cycles: denaturation 94°C for 15 sec, annealing 65°C for 30 sec, elongation 72°C for 40 sec; 30 cycles denaturation 94°C for 15 sec, annealing 55°C for 30 sec, elongation 72°C for 40 sec; final elongation 72°C for 5 min. For the KO allele: initial denaturation at 94°C for 5 min; 10 cycles: denaturation 94°C for 15 sec, annealing 62°C for 30 sec, elongation 72°C for 40 sec; 25 cycles: denaturation 94°C for 15 sec, annealing 57°C for 30 sec, elongation 72°C for 40 sec; final elongation 72°C for 5 min. The PCR products obtained were migrated on a 1.5% agarose gel containing 10 μg/ml of Ethidium Bromide. Migration was performed in a buffer tank filled with TAE buffer containing 40mM Tris, 20mM acetic acid, 1mM EDTA, pH=8 for 20 min at 120 V and visualization of the PCR products under a UV lamp connected to a photographic device.\n\nThe resulting PCR products from multiple KO mice confirmed the absence of the TAP-L gene and the presence of the Neo cassette (Figure 2B), indicating that the TAP-L gene was deleted as expected and that the mice obtained were TAP-L KO/KO. Consequently, the band recognized by the ABCB9 antibody, even though running at the expected molecular weight, could not correspond to the TAP-L protein.\n\n\nConclusion\n\nCollectively, these results show that the commercial ABCB9 antibody recognizes a protein with a molecular weight similar to that of TAP-L. It is impossible to know whether it also recognizes TAP-L. Our findings highlight the importance of verifying commercial antibody specificity using knockout cells. If such cells are not available, lentiviruses encoding target-specific shRNA, which are now readily available for an essentially complete range of proteins, can be used to produce cells that provide informative negative controls.", "appendix": "Author contributions\n\n\n\nML designed, performed and interpreted experiments and wrote the manuscript. PvE designed and interpreted experiments and edited the manuscript.\n\n\nCompeting interests\n\n\n\nBoth authors confirm that they have no conflict of interest.\n\n\nGrant information\n\nSupported by a grant from the Fondation pour la Recherche Médicale (Equipe FRM DEQ20130326539) to PvE.\n\n\nReferences\n\nBangert I, Tumulka F, Abele R: The lysosomal polypeptide transporter TAPL: more than a housekeeping factor? Biol Chem. 2011; 392(12): 61–66. PubMed Abstract \| Publisher Full Text\n\nDemirel O, Waibler Z, Kalinke U, et al.: Identification of a lysosomal peptide transport system induced during dendritic cell development. J Biol Chem. 2007; 282(52): 37836–37843. PubMed Abstract \| Publisher Full Text\n\nHerget M, Kreissig N, Kolbe C, et al.: Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis. J Biol Chem. 2009; 284(49): 33740–33749. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInaba K, Inaba M, Romani N, et al.: Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992; 176(6): 1693–702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolters JC, Abele R, Tampé R: Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9). J Biol Chem. 2005; 280(25): 23631–23636. PubMed Abstract \| Publisher Full Text" }	[ { "id": "9028", "date": "15 Jun 2015", "name": "James Drake", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors report the results of studies in which they attempt to validate the specificity of a commercially available antibody to TAP-Like (TAP-L), which is an endosomal peptide transporter from the same family at the prototypical ER TAP proteins. They report that, unexpectedly, the anti-TAP-L antibody exhibits immunoreactivity on samples prepared from TAP-L knock mice. Suggestions for improvement:It would be helpful to know the number of cell equivalents loaded on each lane of the gels. The gels were loaded for equal protein, so the number of cell equivalents is likely similar, but this point should be addressed.Figure 1 – Since the purpose of the experiment is to test the specificity of the primary western blot antibody (i.e., anti-TAP-L), it would be appropriate to include a control blot that was probed with secondary antibody only. It is possible that the unexpected reactivity on the western blot is due to the secondary antibody (and that the primary antibody is generating no signal). A blot probed with secondary antibody only would address this possibility.The company makes specific note of the peptide used to generate the reagent under analysis. Therefore, it would be interesting of the authors compared this sequence to the protein sequence in the available databases to see if they could identify candidates for the non-TAP-L protein being recognized by the antibody under analysis.", "responses": [] }, { "id": "8943", "date": "15 Jun 2015", "name": "Frank Momburg", "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 in an interesting antibody validation article showing that a rabbit polyclonal antibody raised against a peptide (475-525) within the human TAP-L transporter (84 kDa) apparently cross-reacts with another unknown protein of similar size within TAP-L deficient murine dendritic cells. Since mouse TAP-L has a decent homology to TAP2 (77.5 kDa) and TAP1 (78.9 kDa) the authors should precipitate mTAP1/2 and perform a blot with this polyclonal antibody to prove or disprove that the reported cross-reactivity is to TAP. Please follow this link to view the homology of mTAPL(a.a. 475-525) with mTAP2, and with mTAP1:https://f1000researchdata.s3.amazonaws.com/supplementary/6535/041aa8a5-5260-4b06-929e-b91f6b3c51e9.tif", "responses": [] }, { "id": "9026", "date": "29 Jun 2015", "name": "Malini Raghavan", "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\nThis study relates to antibody specificity for the characterization of ABCB9 (transporter associated with antigen processing-like (TAP-L)) expression. The methods and results are explained in detail and the abstract and title are appropriate for the study. TAP-L knockout/wild type heterozygous mice from a commercial source were crossed to obtain homozygous TAP-L knockout mice. TAP-L deficiency in the knockout is confirmed by PCR. Cell lysates from bone marrow-derived dendritic cells of wild type or TAP-L-deficient mice were subject to immunoblotting analyses with a rabbit polyclonal anti-TAP-L antibody from Abcam (http://www.abcam.com/ABCB9-antibody-ab86222.html). An expected band at 84 kDa is seen. However, similar sized bands are seen in lanes containing lysates from either the wild type or the knockout mice, suggesting that the tested antibody is not specific for TAP-L, at least based on immunoblotting analyses. The commercial vendor should take note of this study. The commercial link also indicates immunoprecipitations and immunohistochemistry as tested applications for the antibody. These applications could also be tested using the knockout cells as negative controls. While the study correctly emphasizes the importance of relevant controls prior to the use of commercial antibodies, it appears that a number of TAP-L-specific antibodies are available from different commercial sources. It would be useful to the reader to know which of the commercial antibodies are in fact specific for TAP-L.", "responses": [] } ]	1	https://f1000research.com/articles/4-125
https://f1000research.com/articles/4-124/v1	22 May 15	{ "type": "Case Report", "title": "Case Report: Pre- and postnatal management of an allantoic cyst with patent urachus and single umbilical artery", "authors": [ "Than Trong Thach", "Vo Duy Quan", "Tran Diem Nghi", "Nguyen Hoang Anh", "Le Phi Hung", "Nguyen Thien Luan", "Nguyen Phuoc Long", "Than Trong Thach", "Vo Duy Quan", "Tran Diem Nghi", "Nguyen Hoang Anh", "Le Phi Hung", "Nguyen Thien Luan" ], "abstract": "Patent urachus is a rare congenital abnormality. Since its first description by Cabriolus in 1550, few cases have been reported. A 26-year-old Vietnamese primigravida presented at 20 weeks of gestation for evaluation of a cystic mass in the umbilical cord, which was first discovered at week 13 of pregnancy by ultrasound scan. The cystic mass originated from the root of the umbilical cord, connected to the urinary bladder, and no intestinal contents were enclosed within. Doppler ultrasound assessment showed that the single umbilical artery existed within the normal range. The progression of the umbilical cyst continued to be screened, but the mass disappeared on ultrasound images at 27 weeks of gestation. This led to the consideration of the cyst’s rupture. After 38 gestational weeks, the pregnant woman delivered a 3350g male infant via cesarean section because of an obstructed vaginal labor. The following days, a stream of urine was recorded leaking out from the umbilical mass whenever he cried. Seven weeks after delivery, an open surgical approach was successfully performed. The baby is now 43 months of age, growing and developing normally. Since an allantoic cyst with patent urachus is a rare clinical entity, early discovery, close monitoring and accurate diagnosis through ultrasound in the prenatal period may consequently allow clinicians to have suitable attitudes towards management when the infant is born.", "keywords": [ "allantoic cyst", "patent urachus", "single umbilical artery" ], "content": "Introduction\n\nUrachus is a fibrous remnant of the allantois which communicates from the apex of the urinary bladder to the umbilicus. Failed obliteration of the urachus can lead to various abnormalities: urachal cyst, urachal diverticulum, sinus or patent urachus - the most common type1.\n\nPatent urachus, which was first described by Cabriolus in 1550, is an extremely rare clinical presentation, occurring in 1 to 2 or 2.5 per 100,000 deliveries2,3. Allantoic cysts in infants with patent urachus can be formed due to the drainage of urine into the umbilical cord4–6, or in uncommon situations, after leakage of hypo-osmotic urine into the Wharton’s jelly7,8.\n\nHere we present a clinical case with a diagnosis of patent urachus. The newborn possessed a single artery in the umbilical cord and one allantoic cyst, and underwent a successful surgical resection.\n\n\nCase report\n\nA 26-year-old Vietnamese primigravida, with no relevant medical, psychosocial or family history of morbidity, toxic exposure or abnormalities, after 1 year of marriage was informed of pregnancy at 7–8 weeks of gestation. The expected time for labor was October 1st 2011. First ultrasound screening at 12–13 weeks of gestation showed a 1.9mm nuchal translucency thickness. Simultaneously, a double test was also undertaken and demonstrated a trisomy 21 risk of 1/61000 and a trisomy 18 risk of 1/100000. Based on the fetal ultrasound exam, clinicians discovered a small cystic mass on the anterior abdominal wall whose nature remained unknown. No specific interventions were made and clinicians intended to keep observing the cyst. The next ultrasound exam at 16 weeks of gestation demonstrated a well-developing fetus with an enlarging cystic mass. However, its nature still remained unidentified due to lack of experience on image observation. Discrimination between allantoic cyst and pseudo-cyst presented a significant challenge for most of the ultrasonographists as well as clinicians at that time. Therefore, they decided to scrutinize it for one more month before deciding on a therapeutic intervention, especially since the triple test also showed low risk of trisomy 21, 18 and 13.\n\nThe morphology ultrasound at 20 weeks of gestation on the fetus showed the presence of a single umbilical artery (Figure 1) and a cystic mass (dimensions = 29mm × 25mm) at the root of the umbilical cord, connecting to the urinary bladder and no bowel contents enclosed within (Figure 2A and Figure 2B). Doppler velocimetry revealed flows around this cyst (Figure 2C). Other structures remained normal. Amniocentesis was indicated later and the result revealed a normal XY karyotype. A pediatric surgeon was invited to consult about this rare clinical entity and the consensus of postnatal operation for the neonate was finally made.\n\nA: Umbilical cord cyst first detected at 20 0/7 weeks of gestation (arrow). B: Umbilical cord cyst was confirmed and a clear image of the connection between the bladder and the umbilicus was found at 22 5/7 weeks of gestation. C: Doppler velocimetry demonstrated flow around the cyst. D: Doppler ultrasound examined the size of the cyst.\n\nAt 23 weeks of gestation, through the second morphology ultrasound, the cyst showed an increase in dimensions to 35mm × 28mm (Figure 2D).\n\nAt 24 weeks of gestation, due to her weight gain reaching 13kg, an oral glucose tolerance test (OGTT) with 75g glucose was performed with a positive result for gestational diabetes. As a result, the patient was instructed to undertake a carbohydrate-restricted diet including 6 small meals plus 2 glasses of unsweetened milk every day. After 2 weeks, her blood glucose level returned to normal.\n\nAt 27 weeks of gestation, the cyst disappeared on ultrasound images, and a 180-minute echographic recording found no urine in the bladder. From the above findings, clinicians suspected that the cyst was ruptured, forming a fistula by which the urine refluxed into amniotic sac. No interventions were considered at this point. The next serial ultrasonography scan showed a little urine in the bladder but no umbilical cyst. Although there was only one single umbilical artery, the fetus still grew well, which was reflected by the growth chart of Hadlock with an estimated fetal weight (EFW) ranging from 70th–90th percentile. A Doppler ultrasound evaluation using the Japan Society of Ultrasonics in Medicine (JSUM) 2001 proposal (More information can be found in the Voluson® E-Series (BT09) Advanced Reference Manual) showed that Resistive Index (RI) and Pulsatility Index (PI) of the umbilical artery and middle cerebral arteries was within the 20th and 50th percentile9.\n\nThe patient had obstructed vaginal labor at 38 and 4/7 weeks of gestation with high suspicion of her limited pelvis. As a result, she underwent a cesarean section under general anesthesia and was administered Suxamethonium, Propofol, Esmeron and Sufentanil, after the failure of epidural block. Forty-five minutes later, the mother gave birth to a healthy male infant weighing 3350g. Post-delivery evaluation demonstrated a herniated sac-like mass (dimensions = 3mm × 4mm) at the root of umbilical cord and confirmed that the umbilical cord contained only one artery. The placenta was in normal condition. The umbilical cord was clamped about 5cm from the root.\n\nIn the following days, we recorded a stream of urine flushing out from the umbilical mass every time the infant cried. The umbilical tape was changed three times a day using normal saline and sterilized gauze. Five days after delivery, the umbilical stump dried and fell off, but the mass still persisted. The umbilical mass was still kept clean and covered by new tapes as described above until the operation at the infant’s seventh week. The open surgical approach was used under general anesthesia. Surgeons made a 2cm incision in the midline below the umbilicus, dissected rectus abdominis muscle and exposed the allantoic duct. They removed the duct, tied surgical knots using absorbable sutures, checked the two ureters and the bladder to ensure there were no further abnormalities and finally, the umbilicus was reconstructed. The patient was discharged 2 days after the operation without any complication.\n\nAt present, the patient is a 43-month-old boy with weight of 14kg, height of 100cm, and normal psychomotor development. He was re-checked several times at 3, 6 and 12 months of age, as well as every year after that with ultrasound examination and no abnormal structure was shown.\n\n\nDiscussion\n\nUrachal anomalies are the general name for several abnormal conditions (urachal cyst, sinus, patent urachus, or diverticulum) resulting from the failure of closing the allantoic duct during the 14th gestational week3. They are divided into two groups: congenital and acquired. Patent urachus, which accounts for 10–15% of all urachal remnant diseases, is usually congenital10. Although newborns with congenital patent urachus have been recognized since the sixteenth century, prenatal diagnosis of this condition has only been carried out since 19882 and our case is one of the first observed in Vietnam. As with other previous patent urachus cases, in our situation the mother underwent a morphology ultrasound examination at 20 weeks of gestation which revealed an extra-abdominal 29mm × 25mm cystoid structure near the umbilical cord root connecting to the fetal bladder. Omphalocele was ruled out because of the absence of fetal bowel contents within the channel. However, remarkably, only one umbilical artery was detected. Next ultrasonography performed at 27 weeks of gestation noticed the disappearance of the cyst. No urine was found in the fetal bladder after 180-minute recording. We suspected a rupture which created a fistula and drained the urine into the amniotic fluid. As the cyst ruptured in its early period, no umbilical cord compression was identified. The fetus developed normally regardless of the cyst and the presence of a two-vessel cord. At about 38 weeks of gestation, the pregnant woman was admitted with uterine contractions, and vaginal delivery was performed. Obstructed labor, however, subsequently took place, which prompted obstetricians to make an emergency cesarean section instead. To the best of our knowledge, only C. Rasteiro et al. has ever mentioned a case of allantoic cyst with patent urachus before11. Unlike ours, in their description, although the cyst grew to 100mm in size, it did not burst and the amniotic fluid volume remained unchanged. Similarly, the bladder was empty but this was because the fetus micturated and urine leaked into the cyst. Although there have been several patent urachus cases reported, our patent urachus case with a ruptured cyst in the setting of single-umbilical-artery fetus is unique.\n\nA cystoid mass perceived through ultrasound may be involved in different conditions, which makes the prenatal diagnosis questionable7. Particularly, differentiation between a pseudo- and a true cyst can regularly confound ultrasound diagnosis, despite the fact that very rare cases have been distinguished based on ultrasound imaging. Nevertheless, there are at least three features which can give us important suggestions. A true cyst is always in close contact with the fetal anterior abdominal wall, surrounded by umbilical vessels which can be verified by color flow imaging, and shows an open channel from the cyst to the bladder and the fetal umbilicus12. The gold standard for differential diagnosis between a pseudo- and a true cyst is histopathology. Under the microscope, a true cyst comprises an epithelial lining and originates from remnants of the allantoic duct while the pseudo-cyst is the result of the degeneration or the edema of Wharton’s jelly12. A pseudo-cyst not only makes up a higher incidence but is also more associated with aneuploidy and other chromosomal disorders than a true cyst13. Hence, obstetricians are usually recommended to perform a karyotype once they discover a cystic lesion. In our current case, we assumed that it was indeed a true cyst. A karyotype was executed but no peculiarities were found. This poses another complicated problem - the best and earliest time for authenticating a true cyst. In medical literature, Waldo Sepulveda et al. reported three cases of patent urachus detection in the first trimester at 11 to 14 weeks’ gestation, based on the findings of megacystis and the large umbilical cord cyst6. In most of the cases, patent urachus was recognized during the second and third trimester. However, clinicians still remained unconfirmed about this condition until the urine dribbles from the umbilicus right after the delivery, and our case was no exception6.\n\nThe recognition of patent urachus is not easy due to a very low frequency of 1- 2.5:100000 live births2,3 and non-specific signs and symptoms. Therefore, specialists should be accustomed to it and be prepared for the treatment. Different methods can be used to manage this condition. Complete surgical removal of the urachus is one of the general recommendations with favorable outcome14. Surgical techniques can avoid malignant conversion and other complications occurring together with increasing age whereas conservative treatment (i.e. drainage and antibiotic therapy) sometimes leads to recurrent infection or cyst formation. Apropos of surgical treatment, experts and researchers up to now have still debated on the optimal method and have not come to any conclusion. Prior to the endoscopic era, open excision was the main method for removing the urachus. A large ten-year retrospective study focusing on open surgery conducted by Mesrobian et al. in 1997 did not show any complications or reoperations afterward15. Open surgery also required shorter hospital stays16. However, since the appearance and universal application of laparoscopy in 199517, several studies have suggested that this method could be a safe and effective alternative with minimal morbidity18. Laparoscopic approach not only provides an all-around visualization of the urachal channel and fetal bladder but also improves patient’s general state and reduces post-operative pain more rapidly, as do all endoscopic procedures16. In our case presented here, surgeons and urologists who participated in that operation opted for open excision. The best time to execute a patent urachus surgical removal also remains controversial19.\n\nAn open surgical approach was carried out when the neonate was seven weeks old. The operation ended with success and the male baby was discharged after two days. He was observed closely over the following days and no further complications were found. The baby grew up normally. At present, he is a 43-month-old boy with physical and psychomotor development corresponding to his age. Ultrasound examination has again verified his normal anatomical structure. Through this case, we are likely to make a conclusion that patients with isolated patent urachus who already underwent a surgical removal may have a far more optimistic prognosis. Medical literature describes several early-treated patent urachus neonates who then developed well with no anomalies14,20, which might support our hypothesis. However, no optimal follow-up time interval was accurately proposed. In the past, Sepulveda et al. reported a case with a three-year follow-up, wherein no abnormalities were ultimately revealed6. Ideally, a randomized, prospective study with larger population should be conducted to achieve a certain conclusion.\n\nOur case demonstrates some limitations. We could have diagnosed patent urachus earlier but in reality, we could not reach the confirmation till 20 weeks of gestation. Our lack of experience may be the major reason, since it was the first time we encountered this urachal remnant disease in Vietnam. Regardless of the inevitable limitation detailed above, the main aim of our paper is to present a new case and provide more information about patent urachus, hence contribute partly to a better and safer treatment of this rare congenital anomaly.\n\n\nConclusion\n\nBriefly, we have described a ruptured allantoic cyst with patent urachus in the setting of single umbilical artery fetus. The majority of patent urachus were discovered in the second and third trimester. Regarding our case, open resection may also achieve optimistic prognosis without any complication, hospital readmission and reoperation. In conclusion, there are still challenges on turning out early diagnosis and better treatment methods.\n\n\nConsent\n\nInformed consent for publication of images and information from this case was obtained from the mother of the infant.", "appendix": "Author contributions\n\n\n\nTTT, NTL, and NPL conceived the case report. TTT, VDQ, TDN, NHA, LPH, NTL, and NPL collected, classified, and analyzed the contents of the case report. TTT, VDQ, TDN, NHA, and LPH prepared the first draft of the manuscript. All authors have read, revised critically, and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSpecial thanks go to Dr. Le Tan Son, Dr. Trinh Huu Phuc, and Dr. Ha To Nguyen for their contribution to taking care of the patients. We thank Ms Pham Huyen Ngan for her help in editing the manuscript. TDN and NHA have been awarded scholarships from the Vietnam Student Development Fund (VNSDF; www.vnsdf.org). The organization had no role in any part of the case report or decision to publish.\n\n\nReferences\n\nYu JS, Kim KW, Lee HJ, et al.: Urachal remnant diseases: spectrum of CT and US findings. Radiographics. 2001; 21(2): 451–61. PubMed Abstract \| Publisher Full Text\n\nPersutte WH, Lenke RR, Kropp K, et al.: Antenatal diagnosis of fetal patent urachus. J Ultrasound Med. 1988; 7(7): 399–403. PubMed Abstract\n\nFuchs F, Picone O, Levaillant JM, et al.: Prenatal diagnosis of a patent urachus cyst with the use of 2D, 3D, 4D ultrasound and fetal magnetic resonance imaging. Fetal Diagn Ther. 2008; 24(4): 444–7. PubMed Abstract \| Publisher Full Text\n\nFrazier HA, Guerrieri JP, Thomas RL, et al.: The detection of a patent urachus and allantoic cyst of the umbilical cord on prenatal ultrasonography. J Ultrasound Med. 1992; 11(2): 117–20. PubMed Abstract\n\nWeichert J, Chiriac A, Kaiser M, et al.: Prenatal management of an allantoic cyst with patent urachus. Arch Gynecol Obstet. 2009; 280(2): 321–3. PubMed Abstract \| Publisher Full Text\n\nSepulveda W, Rompel SM, Cafici D, et al.: Megacystis associated with an umbilical cord cyst: a sonographic feature of a patent urachus in the first trimester. J Ultrasound Med. 2010; 29(2): 295–300. PubMed Abstract\n\nSchiesser M, Lapaire O, Holzgreve W, et al.: Umbilical cord edema associated with patent urachus. Ultrasound Obstet Gynecol. 2003; 22(6): 646–7. PubMed Abstract \| Publisher Full Text\n\nKita M, Kikuchi A, Miyashita S, et al.: Umbilical cord cysts of allantoic and omphalomesenteric remnants with progressive umbilical cord edema: a case report. Fetal Diagn Ther. 2009; 25(2): 250–4. PubMed Abstract \| Publisher Full Text\n\nOkai T: Ultrasound Fetal measurement standardization & Japanese standard proposals. J Med Ultrasonics. 2001; 28(5).\n\nCilento BG Jr, Bauer SB, Retik AB, et al.: Urachal anomalies: defining the best diagnostic modality. Urology. 1998; 52(1): 120–2. PubMed Abstract \| Publisher Full Text\n\nRasteiro C, Ramalho C, Loureiro T, et al.: Bladder emptying into an umbilical cord cyst: prenatal sonographic sign of allantoic cyst with patent urachus. Ultrasound Obstet Gynecol. 2013; 42(2): 239–40. PubMed Abstract \| Publisher Full Text\n\nBunch PT, Kline-Fath BM, Imhoff SC, et al.: Allantoic cyst: a prenatal clue to patent urachus. Pediatr Radiol. 2006; 36(10): 1090–5. PubMed Abstract \| Publisher Full Text\n\nKilicdag EB, Kilicdag H, Bagis T, et al.: Large pseudocyst of the umbilical cord associated with patent urachus. J Obstet Gynaecol Res. 2004; 30(6): 444–7. PubMed Abstract \| Publisher Full Text\n\nTolaymat LL, Maher JE, Kleinman GE, et al.: Persistent patent urachus with allantoic cyst: a case report. Ultrasound Obstet Gynecol. 1997; 10(5): 366–8. PubMed Abstract \| Publisher Full Text\n\nMesrobian HG, Zacharias A, Balcom AH, et al.: Ten years of experience with isolated urachal anomalies in children. J Urol. 1997; 158(3 Pt 2): 1316–8. PubMed Abstract \| Publisher Full Text\n\nWhitehead A, Arthur LG, Prasad R: Laparoscopic vs Open Excision of Urachal Remnants in Children. J Surgery. 2015; 2(2): 3. Reference Source\n\nKurtz M, Masiakos PT: Laparoscopic resection of a urachal remnant. J Pediatr Surg. 2008; 43(9): 1753–4. PubMed Abstract \| Publisher Full Text\n\nJeong HJ, Han DY, Kwon WA: Laparoscopic management of complicated urachal remnants. Chonnam Med J. 2013; 49(1): 43–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchaefer IM, Seeliger S, Strauss A, et al.: A three-step technique for umbilicoplasty in a patent urachus. BJU Int. 2012; 109(4): 640–4. PubMed Abstract \| Publisher Full Text\n\nShima Y, Hayashida M, Hayashi T, et al.: Characteristic prenatal ultrasonographic findings of patent urachus: a case report. J Nippon Med Sch. 2003; 70(2): 172–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8742", "date": "27 May 2015", "name": "John Svigos", "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\nThe article is well written and descriptive of the likely problems encountered with this rare condition.Whilst the problem was corrected at open surgery, laparoscopic surgical excision represents the contemporary management with its reduced post operative morbidity and length of hospital stay although both methods are equally successful in correcting the defect.", "responses": [] }, { "id": "9460", "date": "13 Jul 2015", "name": "Gamal Serour", "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\nThis is a case report of a very rare congenital anomaly. Its indexation would be of interest to obstetricians and gynaecologists as it outlines methods of diagnosis and treatment.", "responses": [] } ]	1	https://f1000research.com/articles/4-124
https://f1000research.com/articles/4-123/v1	22 May 15	{ "type": "Research Article", "title": "Neutrophil Lymphocyte Ratio as a predictor of systemic inflammation - A cross-sectional study in a pre-admission setting.", "authors": [ "Lashmi Venkatraghavan", "Tze Ping Tan", "Jigesh Mehta", "Anil Arekapudi", "Arun Govindarajulu", "Eric Siu", "Tze Ping Tan", "Jigesh Mehta", "Anil Arekapudi", "Arun Govindarajulu", "Eric Siu" ], "abstract": "Background: Neutrophil:lymphocyte ratio (NLR) is an emerging biomarker that is used to predict postoperative mortality and morbidity in cardiac and cancer surgeries. The association of this biomarker with systemic illness and its usefulness in risk assessment of preoperative patients has not been fully elucidated.Objectives: To determine the prevalence of elevated NLR in preoperative patients and to examine the relationship between elevated NLR and the presence of systemic illnesses as well as anaesthesia risk indices such as American Society of Anesthesia (ASA) and the revised cardiac risk index (RCRI) scores. Design: Cross-sectional studySetting: Anaesthesia pre-admission clinic, Toronto Western Hospital, Toronto, CanadaPatients: We evaluated 1117 pre-operative patients seen at an anesthesia preadmission clinic.Results: NLR was elevated (>3.3) in 26.6% of target population. In multivariate analysis, congestive cardiac failure, diabetes mellitus and malignancy were independent risk factors predicting raised NLR. After regression analysis, a relationship between NLR and ASA score (Odds Ratio 1.78; 95% CI: 1.42-2.24) and revised cardiac risk index (RCRI, odds ratio 1.33; 95% CI: 1.09-1.64, p-value: 0.0063) was observed.Conclusions: NLR was elevated (> 3.3) in 26.6% of patients. Congestive cardiac failure and malignancy were two constant predictors of elevated NLR at >3.3 and > 4.5. There was a strong association between NLR and anesthesia risk scoring tools of ASA and RCRI.", "keywords": [ "Neutrophil Lymphocyte Ratio", "Revised Cardiac Risk Index" ], "content": "Introduction\n\nPerioperative major cardiovascular adverse events contribute to significant morbidity and mortality in patients undergoing non-cardiac surgery1. Prospective identification of high risk patients coming for surgery will allow hospitals to prioritise the allocation of valuable resources such as intensive care beds and to avoid unplanned readmissions2. The use of the American Society of Anaesthesia (ASA) physical status as the sole risk stratification score may not give the complete picture of the patient’s underlying inflammatory processes and may be inadequate for identifying risk during the immediate postoperative period. Hence, in addition to risk indices, biomarkers may have a role in assisting risk prediction and aid in developing a management plan. Biomarkers such as B-natriuretic peptide and C-reactive peptide have been effectively used in the past to stratify risk and optimise patient care to minimise the adverse events in the perioperative setting3–6. However, these markers have some limitations in their role in routine clinical practice, as they have been shown to provide either modest or no improvement over conventional risk factors in predicting cardiovascular outcome in non-heart failure populations3.\n\nAn ideal biomarker would be something that can be measured or derived from routine blood work done during the preoperative preparation of the patient for the surgery. It should also have a high predictive value for the risk factors of interest. The role of white cell counts in predicting cardiac morbidity and mortality after surgery is well recognised7–9. An elevated neutrophil count has been shown to correlate with worse outcomes in patients with acute coronary events10.\n\nNeutrophil:lymphocyte ratio (NLR), a ratio of the neutrophil to lymphocyte count, has recently been shown to be predictive of morbidity and mortality in patients with acute coronary syndrome11,12. In addition, elevated NLR is associated with increased in-hospital and post-hospital mortality as well as increased risk in myocardial infarction patients13–15. However, the role of NLR in the risk assessment of preoperative patients in the general surgical population has not been well studied, as prior studies have concentrated on specific cardiac, vascular and oncological diseases.\n\nTherefore, we sought to study the normal values and distribution of preoperative NLR in patients before their surgery, with the primary objective of examining the prevalence of elevated NLR in the non-cardiac surgical population. Secondary objectives included examining the relationship between this biomarker and the presence of systemic illnessess and the anaesthesia risk indices such as the ASA physical status and the RCRI scores16.\n\n\nMethods\n\nEthical approval for this study was provided by the University Health Network Research Ethics Board, Toronto, Canada on 26th May 2013 (Protocol#: 13-6395; REB Co-chair: Anna Gagliard). Informed consent was waived by the REB for this study. This study complies with STrengthening the Reporting of Observational studies in Epidemiology (STROBE) statements for reporting of observational trials17.\n\nThis cross-sectional study investigated all consecutive elective surgical patients who attended the anaesthesia pre-admission clinic at our institution from 1st January 2013 to 30th April 2013. The pre-admission clinic conducts preoperative preparation and anaesthetic evaluation of patients undergoing major orthopedic, neuro-, spine, bariatric and general surgeries. Orthopedic surgeries included knee or hip joint replacements, hand and foot surgeries; neurosurgery included craniotomies for tumors, clipping of aneurysms and transphenoidal pituitary surgeries; spinal surgeries included lumbar and cervical decompression and fusions and tumor resections, and general surgeries included bowel resections, hernia repairs and Roux en Y gastric bypasses.\n\nAll patients in this cohort who were eligible for a complete blood count test were included in this study. As per the hospital policy, all patients who were above 60 years of age or suspected of having anemia, or if they required a cross-matching for the surgical procedure, will undertake a complete blood count test. Patients who are not required to have this blood test preoperatively and those with incomplete blood test results were excluded from the study.\n\nAs per our clinic’s practice, all major surgical patients are assessed by a nurse, and clinical history and examinations are entered in an electronic ‘Clinical Anaesthesia Information System’ (CAIS). The data were identified retrospectively from the CAIS database and extracted for patients demographics (age, sex), anaesthetic variables (ASA status), surgical variables (type of surgery) and presence of systemic illness medications (number of medications, ischemic heart disease, congestive cardiac failure, hypertension, diabetes, chronic kidney disease, cerebrovascular disease, and oncological disease). RCRI was computed based on the variables obtained from the clinical database. The differential white blood cell count, containing the neutrophil and lymphocyte absolute counts, and platelet counts were retrieved separately from the hematology records in the hospital’s electronic patient record (EPR). NLR was calculated from the complete blood counts.\n\nContinuous variables were expressed as mean +/- standard deviation (SD). Categorical data were summarised using absolute values (percentage). Previous studies have shown that an NLR value of > 3.3 and > 4.5 was shown to predict increased risk of myocardial infarction in post-surgical and asymptomatic populations, respectively12,13. Hence we selected both cut-off values for dichotomous comparisons to categorise patients into normal or elevated NLR.\n\nWelch’s t-test was used for comparison between groups for continuous variables. Effects of the different variables on NLR were calculated in univariate analysis. The candidate predictors for the multivariate model were selected by screening with univariate analysis with a threshold for inclusion of p < 0.25. Multiple logistic regressions were used to determine independent predictors for elevated NLR. A p-value of < 0.05 was considered statistically significant. All statistical analyses were performed using R (Version 3.02, The Comprehensive R Archive Network; http://cran.r-project.org, Accessed 17th November 2014).\n\n\nResults\n\nOne thousand, one hundred and seventy-three patients were identified through the CAIS database. Fifty-six patients were excluded from the study as there was either no complete blood count performed or the white cell count subtypes were not performed. Therefore, 1117 patients were included for further analysis.\n\nThe patient population had a mean (±SD) age of 59 (±14.9) years and was comprised of 54.3% females. The majority of patients had an ASA score of 2 or 3 (91.6%). Patients for orthopedic surgery were the largest group seen in the pre-admission clinic (44.0%), followed by neurosurgery (21.5%), spinal surgery (15.4%), bariatric surgery (7.9%) and the rest (11.2%) were for urology and general surgery.\n\nBaseline characteristics of patients grouped according to NLR > 3.3 and NLR > 4.5 are summarised in. The median value of NLR in the study population was 2.20. About one fourth (26.6%) of patients had a NLR value > 3.3 and 9.04% patients had a NLR value > 4.5. In univariate analysis using NLR > 3.3 as a threshold, age, congestive cardiac failure, chronic kidney disease, malignancy and the number of prescribed medications were increasingly associated. Congestive cardiac failure, diabetes mellitus and malignancy were the independent risk factors associated with elevated NLR after multivariate analysis (Table 2).\n\nData layout: Numbers of patients (percentage of the total cohort). Key: ASA - American Society of Anaesthesiologist physical status score; IHD - Ischemic heart disease; CHF - Congestive heart failure; HT - Hypertension; CV - Cerebral Vascular; CKD stage 1–5 - Chronic kidney disease stage 1–5; RCRI - Revised Cardiac Risk Index Score.\n\nKey: OR - odds ratio; CI - confidence interval; IHD - Ischemic heart disease; CHF - Congestive heart failure; HT - Hypertension; CV - Cerebral Vascular; CKD - Chronic kidney disease. Significant results in bold face.\n\nHowever, when NLR > 4.5 were analyzed, polypharmacy emerged as a risk factor along with congestive cardiac failure and malignancy (Table 3). Diabetes mellitus was not an independent risk factor.\n\nKey: OR - odds ratio; CI - confidence interval; IHD - Ischemic heart disease; CHF - Congestive heart failure; HT - Hypertension; CV - Cerebral Vascular; CKD - Chronic kidney disease. Significant results in bold face.\n\nIn addition, NLR was significantly elevated in patients with any type of malignancy compared to patients with no malignancy (mean NLR 3.42 vs 2.70, p = 0.049). Regression analysis showed a strong association between NLR and ASA scores (Odds Ratio 1.78; 95% CI: 1.42-2.24, p-value: <0.001) and also between NLR and RCRI (Odds Ratio 1.33; 95% CI: 1.09-1.64, p-value: 0.0063).\n\n\nDiscussion\n\nTo our knowledge this is the first cross-sectional study on the prevalence of elevated NLR in a preoperative setting. In our patient population, 26.6% of patients had an elevated NLR value of more than 3.3, which had been shown to be associated with increased mortality after cardiac surgery18. We found an independent association between congestive cardiac failure and NLR values > 3.3 and > 4.5. This is in line with established evidence on the utility of NLR in predicting long-term outcome in acutely decompensated heart failure19. Our study also showed a strong association between the presence of malignancy and NLR thresholds of > 3.3 and > 4.5. This confirms the previously known prognostic value of NLR in terms of mortality and recurrence of disease in patients with cancer20.\n\nIn addition, we found a significant association between NLR and ASA physical status and RCRI. This raised the possibility that NLR may be of added value in anaesthetic risk stratification and comparison in a preoperative setting. This may be particularly relevant in a tertiary hospital setting where patients often present with many comorbidities.\n\nOur study also showed that increased use of medication is a risk factor associated with NLR values > 4.5. The increased number of medications reflects the presence of more systemic illnesses. The relationship between polypharmacy and comorbidity is a well-recognised phenomenon especially in the elderly21. This also highlights the relationship between NLR and presence of increased disease burden in patients with chronic systemic illnesses.\n\nHowever, we did not demonstrate an association between the presence of ischemic heart disease and elevated NLR, contrary to previous publications12. It is not clear from our cohort the reason for this discrepancy. It may be possible that the inclusion criteria for ischemic heart disease in our database includes all patients from angina-like symptoms to patients with previous myocardial infarction, thus weakening the association between NLR and ischemic heart disease. Further, it is likely that the majority of patients who presented to the pre-surgical clinic had stable ischemic heart disease and are on statin therapy and therefore have suppression of low-grade inflammation, leading to a lower NLR than anticipated.\n\nPrediction of systemic illness and its severity are crucial in formulating a safe intraoperative plan and postoperative care in surgical patients. Elevated NLR is known to predict all-cause mortality in cardiac and major vascular surgeries18,22. Elevated preoperative NLR is also associated with increased morbidity postoperatively, with prolonged hospitalization and increased intensive care admission18. Further, elevated NLR is associated with increased morbidity and mortality in sepsis23. Hence, the use of NLR as a potential biomarker for preoperative risk assessment and stratification can be pertinent for several reasons. Firstly, NLR is obtained from a complete blood count that is routinely performed pre and postoperatively; it doesn’t necessitate additional investigation and is an inexpensive and readily available marker. Secondly, the application of elevated NLR can be used to plan for the postoperative support and rehabilitation of patients undergoing major surgeries. Another similar finding has been observed in the general population cohort in the recently published post-hoc analysis of the National Health and Nutrition Examination Survey-III that showed that NLR can independently predict cardiac mortality. In analysis of 7363 subjects, Shah et al. showed that NLR can be used as a risk index to reclassify patients from low to intermediate risk categories and improve the Framingham risk score24.\n\nElevated NLR has been shown to be associated with increased tumor necrosis factor (TNF) alpha, and various interleukins (IL-6, IL-7, IL-8, IL-12, IL-17)25,26. These markers are known to be associated with poor outcome in critically ill patients, as well as with increased incidence of recurrent ischemic events in cardiac patients27,28. However, measuring these biomarkers is expensive and tests are not routinely performed. In contrast, NLR is a simple index derived from routine blood tests which might provide equal and valuable information in the preoperative setting.\n\nOur study suffers from some major limitations. This study is not designed to examine the utility of this biomarker for stratifying risk and predicting clinical end-points of mortality and major cardiovascular adverse events. Rather it is designed to provide a cross-sectional “snapshot” of the distribution of NLR in a cohort of patients who attended the anaesthesia pre-admission clinic before their surgery and to correlate this biomarker with the presence of systemic illnesses.\n\nFurther prospective cohort studies are needed to examine the optimal role of NLR as a biomarker in the perioperative setting. Establishing a relationship between the NLR and systemic illnesses in elective preoperative patients sets the stage for evaluating the role of this biomarker in future prospective studies looking at the ideal cut-off value of these biomarkers and the correlation between elevated NLR, and perioperative morbidities and mortalities. Prediction of systemic illness and its severity are crucial in formulating a safe intraoperative plan and postoperative care in surgical patients.\n\n\nConclusion\n\nIn summary, elevated preoperative NLR (> 3.3) was observed in 26.6% of patients attending a pre-surgical anaesthetic clinic at our tertiary hospital. Congestive cardiac failure and malignancy were two constant associations of elevated NLR at > 3.3 and > 4.5 respectively. There was also a strong association between NLR, and ASA and RCRI scores of patients. More studies are needed to determine the utility of NLR as a biomarker in the preoperative risk stratification of patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Neutrophil-lymphocyte ratio study - patient characteristics, co-morbidities, revised cardiac risk index, ASA status and NLR., 10.5256/f1000research.6474.d4734529", "appendix": "Author contributions\n\n\n\nTPT contributed to the study design and assisted in data collection and analysis of the data. JM and AA contributed to data collection. APG contributed to the study design. ES assisted with the data analysis. LV contributed the study concept, content data collection and analysis. All authors were involved in writing the manuscript and have agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe acknowledged the help of Ms. Maria Engleakis, librarian at the University Health Network library for help in retrieving of manuscripts as part of the preliminary systematic review.\n\n\nReferences\n\nDevereaux PJ, Goldman L, Cook DJ, et al.: Perioperative cardiac events in patients undergoing noncardiac surgery: a review of the magnitude of the problem, the pathophysiology of the events and methods to estimate and communicate risk. CMAJ. 2005; 173(6): 627–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlance LG, Kellermann AL, Osler TM, et al.: Hospital readmission after noncardiac surgery: the role of major complications. JAMA Surg. 2014; 149(5): 439–45. PubMed Abstract \| Publisher Full Text\n\nEdwards M, Whittle J, Ackland GL: Biomarkers to guide perioperative management. Postgrad Med J. 2011; 87(1030): 542–9. PubMed Abstract \| Publisher Full Text\n\nChoi JH, Cho DK, Song YB, et al.: Preoperative NT-proBNP and CRP predict perioperative major cardiovascular events in non-cardiac surgery. Heart. 2010; 96(1): 56–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyding AD, Kumar S, Worthington AM, et al.: Prognostic value of brain natriuretic peptide in noncardiac surgery: a meta-analysis. Anaesthesiology. 2009; 111(2): 311–9. PubMed Abstract \| Publisher Full Text\n\nAckland GL, Scollay JM, Parks RW, et al.: Pre-operative high sensitivity C-reactive protein and postoperative outcome in patients undergoing elective orthopaedic surgery. Anaesthesia. 2007; 62(9): 888–94. PubMed Abstract \| Publisher Full Text\n\nDacey LJ, DeSimone J, Braxton JH, et al.: Preoperative white blood cell count and mortality and morbidity after coronary artery bypass grafting. Ann Thorac Surg. 2003; 76(3): 760–4. PubMed Abstract \| Publisher Full Text\n\nColler BS: Leukocytosis and ischemic vascular disease morbidity and mortality: is it time to intervene? Arterioscler Thromb Vasc Biol. 2005; 25(4): 658–70. PubMed Abstract \| Publisher Full Text\n\nAlbert AA, Beller CJ, Walter JA, et al.: Preoperative high leukocyte count: a novel risk factor for stroke after cardiac surgery. Ann Thorac Surg. 2003; 75(5): 1550–7. PubMed Abstract \| Publisher Full Text\n\nAdamsson ES, Smith JG, Melander O, et al.: Incidence of coronary events and case fatality rate in relation to blood lymphocyte and neutrophil counts. Arterioscler Thromb Vasc Biol. 2012; 32(2): 533–9. PubMed Abstract \| Publisher Full Text\n\nZouridakis E, Avanzas P, Arroyo-Espliguero R, et al.: Markers of inflammation and rapid coronary artery disease progression in patients with stable angina pectoris. Circulation. 2004; 110(13): 1747–53. PubMed Abstract \| Publisher Full Text\n\nNúñez J, Núñez E, Bodí V, et al.: Usefulness of the neutrophil to lymphocyte ratio in predicting long-term mortality in ST segment elevation myocardial infarction. Am J Cardiol. 2008; 101(6): 747–52. PubMed Abstract \| Publisher Full Text\n\nGazi E, Bayram B, Gazi S, et al.: Prognostic Value of the Neutrophil-Lymphocyte Ratio in Patients With ST-Elevated Acute Myocardial Infarction. Clin Appl Thromb Hemost. 2015; 21(2): 155–9. PubMed Abstract \| Publisher Full Text\n\nAzab B, Shah N, Akerman M, et al.: Value of platelet/lymphocyte ratio as a predictor of all-cause mortality after non-ST-elevation myocardial infarction. J Thromb Thrombolysis. 2012; 34(3): 326–34. PubMed Abstract \| Publisher Full Text\n\nGhaffari S, Nadiri M, Pourafkari L, et al.: The predictive Value of Total Neutrophil Count and Neutrophil/Lymphocyte Ratio in Predicting In-hospital Mortality and Complications after STEMI. J Cardiovasc Thorac Res. 2014; 6(1): 35–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee TH, Marcantonio ER, Mangione CM, et al.: Derivation and prospective validation of a simple index for prediction of cardiac risk of major noncardiac surgery. Circulation. 1999; 100(10): 1043–9. PubMed Abstract \| Publisher Full Text\n\nVon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet. 2007; 370(9596): 1453–7. PubMed Abstract \| Publisher Full Text\n\nGibson PH, Croal BL, Cuthbertson BH, et al.: Preoperative neutrophil-lymphocyte ratio and outcome from coronary artery bypass grafting. Am Heart J. 2007; 154(5): 995–1002. PubMed Abstract \| Publisher Full Text\n\nUthamalingam S, Patvardhan EA, Subramanian S, et al.: Utility of the neutrophil to lymphocyte ratio in predicting long-term outcomes in acute decompensated heart failure. Am J Cardiol. 2011; 107(3): 433–8. PubMed Abstract \| Publisher Full Text\n\nGuthrie GJ, Charles KA, Roxburgh CS, et al.: The systemic inflammation-based neutrophil-lymphocyte ratio: experience in patients with cancer. Crit Rev Oncol Hematol. 2013; 88(1): 218–30. PubMed Abstract \| Publisher Full Text\n\nPayne RA, Avery AJ, Duerden M, et al.: Prevalence of polypharmacy in a Scottish primary care population. Eur J Clin Pharmacol. 2014; 70(5): 575–81. PubMed Abstract \| Publisher Full Text\n\nBhutta H, Agha R, Wong J, et al.: Neutrophil-lymphocyte ratio predicts medium-term survival following elective major vascular surgery: a cross-sectional study. Vasc Endovascular Surg. 2011; 45(3): 227–31. PubMed Abstract \| Publisher Full Text\n\nDe Jager CP, Wever PC, Gemen EF, et al.: The neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia. PLoS One. 2012; 7(10): e46561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah N, Parikh V, Patel N, et al.: Neutrophil lymphocyte ratio significantly improves the Framingham risk score in prediction of coronary heart disease mortality: insights from the National Health and Nutrition Examination Survey-III. Int J Cardiol. 2014; 171(3): 390–7. PubMed Abstract \| Publisher Full Text\n\nTurkmen K, Guney I, Yerlikaya FH, et al.: The relationship between neutrophil-to-lymphocyte ratio and inflammation in end-stage renal disease patients. Ren Fail. 2012; 34(2): 155–9. PubMed Abstract \| Publisher Full Text\n\nMotomura T, Shirabe K, Mano Y, et al.: Neutrophil-lymphocyte ratio reflects hepatocellular carcinoma recurrence after liver transplantation via inflammatory microenvironment. J Hepatol. 2013; 58(1): 58–64. PubMed Abstract \| Publisher Full Text\n\nGiannoudis PV, Hildebrand F, Pape HC: Inflammatory serum markers in patients with multiple trauma. Can they predict outcome? J Bone Joint Surg Br. 2004; 86(3): 313–23. PubMed Abstract \| Publisher Full Text\n\nRidker PM, Rifai N, Pfeffer M, et al.: Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction. Circulation. 2000; 101(18): 2149–53. PubMed Abstract \| Publisher Full Text\n\nVenkatraghavan L, Tan TP, Mehta J, et al.: Dataset 1 in: Neutrophil Lymphocyte Ratio as a predictor of systemic inflammation - A cross-sectional study in a pre-admission setting. F1000Research. 2015. Data Source" }	[ { "id": "8777", "date": "28 May 2015", "name": "Gareth L. Ackland", "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\nThe authors have performed a cross sectional observational study examining the relationship between NLR, preoperative systemic illnesses and peri-operative risk markers in a large cohort of surgical patients. This makes a contribution to the existing literature by including patients from a variety of surgical specialties rather than focussing on specific surgical pathologies. They found that increased NLR at two cut-offs was independently associated with pre-existing cardiac failure and malignancy (confirming similar findings in other studies) – but not ischaemic heart disease - in a multivariable model. Overall the design, methods and analysis are appropriate and the authors’ conclusions are balanced and justified. The study authors may consider the following points which may strengthen this work:The abstract and results text state that diabetes was one of the pre-existing co-morbidities associated with increased NLR in the multivariate model, however this is not consistent with the results tables. The regression models examining ASA / RCRI and NLR could be described more clearly. Were they separate univariate models or part of the wider multivariate models including co-morbidities? If the latter, the extent of co-linearity between these risk scores and the presence of co-morbidities would be useful to know. As the authors point out, this study has not included clinical outcomes and did not aim to examine NLR as a prognostic marker. With this in mind, could NLR have been examined as a continuous variable in the regression model rather than being dichotomised? Dichotomising continuous data may have led to a loss of statistical power (Altman and Royston, 2006). Also, although the NLR cut-offs examined have been justified based on prior related literature, giving these cut-offs (3.3 and 4.5) might imply to the casual reader that these are prognostically important values, whereas this needs further exploration as the authors acknowledge.", "responses": [] }, { "id": "8736", "date": "03 Jun 2015", "name": "Neeraj N. Shah", "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\nVenkatraghavan et al present an interesting study of preoperative neutrophil to lymphocyte ratio (NLR) in patients undergoing non-cardiac surgeries. While NLR has been studied extensively in acute and chronic cardiac conditions and in the setting of cardiac surgeries, there is scarce data about NLR in non-cardiac surgeries. It is unfortunate that the authors did not look at mortality or morbidity endpoints, rather this is a cross sectional study looking at prevalence of risk factors and correlation with ASA and RCRI scores preoperatively.Suggested revisions:1. Introduction, first paragraph: In the sentence \"Prospective identification of high risk pts..... and to avoid unplanned readmissions\". How will identification of high risk preop patients avoid unplanned readmissions? This sentence is confusing and I would recommend the authors deleting the part which says \"to avoid unplanned readmissions\".2. Results: Use numbers instead of words e.g. use 1,173 instead of one thousand one hundred seventy three.3. Table 1: Please include type of surgery in table 1 (e.g. orthopedic, neurosurgery, etc.)3. Table 1: Medications: Please lump together 4,5, or 6 medications as 4 or more medications, since the definition of polypharmacy is 4 or more medications.4. (Major concern) Results: Table 2 clearly shows that for NLR > 3.3, the independent predictors are age, CHF and malignancy, but the authors mention CHF, DM and malignancy in the text. Kindly address this discrepancy.4. (Major concern) Results: Number of medications: It seems that the authors conducted logistic regression with either NLR>3.3 or NLR>4.5 as the dependent variable and number of medications as an independent, continuous variable. This is incorrect. One cannot assume the number of medications to have a continuous effect. The authors need to change their analysis, and incorporate number of medications as a categorical variable with categories: zero (referent), 1, 2, 3 and 4 or more. There will be separate odds ratios for categories 1, 2, 3 and 4 or more (the authors may elect to choose different categories, but it is important that they use categories rather than a continuous variable). This may change the results, esp for NLR > 4.5.5. (Major concern) Results: The authors need to explain in greater detail how they performed 'regression' analysis with NLR and ASA and NLR and RCRI. Was NLR treated as a continuous variable or in categories>3.3 or >4.5? What was the dependent variable? I am assuming that the authors performed logistic regression analysis with ASA or RCRI as the dependent variable and NLR as the independent variable in a continuous fashion, but this needs to be explicitly stated, since it helps in understanding what the odds ratios mean.6. What about dividing NLR into tertiles or quartiles instead of using the said cut offs, since there are not established cut offs for NLR?7. Discussion, first paragraph: The association that the authors observe in this study between NLR and CHF and NLR and malignancy is just that- an association. The authors cannot infer that this means that either NLR is associated with long term outcomes in CHF (ref 19) or prognostic value of NLR in cancer (ref 20). I would therefore strongly recommend the authors to remove those two lines from the first paragraph of discussion.8. Discussion, Third paragraph (no. of meds): This may have to be re-written after the authors re-analyze the data with no. of meds as a categorical variable.9. The authors do have interesting findings and I was hoping to see if preoperative NLR predicts outcomes (morbidity and mortality or major cardiac events) in the postoperative period. Moreover, if NLR does predict outcomes in the postoperative period, it may be used to re-classify the existing ASA and RCRI indices for non-cardiac surgeries. The authors should mention this in discussion or conclusion as future studies that may be undertaken in this subject.", "responses": [] }, { "id": "9421", "date": "13 Jul 2015", "name": "Edwin Seet", "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\nDr Venkatraghavan and colleagues have undertaken a cross-sectional observation study in a tertiary centre in Toronto, evaluating the utility of a raised Neutrophil:Lymphocyte Ratio as a biomarker for risk stratification in the perioperative period. A raised NLR was compared with conventionally accepted risk assessment tools such as the ASA physical status and the RCRI scores. Multivariate regression analysis was done to investigate independent predictors of a raised NLR. The authors found that a raised NLR was associated with malignancy, congestive heart failure and polypharmacy. A relationship between raised NLR and 2 cutoffs and increased ASA physical status and RCRI scores was observed.Several possible opportunities of improvement of the manuscript may exist:This is a novel study investigating raised NLR and perioperative comorbidities and conventional risk stratification tools. Most readers would not be familiar with the biological plausibility of a raised NLR and perioperative outcomes. More details may be included in the introduction and discussion to make this clearer to the readers.Dr Peter Nagele and colleagues published a landmark paper correlating a raised High-Sensitivity Troponin T test and perioperative outcomes in Nagele et al (2013). This publication should be referenced together with other biomarkers in the introduction and discussion such as the BNP and CRP.The authors have chosen to represent the association between NLR and ASA physical status and RCRI scores in the form of Odds Ratio. I wonder if correlation statistics and/or Receiver Operating Characteristics at various cutoffs may be utilized and represented in the results as well?All in all, Dr Venkatraghavan et al have undertaken an interesting study which answers a novel and fairly important clinical question. I would like to congratulate them on a stellar effort.", "responses": [] } ]	1	https://f1000research.com/articles/4-123
https://f1000research.com/articles/4-17/v1	21 Jan 15	{ "type": "Research Note", "title": "Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes", "authors": [ "Ron Ammar", "Tara A. Paton", "Dax Torti", "Adam Shlien", "Gary D. Bader", "Tara A. Paton", "Dax Torti", "Adam Shlien" ], "abstract": "Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.", "keywords": [ "nanopore", "DNA", "sequencing", "haplotype", "pharmacogenomics" ], "content": "Introduction\n\nAn important aspect of precision medicine is the study of how genes influence individual response to drug therapies, known as pharmacogenomics (PGx). PGx genotyping impacts the choice of drug dosing in many medical contexts. As an example, in acute lymphoblastic leukemia patients the metabolizer status of thiopurine methyltransferase (TPMT) must be considered when calculating the initial drug dose of mercaptopurine (6-MP) to ensure proper treatment and avoid fatal toxicity1,2.\n\nPGx data are typically collected by sequencing a small panel of known PGx genes via traditional Sanger sequencing, or targeted genotyping technologies3. Diagnostic labs are also exploring the use of whole genome or exome sequencing (WGS, WES) for PGx. However, existing methods have various limitations, which may lead to adverse drug responses. WGS and WES methods may fail to capture or provide adequate sequence coverage for certain PGx loci. Targeted genotyping approaches, such as Taqman (Life Technologies), Luminex (Luminex Corp.) or Sequenom (Agena Bioscience), can fail to detect novel loss-of-function mutations due to their selective interrogation of predefined genomic loci. In a diagnostic clinic, where results are often required within days of administering diagnostic tests, some existing technologies can delay the return of clinical results. Current massively parallel technologies have high capital costs (ranging from $100,000–$1,000,000) requiring clinical laboratories to purchase and maintain large instruments to perform in-house genotyping. Alternatively, a laboratory can send these PGx samples to a third party service for a fee, but may wait up to several months for a clinical report.\n\nClinical haplotypes, tightly-linked collections of inherited alleles, that are responsible for a plethora of medical phenotypes including patient drug response are important in many medical sequencing applications. Information about haplotypes, or genotype phase, can be inferred from parental genotypes or genetic pattern frequency in the human population, however, these predictions can be inaccurate if de novo or rare haplotypes are encountered in a patient4,5. Due to the chromosomal distance between alleles, current short read technologies in use for PGx are often unable to resolve haplotype information without supplemental statistical phasing or parental genotypic data.\n\nRecently, Oxford Nanopore Technologies Inc. has developed the MinION, a real time nanopore-based DNA sequencing instrument which is compact, inexpensive and faster than most established DNA sequencing technologies. The time it takes from initiation of library preparation to basecalling the first sequence read is approximately 3 hours and the instrument is capable of detecting long sequence reads in excess of 50kb (according to the manufacturer, ONT). Nanopore-based technology promises major advances in DNA sequencing by offering an inexpensive (e.g. on the order of $1000) pocket-sized device for clinical diagnostics or field experiments.\n\nHere we report nanopore-based sequencing of three clinically relevant PGx genes to identify medically actionable variants and haplotypes without statistical phasing.\n\n\nMethods\n\nPrimer sequences for HLA-A, HLA-B and CYP2D6 are available in Table S1. For CYP2D6, we designed primers to specifically amplify CYP2D6 while not amplifying the 94% identical CYP2D7. PCR primer specificity was verified using UCSC in silico PCR. We used the standard protocol (for fragments up to 8 kb) of the KAPA LongRange HotStart PCR system: 5× KAPALongRange Buffer (without Mg2+) 1×, MgCl2 (25 mM) 1.75 mM, dNTPs (10 mM each dNTP) 0.3 mM, Fwd primer (10 μM) 0.5 μM, Rev primer (10 μM) 0.5 μM 50ng of genomic DNA (1ul of a 50ng/ul preparation), KAPA LongRange HotStart DNA Polymerase (2.5 U/μl) 1.25 U/50 μl, PCR grade water up to 50μl. For HLA-A and HLA-B, genomic sequence was downloaded from UCSC Browser with common SNPs masked. Primers were designed using Primer3 using parameters of 68°C for optimal annealing temp and 26bp minimum primer length6.\n\nThe DNA libraries were prepared using the Oxford Nanopore Genomic DNA Sequencing protocol (SQK-MAP003). 1.5μg of PCR product was used (instead of the suggested 1μg based on improved yield in earlier testing) with equimolar amounts of CYP2D6, HLA-A and HLA-B amplicons in solution. DNA was not fragmented because the PCR amplicons were already at the desired size for sequencing and downstream haplotyping (4–5Kbp; Table S1). In accordance with the protocol, we end-repaired the DNA with the NEBNext end repair module (New England Biolabs, cat. no. E6050) and subsequently dA-tailed the sample using the NEBNext dA-tailing module (New England Biolabs cat. no. E6053), prior to ligation of nanopore-specific adapters. All purifications were accomplished with Agencourt AMPure XP beads (Beckman Coulter Inc., cat. no. A63880). Throughout the library preparation, care was taken not to vortex or vigorously pipette/mix the library to avoid shearing the DNA into smaller fragments.\n\nThe MinION flowcell (R7.3 flowcell chemistry) was run for 24 hours using the MinKNOW software (v47.3) producing 24,859 fast5 files, corresponding to individual reads from base detection events at specific nanopore channels. Online basecalling was performed using the Metrichor software (v2.23). The MinION outputs 3 reads for each dsDNA molecule that passes through a pore. The leading ssDNA is referred to as “1D template” and its complementary ssDNA strand is the “1D complement”. When both 1D template and 1D complement reads are basecalled, a 2D consensus sequence is determined based on complementarity. We observed 19,655 1D template reads, 9,584 1D complement reads and 7,540 2D reads. The mean lengths were 2,693bp for 1D template, 2,706bp for 1D complement and 3,486bp for 2D consensus.\n\nExisting massively parallel sequencing instruments, such as the Illumina HiSeq 2500, produce accurate short reads typically up to ~250bp in length. These sequencers can produce hundreds of millions of reads which need to be rapidly aligned to a reference genome. Current computational methods for accurate alignment of these reads, including BWA7 and Bowtie28 are based on the Burrows-Wheeler Transform FM index, and they are designed to align short reads with minimal variation to a reference assembly. BWT-FM methods are insufficiently sensitive to align much longer reads with higher error rates9. These long reads, generated by single molecule sequencers such as the Oxford Nanopore MinION or the Pacific Biosciences RS II, have a significantly higher error rate, enriched for insertions or deletions (indels) rather than substitutions9. Mapping of these reads is best suited to aligners that were originally designed for whole genome alignments, such as LAST10. We chose to use BLASR, originally developed for the Pacific Biosciences system, to align our data, because it was designed to align long error-prone reads rather than genomes9.\n\nAll reads were aligned to the human genome reference assembly GRCh37.p13 (hg19) using default BLASR parameters (gap open penalty = ten, gap extension penalty = zero, minimum seed length = 12). The use of other parameters, such as a gap-open penalty of zero (with default gap extension penalty = zero) did not alter the results, even though it may be expected to do so given the prevalence of indels expects in single molecule nanopore sequencing. The majority of successfully aligned long read fragments were obtained from 2D basecalls (Table 1), and these were of higher quality because they are consensus reads constructed from corresponding 1D template and complement. For the final alignment data, for each separate read event (1D template, 1D complement and 2D consensus), we selected the 2D if it was available. Since the 1D reads typically had lower mapping accuracy and significantly shorter aligned fragments (Table 1), these were not included in our variant or haplotype calling analysis.\n\nFinally, we performed two separate alignments depending on our desired sequencing application: a) gene targets with highly similar nearby genes; and b) highly polymorphic gene targets. The first analysis only selected the single best alignment for each long read. This was critical for the gene CYP2D6 because the CYP2D locus on chromosome 22 harbors two paralogous pseudogenes CYP2D7 and CYP2D8P11. In particular, CYP2D6 and CYP2D7 are highly similar (94% identity, BLAST E-value = 0.0) and are positioned in tandem on the chromosome. By allowing reads to only map to a single best hit, we were able to verify that the PCR selectively amplified CYP2D6 and not nearby related genes (see coverage in Figure 1). Our second analysis was performed due to the high degree of polymorphism in the MHC locus on chromosome 6. As part of the MHC haplotype project12, multiple reference contigs for this highly variable region are included in the GRCh37 reference assembly as indicated in the release notes (http://www.ncbi.nlm.nih.gov/genome/guide/human/release_notes.html). Since long reads from the NA12878 HLA-A and HLA-B genes mapped to different chromosome 6 reference haplotype contigs, by allowing multiple alignments to the reference (up to 10) for each read, we could gather all reads for a single gene in a single pileup to any of the eight HLA-A/B loci to generate a consensus sequence. For this study, we used the reference chromosome 6 contig NC_000006.11 (not the MHC haplotype project contigs).\n\nThe majority of reads aligned across the entire length of CYP2D6 as was expected by selective PCR amplification. Downstream, an insignificant number of read fragments aligned to CYP2D7 and CYP2D8 (2D8 is located from 42,545,874 to 42,551,097; exon-intron diagram not shown in gene annotation track). Due to the extremely high coverage at CYP2D6, not all reads are shown in this pileup diagram.\n\nDue to the long reads, high error rates and continuously evolving error profile of the MinION basecalls at this early stage of technology roll out, variant callers such as the Genome Analysis Toolkit’s UnifiedGenotyper or HaplotypeCaller13 were unable to identify variants or haplotypes in the MinION sequence data during our trials. Variant and haplotype level information, however, was readily accessible based on coverage of aligned reads, which we extracted using SAMTools via the Pysam wrapper (http://github.com/pysam-developers/pysam)14.\n\nMean coverage was 1236.4× for CYP2D6 (single best hit alignment), 785.5× for HLA-A (multi-hit alignment) and 1416.3× for HLA-B (multi-hit alignment).\n\nVariants were detected using a naïve threshold requiring 1/3 of reads to contain the variant genotype at that position. While this was effective for substitution detection, we are likely to detect many false positive deletions due to the high deletion error rate (Table 1). Haplotype proportions were identified by interrogating clinical marker positions (Table S2) across all reads aligned to a particular gene to establish the proportion of reads corresponding to each haplotype. Pharmacogenomic haplotypes were verified by comparison to diagnostic data sets (see below) using the MedSavant software (www.medsavant.com; manuscript in review) with the pharmacogenomics app that we developed. The PGx app interprets human pharmacogenomic variants with medically actionable output based on published guidelines established by the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Pharmacogenomics Knowledgebase (www.pharmgkb.org).\n\nComplete Genomics WGS data for NA12878 were obtained from the public 69 genomes project (CG analysis pipeline version 2.0.0; http://www.completegenomics.com/public-data/69-Genomes/)15.\n\n10ng of genomic DNA from NA12878 was genotyped for 36 SNP, indel and copy number variants for CYP2D6 using the iPLEX® ADME CYP2D6 Panel v1.0, developed by Assays by Agena (formerly Sequenom) on the MassARRAY4 System. Haplotype assignment and copy number determination was done using Typer software version 4.0 (Agena Biosciences).\n\nIn parallel, copy number estimation of CYP2D6 was performed using the Taqman copy number assays Hs04502391_cn and Hs04083572_cn (Life Technologies) using the manufacturer’s recommended protocol (Figure S1). The assay was performed in quadruplicate on 10ng genomic DNA for each sample in a 96-well plate. The 10μL reaction mix consisted of 5μL 2× Taqman Genotyping Master Mix (Life Technologies), 0.5μL of 20X copy number assay (described above), 0.5μL TaqMan RNAse P Copy Number Reference Assay (Life Technologies cat. no. 4403326), 2μL water and 2μL of 5ng/μL genomic DNA. Cycling conditions for the reaction were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Samples were analyzed using the ViiA™ 7 Real-Time PCR System (Life Technologies) and analyzed using CopyCaller Software (Life Technologies). The HuRef sample was used as a 2-copy calibrator sample.\n\nComplete Genomics WGS and Sequenom MassARRAY genotypes were statistically phased using the BEAGLE software (v4.0) and the 1000 Genomes Project phase 3 reference panel (http://faculty.washington.edu/browning/beagle/beagle.html)4. For the HLA-A/B genes, phase information was obtained from the HapMap phase 2 data (http://hapmap.ncbi.nlm.nih.gov/downloads/index.html.en)16. HLA-A/B alleles were determined using the GATK HLACaller software package (http://gatkforums.broadinstitute.org/discussion/65/hla-caller).\n\n\nResults\n\nTo evaluate the MinION for diagnostic PGx sequencing, we selectively amplified and sequenced the genes CYP2D6, HLA-A, and HLA-B from the CEPH/UTAH pedigree 1463 sample NA12878. CYP2D6 is a pharmacogenetically vital cytochrome P450 gene because it encodes a protein responsible for metabolism of 20% of clinically used drugs11. The diagnostic relevance of 2D6 is derived from its significant polymorphism which contributes to dramatic inter-individual variability in enzyme activity11. Also important are the HLA genes which are clinically relevant for solid organ transplantation and accurate dosing of abacavir, allopurinol and carbamazepine, used to treat HIV/AIDS, hyperuricemia and seizure disorders, respectively17–19. The HLA genes are among the most polymorphic loci in the human genome, making their sequencing and confident typing difficult with current short read DNA sequencing methods. These three genes were also chosen for sequencing due to their length (4–5Kbp), which did not require long range PCR amplification methods.\n\nPCR amplicons of these three genes from NA12878 (CEPH/Utah Pedigree 1463) were sequenced on the MinION instrument yielding 19655 read events. Each read event could be basecalled in multiple forms, as a template or complement strand (1D) or as a consensus of the two (2D), and we obtained 36779 1D and 2D reads in total. For the purpose of diagnostic evaluation, we chose to align only the consensus 2D reads due to their lower error rate and extended length (Table 1; see Methods). Reads were aligned to the human genome (GRCh37), with an abundance of aligned reads 4–5Kb in length representing full-length PCR amplicons. As well, smaller aligned read fragments were observed, some of which are speculated to be byproducts of shearing during experimental DNA handling (Figure 2A, Table S1).\n\nWith depth of coverage of ~1000× for each of the genes, many chromosomal positions were called with 70–90% consensus, demonstrating that as coverage of loci increases on the MinION, confidence improves with regard to specific base calls (Figure 2B). While the MinION basecalls are emitted with a comparatively high error rate (Table 1), the majority of errors appear to be randomly distributed across the length of the reads, which is why increasing coverage can yield a consensus that matches variant calls from existing sequencing and genotyping platforms such as Illumina, Complete Genomes and Sequenom.\n\nMinION-called variants and haplotypes were validated against statistically phased genotypes from multiple platforms including Complete Genomics and Sequenom MassARRAY (see Methods). Based on the statistically phased genotypes, we determined that NA12878 possesses both the 3 and 4 loss-of-function alleles for CYP2D6, and this 3/4 diplotype is interpreted as reduced metabolism of drugs such as codeine (an opiate) and olanzapine (an atypical antipsychotic)20–22.\n\nCYP2D6 haplotype proportions were identified by interrogating clinical marker positions across all aligned reads to establish the proportion of reads corresponding to each PGx haplotype (Table S2). Only reads spanning all clinical markers were included (n = 404), so that haplotypes could be measured by linkage of markers on a single DNA molecule. The MinION data confirmed the statistically-phased haplotypes by direct interrogation of markers from individual reads (Figure 2C). However, we also observed a prominent 2 haplotype, which we could not account for given that our Sequenom MassARRAY and qPCR results indicated that the CYP2D6 locus was diploid (no copy number variation) and could only correspond to a 3/4 diplotype. To determine whether the 2 haplotype could arise from mismatched CYP2D7 DNA, which was not supposed to be PCR amplified (see Methods), we interrogated four positions with different bases between CYP2D6 and CYP2D7 reference sequences and found that all reads corresponded to CYP2D6 (Supplementary File S1). Finally, we hypothesized that the 2 haplotype might arise due to 3 and 4 duplexes forming during PCR (effectively outcompeting the primer binding during the annealing step), but this was ruled out by identifying the 2 haplotype using only 1D reads. It is possible that this 2 haplotype arose either due to early cycle template switching during PCR or sample contamination23. Also, the relative proportion of haplotypes was likely skewed by potential biases during PCR amplification.\n\nA. Length distribution of aligned reads. 4–5Kb reads represent full-length PCR amplicons. Slightly smaller fragments were likely byproducts of shearing during DNA handling in the experimental protocol. B. With depth of coverage of ~1000× for each of the genes, many chromosomal positions were called with 70–90% consensus. This is a short window of aligned reads for the 4 locus of CYP2D6 with over 1200× coverage. The heterozygous 4 allele rs1065852 is indicated with the arrow. C. Proportions of haplotypes of CYP2D6, HLA-A and HLA-B when directly measured from individual reads spanning all haplotype markers.\n\nThe HLA-A and HLA-B haplotypes were determined in the same way as the CYP2D6 haplotypes, using predefined markers from the HapMap project. In HLA-A (only spanning reads, n = 203), the most abundant haplotype matched the transmitted haplotypes of the parents NA12891 and NA12892, which both transmitted an identical haplotype (Figure 2C). Accounting for the errors in MinION sequencing, when allowing for a single mismatch in the haplotype, ~85% of reads confirm the NA12878 diplotype. In HLA-B (only spanning reads, n = 202), the majority of reads corresponded to the transmitted and untransmitted haplotypes of the parent NA12891, with only 8.4% of reads corresponding to the transmitted haplotype of parent NA12892 (Figure 2C). This could be a result of potential contamination suspected earlier in described above with CYP2D6. As suggested for CYP2D6, the relative proportion of HLA haplotypes was likely also affected by PCR bias during amplification. HLA alleles were called with 4-digit resolution using the GATK HLACaller, but due to the high error rates of nanopore reads, HLA alleles did not match with alleles called using HapMap data (Table S3)24.\n\n\nConclusions and discussion\n\nPhasing of genotypes is critical to prevent misinterpretation of PGx variants. The importance of correct phasing of PGx genotypes is illustrated with the gene TPMT, which plays a critical role in the metabolism of thiopurine, a drug used to treat acute lymphoblastic leukemia. In a recent study25, an individual was reported to have a TPMT 3B/3C diplotype, based on observed heterozygous genotypes for the rs1142345 and rs1800460 variants, but this was a misinterpretation due to faulty haplotyping and 1/3A is the correct diplotype. The rs1800460 variant is present in both 3A and 3B haplotypes while the rs1142345 variant is present in both 3A and 3C haplotypes. As a result, it was possible for the individual to have a 1/3A diplotype or a 3B/3C diplotype. Clinically, a 1/3A diplotype corresponds to an intermediate metabolizer, requiring a 30–70% reduction in thiopurine dose, while a 3B/3C diplotype corresponds to a poor metabolizer with a 90% reduction in dose2. An individual who receives a standard dose and is a poor metabolizer can experience fatal toxicity, while a low dose for a normal metabolizer can lead to disease progression. Clinical trials have demonstrated the medical importance of TPMT haplotyping in treatment of myeloid leukemias and non-malignant immunologic disorders2,26.\n\nLong sequence reads aid haplotype identification by determining which genetic variants are in phase (i.e. on the same DNA strand). If the TPMT genotypes from the example above were sequenced using nanopore-based long read technology, the 1/*3A diplotype would likely be called correctly (note that rs1142345 and rs1800460 are only 8,310bp apart).\n\nWhile nanopore sequencing with the MinION is demonstrably error-prone in its current stage of development, we assert that this technology holds promise for clinical applications because accurate consensus sequences can be built with sufficient coverage given the high number of reads generated. As well, we have been able to successfully call haplotypes from long reads de novo in the absence of parental haplotypes or statistical phasing. The MinION device produced sufficiently long mappable reads to phase all variants in the loci examined. As error rates on the MinION decrease, we can expect to deconvolute these data into more accurate diplotypes with less noise and will be able to measure how much multi-sample multiplexing can be supported by a single run.\n\nAccording to the CPIC, 63 genes and 132 drugs have guidelines for pharmacogenomic status (http://www.pharmgkb.org/cpic/pairs), and this list is constantly expanding. With increasing guidelines and demands for PGx in the clinic, affordable and rapid nanopore sequencing may hold great utility.\n\n\nData availability\n\nfigshare: Nanopore reads and alignments, doi: http://dx.doi.org/10.6084/m9.figshare.128971727\n\nRaw nanopore reads and alignment files are available at the NCBI Sequence Read Archive, accession SRP051851 (http://www.ncbi.nlm.nih.gov/sra/?term=SRP051851).", "appendix": "Author contributions\n\n\n\nRA, AS and GDB conceived the study. TAP designed and performed the PCR amplification. RA and DT performed the library preparation. RA performed the sequencing and downstream analysis. RA, TAP and GDB drafted the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nR.A. is a member of the Oxford Nanopore Technologies Inc. MinION Access Programme and the MinION instrument and R7.3 flowcells were received free of charge.\n\n\nGrant information\n\nThis study was funded by a Large-scale Applied Project grant from Genome Canada and the Ontario Genomics Institute (grant ID OGI-068). We confirm that the 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 Stephen Scherer and Peter Ray of the Hospital for Sick Children for contributing diagnostic validation data.\n\n\nSupplementary material\n\nCYP2D6 from sample NA12878 was observed to be diploid.\n\n\nReferences\n\nEvans WE, Hon YY, Bomgaars L, et al.: Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine. J Clin Oncol. 2001; 19(8): 2293–2301. PubMed Abstract\n\nRelling MV, Gardner EE, Sandborn WJ, et al.: Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther. 2011; 89(3): 387–391. 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PubMed Abstract \| Publisher Full Text\n\nFrazer KA, Ballinger DG, Cox DR, et al.: A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007; 449(7164): 851–861. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeckband SG, Kelsoe JR, Dunnenberger HM, et al.: Clinical Pharmacogenetics Implementation Consortium guidelines for HLA-B genotype and carbamazepine dosing. Clin Pharmacol Ther. 2013; 94(3): 324–8. PubMed Abstract \| Free Full Text\n\nMartin MA, Hoffman JM, Freimuth RR, et al.: Clinical Pharmacogenetics Implementation Consortium Guidelines for HLA-B Genotype and Abacavir Dosing: 2014 update. Clin Pharmacol Ther. 2014; 95(5): 499–500. PubMed Abstract \| Free Full Text\n\nHershfield MS, Callaghan JT, Tassaneeyakul W, et al.: Clinical Pharmacogenetics Implementation Consortium guidelines for human leukocyte antigen-B genotype and allopurinol dosing. Clin Pharmacol Ther. 2013; 93(2): 153–8. PubMed Abstract \| Free Full Text\n\nZhou, SF: Polymorphism of human cytochrome P450 2D6 and its clinical significance: part II. Clin Pharmacokinet. 2009; 48(12): 761–804. PubMed Abstract \| Publisher Full Text\n\nCrews KR, Gaedigk A, Dunnenberger HM, et al.: Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update. Clin Pharmacol Ther. 2014; 95(4): 376–82. PubMed Abstract \| Free Full Text\n\nHicks JK, Swen JJ, Thorn CF, et al.: Clinical Pharmacogenetics Implementation Consortium guideline for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants. Clin Pharmacol Ther. 2013; 93(5): 402–8. PubMed Abstract \| Free Full Text\n\nOdelberg SJ, Weiss RB, Hata A, et al.: Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. Nucleic Acids Res. 1995; 23(11): 2049–2057. 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Data Source" }	[ { "id": "7404", "date": "06 Feb 2015", "name": "Martin Kennedy", "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\nGeneral comments:This research note describes preliminary results from the application of a new nanopore sequencing device (the MinIon), under development by Oxford Nanopore Technologies, to the analysis of three amplicons of pharmacogenetic interest.The paper is suitable for a research note. Although the findings are very preliminary, this is a new technology and there is a lot of interest in understanding its current capabilities, longer-term potential and limitations.Positive aspects of the report are description of long-read nanopore sequencing on long amplicons (4-5kb), and description of data handling and bioinformatics approaches this team is using, which may aid others working with the MinIon device. Negative aspects of the report are that all the data are from only one reference sample, and one run on the device; rather than being utterly convincing, the data suggests haplotyping on unknown samples may be possible only once error rates on the MinIon reduce; and there is an unresolved question about the CYP2D6 CNV or haplotype analyses, potentially due to sample contamination.Despite these issues, the report is still of merit and will be of interest to many in the field. Specific comments:MethodsLong PCR has been widely used for specific amplification of CYP2D6, with reaction conditions and primer sequences well established. It is not clear why the authors designed their own primers for this task, or how these novel primers were validated. This should be spelt out more clearly.It would have been useful to have non-diploid control samples for the CYP2D6 CNV assay – for example a haploid (CYP2D65) case or multicopy case, to provide confidence that the assay was working as expected. This is relevant because of the question raised by the 2 haplotype which shows up in the MinIon analyis of NA12878.ResultsPage 6, first para: This description of the possible origins of the mystery CYP2D62 haplotype needs some editing for improved clarity. Not clear what is meant by “3 and *4 duplexes forming”. Also not clear what would cause PCR biases alluded to in the last sentence of this paragraph (and of the following paragraph). Typos/suggested edits:Abstract MinIOn > MinIonShould refer to NA12878 as “reference sample” rather than just “sample”Suggest sentence be modified thus for clarity: “…statistically phased genotype data from Complete Genomics and Sequenom.”Suggest delete “Standalone” in penultimate line.IntroductionSuggest this sentence be changed: However, existing methods have various limitations, which may lead to adverse drug responses. > However, existing methods have various limitations, which may lead to failure to detect variants of pharmacogenetic significance.MethodsPage 3, 2nd para: indels expects > indels expectedPage 4 para 2 – clarify “The HuRef sample…”Figure 1 title: Integrate > IntegrativeResultsPage 5, para 4. First sentence should read thus, for improved clarity: “CYP2D6 haplotype proportions in MinIon data were identified by interrogating clinical marker positions…” Supp File S1It would be helpful to indicate which of the sequences is CYP2D6 and which is CYP2D7.", "responses": [] }, { "id": "7919", "date": "30 Mar 2015", "name": "Thomas Hoenen", "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\nThis manuscript describes the analysis of three PCR-fragments generated from a human reference sample using a MinION sequencing device. Despite the preliminary nature of the data, and the fact that the paper is based on a single run of a single sample, it will nevertheless be of significant interest, and publication as a research note is in my view justified. In fact, given the broad interest in this emerging new technology, and its applicability to all life sciences (not just human (pharmaco-)genetics), the readership might be much wider than anticipated by the authors. This constitutes at the same time the biggest weakness of the paper, since it is in parts rather inaccessible for readers from fields other than human genetics (e.g. virology or microbiology), for whom this paper might nevertheless be highly relevant.In order to address the concern that the manuscript is based on a single MinION run, and given the nature of the MAP program, it should be no problem for the authors to repeat the experiment with another sample, and while doing so specifically address the concerns of Dr. Kennedy. In addition, the authors should strive to improve accessibility to a wider audience wherever possible. Finally, it would be helpful to include additional experimental details that are currently missing.Specific comments:PCR cycling conditions should be provided.The authors refer to the SQK-MAP003 sequencing protocol. As far as I am aware, Oxford Nanotechnologies does not make the detailed protocol available to people outside the MAP program, although there have been indications that a non-technical version of the protocol will be made available for publication purposes. The authors should reference such a protocol (including a link) as soon as possible, and approach ONT about making it available to the general public, if this hasn’t happened already.Did the authors perform a PCR purification prior to the library preparation? If so, what was the volume/ratio of Agencourt beads to sample?How did the authors extract reads from the fast5 files? Did they use poretools, or another tool (which should be referenced)?In general, providing more details about the exact bioinformatics workflow would be helpful.What was the rationale for the cut-off of 1/3 of reads for variant calling?It seems odd that the length of the aligned fragments from the 1D reads is so much shorter than the read length. In our hands (using a similar approach on ~2 kB PCR products amplified from virus genomes, albeit with a later chemistry/protocol version (SQK-MAP004) and using LAST for the alignment) we get much longer average alignments (92% for 2D reads, 82% for template reads, and 85% for complement reads, vs. 85%, 23% and 11% reported by the authors). This could either indicate significant advances in base-calling accuracy since the authors performed their experiments, or that their alignment is suboptimal. It might be very interesting to see whether the authors can get longer alignments using LAST or other alignment softwares in their workflow.It would be very helpful if the authors could repeat the experiment using the newest chemistry/protocol/software versions, which have changed considerably over the last months. At the same time this would allow them to address many of the concerns of Dr. Kennedy.", "responses": [] } ]	1	https://f1000research.com/articles/4-17
https://f1000research.com/articles/4-32/v1	29 Jan 15	{ "type": "Research Article", "title": "Enhancement of COPD biological networks using a web-based collaboration interface", "authors": [ "The sbv IMPROVER project team (in alphabetical order)", "Stéphanie Boué", "Brett Fields", "Julia Hoeng", "Jennifer Park", "Manuel C. Peitsch", "Walter K. Schlage", "Marja Talikka", "The Challenge Best Performers (in alphabetical order)", "Ilona Binenbaum", "Vladimir Bondarenko", "Oleg V. Bulgakov", "Vera Cherkasova", "Norberto Diaz-Diaz", "Larisa Fedorova", "Svetlana Guryanova", "Julia Guzova", "Galina Igorevna Koroleva", "Elena Kozhemyakina", "Rahul Kumar", "Noa Lavid", "Qingxian Lu", "Swapna Menon", "Yael Ouliel", "Samantha C. Peterson", "Alexander Prokhorov", "Edward Sanders", "Sarah Schrier", "Golan Schwaitzer Neta", "Irina Shvydchenko", "Aravind Tallam", "Gema Villa-Fombuena", "John Wu", "Ilya Yudkevich", "Mariya Zelikman", "Stéphanie Boué", "Brett Fields", "Jennifer Park", "Manuel C. Peitsch", "Walter K. Schlage", "Marja Talikka", "Ilona Binenbaum", "Vladimir Bondarenko", "Oleg V. Bulgakov", "Vera Cherkasova", "Norberto Diaz-Diaz", "Larisa Fedorova", "Svetlana Guryanova", "Julia Guzova", "Galina Igorevna Koroleva", "Elena Kozhemyakina", "Rahul Kumar", "Noa Lavid", "Qingxian Lu", "Swapna Menon", "Yael Ouliel", "Samantha C. Peterson", "Alexander Prokhorov", "Edward Sanders", "Sarah Schrier", "Golan Schwaitzer Neta", "Irina Shvydchenko", "Aravind Tallam", "Gema Villa-Fombuena", "John Wu", "Ilya Yudkevich", "Mariya Zelikman" ], "abstract": "The construction and application of biological network models is an approach that offers a holistic way to understand biological processes involved in disease. Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease of the airways for which therapeutic options currently are limited after diagnosis, even in its earliest stage. COPD network models are important tools to better understand the biological components and processes underlying initial disease development. With the increasing amounts of literature that are now available, crowdsourcing approaches offer new forms of collaboration for researchers to review biological findings, which can be applied to the construction and verification of complex biological networks. We report the construction of 50 biological network models relevant to lung biology and early COPD using an integrative systems biology and collaborative crowd-verification approach. By combining traditional literature curation with a data-driven approach that predicts molecular activities from transcriptomics data, we constructed an initial COPD network model set based on a previously published non-diseased lung-relevant model set. The crowd was given the opportunity to enhance and refine the networks on a website (https://bionet.sbvimprover.com/) and to add mechanistic detail, as well as critically review existing evidence and evidence added by other users, so as to enhance the accuracy of the biological representation of the processes captured in the networks. Finally, scientists and experts in the field discussed and refined the networks during an in-person jamboree meeting. Here, we describe examples of the changes made to three of these networks: Neutrophil Signaling, Macrophage Signaling, and Th1-Th2 Signaling. We describe an innovative approach to biological network construction that combines literature and data mining and a crowdsourcing approach to generate a comprehensive set of COPD-relevant models that can be used to help understand the mechanisms related to lung pathobiology. Registered users of the website can freely browse and download the networks.", "keywords": [ "COPD", "Chronic Obstructive Pulmonary Disease", "network model", "signaling pathway", "crowdsourcing", "crowd verification", "jamboree", "online collaboration" ], "content": "Introduction\n\nMolecular networks, such as the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways1,2, aid in understanding the complex interplay of signaling pathways in disease. Biological network models (hereafter referred to as networks) depict the inter-relationships between multiple signaling pathways and how their perturbations may dysregulate biological processes, eventually leading to the disease.\n\nIn previously published reports, we described the construction of a set of 90 networks that captured a large range of biological processes relevant to non-diseased lung tissue3–7. The generation of this set of networks relied on both manual curation of published literature and a data-driven reverse causal reasoning (RCR) methodology8 to augment the causal biological framework underlying the network architecture (Figure 1). We used the Biological Expression Language (BEL) to represent precise biological relationships in a computable and standardized format8. We have built upon this approach and describe here a unique, three-phase systems biology and crowdsourcing approach to construct a comprehensive set of 50 molecular networks that describe the biological processes relevant to chronic obstructive pulmonary disease (COPD) and lung biology (Figure 2). COPD is the fourth leading cause of death worldwide and its incidence is increasing among chronic diseases in the USA9,10. COPD is a chronic, progressive inflammatory disease induced by cigarette smoking, inhalation of pollutants, dust, chemicals, or other foreign matter, which ultimately manifests as tissue destruction in the alveolar compartments and airflow limitation, leading to reduced oxygen exchange11–15. COPD affects a wide spectrum of biological processes in lung tissue, such as oxidative stress, inflammation, apoptosis, proliferation, and senescence16,17. Understanding the mechanisms involved in these processes is important in understanding the onset of the disease and in identifying drug targets to develop effective COPD treatments18,19. As recently reported by the Global Initiative for Chronic Obstructive Lung Disease (GOLD), current pharmacologic therapies cannot cure the disease but only reduce the symptoms, and the frequency and severity of exacerbations, i.e., slow down the rate of disease progression11; thus it appears most efficient to target the COPD-specific pathomechanisms at the earliest distinguishable state, when the extent of irreversible damage is still small, and their molecular processes are not yet convoluted with secondary processes and comorbidities, e.g., bacterial and viral infections, as they occur during the exacerbations typical for later stages of COPD. Since smoking cessation/replacement appears to be the most efficient therapy in smoking-related COPD11, the models of early onset COPD can also be expected to be valuable tools for the development and testing of reduced risk products that may prevent COPD progression in a comparable manner as cessation does.\n\nNetworks were constructed using published literature and data sets, and opened to the public for comment and editing in the Network Verification Challenge. The three phases of COPD network construction are shown. (A and B) Phase 1: COPD augmentation using literature and data. (C and D) Phase 2: Online verification by the public during an “open phase”, and Phase 3: Face-to-face jamboree meeting where scientists and subject matter experts gathered to discuss the networks and make final decisions for the next versions.\n\nThe networks reported here were created first from a literature scaffold and expanded via data enhancement using RCR (Phase 1), then they were made available online to the entire scientific community for critical review during the Network Verification Challenge (NVC) “Open Phase” (Phase 2) under the umbrella of the systems biology verification (sbv) IMPROVER project20 (Figure 1). Finally, a prioritized subset of 15 of these networks was discussed during an in-person jamboree meeting where the crowd-submitted revisions were reviewed and decisions to improve the networks were finalized (Phase 3). The final versions of the networks are available at https://bionet.sbvimprover.com for the public to view, and for registered users in the NVC to continue to discuss.\n\nA variety of COPD networks have been created by various research groups, including networks focused on muscle to study skeletal muscle abnormalities21, networks to compare COPD and asthma22, and a knowledge management framework to integrate COPD clinical and experimental data23. To our knowledge, this is the first set of crowd-verified networks available to the broader scientific community as a unified collection on a freely accessible web-based platform. Ultimately, this interface will allow for continuous input and improvement in the networks, leading to better understanding, diagnosis, and treatment of COPD.\n\n\nMethods\n\n\n\nPhase 1: COPD enhancements using data and literature\n\nNinety non-diseased lung networks published previously in the areas of cell proliferation, cell stress, inflammation, DNA damage, cell death, tissue repair, and angiogenesis were used as the initial scaffolds for COPD enhancement during Phase 13–7. Biological pathways implicated in COPD disease pathophysiology, including B-cell and T-cell activation, airway remodeling, extracellular matrix (ECM) degradation, efferocytosis, mucus hypersecretion, and emphysema were all captured within the modified network models. In total, 200 new nodes and 487 new edges were added: 415 of the edges were added to incorporate COPD mechanisms implicated in the literature, and 72 edges were added to incorporate 100 mechanisms predicted from COPD data by RCR to be relevant to COPD (Figure 3). Because the models were built to represent COPD in humans, human evidence was preferred and made up the majority of the networks (74%).\n\nSummary of nodes and edges added to all networks and to three example networks in each phase. A) Nodes added in each phase. B) Edges added in each phase.\n\nDuring Phase 1, the networks with the most significant number of COPD enhancements in terms of percentage of the network with new nodes were the Mucus Hypersecretion (44%), Th2 Signaling (37%), Macrophage Activation (28%), Fibrosis (25%), Autophagy (11%), and Apoptosis (5%) networks. Networks that were not enhanced with COPD-specific mechanisms from the literature or RCR included the DNA Damage and Notch Signaling networks. Although both these networks relevant to the development of COPD, they were not augmented beyond the original, non-diseased network scaffolds, because no studies on the differences in signaling between non-diseased and diseased states were available.\n\nPhase 2: Networks enhanced with lung- and COPD-relevant mechanisms by the crowd during the open phase\n\nPrior to deploying the COPD-enhanced biological networks on the NVC website for verification by the scientific community, the set of 90 networks was agglomerated by the model-building expert team to yield a more concise set of 50 networks that combined and standardized related/complementary cellular pathways (See Methods for details). For example, a new “Th1 Signaling” network model was created by merging three of the original networks that were relevant to the functional biology present in T-helper 1 cell populations: Th1 Differentiation, Th1 Response, and T-cell Recruitment and Activation. For a list of the original models that correspond to the agglomerated models and a description of the new models, see Dataset.\n\nDuring Phase 2, a global community of scientists participated in the NVC by contributing their expertise to one or several of the network models. Scientists could contribute by verifying existing evidence for network edges using a system that allowed users to vote on evidence to indicate agreement or disagreement with its appropriateness within the network structure and boundary conditions. Participants were also encouraged to add new mechanistic biology in the form of network edges. In total, the 50 network models received 2456 evidence votes, 1795 of which supported the confirmation of evidence and 661 that favored the rejection of evidence (see Dataset). The Neutrophil Signaling network model received the largest share of voting activity, with 241 total votes or approximately 10% of all votes cast. Other network models that received large shares of the votes included the Macrophage Signaling (180 votes) and Th1 and Th2 Signaling network models (105 votes) (see Dataset). In addition to verifying existing literature evidence supporting edges in the network models, NVC participants could add novel biological information in the form of new literature evidence (for an existing edge) or contribute new network edges to incorporate new biological components into the network structure. In this way, the community of participants collectively contributed a significant amount of new information into the networks; among the 50 network models, a total of 885 new pieces of evidence, 351 new nodes, and 451 new edges were added (Figure 3).\n\nPhase 3: Jamboree discussion and final decisions for next version networks\n\nFollowing Phase 2, a jamboree (Phase 3) was organized for a group of invited participants to discuss the network enhancements submitted by the crowd. To represent the crowd community, the top 20 active performers who created the most pieces of evidence and submitted at least 20 votes during the NVC were invited to an in-person jamboree to discuss network refinements as a group. Additional subject matter experts in the network biology, COPD, lung biology, and biological processes represented by the networks were invited to participate in the discussions and contribute their expert feedback independent from the network-building experts. Among the 50 network models evaluated during the online NVC, 15 were prioritized and selected for discussion during Phase 3 based on the level of crowd-sourced activity and their importance in COPD onset as considered by the network-building experts (see Dataset). The goal of Phase 3 was to provide an additional layer of “verification” for the online enhancements and to provide holistic comments on the network models at the molecular/biological entity level. In doing so, the three network models that had received the largest amounts of crowd activity (Neutrophil Signaling, Macrophage Signaling, and Th1 Signaling) also underwent significant additional enhancements to improve granularity with respect to COPD onset and pathogenesis. In total, 167 nodes and 296 edges were added among all the network models reviewed during the jamboree sessions, and the three inflammatory networks received 89% of the nodes and 89% of the edges (148 nodes and 263 edges) (Figure 3). Many of these changes came from the identification of missing mechanistic details of processes that occur in COPD (e.g. chemotaxis mechanisms in the Macrophage Signaling network model described in the examples in the “Macrophage signaling” section below).\n\nIn addition to adding mechanistic details of processes that occur in COPD, enhancements were incorporated to improve the granularity and connectivity within the network structures. In several instances, the improvements involved the creation of more detailed linear pathways connecting biological components. In one example, in the Apoptosis network model, the original network pathway indicated that the X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6) protein decreased the process of apoptosis24. During the Phase 3 discussions, additional literature evidence provided a more detailed mechanistic understanding of this phenomenon: XRCC6 was reported to decrease the activity of the BCL2-associated X protein (BAX) protein, which is known to increase mitochondrial permeability and therefore promote apoptosis (Figure 4A). The overall effect of the negative regulation of BAX by XRCC6 was therefore a decrease in apoptotic cell death25. By improving the granularity of this pathway in the Apoptosis network, a more comprehensive representation was achieved for components that are related to critical cellular processes mediating disease onset.\n\nDuring Phase 3 of COPD network construction, improvements were made by adding mechanistic details to over-simplistic edges. A) In the Apoptosis network model, the original connection (left) simply indicated that XRCC6 decreased the process of apoptosis. The improved pathway connection (right) indicates that XRCC6 decreases the activity of BAX, which normally functions to facilitate the transport of calcium ions through the mitochondrial pores and thereby increases apoptosis. B) In the Mechanisms of Cellular Senescence network model, the original connection (left) simply indicated that acrolein increased the process of cellular senescence. The improved pathway connection (right) indicates acrolein mediates its effects on senescence via the activity of SIRT1 and the FOXO3 transcription factor. Triangle denotes activity, diamond denotes biological process or pathology, circle denotes abundance, rounded square represents transport, and square denotes protein abundance nodes. Solid edges denote causal relationships, dotted edges denote non-causal relationships such as a protein connected to its own activity.\n\nA similar improvement was incorporated into the Mechanisms of Cellular Senescence network model: the original network pathway indicated that the chemical acrolein (a common component of cigarette smoke) increased cell senescence26. During Phase 3 discussions, the pathway connecting these two components was expanded using additional literature evidence. In several studies, acrolein was found to decrease the activity of sirtuin 1 (SIRT1), which is a known negative regulator of the forkhead box O3 (FOXO3) transcription factor, and FOXO3 activity is known to promote cellular senescence (Figure 4B)26–28. Therefore, the overall observed effect was acrolein acting to potentiate cellular senescence in exposed cells, which is a well-characterized mechanism of action for this toxic chemical. Again, the generation of more comprehensive network models of biological processes in close proximity to disease onset allowed for a greater mechanistic understanding of how environmental factors can contribute to COPD development.\n\n\nExemplary outcomes of the three-phase COPD network building process\n\nAs part of the pulmonary inflammatory process network building6, five networks (T-cell activation and recruitment, Th1 differentiation, Th2 differentiation, Th1 Response, Th2 response) were built to describe Th1 and Th2 signaling in the non-disease lung context. As described previously, during the preparation phase to NVC, two networks were built around the Th1 and Th2 cells.\n\nPhase 1: COPD augmentation of T-helper cell networks\n\nMechanisms that describe T-cell activation and recruitment induced by neutrophils, macrophages, and dendritic cells were added to the T-cell networks during Phase 1. These immune cells secrete various chemokines that were reported to recruit T-cell populations (i.e. CD8+ cytotoxic T-cells) to injured tissue in an acute inflammatory state29. Alveolar macrophages secrete interleukin 15 (IL15), which is capable of activating both the interleukin 2 (IL2) and IL15 receptors on T-cells and acts as a potent inducer of cell migration to the lung. Dendritic cells within the lung play an important role in this process by secreting chemokine (C-C motif) ligand 3 (CCL3) in response to cigarette smoke, which helps recruit CD8+ T-cells to the lung29. Chemokine (C-C motif) receptor 5 (CCR5) is the receptor for CCL3 and its presence in the lung has been shown to correlate with the severity of COPD30. CCL3 is one example of a node that was added during the literature-based COPD enhancement process in Phase 1 (Figure 5A). Many of the disease-relevant mechanisms identified in the literature curation phase were corroborated by mechanisms predicted from COPD-relevant data sets using RCR (see Methods), including T-cell activation mechanisms (CD28 molecule (CD28) and T cell receptor beta locus (T\\RB), and chemokines and cytokines that activate and are secreted by T-cells (chemokine (C-C motif) receptor 3 (CCR3), CCR5, IL2, interleukin 4 (IL4), interleukin 6 (IL6), interleukin 10 (IL10) and interleukin 13 (IL13)). The prediction of these mechanisms in COPD data sets showed that T-cell activation and migration in response to smoke-exposed lung represents an important process in the innate immune response. In total, 30 nodes and 34 edges were added to the Th1 and Th2 networks during the internal COPD enhancement process.\n\nA) During the literature-based COPD enhancement process in Phase 1, the protein CCL3, important for leukocyte migration and activation of T-cells, was added to the T-cell networks. B) During the open phase in Phase 2, the negative regulation of EGR2 on T-cell activation is a mechanistic detail that was added by the crowd. Overexpression studies demonstrated that EGR2 increased the activity of the E3 ubiquitin ligase CBL-B, which subsequently inhibited T-cell activation. C) During the jamboree discussions in Phase 3, the IFNG/IL-4 feedback loop mediating differentiation of Th1 vs. Th2 cellular subtypes via the activities of IRF1 and IRF2 was added to the new Th1-2 Signaling network model. D) During the jamboree discussions in Phase 3, the T-helper cell-produced chemokine effect on immune cells (e.g. IL-25 activates memory T-cells) was added to the new Th1-2 Signaling network. Triangle denotes activity, diamond denotes biological process or pathology, and square denotes protein abundance nodes. Solid edges denote causal relationships, dotted edges denote non-causal relationships such as a protein connected to its own activity.\n\nPhases 2 and 3: T-cell network crowd improvements\n\nDuring the open phase (Phase 2), the Th1 and Th2 networks received 105 votes from the scientific community, as well as 10 new nodes, 9 new edges, and 13 new pieces of evidence. One such addition to the Th1 Signaling network was the regulatory influence of early growth response 2 (EGR2) on T-cell activation; the submitted evidence demonstrated that overexpression of EGR2 promoted increased activity of the E3 ubiquitin ligase CBL-B and subsequent inhibition of T-cell activation31 (Figure 5B).\n\nDuring the Phase 3 jamboree sessions, the group decided to combine the individual Th1 Signaling and Th2 Signaling networks into a single, unified network model titled Th1-Th2 Signaling to better represent the interplay between the T-helper cell populations in vivo. It was also decided to add granularity to transcriptional pathways mediating Th1 versus Th2 cellular activation and differentiation; one example was the addition of two transcription factors, interferon regulatory factors 1 and 2 (IRF1 and IRF2), that are known to act downstream of interferon-gamma (IFNG) to suppress IL4 expression in Th2 cell populations32. IFNG is secreted by Th1 cells and this pathway potentiates Th1 responses while suppressing Th2 responses in the tissue. The addition of this feedback mechanism during Phase 3 contributed to a more comprehensive network describing the interactions between Th1 and Th2 cells (Figure 5C). Further network enhancements discussed in the jamboree largely emphasized the downstream effects of T-helper cells in potentiating inflammatory signaling by activating additional immune cells in a disease context. For example, secretion of IL5 activates eosinophils, whereas secretion of IL10 and IFNG activates macrophages in the diseased tissue33–35. This interplay between immune cell populations was incorporated into the new Th1-Th2 Signaling network model and better captures the signaling interconnectivity present during disease development (Figure 5D). In total, 12 new nodes and 28 new edges were added to the Th1-Th2 Signaling network model during the jamboree discussions, thereby creating a comprehensive biological network of T-helper cell activity and their interactions with other immune cells in the context of COPD.\n\nAs part of the pulmonary inflammatory process network building6, three networks (Macrophage Differentiation, Macrophage Activation, and Macrophage-mediated Recruitment of Neutrophils) were built to describe macrophage biology in the non-disease lung context. During the preparation phase to NVC, these three networks were merged to obtain an overall picture of macrophage biology.\n\nPhase 1: COPD augmentation of macrophage networks\n\nMacrophages play roles in many COPD disease processes such as clearance of apoptotic neutrophils, tissue destruction, and recruitment of other immune cells by their secretion of cytokines36. Macrophage signaling mechanisms were added to the network in Phase 1, with a focus on components related to efferocytosis (Figure 6A). Efferocytosis is a well-conserved mechanism for the phagocytic removal of apoptotic cells by innate immune cells, such as macrophages, and the process is critical for the resolution of inflammation via the removal of dying cells and antigenic cellular debris. Phagocytically impaired macrophages have been shown to display decreased expression of peroxisome proliferator-activated receptor gamma (PPARy) and efferocytosis-specific bridge molecules, such as growth arrest-specific 6 (GAS6) and milk fat globule-EGF factor 8 protein (MFGE8)37. The number of apoptotic cells was shown to increase in COPD because of exposure of lung tissue to toxic chemicals present in cigarette smoke; for example, and their accumulation was exacerbated by the simultaneous smoke-induced impairment of the phagocytic ability of alveolar macrophages38. Apoptotic cells exhibit surface changes that distinguish them from viable cells, and these changes were recognized by efferocytic receptors including CD36 molecule (CD36), CD14 molecule (CD14), and Stabilin-1/2 (STAB1:STAB2)39. Reduced efferocytosis observed in COPD because of oxidant-driven and Rho-mediated inactivation increased the likelihood of aberrant antigen exposure from apoptotic cells, thereby perpetuating the chronic inflammatory state that is a hallmark of COPD40–42. In adding efferocytosis mechanisms to the macrophage network, we focused on the surface receptors and bridge proteins such as CD36 and GAS6. In total, 45 nodes and 61 new edges were added to the macrophage model during the internal COPD enhancement phase.\n\nA) During the literature-based COPD enhancement process in Phase 1, efferocytosis mechanisms were added to the macrophage networks to take into account its dysregulation effect in COPD. B) During the jamboree discussions in Phase 3, chemotaxis and differentiation mechanisms were identified and subsequently added to the latest version of the Macrophage Signaling network. Triangle denotes activity, diamond denotes biological process, and square denotes protein abundance nodes. Solid edges denote causal relationships, dotted edges denote non-causal relationships such as a protein connected to its own activity.\n\nPhases 2 and 3: Macrophage network crowd improvements\n\nDuring the open phase (Phase 2), 180 total votes were cast for network evidence, with 23 new nodes and 39 new edges added by the crowd. In addition, 72 new pieces of evidence were contributed to support pre-existing edges in the network. The surfactant protein A1 (SFTPA1), which was observed to be increased in COPD43, was added to the network. Its effect on macrophages of increasing interleukin-1 receptor-associated kinase 3 (IRAK3) and interleukin 1, beta (IL1B) were also added to the network during the open phase. Granularity enhancements around IFNG and nucleotide-binding oligomerization domain containing 2 (NOD2), both components of inflammatory signaling, were also added to augment the network models with causal relationships proximal to COPD.\n\nDuring the Phase 3 jamboree discussions, several network enhancements were made in macrophage chemotaxis and differentiation (Figure 6B). Within the chemotaxis process, the nodes chemokine (C-C motif) ligand 2 (CCL2) binding to chemokine (C-C motif) receptor 2 (CCR2) and leading to macrophage chemotaxis were added. The CD69 molecule (CD69) associated with macrophage activation by cigarette smoke was also added. In addition, the effects of activated macrophages on other immune cells were expanded within the network model, including chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-X-C motif) ligand 2 (CXCL2) leading to neutrophil chemotaxis, and chemokine (C-X-C motif) ligand 9 (CXCL9) and chemokine (C-X-C motif) ligand 10 (CXCL10) binding to CXCR3 and leading to T cell recruitment. In total, 30 new nodes and 48 new edges were added to the Macrophage Signaling network during Phase 3, thereby providing a more comprehensive network of macrophage activation and its effect on other immune cells active in COPD.\n\nAs part of the pulmonary inflammatory process network building6, two networks (Neutrophil Response and Neutrophil Chemotaxis) were built to describe neutrophil biology in the non-disease lung context. During the preparation phase to NVC, these two networks were merged to constitute the Neutrophil Signaling network.\n\nPhase 1: COPD augmentation of neutrophil networks\n\nDuring Phase 1, the Neutrophil Signaling network was enhanced primarily with components related to lipid-response pathways. In response to lung damage, leukocytes and tissue-resident cells were reported to interact to generate lipid mediators that enhance the airway immune response and engage defense mechanisms44. Neutrophils, endothelial cells, and macrophages generate prostaglandins and leukotrienes from arachidonic acid during the initial inflammatory response, which amplifies the inflammation signals in the local area and potentiates the process of tissue destruction45. Subsequently, the prostaglandins PGE2 and PGD2 are generated in a cyclooxygenase-dependent way to promote synthesis of lipid mediators with anti-inflammatory activity, such as the lipoxins. Lipoxins inhibit neutrophil recruitment to inflamed sites and suppress their pro-inflammatory actions, but promote recruitment of macrophage precursors46. Lipoxin A4 stimulates macrophages to phagocytose apoptotic neutrophils, and resolvins and protectins, which represent another class of lipid mediators, activate anti-inflammatory pathways and stimulate clearance of inflammatory infiltrates by macrophage phagocytosis47–49. In total, 9 nodes and 20 edges were added to the network model including lipid mediators such as lipoxin A4, resolvin E1, and neuroprotectin D1 (Figure 7A).\n\nA) During the literature-based COPD enhancement process in Phase 1, lipids and their effects on neutrophil chemotaxis were added to the new Neutrophil Signaling network. B) During Phases 2 and 3, neutrophil adhesion and chemotaxis mechanisms were added to the new Neutrophil Signaling network. Triangle denotes activity, diamond denotes biological process, circle denotes abundance, and square denotes protein abundance nodes. Solid edges denote causal relationships, dotted edges denote non-causal relationships such as a protein connected to its own activity.\n\nPhases 2 and 3: Neutrophil network crowd improvements\n\nThe Neutrophil Signaling network was the network most edited by the crowd during the open phase, with the addition of 116 new nodes, 160 new edges, 181 new pieces of evidence, and 241 votes cast. The new edges described neutrophil chemotaxis including new nodes like platelet factor 4 (PF4) and protease-activated receptor 2 (F2RL1). Chemokines such as chemokine (C-X-C motif) ligand 8 (CXCL8) and chemokine (C-X-C motif) ligand 12 (CXCL12), and members of the serine/threonine kinase (AKT) family that have also been shown to induce neutrophil chemotaxis were added to the network (Figure 7B)50.\n\nFollowing the jamboree discussions, additional signaling that described cytoskeletal and adhesion mechanisms necessary for neutrophil chemotaxis, and additional neutrophil activation mechanisms, were incorporated in the new Neutrophil Signaling network (Figure 7B). The role of the CDC42-WASp complex in regulating neutrophil chemotaxis at the cytoskeletal level was incorporated51, as well as other mechanisms of neutrophil chemotaxis including the role of the complement component 5 (C5) in regulating integrin, alpha M (ITGAM)52, and the role of CCL3/CCR5 in stimulating neutrophil migration53. In all, 69 nodes and 129 edges were added. The new mechanisms that were incorporated into the Neutrophil Signaling network added significant granularity to the neutrophil chemotaxis process, which is a key driver of the inflammatory cascade that promotes the development of COPD.\n\n\nDiscussion\n\nHere we report the construction of a COPD-enhanced network model set using a novel methodology that combined traditional manual literature curation and data-driven approaches with a global crowdsourcing endeavor to generate the most comprehensive representation of biological phenomenon proximal to the onset of COPD that is available to date. The three phases of network construction each contributed in different ways to building a more comprehensive network. The Phase 1 literature and data-driven enhancement of the already existing non-diseased networks resulted in the addition of COPD biomarkers and disease drivers known to be associated with COPD, while the Phase 2 crowdsourcing largely focused on contributions to cell-specific networks, and the Phase 3 jamboree discussions uncovered missing signaling processes relevant to COPD.\n\nDuring Phase 1, the non-diseased networks were expanded within the COPD context by the addition of biomarkers, disease drivers, and processes that were reported to increase in COPD, as well as mechanisms predicted in COPD data sets. Most of the edges added to the networks were lung relevant but not specifically investigated in a COPD background. Because of the limited number of mechanistic studies in COPD models that have been published, network construction was focused on adding COPD-known processes and biomarkers in tissue and experimental contexts relevant for COPD (lung, smoking) to the existing non-disease networks.\n\nModeling the process of efferocytosis is an example of the addition of COPD processes to the non-disease networks. The efferocytosis process of phagocytic uptake of apoptotic cells by macrophages is frequently disrupted in COPD tissue, and this disruption is thought to potentiate the chronic state of inflammation in the diseased lung40–42. A new network model detailing components related to efferocytosis was constructed from information available in the published literature with the majority of edges coming from general macrophage experiments. Th2 activation cascades and macrophage signaling events were also implicated generally in the context of COPD, and therefore the non-diseased network models were enhanced by the addition of these pathways from lung-relevant studies. Network models detailing other processes not widely implicated in COPD, such as DNA damage and Notch signaling, which are more generalized conserved biological phenomenon, received very few, if any, enhancements during the COPD literature curation phase.\n\nIn addition to adding COPD processes during Phase 1, we also added COPD biomarkers and mechanisms predicted by RCR to be active in COPD data sets. Biomarkers associated with COPD included chemokines, cytokines, matrix metalloproteinases (MMPs), and other matrix degradation products. Examples of cellular mechanisms uncovered by the data-driven approach included the cytokines IL19 and IL3, as well as the serine protease inhibitor SERPINA1. IL3 is a growth-stimulating cytokine for many inflammatory cells, including macrophages, and IL19 is produced by monocytes and activates the inflammatory STAT3 pathway in several cell types. SERPINA1 is a potent elastase inhibitor, the presence of which plays a critical role in controlling the protease cascade leading to tissue destruction and emphysema. Overall, the RCR approach yielded a diverse range of biological features that were incorporated among a large percentage of the network models, thereby broadening the scope of many networks to include components with potential connections to disease that have not been investigated previously in the COPD context.\n\nDuring the NVC, scientists from around the world browsed the publically available networks on a website, voted on and submitted new evidence, and created new nodes and edges. As may have been expected, several of the more well-studied processes in the literature (e.g. NF-kB pathways leading to inflammatory signaling) attracted a great deal of voting activity within the networks and primarily corroborated known biology. However, participants were incentivized to create new evidence to support existing edges based on the large number of points received by them for this activity. It was this aspect of the challenge that truly demonstrated the power of crowdsourcing because, in many instances, the community of users located lung-relevant and/or more recent publications to better support the existing network architecture and improve the overall relevance of the network models to COPD. With nearly 900 new pieces of evidence added by the challenge crowd, a significant overall enhancement of the networks was achieved in a relatively short time (5 months), which demonstrated the remarkable utility of harnessing knowledge from the global scientific community for a specific application. Specifically, 30% (266/885) of all the new pieces of evidence and 46% (208/451) of all the new edges that were contributed fell within three network models, namely the Neutrophil Signaling, Macrophage Signaling, and Th1-Th2 Signaling networks. These networks were edited more than other networks because of their clear boundaries, which allowed scientists to narrow their search to a particular cell type. Networks such as Clock, Wnt, mTor, and Regulation of CDKN2A expression were edited minimally and received more ‘Down’ votes than the cell-specific networks, possibly because of the more ambiguous boundaries of which cell types could be included. This observation emphasizes the need for clear boundaries in a crowdsourcing effort. In the case of general networks such as Cell Cycle, Response to DNA Damage, and Oxidative Stress, many experiments concerning these processes have been performed in cell types that were excluded in our boundaries (i.e. tumorigenic cell lines). Perhaps boundary conditions could be loosened for networks such as these if it is assumed that signaling is conserved across different cell types.\n\nThe final phase of network improvements emphasized the discussion and consolidation of all submissions from the challenge crowd to synthesize more holistic changes within the set of network models. During the challenge, participants worked individually on the website adding individual edges, but did not have the ability to make major changes to the structure of the network models. The in-person jamboree discussions were therefore an opportunity to implement broader changes to better represent the biological processes as they related to COPD. These discussions were led by experts in the subject matter of the processes that the networks represented. During these sessions, missing pieces of biology and the interactions of different cell types in COPD were identified. In this manner, the jamboree was very conducive to broader network structural changes that made the set of network models more informative and representative of processes implicated in COPD and, therefore, more useful to a broader group of scientists.\n\nIn recent years, crowdsourcing has emerged as a powerful tool to address topics related to “big data” in the domain of the life sciences, particularly in topics related to systems biology. For example, the series of DREAM challenges empowered the global scientific community to build application-specific, clinically relevant predictive biological networks using vast quantities of genomic data54. Similarly, the recent sbv IMPROVER challenges allowed researchers to participate in collaborative competitions to validate systems biology research, for example, by testing and validating computational approaches that are used to classify clinical samples based on transcriptional data55–57. In the current approach, we describe a unique paradigm for biological network construction that combines a predictive computational methodology with a large-scale crowd sourcing approach to generate very comprehensive network models describing COPD pathogenesis.\n\nCompared with other published COPD networks, the networks described here are more comprehensive in scope, are focused on molecular pathways that can drive disease rather than on descriptions of more general clinical or physiological measures, and have been improved using crowdsourcing21–23. The Synergy-COPD European project is similar in its goal of creating a model of COPD for better understanding of the disease by combining information from many different sources. However, Synergy-COPD comprises seven physiological-focused mathematical networks rather than the 50 molecular networks described here, and does not currently have an intuitive web interface that allows users to freely navigate the resulting networks23.\n\nCompared with other more general pathway approaches such as KEGG1, the networks we describe contain edges that have one or more detailed evidences supported by a specific literature reference and contain tissue and species-level metadata. In our approach each of these pieces of evidence under an edge can be validated with the potential for a larger crowd with wide expertise, compared to a non-crowdsourced approach where the small group constructing the networks may not be able to sufficiently cover all the expertise necessary to verify every pathway within these networks. The BEL language syntax allows many participants to contribute by standardizing the biological representation and requiring that each node be associated with a namespace, which standardizes the representation of gene names and biological processes.\n\nThe web-based platform captures network provenance, allowing for a transparent record of what has been validated with a full revision history. The uncertainty for specific edges based on voting patterns can be demonstrated with the full voting history being captured in the network versions. By incorporating a continuous “feed” of real time enhancements submitted on the website, users are able to view the most up-to-date networks at any time; network models created using other platforms not available for crowdsourced editing remain static representations of biology and frequently do not include the most recent findings from the scientific literature. Currently networks with the most recent crowd edits can be viewed, but not downloaded. Networks with changes from the most recent Jamboree meeting are made available for download.\n\nAnother novel component of these networks is the incorporation of RCR predictions to enhance the overall biological representation within the network models. RCR analysis was performed on human COPD gene expression data sets in the public domain in order to predict potential mechanisms implicated in COPD onset and include as nodes in the networks. This unbiased approach resulted in the addition of many new nodes among the networks predicted to be active based on COPD gene expression footprints that may have less well-established or direct connections to disease etiology. As such, this important aspect of network construction potentially captures those biological components that may have “emerging” roles in disease progression. The iterative nature of the network enhancement process facilitated by the Bionet platform allows for new biology and supporting evidence to be incorporated into the networks as new findings emerge in the literature and therefore generate the most comprehensive, up-to-date COPD model sets available to the scientific community.\n\nThe enhanced crowd-verified models are publicly available on the sbv IMPROVER website (https://bionet.sbvimprover.com/) and remain open to receive further enhancements from the online community. Because the first iteration of the NVC proved the effectiveness of this approach and because the networks can continue to be reviewed by the crowd, a second iteration of the NVC (NVC2) has been started so that additional modifications and recently published literature can be incorporated. This will help to continually refine the network models and strengthen the relevance to the processes that underlie the development of COPD. The crowd verification approach continues to be refined, so, in addition to disease process-centered networks, other networks including chemical-centered networks can be built using a similar approach. These networks can aid in the development of more efficient interventions and enhance toxicological assessment of environmental exposures that may also contribute to the development of COPD.\n\n\nConclusion\n\nHere we describe a novel approach to biological network construction and have generated a suite of COPD-relevant network models that the larger scientific community is free to edit and explore. Networks are available for download from the sbv IMPROVER website (https://bionet.sbvimprover.com/) upon registration and taking a certain number of actions as a participant (e.g., voting on an evidence). Scientists from all backgrounds are encouraged to submit additional network enhancements as participants in the NVC258. By building the network model set in the BEL language format, we have generated a model framework suitable for biomarker discovery and for the interpretation of transcriptomic signatures found in human lung tissue. More generally, this large assembly of biological knowledge relevant to human lung will be of great use to both academic and industry users in promoting future research in this area of great therapeutic importance.\n\n\nMethods\n\nNetworks that described molecular mechanisms of five broad biological processes were constructed previously using a literature and data mining approach. These networks cover mechanisms of cell proliferation5, cell stress4, DNA damage, autophagy, cell death and senescence3, pulmonary inflammation6, and tissue repair and angiogenesis7 in the non-diseased pulmonary context. To create COPD-relevant networks, these non-diseased networks were enhanced by incorporating COPD mechanisms sourced using a literature and data set approach (Figure 1) in an iterative approach, as described in detail for the non-diseased network model construction, by a team of subject matter experts in computational biology, molecular biology, inhalation toxicology, and COPD.\n\nBecause the goal of the research was to understand COPD onset, the focus of these networks was on early stage COPD mechanisms (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I and II). When supporting literature from early COPD studies was not available, stage-independent COPD studies were used. When COPD studies were not found, the inclusion criteria were expanded to studies from non-diseased context, and mechanisms active in processes implicated in COPD were incorporated into the disease models. Literature describing the processes active in acute exacerbation in COPD patients was excluded from the supporting edges of the network models. In order to focus on the molecular mechanisms most specific to early stage COPD, we also excluded context from diseases with different pathogenesis and differential diagnosis: lung cancer and non-cancerous lung diseases, such as cystic fibrosis, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, septic pneumonitis, obliterative bronchiolitis, pneumoconiosis, bronchiectasis, viral and bacterial infections, and, allergic responses/asthma, bronchitis. Animal inhalation studies with solid particles (e.g. titanium dioxide, quartz, asbestos, carbon black, and diesel exhaust) were also excluded due to their specific mode of action. Ideally, all nodes and edges of the network model would be supported by published data from experiments conducted in the tissues and cell types found in the lung under the conditions of early COPD, e.g., airway and alveolar epithelial cells, lung fibroblasts, resident and recruited immune cells, and microvascular cells. These were prioritized but the respective cell types were also considered from other tissue origin if such lung specific context was not reported in the literature. For in vitro-specific exclusion criteria, tumor-derived cell lines, immortalized cell lines, neuronal cells, and cell types that are not found in the respiratory/vascular system were excluded. In some cases, we made exceptions and included non-lung cell types for canonical mechanisms for which there was additional evidence from the literature that the relationship was not tissue-specific but could also take place in the lung. Human-specific connections were prioritized, but where human data were not available, knowledge has been augmented with orthologous causal assertions derived from rat and mouse sources included after homologization in the Selventa knowledgebase where human data were not available5.\n\nThe 90 previously published non-diseased network models used for the initial substrate included networks involved in cell proliferation5, cell stress4, DNA damage, apoptosis, senescence, autophagy, necroptosis (DACS)3, pulmonary inflammation (IPN)6, and tissue repair and angiogenesis (TRAG)7. The Endothelial Shear Stress network from the cell stress model was excluded because the focus of the COPD Network was to describe lung biology.\n\nWe conducted a broad survey of the literature to locate studies that had investigated the mechanistic biology of COPD pathogenesis and processes involved in COPD. Potential COPD biomarkers from sputum, bronchoalveolar lavage, and mouse and human blood samples, and mechanisms that regulate COPD processes were gathered from the literature and curated. Because only a small number of the studies had focused on early COPD, we expanded our searches to include stage-independent COPD studies, but excluded late-stage processes. Some processes known to be closely linked to COPD pathogenesis (e.g. B-cell activation and T-cell recruitment to lung tissue) have not been studied directly in the disease context; however, literature that detailed cell-type-specific canonical biology was sourced irrespective of the disease context.\n\nRCR was performed using Gene Expression Omnibus (GEO) COPD and emphysema data sets from lung, small airway, and alveolar macrophages of early COPD patients and healthy smokers (see Dataset)59–63. RCR has been used previously to predict upstream regulators from transcriptomic data8. Mechanisms that were predicted by RCR to be active and that were not already incorporated in the non-diseased networks were vetted on an individual basis to locate supporting literature for their potential involvement in COPD pathogenesis. Mechanisms that had not been studied directly in a COPD context were evaluated in an expanded tissue context to consider tissue deemed disease-relevant (e.g. alveolar macrophages). Mechanisms that were deemed relevant were connected in the most appropriate network based on their probable roles in COPD or lung biology.\n\nTo generate a more concise model set for presentation to the crowd during the NVC, we consolidated networks associated with related biological processes among the 90 COPD-enhanced networks. An example of this consolidation is the merging of three non-disease networks related to T-helper 1 cells (Th1 Differentiation, Th1 Response, and T-cell Recruitment/Activation) into a single new Th1 Signaling network. Fifty-six of the original 90 networks were combined into a concise set of 16 network models; the remaining 34 networks remained as standalone network models (see Dataset), yielding a final set of 50 models that were posted on the NVC website for review by the scientific crowd. In addition to the network agglomeration, protein, gene expression, and secretion edges were agglomerated to reduce the number of edges required for verification.\n\nThe crowd verification process of improving biological networks has been published previously20. Briefly, the full set of 50 COPD-relevant network models was posted on the BioNet web portal58 for a period of 20 weeks (the “Open Phase”), during which time a global community of participants were invited to submit biological improvements to the models. The improvements included submission of new evidence, additional literature publications to support existing network edges, and submission of new biological edges with supporting evidence for relationships that were not represented in a network. Users could also vote on evidence to indicate agreement or disagreement with its appropriateness within the network structure; disagreements often indicated improper tissue or experimental context for the given network. Evidence that received at least four ‘Up’ votes was “locked” to indicate crowd approval and evidence that received at least four ‘Down’ votes was “locked” to indicate rejection by the crowd. Depending on the frequency and type of submitted improvements, participants received credit points and were assigned a dynamic ranking on the community Leaderboard. For more information about the NVC challenge, see the 5-minute overview videos at https://sbvimprover.com/challenge-3/videos or the 1-hour webinars at https://sbvimprover.com/challenge-3/tutorials.\n\nWhen the open phase was closed, the top-ranked participants were invited to a 3-day-long in-person jamboree to discuss improvements submitted by the community and to further refine the network models. Subject matter experts in lung, COPD, and network biology, as well as experts in other related biological processes, were also invited to guide the discussions and to provide expert feedback of missing or misrepresented signaling. Scientists involved in the construction of the original non-disease networks and Phase 1-enhanced networks were present to provide feedback for the rationale behind the boundary conditions and the mechanics of network construction and BEL. During the jamboree, 15 networks were prioritized to discuss in small groups of 6–10 people focusing on one network at a time. At the end of each session, final decisions were made about follow-up actions for each network and these actions were carried out subsequently by the scientists who constructed the original networks because of their familiarity with the mechanics of network construction and BEL.\n\nThe changes to the 15 networks that were discussed during the jamboree are posted online58 in open-source XGMML (eXtensible Graph Markup and Modeling Language) format.\n\nThe networks were built using the Biological Expression Language (BEL), which is an open source language that can represent scientific findings in the life sciences in a computable form64. BEL was designed to represent scientific findings by capturing causal and correlative relationships in context, where context can include information about the biological and experimental system in which the relationships were observed and the supporting publication citations. The structure of a BEL node, which includes the biological entity, the namespace or database to standardize the nomenclature of the entity, and the function that describes the type of entity (protein, chemical, biological process, family, complex, etc), is shown in Figure 8. Table 1 and Table 2 show the definition of the prefixes for BEL namespaces and functions that appear in the networks.\n\nA BEL term is the standard way a node is described. It includes an entity that is described using standard nomenclature in the Namespace and the Function fields of the entity.\n\nUnofficial BEL namespace to be formalized in BEL 2.0\n\n\nData availability\n\nUp-to-date networks including all users’ activity can be browsed freely on the Bionet website (https://bionet.sbvimprover.com/). Permanent URLs to each network are listed in the associated Data Set (Original networks, NVC networks and their descriptions). Networks can be downloaded by logged in users who had a few actions on the site as XGMML file for offline use in the version that started a verification phase, i.e. after review and QC by experts. The 15 networks discussed in the jamboree are available in a post-jamboree version. Moreover, different versions of the networks are available to browse and download in diverse formats from the CBN database available at causalbionet.com.\n\n\nData availability\n\nFigshare: Original networks, NVC networks and COPD data sets used in: Enhancement of COPD biological networks using a web-based collaboration interface http://dx.doi.org/10.6084/m9.figshare.1284583 65", "appendix": "Author contributions\n\n\n\nWKS, MT, SB, JP, and BF constructed and reviewed the networks. JH and MCP conceived the idea and managed the project. JP and BF wrote the manuscript text. JP, BF, and SB developed the figures. The Challenge Best Performers: IB, VB, OVB, VC, NDD, LF, SG, JG, GIK, EK, RK, NL, QL, SM, YO, SCP, APM ES, SS, GSN, IS, AT, GVF, JW, IY, and MZ participated in the network verification process and in the review of the manuscript. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nSelventa and PMI authors performed this work under a joint research collaboration funded by PMI. The Network Verification Challenge was funded by PMI.\n\n\nGrant information\n\nThe research described in this article was funded by Philip Morris International in a collaborative project with Selventa.\n\n\nAcknowledgements\n\nThe authors thank IBM for their help in organizing the Network Verification Challenge and jamboree, and Michael Maria and Jean Binder for their help in project management and preparation of this manuscript. The project team expresses their gratitude to the subject matter experts and moderators who actively participated in the jamboree: Maria Laura Belladonna, Michael Borchers, Maciej Cabanski, Natalia Boukharov, Stephan Gebel, Ignacio Gonzalez Suarez, Daniele Guardavaccaro, Anita Iskandar, Ulrike Kogel, Katica Jankovic, David Kling, Sophia Kossida, Hector de Leon, Karsta Luettich, Yukiko Matsuoka, Dragana Mitic Potkrajac, Michael Peck and Carine Poussin.\n\n\nReferences\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Goto S, Sato Y, et al.: Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014; 42(Database issue): D199–205. 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PubMed Abstract \| Publisher Full Text\n\nWang X, Bu HF, Zhong W, et al.: MFG-E8 and HMGB1 are involved in the mechanism underlying alcohol-induced impairment of macrophage efferocytosis. Mol Med. 2013; 19: 170–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrusselle GG, Joos GF, Bracke KR: New insights into the immunology of chronic obstructive pulmonary disease. Lancet. 2011; 378(9795): 1015–1026. PubMed Abstract \| Publisher Full Text\n\nKorns D, Frasch SC, Fernandez-Boyanapalli R, et al.: Modulation of macrophage efferocytosis in inflammation. Front Immunol. 2011; 2: 57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCosio MG, Saetta M, Agusti A: Immunologic aspects of chronic obstructive pulmonary disease. N Engl J Med. 2009; 360(23): 2445–2454. PubMed Abstract \| Publisher Full Text\n\nHodge S, Hodge G, Ahern J, et al.: Smoking alters alveolar macrophage recognition and phagocytic ability: implications in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2007; 37(6): 748–755. PubMed Abstract \| Publisher Full Text\n\nRichens TR, Linderman DJ, Horstmann SA, et al.: Cigarette smoke impairs clearance of apoptotic cells through oxidant-dependent activation of RhoA. Am J Respir Crit Care Med. 2009; 179(11): 1011–1021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIshikawa N, Hattori N, Tanaka S, et al.: Levels of surfactant proteins A and D and KL-6 are elevated in the induced sputum of chronic obstructive pulmonary disease patients: a sequential sputum analysis. Respiration; international review of thoracic diseases. 2011; 82(1): 10–18. PubMed Abstract \| Publisher Full Text\n\nHerold S, Mayer K, Lohmeyer J: Acute lung injury: how macrophages orchestrate resolution of inflammation and tissue repair. Front Immunol. 2011; 2: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFunk CD: Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001; 294(5548): 1871–1875. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRose JJ, Foley JF, Yi L, et al.: Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils. J Biomed Sci. 2008; 15(4): 441–461. PubMed Abstract \| Publisher Full Text\n\nKumar S, Xu J, Perkins C, et al.: Cdc42 regulates neutrophil migration via crosstalk between WASp, CD11b, and microtubules. Blood. 2012; 120(17): 3563–3574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmid E, Warner RL, Crouch LD, et al.: Neutrophil chemotactic activity and C5a following systemic activation of complement in rats. Inflammation. 1997; 21(3): 325–333. PubMed Abstract \| Publisher Full Text\n\nOttonello L, Montecucco F, Bertolotto M, et al.: CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2. Cell Signal. 2005; 17(3): 355–363. PubMed Abstract \| Publisher Full Text\n\nPrill RJ, Saez-Rodriguez J, Alexopoulos LG, et al.: Crowdsourcing network inference: the DREAM predictive signaling network challenge. Sci Signal. 2011; 4(189): mr7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer P, Alexopoulos LG, Bonk T, et al.: Verification of systems biology research in the age of collaborative competition. Nat Biotechnol. 2011; 29(9): 811–815. PubMed Abstract \| Publisher Full Text\n\nMeyer P, Hoeng J, Rice JJ, et al.: Industrial methodology for process verification in research (IMPROVER): toward systems biology verification. Bioinformatics. 2012; 28(9): 1193–1201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTarca AL, Lauria M, Unger M, et al.: Strengths and limitations of microarray-based phenotype prediction: lessons learned from the IMPROVER Diagnostic Signature Challenge. Bioinformatics. 2013; 29(22): 2892–2899. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n[https://bionet.sbvimprover.com/].\n\nEzzie ME, Crawford M, Cho JH, et al.: Gene expression networks in COPD: microRNA and mRNA regulation. Thorax. 2012; 67(2): 122–131. PubMed Abstract \| Publisher Full Text\n\nGemelli C, Orlandi C, Zanocco Marani T, et al.: The vitamin D3/Hox-A10 pathway supports MafB function during the monocyte differentiation of human CD34+ hemopoietic progenitors. J Immunol. 2008; 181(8): 5660–5672. PubMed Abstract \| Publisher Full Text\n\nAmmous Z, Hackett NR, Butler MW, et al.: Variability in small airway epithelial gene expression among normal smokers. Chest. 2008; 133(6): 1344–1353. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaykhiev R, Otaki F, Bonsu P, et al.: Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo. Cell Mol Life Sci. 2011; 68(5): 877–892. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaykhiev R, Krause A, Salit J, et al.: Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease. J Immunol. 2009; 183(4): 2867–2883. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n[http://www.openbel.org/].\n\nBoue S, Fields B, Hoeng J, et al.: Original networks, NVC networks and COPD data sets used in: Enhancement of COPD biological networks using a web-based collaboration interface. Figshare. 2014. Data Source" }	[ { "id": "7528", "date": "06 Feb 2015", "name": "Winston A. Hide", "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\nThis work uses a hybrid approach network modelling approach to incorporate predictive methodology with empirical knowledge and crowd sourcing for models of COPD pathogenesis. It is a good idea, thoroughly implemented and has produced a potentially useful set of pathways. The value of the resulting pathways is not clear as they do not have community validation, only community design. The manuscript is exhaustive in its descriptions and the process of developing the models is clear.The work represents the first phase of understanding for knowledge driven development of network models of COPD - the process of building the models is well described and the actual outcomes of the interactions with community are informative. The question of the actual true value of the models in terms of their accuracy, adoption and accessibility is not yet convincingly addressed. That may be expected as the purpose of this work appears to be a description of the first part of the process of developing knowledge based models for a disease. The models as presented appear unvalidated and without a description of the framework for assessing the value and actioning of the networks, it is not clear how their uptake by the community will be assured.This is a unique effort but the manuscript should make more reference to existing pathway based community annotation efforts e.g.: wikipathways and/or open science initiatives such as those promoted by community interaction leaders such as Andrew Su. It should show how the value of this approach differs to existing efforts.In terms of access to expertise, it is not clear how an uninvited scientist would contribute to an existing pathway model - except through the open but time-limited crowdsourcing venue.Straightforward validation of the models network is not tested in terms of their consistency or cross-valdiation within COPD high dimensional assays - where it should be possible to see evidence of enrichment for co-expression etc.Contextual nature of networks is mentioned and attempts are made to address contextual pathway structures, but the context is not tested.As a suggestion the authors should consider community validationPathway accessibility and distribution is described but it is not clear as to how these models are available in any format except web browsing. For the models to be tested by the community, value would come from making them openly available as downloadable instances in several of the most popular formats. Feedback on their accuracy could then be encouraged.", "responses": [ { "c_id": "1347", "date": "20 May 2015", "name": "Stephanie Boue", "role": "Author Response", "response": "This work uses a hybrid approach network modelling approach to incorporate predictive methodology with empirical knowledge and crowd sourcing for models of COPD pathogenesis. It is a good idea, thoroughly implemented and has produced a potentially useful set of pathways. The value of the resulting pathways is not clear as they do not have community validation, only community design. The manuscript is exhaustive in its descriptions and the process of developing the models is clear.The work represents the first phase of understanding for knowledge driven development of network models of COPD - the process of building the models is well described and the actual outcomes of the interactions with community are informative. The question of the actual true value of the models in terms of their accuracy, adoption and accessibility is not yet convincingly addressed. That may be expected as the purpose of this work appears to be a description of the first part of the process of developing knowledge based models for a disease. The models as presented appear unvalidated and without a description of the framework for assessing the value and actioning of the networks, it is not clear how their uptake by the community will be assured.Authors’ response: The point of the reviewer is absolutely relevant and we acknowledge that it will be of utmost importance to critically assess how the usefulness of the networks changed through each phase of the project. Whenever possible, orthogonal data sets were used to validate the network model during the building process. In the paper Systematic verification of upstream regulators of a computable cellular proliferation network model on non-diseased lung cells using a dedicated dataset, we have done just that by using a specifically designed, independent lung cell proliferation dataset to verify the correctness of the cell cycle network model 1. The validation of all available networks requires an extensive analysis leveraging multiple relevant datasets and to be reported thoroughly would dissolve the intended content of this manuscript that concentrates on the way networks were built and later on verified and refined through a crowdsourcing approach. We will conduct such an analysis and make sure to reference it here as soon as it will be available.This is a unique effort but the manuscript should make more reference to existing pathway based community annotation efforts e.g.: wikipathways and/or open science initiatives such as those promoted by community interaction leaders such as Andrew Su. It should show how the value of this approach differs to existing efforts.Authors’ response: We have discussed the comparison of our network models with other resources in other articles2,3 and in a book chapter4. We have added this statement in the discussion for readers who wish to find more background information about the network models and see how they compared with other approaches to interpret data.In terms of access to expertise, it is not clear how an uninvited scientist would contribute to an existing pathway model - except through the open but time-limited crowdsourcing venue.Straightforward validation of the models network is not tested in terms of their consistency or cross-valdiation within COPD high dimensional assays - where it should be possible to see evidence of enrichment for co-expression etc.Contextual nature of networks is mentioned and attempts are made to address contextual pathway structures, but the context is not tested.As a suggestion the authors should consider community validationPathway accessibility and distribution is described but it is not clear as to how these models are available in any format except web browsing. For the models to be tested by the community, value would come from making them openly available as downloadable instances in several of the most popular formats. Feedback on their accuracy could then be encouraged. Authors’ response: The networks can be browsed on the bionet.sbvimprover.com website, including latest votes and modification. More stable versions are stored in the causalbionet.com database2. References 1. Belcastro V, Poussin C, Gebel S, Mathis C, et al.: Systematic verification of upstream regulators of a computable cellular proliferation network model on non-diseased lung cells using a dedicated dataset. Bioinformatics and biology insights. Bioinformatics and biology insights. 2013; 7: 217-230 PubMed Abstract \| Free Full Text \| Publisher Full Text 2. Boue S, Talikka M, Westra JW, Hayes W, et al.: Causal Biological Network (CBN) database: a comprehensive platform of causal biological network models focused on the pulmonary and vascular systems. Database. 2015; 2015 (bav030): 1-14 PubMed Abstract \| Publisher Full Text 3. sbv IPT, Binder J, Boue S, Di Fabio A, et al.: Reputation-based collaborative network biology. Pacific Symposium on Biocomputing. 2015. 270-281 PubMed Abstract \| Publisher Full Text 4. Hoeng J, Talikka M, Martin F, Ansari S, et al.: Toxicopanomics: Applications of Genomics, Transcriptomics, Proteomics, and Lipidomics in Predictive Mechanistic Toxicology. In Hayes' Principles and Methods of Toxicology, Sixth Edition. 2014; Edited by Hayes AW, Kruger CL (CRC Press): 295-332 Reference Source" } ] }, { "id": "7526", "date": "02 Mar 2015", "name": "Gary D Bader", "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\nHigh quality curation by trained database curators is needed in our community to convert the literature to computable models, but it is difficult to imagine how manual curation will scale to handle the ever-growing data generation rate in biology. Thus, the biological research community needs to figure out how to get crowdsourcing working for everyone as a tool to improve access to computable data. This paper does a very good job of describing how a set of COPD networks were constructed and enhanced (they grew in size and level of detail) through an interesting three-phase process. However, it would be useful to better describe the utility of the resulting networks and to further analyze the crowdsourcing process itself. Addressing these points will give the work a broader impact. Utility of networks:It is not clear what advantages the use of causal networks brings compared to more established models in the community, such as molecular interaction networks used by many algorithms (e.g. gene function prediction, module detection, interpretation of molecular profile data, network biomarkers) or detailed biochemical pathway models (used by most textbooks and pathway databases). While many results are published in terms of causal networks (e.g. A activates B), one important issue with networks constructed by collecting these relationships is that they may be difficult to integrate across resources since they are context specific: A may activate B in the lung, but inhibit B in the heart and when these are integrated, a conflict arises. Many computational analysis methods require integration of networks from multiple sources to construct the largest available network and integrate this data with disease-specific molecular profile data (e.g. gene expression data) to gain context (as it seems is done in the RCR approach). It would be useful for the authors to further discuss the utility of context-specific causal networks for follow on discovery. I only noticed one sentence mentioning use: “By building the network model set in the BEL language format, we have generated a model framework suitable for biomarker discovery and for the interpretation of transcriptomic signatures found in human lung tissue.” However, this sentence is not clear and doesn’t cite any prior literature. How does using the BEL format create models suitable for biomarker discovery? Can’t molecular interaction or other types of networks also be used for biomarker discovery? What type of biomarker discovery is referred to here? How are transcriptomic signatures interpreted and analyzed? Crowdsourcing comments:“Networks that were not enhanced with COPD-specific mechanisms from the literature or RCR included the DNA Damage and Notch Signaling networks. Although both these networks relevant to the development of COPD, they were not augmented beyond the original, non-diseased network scaffolds, because no studies on the differences in signaling between non-diseased and diseased states were available.” How do the authors know that no relevant studies were available? It seems that many papers at least have discussed links between COPD and DNA damage or Notch signaling (e.g. PMID: 19106307 published in 2009 “Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.”) “In total, 12 new nodes and 28 new edges were added to the Th1-Th2 Signaling network model during the jamboree discussions, thereby creating a comprehensive biological network of T-helper cell activity and their interactions with other immune cells in the context of COPD.” How is ‘comprehensive’ measured? How do we know how much of the available literature was covered by the crowdsource process? That is, what is the sensitivity of the crowdsourcing process? How many contributors were involved in enhancing each network in phase 2? Where were they from e.g. academia, industry? What incentivized them to contribute – for instance, were they COPD researchers? For the sake of research into crowdsourcing in biology, it would be very useful to provide additional analysis of the contributor community. We need to learn more about what works and what doesn’t in crowdsourcing initiatives so future generations of these approaches can be improved. The authors state “With nearly 900 new pieces of evidence added by the challenge crowd, a significant overall enhancement of the networks was achieved in a relatively short time (5 months),” How many papers (PMIDs) supported the 900 pieces of evidence? Questions about use of BEL:Figure 4. The shorthand BEL notation is not widely recognized as a visual format and difficult to read in general. An easy to read visualization format would make the network figures much easier to understand. Also, what do the different edge end symbols (e.g. arrow, dot, diamond) mean? Part C of Figure 1 mentions “BEL to openBEL conversion”. What’s the difference between BEL and openBEL? Other comments:A broader review of the literature of pathway databases and crowdsourcing efforts should be included in the introduction.", "responses": [ { "c_id": "1346", "date": "20 May 2015", "name": "Stephanie Boue", "role": "Author Response", "response": "High quality curation by trained database curators is needed in our community to convert the literature to computable models, but it is difficult to imagine how manual curation will scale to handle the ever-growing data generation rate in biology. Thus, the biological research community needs to figure out how to get crowdsourcing working for everyone as a tool to improve access to computable data. This paper does a very good job of describing how a set of COPD networks were constructed and enhanced (they grew in size and level of detail) through an interesting three-phase process. However, it would be useful to better describe the utility of the resulting networks and to further analyze the crowdsourcing process itself. Addressing these points will give the work a broader impact. Authors’ response: We have previously published several papers introducing use cases where the biological signal is interpreted in a meaningful manner using the causal network models 1-7 and have added these references to support the statement in the text. The point of the reviewer is absolutely relevant and we acknowledge that it will be of utmost importance to critically assess how the usefulness of the networks changed through each phase of the network verification project. As a first step, the previously published analyses can be repeated with the crowd-verified networks to assess the impact of network verification on data interpretation. A thorough assessment of the impact of crowd verification, requires however an extensive analysis leveraging multiple relevant datasets and to be reported thoroughly would dissolve the intended content of this manuscript that concentrates on the way networks were built and later on verified and refined through a crowdsourcing approach. We will conduct such an analysis and include the reference as soon as it will become available. We have now addressed these points in the discussion.Utility of networks:It is not clear what advantages the use of causal networks brings compared to more established models in the community, such as molecular interaction networks used by many algorithms (e.g. gene function prediction, module detection, interpretation of molecular profile data, network biomarkers) or detailed biochemical pathway models (used by most textbooks and pathway databases). While many results are published in terms of causal networks (e.g. A activates B), one important issue with networks constructed by collecting these relationships is that they may be difficult to integrate across resources since they are context specific: A may activate B in the lung, but inhibit B in the heart and when these are integrated, a conflict arises. Many computational analysis methods require integration of networks from multiple sources to construct the largest available network and integrate this data with disease-specific molecular profile data (e.g. gene expression data) to gain context (as it seems is done in the RCR approach). It would be useful for the authors to further discuss the utility of context-specific causal networks for follow on discovery. Authors’ response: The usage of causal networks allows all applications that other network models would have, and in addition eases the biological interpretation of the results in a mechanistic, cause and effect fashion. The new, sophisticated algorithms that have been developed to analyze molecular data using the causal network models fully exploit the specific structure of two-layer cause-and-effect network models, providing evidence that causality adds precision on top of interaction1,2,8. However, as the reviewer points out, causality may differ across conditions (space and time), and the usage of BEL is therefore particularly relevant, as it allows for detailed context annotation of each piece of evidence linked to a causal edge. To fully make use of this property, it is important that as much of the literature evidence are collected in a knowledgebase, which will only really be doable thanks to new text mining methods assisting the biologists with the creation of BEL evidences or via crowdsourcing efforts such as the one described here. Because it is a very large undertaking, we have so far tried to restrict the evidences to respiratory and cardiovascular context. It is not excluded, however, that as the crowd and interest for the network grows, a more comprehensive annotation of the networks are achieved, making them usable in a specific context. Furthermore, BEL is being used in both academic and industry settings and BEL converters are being developed that can translate information from other sources such as BioPAX and SBML to facilitate comprehensive aggregation of networks.I only noticed one sentence mentioning use: “By building the network model set in the BEL language format, we have generated a model framework suitable for biomarker discovery and for the interpretation of transcriptomic signatures found in human lung tissue.” However, this sentence is not clear and doesn’t cite any prior literature. How does using the BEL format create models suitable for biomarker discovery? Can’t molecular interaction or other types of networks also be used for biomarker discovery? What type of biomarker discovery is referred to here? How are transcriptomic signatures interpreted and analyzed? Authors’ response: We have previously published several papers introducing use cases where the biological signal is interpreted in a meaningful manner using the causal network models1-7 and have added these references to support the statement in the text. Martin et al. describes the development of network signatures that identify mechanisms that may explain differential drug treatment response between individuals, demonstrating that the causal two layered networks allow analyses which go beyond what normal networks can provide, i.e. provide classification power coupled with mechanistic detail8.Crowdsourcing comments:“Networks that were not enhanced with COPD-specific mechanisms from the literature or RCR included the DNA Damage and Notch Signaling networks. Although both these networks relevant to the development of COPD, they were not augmented beyond the original, non-diseased network scaffolds, because no studies on the differences in signaling between non-diseased and diseased states were available.” How do the authors know that no relevant studies were available? It seems that many papers at least have discussed links between COPD and DNA damage or Notch signaling (e.g. PMID: 19106307 published in 2009 “Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.”) Authors’ response: We reformulated the sentence. Although there may be papers that report on the correlation between COPD and these processes like the Notch paper you mention, we are referring to mechanistic papers that will provide causal links within the model. For example, a paper from a NOTCH1 knockout experiment in a COPD animal model that shows a particular protein being decreased will allow us to add the causal link of NOTCH1 activity increasing that protein in the Notch signaling COPD model. These are the types of causal mechanistic papers we have searched for and have not found in the context of COPD.“In total, 12 new nodes and 28 new edges were added to the Th1-Th2 Signaling network model during the jamboree discussions, thereby creating a comprehensive biological network of T-helper cell activity and their interactions with other immune cells in the context of COPD.” How is ‘comprehensive’ measured? How do we know how much of the available literature was covered by the crowdsource process? That is, what is the sensitivity of the crowdsourcing process? Authors’ response: As to avoid any confusion, and because the sensitivity of crowdsourcing is not easily measurable (as it would require to assess all possible literature), we reformulated to “more comprehensive”.How many contributors were involved in enhancing each network in phase 2? Where were they from e.g. academia, industry? What incentivized them to contribute – for instance, were they COPD researchers? For the sake of research into crowdsourcing in biology, it would be very useful to provide additional analysis of the contributor community. We need to learn more about what works and what doesn’t in crowdsourcing initiatives so future generations of these approaches can be improved. Authors’ response: A specific publication addresses the statistics related to participation9. Clearly, the most difficult part of such a crowdsourcing project is to get the right incentives for people to participate. We acknowledge that showing the usefulness of the networks and their refinements should allow for a bigger buy-in from the scientific community, and likely more participation. The authors state “With nearly 900 new pieces of evidence added by the challenge crowd, a significant overall enhancement of the networks was achieved in a relatively short time (5 months),” How many papers (PMIDs) supported the 900 pieces of evidence? Authors’ response: 479 unique PMIDs supported the 886 new pieces of evidence. We have included this detail in the text. Questions about use of BEL:Figure 4. The shorthand BEL notation is not widely recognized as a visual format and difficult to read in general. An easy to read visualization format would make the network figures much easier to understand. Also, what do the different edge end symbols (e.g. arrow, dot, diamond) mean?Authors’ response: We have added a legend to the figure. Please note that the bionet website also has a legend for the network visualization part. Part C of Figure 1 mentions “BEL to openBEL conversion”. What’s the difference between BEL and openBEL? Authors’ response: BEL was a proprietary language developed by Selventa. In the interest of the growing community of researchers using BEL, an openBEL language derived from BEL has been developed and released as open source http://www.openbel.org/. One of the main differences between the two is that in the openBEL, the namespace (i.e. databases in which the biological entity is defined) is clearly stated, allowing for a better standardization of used ontologies and databases. We have added this specification in the figure legend. Other comments:A broader review of the literature of pathway databases and crowdsourcing efforts should be included in the introduction. Authors’ response: We have discussed the comparison of our network models with other resources in other publications2,9,10. We have added this statement with appropriate references in the discussion for readers, who wish to find more background information about the network models and see how they compared with other approaches to interpret data. References 1. Hoeng J, Deehan R, Pratt D, Martin F, et al.: A network-based approach to quantifying the impact of biologically active substances. Drug discovery today. 2012; 17 (9-10): 413-418 PubMed Abstract \| Publisher Full Text 2. Hoeng J, Talikka M, Martin F, Ansari S, et al.: Toxicopanomics: Applications of Genomics, Transcriptomics, Proteomics, and Lipidomics in Predictive Mechanistic Toxicology. In Hayes' Principles and Methods of Toxicology, Sixth Edition. 2014; Edited by Hayes AW, Kruger CL (CRC Press): 295-332 Reference Source 3. Kogel U, Schlage WK, Martin F, Xiang Y, et al.: A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food and Chemical Toxicology. 2014; 38: 204-217 PubMed Abstract \| Publisher Full Text 4. Phillips B, Veljkovic E, Peck MJ, Buettner A, et al.: A 7-month cigarette smoke inhalation study in C57BL/6 mice demonstrates reduced lung inflammation and emphysema following smoking cessation or aerosol exposure from a prototypic modified risk tobacco product. Food and Chemical Toxicology. 2015; 80: 328-345 PubMed Abstract \| Publisher Full Text 5. Schlage WK, Iskandar AR, Kostadinova R, Xiang Y, et al.: In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures. Toxicology mechanisms and methods. 2014; 24 (7): 470-487 PubMed Abstract \| Free Full Text \| Publisher Full Text 6. Talikka M, Kostadinova R, Xiang Y, Mathis C, et al.: The Response of Human Nasal and Bronchial Organotypic Tissue Cultures to Repeated Whole Cigarette Smoke Exposure. International Journal of Toxicology. 2014; 33 (6): 506-517 PubMed Abstract \| Publisher Full Text 7. Thomson TM, Sewer A, Martin F, Belcastro V, et al.: Quantitative assessment of biological impact using transcriptomic data and mechanistic network models. Toxicology and Applied Pharmacology. 2013; 272 (3): 863-878 PubMed Abstract \| Publisher Full Text 8. Martin F, Sewer A, Talikka M, Xiang Y, et al.: : Quantification of biological network perturbations for mechanistic insight and diagnostics using two-layer causal models. BMC Bioinformatics. 2014; 15: 238 PubMed Abstract \| Free Full Text \| Publisher Full Text 9. sbv IPT, Binder J, Boue S, Di Fabio A, et al.: Reputation-based collaborative network biology. Pacific Symposium on Biocomputing. 2015. 270-281 PubMed Abstract \| Publisher Full Text 10. Boue S, Talikka M, Westra JW, Hayes W, et al.: Causal Biological Network (CBN) database: a comprehensive platform of causal biological network models focused on the pulmonary and vascular systems. Database. 2015; 2015 (bav030): 1-14 PubMed Abstract \| Publisher Full Text" } ] }, { "id": "7527", "date": "02 Mar 2015", "name": "Patrick Ruch", "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 would welcome more information about the original networks, which were generated out of text/data mining. In particular, I would like to know what keywords were used to fetch the source articles (a set of genes/gene products, e.g. AQP5, MUC5...; a list of pathologies and synonyms, e.g. COPD Chronic Obstructive Pulmonary Disorders, early-stage COPD; a list of chemical compounds...) , what search engines were used (e.g. PubMed), what collection (MEDLINE, PubMed Central full-texts). Additionally quantitative details would be welcome also: how many abstracts or full-text articles were collected first?", "responses": [ { "c_id": "1345", "date": "20 May 2015", "name": "Stephanie Boue", "role": "Author Response", "response": "Networks were built in multiple phases, as briefly described in M&M and in figure 1. The “original” networks were built in a non-disease context as described in detail in the respective publications [1-5]. Based on relevant reviews in the area and context of interest, research papers that report the causal mechanistic relationships relevant for a specific biological process were identified and the causal relationships were extracted and added to the model. Mechanistic relationships were also added from an existing Knowledgebase, a collection of causal relationships curated over more than ten years from over 40,000 papers and over 500,000 statements." } ] } ]	1	https://f1000research.com/articles/4-32
https://f1000research.com/articles/4-122/v1	20 May 15	{ "type": "Opinion Article", "title": "ORCID for funders: Who’s who - and what are they doing? - ORCID IDs as identifiers for researchers and flexible article based classifications to understand the collective researcher portfolio", "authors": [ "Christian Herzog", "Giles Radford", "Giles Radford" ], "abstract": "For science funders, ORCID provides a persistent identifier that distinguishes one researcher from the others, and can facilitate workflows in grant submission, career tracking, and research impact(s). It makes life easier for the researcher – they can update their information in ORCID and make his/her past publications available to a funder as an ongoing service by just allowing this access as a one-time agreement. With these newly launched persistent tokens, researchers can grant a funder the right to update their grant record on ORCID once awarded – the metadata goes on an automatic roundtrip – effortless for the researcher, but the researcher stays in control, and can remove this right at any stage.\n\nHaving and sharing data is one aspect – but being able to understand true researcher activity is another – and even more challenging is to understand research activity in the aggregate. What are hundreds or thousands of researchers doing? Often a standard search will only answer or provide insights into a slice of the data. Research classification systems - like the Fields of Research (FOR) - provide sufficient aggregation, but these normally require manual tagging and curation of all the documents in a dataset. However, by using machine learning to automate tagging, it becomes possible to answer the ‘what’ question easily. This ‘article-based classification’ is realized using Natural Language Processing (NLP) technology.\n\nWith Dimensions, a portfolio analysis tool for research funders these capabilities are combined for research funders: allowing the researcher to provide controlled access to their ORCID profile and a solution environment for flexible article based classification, providing immediate access to analytical information on the researcher and institutional level – answering the questions ‘who is who’ and ‘what are they doing’?", "keywords": [ "ORCID-CASRAI", "Researcher Identification", "Name ambiguity" ], "content": "Challenges on different levels - who, how and what?\n\nResearcher identification has always been challenging for all research systems in a number of ways - one of them has been that researcher names are, obviously, not always unique. In addition they tend to express themselves in different permutations, especially over time. They may use a full first name, then a shortened first name, or just an initial only. And sometimes they might add a middle initial, sometimes not etc. This makes identifying the same person, over time, based on their name variations exceedingly difficult. Additionally, in an increasingly data-driven context of science funding and evaluation, the ability to attribute grants and publications to the individual researcher is in everyone’s interest, and is now often required by employing institutions or funders. The answer of how to allow researchers to manage and share their research activities and outputs accurately is known as the ORCID ID system. Each researcher gets a unique identifier. It allows the individual researcher to manage their inputs and outputs in a single place and control and allow which organization/system can access it automatically (e.g. to provide the information in the context of a grant application without any effort other than providing the reading permit to the respective system - the rest then happens automatically). The question of how research activities and outputs can be related to a person is solved with the ORCID ID. It solves the ‘who’ question.\n\n\nDriving adoption\n\nHowever, obviously ORCID requires adoption in order for it to work as envisioned, which relates to the ‘how’ question. How can we make ORCID adoption universal? There are obviously a few gatekeepers in the research process with a critical role to play - primarily funders, publishers and research organizations. The benefits are obvious – having reliable information on researchers and their related documents and activities is critical for funding decisions, saves costs and enables more specific support services.\n\nSome areas have even more need than others. Organizations in Asian countries often have even greater challenges in this area of name ambiguity, due to a lot of synonymous names, and confusion of ‘first’ and ‘last’ name which often leads to data transposition. These countries are therefore able to profit from ORCID – allowing the researcher to distinguish their artifacts from others. The emergence of community and industry solutions like ORCID; CrossMark and FundRef provides a very cost efficient, standard based and effective implementation1.\n\nFunding organizations are key players, in that they are generally at the start of the research process – and in a unique position to drive the adoption of systems like ORCID. They can apply rules making it mandatory to have an ORCID ID. Creating an ORCID D isn’t difficult and we have already seen the ongoing benefits, like the ability to extract previous publication lists (required for the application process) from ORCID rather than the applicant manually submitting them, but there are downstream benefits too; for instance when other parties, such as publishers require the same type of information in the context of publishing the results of the funded research.\n\nRequesting, or even insisting, that the researcher must have an ORCID ID is one thing – but how do the relevant publications, activities and grants get into the ORCID record? It still requires effort from the researcher to tag their publications and grants to their profile, and they have to remember to do this regularly, which is a burden and easily forgotten – so it needs to be made as simple as possible to reach two goals: completeness and quality of the data associated with an ORCID ID (Figure 1).\n\nOne answer to the challenge of how to make things easier for the researcher is the ÜberWizard for ORCID. Developed by ÜberResearch when ORCID introduced funded grants as a data type, it allowed the researcher to add their grants from many different funders to their ORCID record in one simple step. The advantages are clear: the researcher can assign all their grants from participating funders in one step (and one wizard rather than searching for the right wizard per funder) and the data in the ORCID record is therefore correct and complete since it has been pulled from a consolidated grant database compiled by ÜberResearch and authenticated by the researcher.\n\nEvery funder can integrate their own grant portfolio into this database to save on the costs of developing their own routines of how to expose their funded grants for integration into ORCID records – but the more important aspect is to simplify the process for the researcher. ÜberResearch provides this service free of charge to support the integration ORCID identifiers in the funding workflow and to support the researcher. In addition funding organizations can make use of the global grant database for portfolio comparison and analysis purposes (The global award database can be analysed with Dimensions for Funders http://www.uberresearch.com/dimensions-for-funders/, which is available at no cost for small funders, see http://www.uberresearch.com/ubershare/).\n\nHowever, this interaction model with the ÜberWizard still asks a lot from the researcher – they have to use the ÜberWizard to bring the data into their ORCID record. But with new functionality launched by ORCID in 2015 (http://orcid.org/blog/2014/11/21/new-functionality-friday-auto-update-your-orcid-record), this can be made much easier – based on trusted relations and automation (Figure 2). The grant application is normally the first step in a research cycle, and with the functionality of a ‘long-lived token’ the funding organization can request permission from the applicant to read and update their ORCID record: This permission starts to send the metadata on a round trip. ORCID starts to become a (hidden) infrastructure working for the researcher; an awarded grant or related information can be pushed automatically into the researcher’s ORCID record once the grant appears in the global grant database fueling the ÜberWizard for ORCID (Figure 3).\n\nThe Portuguese Fundação para a Ciência e a Tecnologia (FCT) made it mandatory for funded researchers to have an ORCID ID. This obviously results in a high adoption rate and becomes a de facto national roll out – making it far easier for the researcher to share their inputs and outputs going forward, and enabling the national Portuguese funder oversight on all Portuguese researchers’ activity. In a recent research assessment exercise, 15,000 researchers registered their ORCID ID and about 10% also added funded projects to their record, using the ÜberWizard for ORCID. This is expected to increase during 2015 when scholarship grants are included and the connection to the national CV system is realized (Personal communication with João Moreira, FCT).\n\n\nUnderstanding the research activities – ‘the what?’\n\nWith the ORCID ID in place the relation between input/output and researcher is in place – the ‘who’ question is solved. But this is still on the metadata level and does not create insights into ‘what’ the researcher or an entire population of researchers is doing. What would be required is an identifier system to tag the content of the documents – preferably automatically. It is clear that the use case is not to understand every article or publication, but to be able to cluster large numbers of documents in high-level categories in order to understand distribution across research topics, disciplines and trends. This is currently done in some areas with journal classifications where subject categories are assigned on the journal level. This works as expected - quite well for highly specialized journals, but not at all well for multidisciplinary journals, and it is not possible to have the same classification for non-journal documents2.\n\nHowever, given that the content is available in the document itself, why not taken the approach of deriving the ‘tags’ or classifications from the document itself? Which classification systems could be used?\n\nResearch classification systems are used for structuring and simplifying portfolios for use cases like trend analysis, reporting and strategic decision making. The systems can span the entire science portfolio, like the Fields of Research (FOR) codes as part of the Australian and New Zealand Standard Research Classification (ANZSRC) system, the OECD Frascati classification of science and technology (FOS) or discipline specific like the Research, Condition and Disease Categorization (RCDC) system used by the National Institutes of Health.\n\nSome of these systems are in use in some countries with some funders, meaning a small subset of grants can be interrogated by a small subset of classification systems. Most have been assigned manually to documents, but some have been assigned using semantic routines (e.g. the RCDC system, albeit only on NIH grants). Based on the use case of being able to get insights on the portfolio level in large document databases without the unmanageable burden of the manual effort in reading and classifying all the documents, ÜberResearch started to work with funding organizations, as development partners, to develop the routines and tools to be able to assign various classification systems to document databases – for example the FOR coding system from Australia/New Zealand. Using machine learning approaches and a large dataset of manually coded documents as a training set, we were able to derive a model which can now be applied on a document level to any document – achieving a consistent ‘tagging’ without the bias normally introduced by different human coders or professional groups. In addition to the FOR codes, Dimensions has automated the RCDC classification system used by the NIH; the health categories of the Health Research Classification System (HRCS) and a first implementation of the Common Scientific Outline (CSO) coding. The approach used to derive the model using machine learning routines will be discussed in a separate paper once the evaluation of several of the classification systems has been concluded.\n\nAs a result of these efforts, it is possible to assign different research classification systems automatically to all documents – grants, publications, and others - at marginal costs, which allows funders or research organizations to use different ones for different purposes. The research classification approach is implemented in ÜberResearch’s Dimensions – together with the corresponding analytical functionalities. Dimensions has been developed to serve as an ‘applied babel fish’ system (see 3) for research classifications.\n\nThe examples below (see Figure 4 and Figure 5) have been taken from ÜberResearch’s analytical tool Dimensions, analyzing a global grant database covering more than 1.4 million grants with a total funding volume of more than $760 billion US. The examples show how article-based classifications can be surfaced for end users in an application to provide strategic insights on the global funding landscape. These approaches can also be realized in other tools – they should be seen as illustrations and examples.\n\nThe screenshot shows all grants of the European Research Council (ERC) and the most funded organizations –together with the FOR codes receiving the highest funding amounts. It allows a user to understand quickly the focus of a group of research institutions, especially if looked at over time. Screenshot taken from ÜberResearch’s application Dimensions for Funders.\n\nThe aggregated profile for a researcher (in this case only based on grants) shows on the right hand a profile across different classification systems – indicating clearly the different use case: FOR codes are relatively general, the RCDC systems provides as expected more details and the HRCS system adds a broad classification in areas – for what it has been built. Screenshot taken from ÜberResearch’s application Dimensions for Funders.\n\n\nConclusions\n\nWith the open researcher and contributor ID, coupled with the corresponding infrastructure, a power approach has been established, which is constantly refined to make it easier for the researcher. This ‘hidden’ piece of infrastructure is ‘doing the right things automatically’ – while at the same time keeping the researcher in the driving seat in terms of who sees and gets access to his or her data.\n\nThis will help finally solve the challenge of name ambiguity and lack of links between inputs and outputs of researchers, whilst also removing much of the burden from the individual researcher. But it requires adoption and that is only possible with incentives or some ‘light’ pressure – by, for example, funding organisations making it mandatory (even if that feels, initially, like additional effort) – but it will pay off for the researcher downstream: for example when he or she submits their next manuscript, applies for their next grant or moves between organizations. Publishers are in a similar gatekeeper role for strengthening the ORCID approach – creating the scenario that researchers require an ORCID ID. Researchers, too, can immediately see the benefit: having ones grants and publications underrepresented can be both frustrating and, potentially, damaging. A full and complete record will help in all that they want to do.\n\nThe ‘who’ question is increasingly solved by ORCID – and with the right incentives, and more and more gatekeepers adopting it – it will solve the challenge of knowing the relations between works and the individual. But what about the second approach of establishing flexible identifiers for the ‘substance’ or content of the research activities which are generated from the works directly using computational routines?\n\nAgain science funders have a critical role to play here – since they are driving most of the use cases: portfolio classification and reporting on how research funds have been distributed and the analysis of the input, output and impact are at the core of their mission. To know what has been funded in any given research topic can drive effective strategic decisions.\n\nSuch use cases - generating or assigning classifications based on the content of the documents -enable comparisons and interactions between funders and research organizations. To know how much has been funded in a given research topic area requires a conversation using the same classification language, applied in the same consistent way. The approach is still in its infancy, since it takes time to replace established routines (primarily manual tagging of documents), but the increase in attention to the field and approach hint at a near future, where the classification routines are part of a hidden and effective infrastructure, like the ORCID system. That would solve the ‘what’ question. And if both ‘who’ and ‘what’ can be solved using (mostly) automatic routines then the research funding landscape overview can help drive funding policy decisions based on reliable data, which has to be a good thing for science in general.", "appendix": "Author contributions\n\n\n\nBoth authors have equally contributed to the article. Both authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nChristian Herzog is the CEO and co-founder of ÜberResearch, a Digital Science Portfolio company, Giles Radford is employed by ÜberResearch.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHuh S: Application of new information technologies to scholarly journals: ORCID, CrossMark, and FundRef. J Korean Med Assoc. 2014; 57(5): 455–462. Publisher Full Text\n\nGlänzel W, Schubert A, Czerwon HJ, et al.: An item-by-item subject classification of papers published in multidisciplinary and general journals using reference analysis. Scientometrics. 1999; 44(3): 427–439. Publisher Full Text\n\nTerry RF, Allen L, Gardner CA, et al.: Mapping global health research investments, time for new thinking--a Babel Fish for research data. Health Res Policy Syst. 2012; 10: 28. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "9075", "date": "16 Jun 2015", "name": "Johanna R. McEntyre", "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\nThis article is about two tools implemented within UberResearch, a small company operating as part of the Digital Science portfolio. The first is a tool that allows researchers to add funding awards to their ORCID record (the UberWizard for ORCID); the second is a tool for funders (\"Dimensions\") that performs analytics on funding decisions using research classification systems. A machine-learning method has been uses to tag documents (grant awards) with various categories of the research classification systems. The two come together as grants claimed by an individual researcher can be tagged and therefore categorized on an individual basis as well as for funders. I wish the authors could have been clearer on this from the outset. I think this context would help orient readers better. Much of the abstract focuses on the details of funders being able to automatically update a researcher's ORCID with funding information and an abstract discussion on automatic tagging of research classification systems. The writing and narrative style could be improved - some sentences extremely long e.g. p3. \"Creating an ORCID D (sic) isn't difficult .... funded research.\" However the major comment I have on the article is that it describes outcomes without describing how they were achieved, and furthermore there is no way for readers to evaluate Dimensions as a product. More specifically:Did UberResearch need to be a member of ORCID to provide the grant linking service? How has the \"global grant database\" been generated - from where? What is its scope? What protocols and standards are used to make the transactions between ORCID and the UberWizard? Are there any other tools available? What methods of machine learning were used, how were they validated? The given info is: \"The approach used to derive the model using machine learning routines will be discussed in a separate paper once the evaluation of several of the classification systems has been concluded.\" I would be happier if this were published already. As a reviewer or a reader, I can't access Dimensions so I have no means to evaluate it. The article seems to be aimed primarily at funders, as is the Dimensions product - what is in it for the researcher, who must be the primary readership of F1000Research?I have personally used the UberWizard for ORCID and it worked fine; I also saw a brief preview of Dimensions a few months ago, and was impressed. I see this article is an \"Opinion\" article - but nevertheless it is tough to evaluate when the methods and outcomes are not publicly available. I would agree that ORCIDs are important for effective research assessment, but it would be a more powerful position if there were some means to evaluate Dimensions directly. Would a product review be a more appropriate route?", "responses": [] } ]	1	https://f1000research.com/articles/4-122
https://f1000research.com/articles/4-121/v1	19 May 15	{ "type": "Research Article", "title": "A reanalysis of mouse ENCODE comparative gene expression data", "authors": [ "Yoav Gilad", "Orna Mizrahi-Man", "Orna Mizrahi-Man" ], "abstract": "Recently, the Mouse ENCODE Consortium reported that comparative gene expression data from human and mouse tend to cluster more by species rather than by tissue. This observation was surprising, as it contradicted much of the comparative gene regulatory data collected previously, as well as the common notion that major developmental pathways are highly conserved across a wide range of species, in particular across mammals. Here we show that the Mouse ENCODE gene expression data were collected using a flawed study design, which confounded sequencing batch (namely, the assignment of samples to sequencing flowcells and lanes) with species. When we account for the batch effect, the corrected comparative gene expression data from human and mouse tend to cluster by tissue, not by species.", "keywords": [ "ENCODE", "RNA-seq", "developmental pathways", "flowcells", "sequencing" ], "content": "Introduction\n\nThe mouse ENCODE Consortium has collected multiple types of genomic and functional data in order to better understand the potential utility of the mouse as a model system for biomedical research. To study gene expression levels, the Consortium collected RNA sequencing data from multiple tissues from human and mouse. Their comparative analysis revealed that gene expression patterns tend to support clustering of the data by species, rather than by tissue (Figure 2a in reference 1).\n\nThis pattern was confirmed and discussed in greater detail in a companion paper by Lin et al.2, which also acknowledged that this observation is somewhat unexpected. Indeed, previous comparative studies reported that gene expression data from human and mouse (and across other species more generally) tend to cluster by tissues, not by species. Lin et al. proposed that previous studies might have been biased in their focus on a few ‘specialized’ tissues that tend to express the largest number of ‘tissue-specific genes’, while the overall pattern supports less tissue specificity.\n\nThe implications of the observation that human and mouse gene expression data may be clustering by species more than by tissues can be profound. To a large degree, modern biology is built upon the empirical observation that homologous gene regulatory networks establish the identities of homologous cell-types, tissues, and organs across species – the results of Lin et al., if true, challenge these observations and the biological basis of homology. From a more practical perspective, the mouse is arguably the most important animal model for biomedical research. If gene regulation in any mouse tissue is markedly more representative of a general mouse regulatory network than the regulatory network of a corresponding human tissue, this would call into question the utility of the mouse, and perhaps any other non-human animal, as a useful model system for biomedical research.\n\nHere, we present a reanalysis of the mouse ENCODE Consortium comparative RNA sequencing data. We argue that a flaw in their study design raises doubt regarding their conclusions.\n\n\nMethods\n\nIn December 2014 we asked and were kindly provided by the authors of Lin et al.2 the names of the sequence files used in their comparative analysis. Based on this information we obtained sequence files in FASTQ format (Supplementary Table 1) from the ENCODE project1 site (https://www.encodeproject.org/; some of the files were only available from early January 2015).\n\nFor our analysis, we used the same genome build and gene annotation files as in Lin et al.2. The ENSEMBL3 genome build Mus musculus GRCm38.68 was downloaded from ftp://ftp.ensembl.org/pub/release-68/fasta/mus_musculus/dna/Mus_musculus.GRCm38.68.dna_sm.toplevel.fa.gz; the corresponding transcript annotation file was downloaded from ftp://ftp.ensembl.org/pub/release-68/gtf/mus_musculus/Mus_musculus.GRCm38.68.gtf.gz. The Homo sapiens genome build provided by ENSEMBL3 contains haplotypic regions that are not part of the primary assembly. To avoid these regions, genome build Homo sapiens GRCh37 was downloaded from the Illumina iGenomes page: (http://support.illumina.com/sequencing/sequencing_software/igenome.html). The GENCODE4 Release 14 transcript annotation file for human was downloaded from ftp://ftp.sanger.ac.uk/pub/gencode/release_14/gencode.v14.annotation.gtf.gz. The chromosome names in the GENCODE gtf file did not match those in the genome sequence file, and were thus modified.\n\nBased on the sequence identifiers found in the FASTQ files, we reconstructed the sequencing study design used to collect the gene expression data in Lin et al.2. The sequence identifier line in a FASTQ file generated from an Illumina sequencing run can take two formats, depending on the version of the Consensus Assessment of Sequence and Variation (CASAVA) pipeline used to generate it. Prior to version 1.8 of this pipeline the sequence identifier line was of the following format (CASAVA v1.7 user guide p.88; downloaded from: http://support.illumina.com/downloads/casava_software_version_17_user_guide_(15011196_a).html\n\n@<machine_id>:<lane>:<tile>:<x_coord>:<y_coord>#<index>/<read_#>\n\nStarting from version 1.8 the sequence identifier line is of the format http://support.illumina.com/help/SequencingAnalysisWorkflow/Content/Vault/Informatics/Sequencing_Analysis/CASAVA/swSEQ_mCA_FASTQFiles.htm\n\n@<machine_id>:<run number>:<flowcell ID>:<lane>:<tile>:<x-pos>:<y-pos> <read>:<is filtered>:<control number>:<index sequence>\n\nBelow is a sequence identifier line from the mouse pancreas read1 FASTQ file (sequence identifier lines from the remaining FASTQ files were of similar format):\n\n@D4LHBFN1:276:C2HKJACXX:4:1101:3448:12374 1:N:0:AGTTCC\n\nBased on this information we inferred that the FASTQ files were generated by CASAVA version 1.8 or higher. Thus, we could extract from the sequence identifiers the following details that pertain to the sequencing study design: machine identifier, run number, flowcell identifier, and flowcell lane number. We found that the sequencing was performed in five batches, each consisting of a multiplexed single run on a single lane on one of four sequencers (Figure 1; note that two of the batches, composed of human samples only, differed only in their lane number). The design was such that only one batch contained samples from both species. The remaining four batches could be divided into pairs where each of the two batches had a nearly identical tissue composition, but a different species.\n\nSequencing batches as inferred based on the sequence identifiers of the RNA-Seq reads.\n\nFollowing Lin et al.2, we used the protein-coding ortholog list generated by the modENCODE and mouse ENCODE consortia5. A file containing all orthologs from human, mouse, fly and worm was downloaded from http://compbio.mit.edu/modencode/orthologs/modencode.common.orth.txt.gz. From this list we extracted 14,744 human-mouse one-to-one ortholog pairs, for which both members were included in the transcript annotation files we used. We note that this number is lower than the ~15,106 ortholog pairs reported in Lin et al. We are not certain of the meaning of the ‘~’ in the report of the number of ortholog pairs analyzed by Lin et al. Nevertheless, we believe that a possible explanation for this disparity is a parsing error. The last two columns of the ‘modENCODE ortholog file’ represent the number of genes from each species in the ortholog group. One of the steps required to obtain the subset of ortholog groups for analysis is to select those records where the two last columns have a value of 1 (i.e. one-to-one ortholog pairs). We found that if this selection is done through a command line search that does not require that the value in the last column be exactly “1”, but rather just begins with “1”, then the result is 15,104 putative human-mouse ortholog pairs.\n\nWe used the FastQC software v0.10.0 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to assess the quality of the individual FASTQ files (Supplementary Table 2–Supplementary Table 6). We were concerned by evidence for GC content bias and overrepresented sequences. To examine the latter in greater detail, we mapped the sequences overrepresented in at least one sample to the genome of the respective species, using BLAT searches6 against the hg19 (human) and mm10 (mouse) assemblies at the UCSC genome browser site (http://genome.ucsc.edu/)6. We found that in both species many of the overrepresented sequences mapped perfectly to the mitochondrial genome (Supplementary Table 3–Supplementary Table 6). For the mouse pancreas sample only, we also found many overrepresented sequences mapped to regions with rRNA repeats from the SSU-rRNA_Hsa and LSU-rRNA_Hsa families.\n\nWe mapped the RNA-Seq reads to their respective genomes using Tophat v2.0.117 with the following options: “--mate-inner-dist 200” (i.e. inner mate distance is 200nt, based on paired-end reads with length 100nt each and an insert size of 350-450nt ); “--bowtie-n” (i.e. the “-n” option will be used in Bowtie8 in the initial read mapping stage); “-g 1” (i.e. multi-mapping reads will be excluded from alignment); “-m 1” (i.e. one mismatch is allowed in the anchor region of a spliced alignment); “--library-type fr-firststrand” (the libraries had been constructed using the Illumina TruSeq Stranded mRNA LT Sample Prep Kit2). An exception was the mouse pancreas sample, for which the mapping process stalled consistently at the same stage. For this sample we used Tophat v1.4.18 with the same options as above. Tophat requires a Bowtie8 index. For human we used the Bowtie index that was packaged with the genome sequence in the file downloaded from the Illumina iGenomes page (http://support.illumina.com/sequencing/sequencing_software/igenome.html). For mouse we built an index using the bowtie-build utility from Bowtie v2.2.1 (v 0.12.7 for the index used with Tophat v1.4.1).\n\nFor each of the two species we used the appropriate GTF file to generate a table, which contains for each gene its ENSEMBL gene identifier its common name, and the GC content of the sequence covered by the union of the gene’s transcripts. To this end, we first generated a GTF file where overlapping exons from different transcripts of the same gene were merged into a single “exon” with the same sequence coverage, retaining the association with the gene identifier. Next, we computed the nucleotide content of the exons in this new GTF file using the ‘nuc’ utility from bedtools v2.17.09. Finally, we computed the GC content for each gene identifier by summing the number of ‘G’ and ‘C’ nucleotides in its merged exons and dividing by the sum of counts of unambiguous nucleotides in these exons.\n\nWe used Cufflinks v2.2.110 to compute fragments per kilo base of transcript per million (FPKM) values and aggregate them per gene. The only option used was “--library-type fr-firststrand”. For the required transcript annotation file (“-G” parameter) we used the GTF file for the respective species described in the “Genome and gene annotation files” section. We then generated a matrix of 14,744 by 26 FPKM values for each gene (in the ortholog table) and sample. While generating this table we noticed that some of the common gene names were associated with more than one ENSEMBL gene identifier. In some cases we determined that this was due to gene identifiers that have been retired from the ENSEMBL database3 but were retained in the GTF file (27 and 64 retired identifiers for human and mouse, respectively). These retired identifiers were ignored when constructing the FPKM matrix. For the remaining such cases we incorporated the value from the first appearance of the common name.\n\nTo compute per gene raw counts from the alignment files produced by Tophat7, we used the program featureCounts v1.4.411 with the respective species’ GTF file specified in the “Genome and gene annotation files” section. For all runs we used the following options: “-p” - indicates that fragments rather than reads should be counted; “-C” - indicates that chimeric fragments will not be included in the summarization process; and “-s 2” - indicates that the paired-ends are reversely stranded. We next generated a matrix of 14,744 by 26 raw counts for each gene (in the ortholog table) and sample. Since the output from featureCounts identifies genes by their gene identifier (the ENSEMBL identifier in our case), whereas the ortholog table uses the gene’s common name to identify it, we used the GC content table, which contains both these identifiers to match counts to the correct row in the ortholog table. As we did when generating the FPKM matrix, we ignored the values from retired ENSEMBL identifiers, and if there were still multiple identifiers for the same common name, we used the value from the identifier that appeared first.\n\n\nResults\n\nIn this reanalysis effort, we focused solely on the RNA sequencing data that can be mapped to coding regions. Lin et al.2 reported additional results, related to data on the expression of non-coding transcripts and histone marks. We did not reanalyze these additional data types.\n\nLin et al.2 analyzed both previously published and newly collected human and mouse gene expression data. The previously published data consist of RNA sequencing from ENCODE, the Illumina Human BodyMap 2.0, and the Roadmap Epigenomics Mapping Consortium. In these previously collected data sets, human and mouse samples were analyzed by different labs at different times, such that there is a clear batch effect that is confounded with species. Lin et al.2 clearly explains this limitation of the previously published data. They state that in order to address this issue they focus on the analysis of only the newly collected data – RNA sequencing data of samples from 13 human and mouse tissues that were collected by the same lab, using the same sample processing protocol. We focus our reanalysis study on the same newly collected data set (see Methods).\n\nAs a first step of our study we set out to replicate the analysis of Lin et al.2. To do so, we started with the matrix of FPKM values (computed, using Cufflinks10, based on the read alignments to the genome). This analysis was done within R environment v 3.1.3 GUI 1.65 Snow Leopard build (6912)12. See Supplementary Text 1 for detailed commands, and a supplement zip file for the R input (available in Zenodo: http://dx.doi.org/10.5281/zenodo.17606).\n\nWe log2-transformed the FPKM matrix (after adding 1 to avoid undefined values). To visualize the data, we used an approach that is similar in principle to that used by the ENCODE mouse consortium and Lin et al. Specifically, we used the function ‘prcomp’ (with the ‘scale’ and ‘center’ options set to TRUE) to perform principal component analysis (PCA) of the transposed FPKM matrix (so that samples were now in rows and genes in columns), after removal of invariant columns (genes). Scatter plots of the PCA results were generated using the ggplot2 package13. In agreement with the findings of Lin et al.2 the samples cluster mostly by species (Figures 2a, Figure S1 and Figure S2). We also plotted the heatmap of the matrix of Pearson correlations between the 26 samples, using the pheatmap function from the pheatmap package v1.0.214 with default settings (i.e. complete linkage hierarchical clustering using the Euclidean distances). Again, samples from the same species tend to cluster together (Figure 2b).\n\na. Two-dimensional plots of principal components calculated by performing PCA of the transposed log-transformed FPKM values (from 14,744 orthologous gene pairs) for the 26 samples, after removal of invariant columns (genes). b. Heatmap based on pairwise Pearson correlation of expression data used in panel a. We used Euclidean distance and complete linkage as distance measure and clustering method, respectively.\n\nA previous evaluation of normalization methods for RNA-Seq data15 suggested that FPKM values were not optimal for clustering analysis. Therefore, as a basis for our reanalysis, we used the matrix of per-gene raw fragment counts. The entire analysis was done within R environment v 3.1.3 GUI 1.65 Snow Leopard build (6912)12. See Supplementary Text 2 for detailed commands, and a supplement zip file for the R input (available in Zenodo: http://dx.doi.org/10.5281/zenodo.17606).\n\nFollowing Li et al.16, we removed the 30% of genes with the lowest expression as determined by the sum of fragment counts across all samples. Next, due to the presence of mitochondrial genes among the overrepresented sequences in the data, we also removed reads that map to the 12 mitochondrial genes. This left us with expression data from 10,309 genes for analysis. We note that merely limiting the analysis to this subset of genes does not have a marked effect on the patterns reported by Lin et al. (Figure S3; detailed commands in Supplementary Text 3, and a supplement zip file for the R input (available in Zenodo: http://dx.doi.org/10.5281/zenodo.17606)). We performed within-column normalization to remove the GC bias in the data, indicated by the initial quality assessment. To this end, we applied the ‘withinLaneNormalization’ function from the EDASeq package v2.0.017 to each column in the matrix, using the gene GC values for the species associated with the column. Next, we used the ‘calcNormFactors’ from the edgeR package v3.8.618, with the trimmed mean of M-values (TMM) method19, to calculate normalization factors for the library sizes for the samples. We used these normalization factors in the depth normalization of the columns (using the column sums of the original, unfiltered, counts matrix as a proxy for library sizes). The normalized data were log2-transformed (after adding ‘1’ to each value in the matrix to avoid undefined values).\n\nWe then considered how to account for the fact that the assignment of samples to sequencing flowcells and lanes was nearly completely confounded with the species annotations of the samples (Figure 1). The consideration of ‘batch effect’ was the most important difference between the analysis that recapitulated the patterns reported by the mouse ENCODE papers (the previous ‘Results’ section) and the current reanalysis effort. Specifically, we accounted for the sequencing study design batch effects using the ‘ComBat’ function from the sva package v3.12.020, with a model that includes effects for batch, species and tissue. For this purpose the samples were classified into five batches, based on the sequencing study design (see methods and Figure 1).\n\nTo visualize the data, we used the function ‘prcomp’ (with the ‘scale’ and ‘center’ options set to TRUE) to perform principal component analysis (PCA) of the transposed log-transformed matrix of ‘clean’ values (after removal of invariant columns, i.e. genes), and the ggplot2 package13 to generate scatter plots of the PCA results. None of the first five principal components (accounting together for 56% of the variability in the data) support the clustering of the gene expression data by species (Figure 3a and Figure S4–Figure S5). However, the sixth principal component, which accounts for 6% of the variability in the data, does support such a clustering, suggesting that even though the ‘species’ and ‘batch’ variables are confounded, accounting for ‘batch’ does not remove completely the variability due to ‘species’ (Figure S5). We also plotted a heatmap of the matrix of Pearson correlations between the 26 samples, using the pheatmap function from the pheatmap package v1.0.214 with default settings (i.e. complete linkage hierarchical clustering using the Euclidean distances). This time the heatmap shows considerable clustering of the comparaive gene expression data by tissue (Figure 3b).\n\na. Two-dimensional plots of principal components calculated by applying PCA to the transposed matrix of batch-corrected log-transformed normalized fragment counts (from 10,309 orthologous gene pairs that remained after the exclusion steps described in the results) for the 26 samples, after removal of invariant columns (genes). b. Heatmap based on pairwise Pearson correlation of the expression data used in panel a. We used Euclidean distance and complete linkage as distance measure and clustering method, respectively.\n\n\nDiscussion\n\nIn our reanalysis we have made a number of specific choices, including the exclusion of a certain subset of lowly expressed genes, the specific approach we chose to summarize the count data, the standardization and normalization methods we used (for example, we chose to standardize by the total count of reads that mapped to the ortholog gene pairs), the approach we used to account for the GC content bias, and the method we used to account for the sequencing design batch effect. Moreover, we excluded the sequencing data from 12 mitochondrial genes from both species, a step that – to the best of our ability to determine – was not taken by the original studies. In addition, our definition of ortholog gene pairs differs slightly from that of the original study, as we discussed in the methods. In practice, only the correction for the sequencing design batch effect had a drastic impact on the results. For example, without accounting for batch, using per-gene raw fragment counts instead of FPKM values does not seem to impact the degree to which the uncorrected data support clustering by species (Figure S6).\n\nVisualizing or plotting the data is another important area where different choices can sometime lead to quite distinct conclusions. We chose to display, in addition to the PCA plots, heatmaps based on the correlations among the samples. We note that if the actual data (not pairwise correlations) are clustered, the observed patterns (by species or by tissues, in the respective analyses), seem practically identical (Figure S7). The heatmaps shown in the main figures are based on Pearson pairwise correlations, which provide the highest level of clustering by tissue in the analysis that takes into account batch effects. Alternative heatmap plots based on either Spearman pairwise correlations or other distance measures and clustering methods look similar in principle (Figure S8 to Figure S10), but the clustering by tissue is somewhat less pronounced (clustering by species, when batch is not accounted for, is more pronounced).\n\nIt is important to note that most of the analysis and plotting decisions we have made contributed to a somewhat better clustering of the expression data by tissue, both visually and empirically. We have made these – mostly standard - analysis and plotting choices regardless of the end result (namely, we believe that these are objectively reasonable choices). Importantly, we made identical choices for the clustering analysis and plot types for the data with and without batch correction, and our conclusions are robust with respect to a wide range of possible alternative approaches (Figure S7–Figure S10).\n\nThat said, we do acknowledge that we find the clustering of the data by tissue to be a more intuitive pattern. In other words, we believe that the clustering of comparative gene expression data by species – a result that contradicts previous observations – is a surprising outcome. Hence, we would have intuitively accepted as more correct most reasonable choices of analysis pipelines and data visualizations that supported a greater degree of clustering by tissue.\n\nAs we mentioned above, most of the choices we made resulted in little difference to the overall pattern. It was only the correction for the sequencing design batch effect that had a profound impact. Once we accounted for the batch effect by using ComBat, the comparative gene expression data no longer clustered by species, and instead, we observed a clear tendency for clustering by tissue. This is not surprising, as the sequencing batch, which we corrected for, was nearly entirely confounded with species. It stands to reason that some individual gene expression levels do cluster by species and some by tissue (see for example, Figure S5). While previous data sets strongly support a general clustering of gene regulatory phenotypes by tissue21, we expect the degree of clustering of the gene expression data to differ somewhat across tissues. Yet, in this particular case, by removing the confounding sequencing batch effect we also removed most of the species effect on gene expression levels (a similar case of confounding batch and main effect of interest was discussed a few years ago, with respect to gene expression differences between human populations22).\n\nOne could potentially employ more sophisticated modeling approaches to try and estimate separately the batch and species effects. One idea would be to rely on the fact that there are five sequencing batches, but only two species. This, however, is complicated by the fact that the two sequence batches specific to the human samples share the same run and flowcell (potentially a smaller batch effect), while the two sequence batches specific to the mouse samples are extend over different instruments (potentially a larger batch effect). In any case, we feel that such modeling is beyond the scope of this reanalysis effort. Instead, we conclude that the study design used by the mouse ENCODE consortium was flawed with respect to the questions they set out to address.\n\nIn summary, we believe that our reanalysis indicates that the conclusions of the Mouse ENCODE Consortium papers pertaining to the clustering of the comparative gene expression data are unwarranted. In the narrow context of our reanalysis effort, we state that their conclusions are unwarranted, not wrong, because the study design was simply not suitable for addressing the question of ‘tissue’ vs. ‘species’ clustering of the gene expression data. That said, a large body of independent previous work supports general clustering of comparative gene expression data by tissue.\n\nFinally, we note that in this reanalysis effort, we have only focused on the RNA sequencing data collected by the mouse ENCODE consortium. We have not considered information with respect to the study design used to collect the many other types of data reported by this consortium. Given our findings, we believe that it is appropriate to call for a careful review of these other data sets as well.\n\n\nData availability\n\nAll data are available from the Mouse ENCODE consortium; see Table S1 for specific source URLs and accession numbers.\n\n\nSoftware availability\n\nWe provide supplementary files of the python codes used to process and prepare the data for analysis with R, and the data files for the python codes. We also provide the R codes we used to perform the different analyses as supplementary files, as well as the input for the R codes.\n\nZenodo. Data files and codes used in the reanalysis of the mouse encode comparative gene expression data. DOI: 10.5281/zenodo.17606\n\nThese codes are provided under the MIT license.", "appendix": "Competing interests\n\n\n\nThe authors have no conflicts of interest or competing interests to disclose.\n\n\nGrant information\n\nThis work was supported by NIH grant MH084703.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgment\n\nWe thank J. Lieb, V. Lynch, J. Novembre, L. Pachter, D. Graur, M. Stephens, N. Banovich, and J. Leek, for comments on the manuscript. We also thank 5 additional colleagues, who asked us not to reveal their names, for their valuable comments.\n\n\nSupplementary tables\n\nTable S1. Source of RNA-Seq data.\n\nTable S2. Summary of test scores for the 52 FASTQ files analyzed.\n\nTable S3. Overrepresented sequences in read1 human files.\n\nTable S4. Overrepresented sequences in read2 human files.\n\nTable S5. Overrepresented sequences in read1 mouse files.\n\nTable S6. Overrepresented sequences in read2 mouse files.\n\n\nReferences\n\nYue F, Cheng Y, Breschi A, et al.: A comparative encyclopedia of DNA elements in the mouse genome. Nature. 2014; 515(7527): 355–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin S, Lin Y, Nery JR, et al.: Comparison of the transcriptional landscapes between human and mouse tissues. Proc Natl Acad Sci U S A. 2014; 111(48): 17224–17229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCunningham F, Amode MR, Barrell D, et al.: Ensembl 2015. Nucleic Acids Res. 2015; 43(Database issue): D662–669. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrow J, Frankish A, Gonzalez JM, et al.: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012; 22(9): 1760–1774. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeek JT, Johnson WE, Parker HS, et al.: The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics. 2012; 28(6): 882–883. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan ET, Quon GT, Chua G, et al.: Conservation of core gene expression in vertebrate tissues. J Biol. 2009; 8(3): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkey JM, Biswas S, Leek JT, et al.: On the design and analysis of gene expression studies in human populations. Nat Genet. 2007; 39(7): 807–808; author reply 808–809. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8732", "date": "26 May 2015", "name": "Rafael A. Irizarry", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this PNAS paper is is found that the first three principal components obtained from mouse and human gene expression data correlate with species and not with tissue. This is interpreted to imply that \"tissues appear more similar to one another within the same species than to the comparable organs of other species\".Gilad and Mizrahi-Man (the authors) downloaded all the data from this paper and reanalyzed it carefully. The majority of their F1000Research article is dedicated to describing, in full detail, how they analyzed the data. The choices made all seem sound and they are able to reproduce the figures of the original PNAS article.An important discovery made and reported by the authors is that mice and human samples were run in different lanes or different instruments. The confounding was near perfect (see Figure 1). The authors then apply a linear model (ComBat) to account for the batch effect and find that, after the correction, samples cluster almost perfectly by tissue (see Figure 3). They conclude that \"Once we accounted for the batch effect by using ComBat, the comparative gene expression data no longer clustered by species, and instead, we observed a clear tendency for clustering by tissue. This is not surprising, as the sequencing batch, which we corrected for, was nearly entirely confounded with species.\"There are three issues I recommend the authors consider:As the authors suggest, with the observed level of confounding, if there is in fact a species effect, applying an approach that models batch as a linear effect will also account for species. Although pointing out that there is almost perfect confounding is an important contribution, I don't see why ComBat should be applied here. If a model that removes species is applied, it is no surprise that the data will no longer cluster by species. As mentioned, with the existing study design it is impossible to completely tease out species from batch. However, there is a relatively simple data analysis that can be performed to explore the possibility that instrument or lane are a large enough source of variability to overcome the tissue effect, which is know to be large. The analysis is:i) perform the same PCA analysis on the mouse data and compare the two instruments and thenii) perform PCA analysis on the human data and compare the two lanes.If in fact lane and instrument are a large sources of variability we should see it here. Of course, there is still the possibility that the instruments used for humans was very different to the one used for mice, while the two instruments used for mice were similar. Due to confounding we won't know for sure, but the analysis described here will at least give us at least a lower bound on how large these effects can be. There is a comment in the F1000Research article from the first author of the PNAS article describing a second experiment in which confounding with instrument or lane was not present. In this analysis species continues to be the first few PCs. In a second version of this article, the authors can perhaps comment on this, as well as some of the other comments that suggest other possible sources of variability that may be confounded with species.As a final remark, I am interested in reading the authors/readers thoughts on the biological interpretations that are being assigned to mathematical (euclidean) distance. Specifically, what does the word \"similar\" mean exactly. I understand what means in mathematics, but I am not sure what it means in biology when points are log (FPKM + 1) values for thousands of genes.", "responses": [ { "c_id": "1389", "date": "26 May 2015", "name": "Yoav Gilad", "role": "Author Response", "response": "Dr. Irizarry, Thank you for spending the time to provide a review of our work. We agree with you that given the study design used by the mouse ENCODE consortium, applying a batch correction is futile. Indeed, we explicitly explain that in our discussion (you referred to that section of the text in your review). We further agree that it would be intellectually interesting to research the extent of the batch effect further – for example, by following your suggestion on how to test for the effect of instrument and lane. However, we feel that this additional effort is beyond the scope of our study. The mouse ENCODE consortium papers did not discuss (or account for) the sequencing study design. We spent considerable effort tracking the details that allowed us to reconstruct their design. We pointed out in our paper that given this study design, the unusual biological result reported by the mouse ENCODE consortium might have a technical explanation. We believe it is the responsibility of the mouse ENCODE consortium authors to provide evidence that excludes this technical possibility, rather than us having to prove that it is indeed the likely explanation. Which leads us to your third point: Indeed, the mouse ENCODE consortium authors commented that they have now collected additional sequence data, using a different design, and that their results held. In that sense, we believe that this means that the mouse ENCODE consortium authors accepted our claim that their original design was flawed. Yet, as mentioned in a few other comments here, there is an additional technical batch effect that was not yet excluded – related to tissue extraction and sample preparation. We plan to discuss this additional technical batch effect in a revised version of the text (we will wait to see additional reviews before we provide a revised version of the paper). Again, thank you for your time and thoughts." } ] }, { "id": "8942", "date": "22 Jun 2015", "name": "Michael B. Eisen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper Gilad and Mizrahi-Man reanalyze a high-profile dataset from the ENCODE consortium that was used to argue that gene expression levels are more similar for different tissues from the same species than the same tissues from different species, a somewhat counterintuitive result that contradicts earlier claims (including those by Gilad).The main result of this new work is a simple observation: in the original experiment described in Lin et al., samples from the same species were run in the same sequencing batch. Since there are well-known batch effects, this is poor experimental design that calls into question the claim by Lin et al. that data clusters by species, since the data could instead by clustering by sequencing batch.There is always a bit of a challenge in figuring out what to do with an observation. As the authors point out, this aspect of the experimental design effectively renders the data useless for asking questions about the relative contribution of species and tissue to gene expression variation. Yet it would seem like too light of a paper to simply say \"The original authors messed up. Their claims are therefore invalid. QED.\" So this paper contains a few analyses designed to ILLUSTRATE the point they make. They are not exactly results, since, once you realize that batch and species are completely confounded, correcting for batch will inevitably remove the species signal. In this context, it's a bit weird to present such an analysis as if it is a result, but I don't really see a way around it, and the authors are forthright in pointing out that their main observation is that the data from Lin et al are useless for addressing this issue, and that they can make no claims, even after correcting for batch effects, about what the data actually do say.", "responses": [] }, { "id": "8832", "date": "25 Jun 2015", "name": "Mick Watson", "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\nThe study is carried out well and the results support the conclusion.The paper would benefit from including some of the discussion points made in the comments made to v1 of the paper. Lin et al. have re-sequenced the samples and removed the sequence lane batch effect, and reproduced the same result; however, the samples themselves are confounded, in that they were treated differently prior to sequencing. This discussion should be added to the paper.I would also like to see a discussion of artifacts which are discoverable within the data, for example:the human samples have significant numbers of rRNA reads compared to the mouse samples. This should not happen with mRNA-Seq which includes a polyA selection. the human samples have a hugely varied number of reads per sample, compared to the mouse samples one of the mouse samples has over 1.8M reads that map to a single rRNA transcript. This is an outlier for mouse, as the other mouse samples have low numbers of rRNA reads The mitochondrial genes are turned on in one species but not the otherThese points are all indicators of different sample extraction techniques, which also confound with species.The authors may also wish to discuss use of FPKM, which may not be the most useful measure of gene expression in this study, as the human and mouse orthologues have different lengths.See https://haroldpimentel.wordpress.com/2014/05/08/what-the-fpkm-a-review-rna-seq-expression-units/", "responses": [] }, { "id": "8710", "date": "30 Jun 2015", "name": "Lior Pachter", "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\nThe article \"A reanalysis of mouse ENCODE comparative gene expression data\" by Gilad and Mizrahi-Man examines a claim, recently published in the pair of papersYue F, Cheng Y, Breschi A, et al.: A comparative encyclopedia of DNA elements in the mouse genome. Nature. 2014; 515(7527): 355–364. Lin S, Lin Y, Nery JR, et al.: Comparison of the transcriptional landscapes between human and mouse tissues. Proc Natl Acad Sci U S A. 2014; 111(48): 17224–17229.that expression data from human and mouse cluster more by species than by tissue.The Gilad--Mizrahi-Man paper consists of three \"results\":A report of the experimental design in Lin et al. An attempt to reproduce the results of Yue et al. and Lin et al. that pertain to the claim about species vs. tissue clustering of expression data. A re-analysis of the Lin et al. data in a manner that addresses shortcomings in the original experimental design.The first is the observation that Lin et al. improperly designed their experiment by confounding species with batches sequenced, thereby leading to a possible \"batch effect\" affecting their results. This observation was already published as a preprint by the first author on the pre-print server Twitter (see https://twitter.com/Y_Gilad/status/593088451462963202). Having noted \"a flaw in their [Lin et al.] study design\" Gilad--Mizrahi-Man turn to the question of whether the flaw affected the conclusions in Yue et al. and Lin et al. about expression differences as pertaining to tissues vs. species. To this end, the authors attempted to reproduce the analysis of Lin et al.It is evident that while the Lin et al. results may, in some technical sense, be \"reproducible\" they were certainly not \"usable\" as published. Gilad--Mizrahi-Man carefully expose a vast number of choices in software and processing options poorly described in Lin et al., and whose effect on the final result(s) is unclear. To quote just one example, they write that \"An exception was the mouse pancreas sample, for which the mapping process stalled consistently at the same stage\", a problem that led them to use TopHat v1.4.1 instead of TopHat v2.0.11. One may wonder whether software choices and other decisions in analysis affect final results, and Gilad--Mizrahi-Man address this question (although only partly).For example, one fundamental analysis choice is whether to quantify abundances of genes by summing raw \"fragment counts\" from alignments to gene regions, or via the summing of abundances as quantified by probabilistic assignment of ambiguously mapped reads. Gilad--Mizrahi-Man cite a paper by Dillies et al. (and the French StatOmique Consortium) suggesting that \"FPKM values were not optimal for clustering analysis\" to argue for using \"fragment counts\". I strongly disagree with this choice because transcript abundances are necessary to accurately estimate gene-level abundances, a point that Dillies et al. fail to realize. As pointed out in my own paper on Cufflinks 2 (Trapnell et al. 2012) wrong does not cancel wrong for differential analysis, nor does it for the purpose of clustering.In any case, Gilad--Mizrahi-Man do examine whether quantification by EM affects results and in a later statement they state that \"using per-gene raw fragment counts instead of FPKM values does not seem to impact the degree to which the uncorrected data support clustering by species\", a result summarized in their Figure S6. While I applaud them for checking the dependence of results on this choice, without further analysis the question remains of whether other analysis choices affect results (although to be fair to the authors, the number of tests that would have to be conducted is enormous and quite possibly practically intractable). Nevertheless, it would be interesting if, for the purpose of future transcriptomics analyses, Gilad--Mizrahi-Man were to investigate some key steps as to their effect (e.g. annotation, an issue discussed recently by Ongen and Dermitzakis, or mapper choice).The final result of Gilad--Mizrahi-Man is a re-analysis of the Lin et al. data from which they observe that a basic correction for batch effect removes the strong clustering of expression profiles by species touted in Yue et al. and Lin et al. The question of the relative species/tissue contribution to expression profile is of course fundamental and interesting, and obviously further data, carefully curated and analyzed, will answer the question definitively. As far as the Lin et al. paper goes, the Gilad--Mizrahi-Man paper certainly casts doubt on the suitability of the data for answering the question. For one thing, the term \"batch effect\" is unfortunately rather generic and in this specific case that has become a problem. After initial posting of the preprint by Gilad on Twitter, the authors of Lin et al. resequenced their libraries in a different configuration, but further investigation of the experimental design by Gilad et al. (subsequent to initial posting of a preprint of the article I'm reviewing) appears to have revealed additional problems. For example, tissues in human and mouse were selected from males vs. females respectively (except in ovaries and testis) resulting in another potential bias that would skew expression profiles to cluster by species rather than tissue. In other words, with their paper and subsequent analysis Gilad and Mizrahi-Man have convinced me to be skeptical of the data and conclusions of Lin et al. (and insofar as it pertains to the results in Lin et al., the paper by Yue et al.).But that is not really the point. What matters now are the carefully documented serious shortcomings in the computational and experimental methodology of Lin et al. and for this reason I have approved the Gilad--Mizrahi-Man manuscript. Hopefully the issues raised will be properly addressed by Lin et al. (in a manner equally rigorous to that of Gilad--Mizrahi-Man).", "responses": [] } ]	1	https://f1000research.com/articles/4-121
https://f1000research.com/articles/3-300/v1	09 Dec 14	{ "type": "Opinion Article", "title": "A critical evaluation of science outreach via social media: its role and impact on scientists", "authors": [ "Craig McClain", "Liz Neeley", "Liz Neeley" ], "abstract": "The role of scientists in social media and its impact on their careers are not fully explored. While policies and best practices are still fluid, it is concerning that discourse is often based on little to no data, and some arguments directly contradict the available data. Here, we consider the relevant but subjective questions about social media for science outreach (SOSM), specifically: (1) Does a public relations nightmare exist for science?; (2) Why (or why aren’t) scientists engaging in social media?; (3) Are scientists using social media well?; and (4) Will social media benefit a scientist’s career? We call for the scientific community to create tangible plans that value, measure, and help manage scientists’ social media engagement.", "keywords": [ "Recently", "both scientists and science communicators have issued numerous calls to the scientific community to engage in social media to both connect with other scientists (inreach) and to connect with the public (outreach). Many of the arguments made in support of using social media for science outreach (SOSM) are simply special applications of arguments for outreach in general. However", "researchers often unknowingly approach engagement and outreach with a variety of counterproductive misunderstandings and assumptions", "including those about whether and why their peers do or do not engage. While we are limited by the scarcity of research on this topic", "we are concerned that this leads to inaccurate conceptions of the value of SOSM based in assumptions and anecdotes as opposed to data. Here", "we draw upon a variety of disciplines to address what is known of the role and impact of social media on scientists." ], "content": "Introduction\n\nRecently, both scientists and science communicators have issued numerous calls to the scientific community to engage in social media to both connect with other scientists (inreach) and to connect with the public (outreach). Many of the arguments made in support of using social media for science outreach (SOSM) are simply special applications of arguments for outreach in general. However, researchers often unknowingly approach engagement and outreach with a variety of counterproductive misunderstandings and assumptions, including those about whether and why their peers do or do not engage. While we are limited by the scarcity of research on this topic, we are concerned that this leads to inaccurate conceptions of the value of SOSM based in assumptions and anecdotes as opposed to data. Here, we draw upon a variety of disciplines to address what is known of the role and impact of social media on scientists.\n\n\nA “public relations nightmare”?\n\nNumerous calls for the scientific community to engage in science outreach via social media (SOSM) have been issued1,2 as part of a broader agenda for researchers to engage the public3–5. In particular, SOSM is presented as a promising new tool for science outreach in the midst of a perceived public relations crisis. Specific discussions of the public’s trust6 and perceived legitimacy of scientists7 have been enfolded into a “public relations nightmare” narrative1,8,9. However, does science actually face diminished support or is the “public relations nightmare” an exaggeration reflecting a “institutional neurosis” of science practitioners?10\n\nGiven the prominence of vocal anti-science movements in debates around high profile issues such as vaccines11,12, climate change13, and evolution14,15—and especially considering the intensity and frequency of personal attacks on scientists in these arenas16,17—it is reasonable to ask whether the public fundamentally distrusts scientists. Despite the coverage of these anti-science movements, polls consistently find that scientists out-perform clergy, artists, journalists, business executives, and lawyers in perceived “contribution to society”18. Even in the wake of the ClimateGate controversy, climate scientists remained more trustworthy than competing sources like weather reporters, political and religious leaders, or the mainstream media19. Scientists are in demand for media interviews, political testimony, and public address, and their influence in policy arenas “is well documented, and so is public support for this arrangement”20. In short, despite the evidence of growing political polarization21, the cultural authority of organized science seems to have remained stable.\n\nSo why is the “public relations nightmare” narrative uncritically and continuously repeated? Besley and Nisbet22 argue that scientists misunderstand public attitudes—despite the available data—because they regard non-scientists as hostile “others” who are ill-informed and highly susceptible to media messages, which they also perceive to be slanted against science. Some evidence does exist that these individual movements received disproportionate media coverage given their participatory numbers and impact/importance; and, in regard to both climate change denial and anti-vaccine movements, that “balanced” coverage sustains the claims in the public and disproportionately favors ideas running against scientific consensus23,24. However, the tendency to lump all opposition to science, despite topic, into a common “anti-science” camp with a single root cause, rather than recognizing the diversity of motivations for challenges and rejections creates further problems14,25. This them-versus-us narrative further solidifies a group entrenchment and contempt for outsiders which ultimately develops into a deeply emotional response. Interestingly, this group mentality does not match individual perceptions of the media; scientists rate personal interactions with media highly22. With social media most scientists surveyed felt that the online public would likely benefit their career and the online audience would listen and treat them respectfully26.\n\n\nAre scientists engaging in social media?\n\nIt is common to describe the scientific establishment as resistant to public engagement, and scientists tend not to believe that their colleagues actually engage in outreach27. Yet surveys suggest that scientists engage in public communication activities more often than is commonly assumed28. One survey of scientists found that nearly half of all academic scientists were engaged in some type of outreach29 and interactions with mainstream media have become a standard expectation30,31.\n\nData and analyses on social media outreach are less forthcoming, but studies appear to indicate that uptake of social media by scientists is low. In a University of Michigan study, approximately 60% of the responding scientists engaged the media, public, and government agencies through traditional channels. Yet, nearly 40% of respondents stated they would never use Twitter for academic or professional work32. Wilkinson and Wietkamp33 surveyed researchers who highlighted their work in policy-relevant newsletters, reporting that, “For the majority of researchers, there has been little change in the use of [social] media to communicate with non-academic audiences over the past five years”. Of the respondents, 73% had never used Twitter, 64% had never used blogs, and 51% never engaged online news forums. These patterns of adoption offer a strange mismatch with the expressed beliefs of nearly 44% of German and 65% of American scientists who thought that social media channels, “can strongly influence how the public thinks about science”34. It may be less a question of the platforms than intent. A 2010 survey of biology professionals and students, found that 73% of respondents did actively engage in social networking (broadly defined) as a research tool35. A recent study36, also confirms the relatively low use of Twitter, Facebook, and Google+ compared to the active use of Google Scholar (~60%) and ResearchGate (~40%).\n\nUntil recently, we have known little about the demographics of scientists engaging in public outreach through social media. In a 2012 survey of AAAS members35, younger respondents were willing to participate in online engagement while women “were less likely to say they would be willing to write online articles, blog posts, or online responses”. Previous studies suggest that career, age, and gender differences lead to differences in outreach commitment. Twice as many female (83%) than male (43%) graduate students were involved in outreach37. Ecklund et al.29 found that women are markedly more involved in outreach work than men (72% versus 43%) and marked drop offs in public engagement occurs from graduate students to postdoctoral fellows and faculty. In contrast, male and female faculty members were more equally represented in science outreach based on their representation in the disciplines37. Another study found that higher status and organization autonomy was also associated with greater outreach but found no effects with gender38.\n\nOverall, the best predictor of outreach by scientists appears to be linked to their perceptions of outreach. In general, scientists have a positive attitude toward participating in public engagement and fear associated with participation is low29. There is also “high overall satisfaction of researchers with their own media contacts”30,31. Furthermore, scientists with more positive attitudes, higher perceived communication skills, and more formal communication training, are more likely to engage in these activities38. Increased positive views toward public outreach also increase with increasing participation in these activities39.\n\nPerhaps the biggest obstacles to engagement with outreach in general and social media outreach specifically is a lack of time. Andrews and Weaver37 found that time constraints were the unanimous primary impediment to scientists’ involvement in outreach. Participants’ availability to do outreach was limited by their other priorities, which were given precedence. Ecklund et al.29 noted that for more than half of all scientists, a lack of time is the most insurmountable barrier to doing more outreach and perceived time constraints are associated with a more negative impression of doing outreach activities. For engagement with social media, Rowlands et al.35 found that the most cited inhibitor was a lack of time.\n\n\nAre scientists communicating through social media well?\n\nThe view of scientists as poor communicators has been reinforced by media portrayals of scientists as eccentric and antisocial, possibly dangerous, and ultimately separate from the public40. While negative stereotypes of scientists among American adults have decreased since the 1980s, about one quarter still agree that scientists are, “odd and peculiar”41. Researchers are aware of these attitudes. Nearly 30% of scientists in one study agree that scientists are poor interpersonal communicators and more (37%) placed the blame on scientists themselves29. However and importantly, no quantitative data or analyses support the perception that scientists are poor public communicators. We caution against the reliance on stereotypes. Arguing over anecdotal examples of scientists as both effective and ineffective communicators does little to identify legitimate deficiencies and offer effective remedies. What the scientific community desperately needs is objective studies on communication effectiveness of scientists and a clear definition of “poor communicator”. One tool of this nature is that of Baram-Tsabari and Lewenstein42 that aims to assess scientists’ written skills in public communication. The framework includes assessment of clarity, content, knowledge organization, style, analogy, narrative, and dialogue.\n\n\nWill social media outreach benefit a scientist’s career?\n\nWith regard to social media outreach, the biggest reason given in informal discussions for a scientist’s involvement in social media outreach is that engagement will directly benefit a scientist’s career. We feel it important to reiterate that social media is a tool that can be utilized for purposes other than public engagement. Discussion of the benefits of social media inreach (engagement within the scientific community) and outreach (engagement with the public) are muddled.\n\nSocial media may be an important tool in quickly connecting with other researchers36,43. “This is the dilemma faced by researchers in the digital age. How can we be expected to produce both quality and quantity and to yield influential research? We simply cannot—at least not on our own. Instead, we must rely on networking and collaborations to build our research programs and to remain influential in our fields in order to advance scientific knowledge. With this collaborative view in mind, scientific influence involves the body of work of both individual researchers and of research groups as a whole”43. Darling et al.44 demonstrated that Twitter followers for scientists were always considerably larger than their departments and these large virtual departments help to rapidly generate, share and refine ideas. Moreover blogs written by scientists for scientists are becoming common and important places for the exchange of ideas45.\n\nIn terms of social media outreach, or outreach in general, the impact on a scientist’s career remains largely unquantified and quite possibly indirect. “Many faculty members identified their primary job responsibilities as research and post-secondary teaching. They felt that outreach participation hindered their ability to fulfill those responsibilities and might be an ineffective use of their skills and time, and that it was not a valid use of their research funding37”. In the survey by Ecklund et al.29, 31% of scientists felt that research university systems value research productivity, as indexed by grants and published papers, over everything else, including outreach. With this prioritization structure in place outreach may be perceived as unrelated to a scientist’s academic pursuits. Additionally, the lack of funding and opportunity can make outreach labor intensive. Although social media outreach is perceived to be easy and low cost in terms of money and time, success of such endeavors may require considerable investment. Using Deep-Sea News, which garnered 2.5 million hits in 2013 as an example, the lead author spent approximately 588 hours in 2013 on social media outreach translating into 14.7 40-hour workweeks.\n\nThe “Sagan Effect” - professional stigma associated with public engagement – is perceived to be widespread29. Even those who support active engagement frequently assume that a scientist’s research quality is inversely proportional to the amount of outreach work he or she does. Interestingly, Sagan’s own research productivity in terms of published scientific articles remained constant, ~10 annually, throughout his career and his total scientific body of work is comparable to other eminent scientists46. However, recent changes by funding agencies, professional agencies, and scientist’s views toward public outreach may help to diminish the “Sagan Effect”. Moreover, the number of scientific citations is greater among French scientists who engage in greater public dissemination activities47. In terms of social media, there also appears to be an overall positive correlation between the number of citations and number of Twitter followers among scientists48.\n\nHowever, one way that the social media appears not to impact a scientific career is a direct link of social media mentions and citations on a scientific article. In an analysis of 1.4 million documents in PubMed and Web of Science published from 2010 to 2012, Haustein et al.49 found no correlation between a paper or a journals citation count and Twitter mentions. As argued by the authors of the study, this suggests that Twitter mentions do not reflect traditional research impact. Rather, social media mentions may capture a previously unquantified impact of a scientist’s career50.\n\n\nCan and should SOSM be integrated into a scientist’s career?\n\nWe argue that actions and rhetoric intended to support or encourage SOSM must focus on the return on investment. For the equation to work out in favor of wide-spread and career-long engagement, we propose the following three elements are necessary:\n\n1. It must be valued: The reward for SOSM is often seen as a direct impact on the metrics that are used in gauging scientific success, e.g. gaining tenure or earning promotion. As outlined above, the sparse data available suggest these impacts on publications, citations, and grants may be minimal and indirect. A conversation about SOSM, and outreach in general, is needed that focuses on the metrics of scientific success used by both the community and institutions and developing new ones. Recently, the University of Wisconsin-Madison Division of Biological Sciences, WI, United States, has enacted new tenure guidelines that place greater reward and priority for outreach.\n\n“Excellence can be documented in [these] clearly defined areas, and/or through synergistic combinations of research, teaching, and outreach or service… A key component for excellence in outreach is the dissemination of information derived from scholarly inquiry for the benefit of society. Successful outreach will involve innovative practices, program developments, impacts and applications that have made continuing and substantial contributions at the local, regional, national or international level. It may also lead to transformative practices derived from clinical programs or community engagement for the benefit of society. A demonstrated capability to develop and sustain an independent, cohesive, and impactful outreach program is essential. Dossiers must include documentation of the outcomes of outreach and its impacts and, in addition, include evaluations by recognized outreach specialists in the candidate’s field outside UW-Madison”.\n\nTenure guidelines for the University of Illinois-Urbana Champaign, IL, United States, also place an increasing emphasis on “valuable public engagement” though excellence in outreach is not enough to warrant tenure.\n\nWe must expand the conversation beyond “traditional” tenure-track positions. Scientists working in the private and government sectors often do not enjoy the same freedom to actively engage the public. Scientists employed by the government are often not allowed, or highly constrained, in how they engage the public especially in the new online frontier that is social media. In the most severe case, the Environmental Protection Agency has sought to increase restriction even on those independent scientists who advise the agency, a movement opposed by journalists and scientists alike51. At the United States Geological Service, the use of social media “to regularly talk about your area of expertise” must be “approved by Office of Communications and Publishing and your supervisor”.\n\nOne area of information needed is whether institutions and scholars treat SOSM activities as actual academic and beneficial pursuits. In terms of social media as inreach, many researchers within academia may not treat blogs as serious academic products because they are not peer reviewed52. Geoffrey North in Current Biology stated, “But there is also, I think, a danger here, which lies in the very speed of response, and the way that blogs are essentially “vanity publications” which lack the constraints of more conventional publishing — they are not reviewed, and do not even have to pass the critical eye of any editor53”.\n\nConversely there may be valid reasons to keep, social media, particularly inreach blogging, free from academic inclusion. Blogging may allow a circumvention of hierarchy and power dynamics of academia54 and provide a space “distinct from the parent culture of institutions”55. In terms of both inreach and outreach, anonymity or pseudo-anonymity have often provided underrepresented groups and junior scientists the sense of security needed to discuss issues in science openly. However, while bringing needed voices to the conversation, these scientists do not get to reap any formal rewards within the halls of academia. Clearly SOSM and more generally the usage of social media require reassessment of what defines academia, scholars, scholarship, and institutions. Part of this will also have to address power dynamics in academia that prevent underrepresented groups and junior faculty from pursuing innovative online models for outreach.\n\n2. It must be measured: We have watched with great interest the recent emergence of Altmetrics as a solution to accurately representing the totality of a scientist’s scholarly impact in a web-native world50. Quantification of total downloads and social media mentions for individual scientific papers have provided an exciting advancement in measuring influence. However, these metrics do not yet capture the total social media and web presence for a researcher, e.g. blog posts or Tweets, meaning there is not yet digital equivalency for the H-index. Furthermore, as with traditional metrics, current tools cannot discriminate between positive and negative mentions (citations), and with the dynamics of social media sharing at play, great care must be taken not to conflate ‘activity’ metrics with proof of influence or approval. The need to consider both quality and quantity was ostensibly the intent of the satirical attempt48 to calculate the so-called Twitter Kardashian Index by plotting individual researchers’ numbers of followers against the number of citations of their scientific output and thereby identify those who are more “famous” than their publication record merits. We find the development of a K-Index, whether in satire or not, to be counterproductive to meaningful understanding of the role of social media and fraught with methodological problems. Indeed, instead of criticizing scientists with large number of Twitter followers, we should be studying them as exemplars for that dimension of social media success.\n\n3. It must be manageable: The most identified issue by scientists preventing them from engaging in SOSM, and outreach in general, is a lack of time. This issue remains relatively unaddressed. If the goal is ultimately to have more scientists engaged more fully in outreach through social media there is a need to target the conflict of the clock. One possible mechanism for this is a reprioritization and integration of research and outreach.\n\nOne way forward may be finding opportunities where effort and products can serve more than one purpose. Several existing examples demonstrate that a research program and SOSM can be integrated. For example, blog posts on scientific literature already being read for journal clubs, classes, grants, and publications represent both a way to more actively engage the literature scientifically while producing online content for public consumption. Crowd funding of science both directly engages the public while also serving as a mechanism to generate smaller funds for research56,57. Online content can also be generated as a product of classroom activities. For example, the “Sizing Ocean Giants” project (http://www.storyofsize.com/sizing-ocean-giants/), an undergraduate directed research course, focused on the body sizes of marine organisms and included a heavy social media component. Undergraduates were directed to use social media to engage the public about their specific animals, leveraging online platforms as a pedagogical tool to increase their understanding of their organisms and the scientific process. As with traditional courses, writing assignments on a variety of scientific topics were included, but these were simply migrated online as blog posts. Twitter assignments included #sizeme where the public was encouraged to Tweet height or weight and students in the course responded with how the measurements compared to a species of marine megafauna effectively illustrating scale. Ultimately, the research by students benefited from online interactions with other scientists and lead to a peer-reviewed publication.\n\nMany programs also exist within and outside of universities for scientists to engage in science communication and SOSM. These require less effort on the part of individual academics because infrastructure, and often training, are already in place. For example, Experiment.com provides a mechanism for scientists to launch crowd-funding campaigns. Setting out to create a new blog may not be as effective as providing a blog post to an established blog or joining a group blog.\n\n\nConclusion\n\nWe clearly need a new way to consider, conduct, and evaluate science outreach, online and off. This new model must be informed by theory and grounded in data analysis, as opposed to predicated on anecdote and assumption. We have shown here that many popular propositions about the attitudes, behaviors, and actions of scientists engaged in SOSM are incorrect. These conceptual errors may slow or impede progress by diverting efforts into solutions for the wrong problems. We propose efforts should concentrate on making SOSM a valued and worthwhile endeavor within and outside academia, an actual quantification of the value of SOSM by individuals, and creating programs that make SOSM time manageable for scientists.", "appendix": "Author contributions\n\n\n\nCRM and LN contributed equally to the conception, research, and writing of the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests are declared.\n\n\nGrant information\n\nCRM is supported by the National Evolutionary Synthesis Center (NESCent), NSF #EF-0905606.\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\nMichelle Gaither-McClain provided editorial assistance and loving patience with the first author.\n\n\nReferences\n\nWilcox C: Guest editorial. It’s time to e-volve: taking responsibility for science communication in a digital age. Biol Bull. 2012; 222(2): 85–7. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "7573", "date": "18 Feb 2015", "name": "Paige Brown Jarreau", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides a much needed point of departure for future research regarding scientists' engagement in social media and public engagement. This article raises great questions that will need answering through future empirical evidence.Previous studies have often queried scientists' engagement in social media and public outreach on a broad level. However, \"scientists\" is an extremely broad grouping. There has been some work looking at scientists' engagement in social media and public outreach by discipline and demographic variables including age and gender, but we need to go further than this. Why do some scientists make the time to engage in outreach or social media, such as tweeting and blogging, while others do not? Logistical constraints are probably involved, but are likely not the major constraint, as many extremely busy scientists find time or make time to engage in outreach via social media. How does the culture at specific organizations/institutions affect this engagement, or scientists' decisions of whether it is \"worth it\"? And how can social scientists uncover these nuances of behavior with regards to scientists' use of social media for outreach? I think it would be valuable it the authors addressed these questions, and addressed how future research should focus on nuances vs. overly broad descriptions of scientists' use of social media for outreach.In the section on how social media outreach will benefit scientists' careers, the authors focus on tangible and professional benefits that are compatible with traditional measures of scientific/academic performance (such as citations and tenure). This leaves out more intangible benefits that scientists may experience from engaging in social media outreach, such as being able to see the practical applications of their research, or becoming more well-rounded in terms of their scientific expertise outside of their narrow fields of research. The authors should address how less traditional \"benefits\" of social media outreach can be measured and communicated to scientists in various fields. A look at the degree of collaboration that occurs for researchers who don't engage in social media vs. those who do would also be interesting.A few other notes:\"the best predictor of outreach by scientists appears to be linked to their perceptions of outreach\" - why isn't this explained in the context of the theory of planned behavior, or the value-belief-norm model for explaining scientists' behavior toward social media engagement / outreach? \"Moreover blogs written by scientists for scientists are becoming common and important places for the exchange of ideas\" - I would caution against broad statements about the impacts of blogs, or what kind of blog genres are growing. In fact, there is some evidence, including from my own ongoing research (although more research is needed), that blogs geared toward outreach and explaining science to broader audience are outnumbering / growing past those blogs that are written by scientists for other scientists (these types of blogs are generally in the minority). Just as in everything else, we need more data about science blogging approaches and the impacts of those approaches. For example, which different social networks (blogs? Twitter? ResearchGate and similar science-focused social networks) are being used by researchers to talk to other researchers, and which are being used in a more outreach type of capability, and why?I think the authors could round-out their discussion of needed research in this and related areas through a modest revision of their original article. I also encourage the authors to discuss how theories of mass communication, psychology and behavior could fit into the areas of research they call for.", "responses": [ { "c_id": "1353", "date": "18 May 2015", "name": "Craig McClain", "role": "Reader Comment", "response": "The reviewer provides many helpful suggestions for the manuscript. We thank them for the insightful review. We have addressed many of these in the article. The reviewer suggests that manuscript include more theory. Indeed, this is interesting topic and a paper that synthesizes these different field's theories and applies them to SOSM is desperately needed. Indeed, to be able to due this topic justice it would require an additional paper with multiple pages of text. This is currently beyond the scope of the manuscript. In addition, the scope of this manuscript was to specifically examine empirical evidence, and not the theory, that addresses these questions. Indeed, the impetus for the work was that several conferences talks and papers seem to ignore much of this empirical evidence." } ] }, { "id": "7923", "date": "20 Mar 2015", "name": "Kathryn B. H. Clancy", "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\nMcClain and Neeley make an important contribution to the science communication literature with their opinion article “A critical evaluation of science outreach via social media: its role and impact on scientists.” The authors do a great job dismantling many of the misconceptions held by those who naysay science communication. I also loved their manifesto at the end of the three necessary elements to support social media for science outreach (particularly the last element, which is very consistent with some initiatives on work allocation I am currently attempting at my own institution!). I have only a few comments about ways in which these authors could strengthen their manuscript:The introduction could more clearly state the authors’ argument. As experts in this field, the authors could really frame their argument for the reader by offering it from the beginning. Right now the introduction does not make clear what side of the issue for which the authors advocate. Related to this: the headings, instead of asking the question, could just make your point. So for instance, the first could read “Science outreach is not a “social media nightmare.”” For the browsing reader, making your point and THEN iterating it can be powerful. I love how strongly cited this article is. I would love to see a little more of the extended explanation of the empirical articles to which the authors cite in their first section – they do a great job of this in the other sections.Minor issues:The acronym “SOSM” appears to be out of order, since it stands for Social Media for Science Outreach (SMSO). Personally I’m not crazy about using “impact” when the intended meaning is “effect,” as is used in the title, but I leave that very minor decision up to the authors. When there is an extended quote, it really should be turned into a blockquote so that it’s clear to the reader the authors are using the words of another to strengthen their argument (examples: first and second paragraphs in right column of page 3). Though I also felt these extended quotes might be better paraphrased by the authorsso as to avoid patchwriting. The use of the term “academic inclusion” (page 4, second full paragraph, right column) is confusing. Inclusion in academic circles is usually a term to discuss diversity initiatives.Thanks for the opportunity to review this interesting piece.", "responses": [ { "c_id": "1352", "date": "18 May 2015", "name": "Craig McClain", "role": "Reader Comment", "response": "We appreciate the efforts of the reviewer to strengthen the manuscript. The main recommendation is take a stronger stance. Our goal is with the paper is not take an opinion but rather to raise specific questions and review the empirical evidence addressing these questions. Indeed, this area has been traditionally opinion rich while data poor. We specifically chose to take a more objective stance. However, we do make recommendations in the final section of the paper. We have addressed all the minor issues in our revision of the paper." } ] }, { "id": "7921", "date": "26 Mar 2015", "name": "Sharon L. Dunwoody", "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\nReaders of this opinion piece will appreciate its ability to locate and reflect on much of the recent literature dealing with the extent and perceived impact of scientists' outreach behaviors. That literature gives social media only a glancing blow, so the authors are correct to note that we know little about scientists' use of social media and about perceived and actual outcomes of such use. We do know that social media use is growing among scientists, and mainstream empirical studies - which typically generate snapshots in time that won't become available in published form for months (or years) - will have a hard time staying on the front edge of such changes. McClain and Neeley's analysis leads me to suggest that scholars pay particular attention to the impacts of social media outreach on scientists' careers. While we know that rendering one's research publicly visible via such traditional means as mass media stories has a salutary impact on the number of citations to that work in the peer-reviewed literature, we are just beginning to track the impact of scientists' \"unmediated\" signaling via tweets, blogs posts, etc. Some recent work found that TED talks did not appear to make much of a difference. But a very recent article in Journalism & Mass Communication Quarterly (Liang et al., 2014) did find that, among a sample of nanoscientists, tweeting was associated with more frequent interactions with reporters and other non-scientists and that those interactions were then linked to enhanced scientific impact. I would implore the authors to \"step away\" from the acronyms. SOSM (science outreach via social media) deserves a quick burial. As is the case with so many acronyms, it obfuscates rather than clarifies.", "responses": [ { "c_id": "1351", "date": "18 May 2015", "name": "Craig McClain", "role": "Reader Comment", "response": "We thank Dr. Dunwoody for their review. While we appreciate the reviewer's opinion on acronym, science outreach via social media (SOSM) was introduced for two reasons.One, previous discussion and research in this area has confused multiple areas including lumping in use of social media of scientists as outreach. Clearly this is incorrect. We wish to specifically bring attention to a tangible issue that is at the intersection of social media usage and engaging the public and online. Specifically providing a term and acronym removes semantic issues and focuses the dialogue. Second, the acronym was introduced for pragmatic reasons. Science outreach via social media is used 20 times in the manuscript. Using the full term would make the language unwieldy for readers." } ] } ]	1	https://f1000research.com/articles/3-300
https://f1000research.com/articles/3-123/v1	10 Jun 14	{ "type": "Research Note", "title": "Low and stable rates of antenatal syphilis and HIV in migrant and refugee women on the Thai-Myanmar border: a descriptive study", "authors": [ "Rose McGready", "Joy Kang", "Isabella Watts", "Mary Ellen G Tyrosvoutis", "Miriam B. Torchinsky", "Aung Myo Htut", "Nay Win Tun", "Lily Keereecharoen", "Chirapat Wangsing", "Borimas Hanboonkunupakarn", "François H. Nosten", "Joy Kang", "Isabella Watts", "Mary Ellen G Tyrosvoutis", "Miriam B. Torchinsky", "Aung Myo Htut", "Nay Win Tun", "Lily Keereecharoen", "Chirapat Wangsing", "Borimas Hanboonkunupakarn", "François H. Nosten" ], "abstract": "Objective: The antenatal prevalence of syphilis and HIV/AIDS in migrants and refugees is poorly documented. The aim of this study was to audit the first year of routine syphilis screening in migrant and refugee women on the Thai Myanmar border.Methods: From August 2012 to July 2013, 3600 pregnant women were screened for HIV (ELISA) and syphilis (VDRL with TPHA confirmation) at clinics along the Thai-Myanmar border.Results: Seroprevalence for HIV 0.47% (95% CI 0.30-0.76) (17/3,599), and syphilis 0.39% (95% CI 0.23-0.65) (14/3,592), were low. Syphilis was significantly lower in refugees (0.07% 95% CI 0.01-0.38) (1/1,469), than in migrants (0.61% 95% CI 0.36-1.04) (13/2,123). The three active (VDRL≥1:8 and TPHA reactive) syphilis cases with VDRL titres of 1:32 were easy to counsel and treat. Women with low VDRL titres (>75% were < 1:8) and TPHA reactive results, in the absence of symptoms and both the woman and her husband having only one sexual partner in their lifetime, and the inability to determine the true cause of the positive results presented ethical difficulties for counsellors.Conclusion: As HIV and syphilis testing becomes available in more and more settings, the potential impact of false positive results should be considered, especially in populations with low prevalence for these diseases. This uncertainty must be considered in order to counsel patients and partners accurately and safely about the results of these tests, without exposing women to increased risk for abuse or abandonment. Our findings highlight the complexities of counselling patients about these tests and the global need for more conclusive syphilis testing strategies.", "keywords": [ "HIV syphilis pregnancy yaws refugees migrants Myanmar" ], "content": "Introduction\n\nThe global health impact of sexually transmitted infections (STIs) including HIV/AIDS and syphilis is well recognized1. Both syphilis and HIV/AIDS pose major health risks in the developing world, impacting maternal and infant health due to vertical transmission via congenital infection and/or through breastfeeding2. This is estimated to cause over 500,000 adverse pregnancy outcomes per year, including stillbirth and congenital infection1. Displaced populations of migrants and refugees within developing regions are particularly vulnerable to disease although data on the prevalence of infection is scarce3.\n\nThe Tak province on the Thai-Myanmar border is home to a diverse population comprised of local Thai and members of Thailand’s ethnic minorities as well as foreign migrant workers and refugees of multiple ethnicities from Myanmar4. Members of the Karen ethnic group represent a large proportion of the ethnic minority people from both countries. The estimated 2 million displaced Burmese living in Thailand3 are vulnerable to STIs and HIV/AIDs5, due to the lack of access to health services, poor education and low income3,5,6. In 2005, we reported on cross sectional surveys of HIV and syphilis in pregnant refugee and migrant women from the Thai-Myanmar border which showed low rates of HIV (0.4%) and syphilis (0.4%)7. While HIV screening in pregnancy was routine since the cross sectional surveys syphilis screening was only introduced when funding became available. The aim of this study was to audit the first year of routine syphilis screening in the same population and reassess the trends in HIV rates.\n\n\nMethods\n\nThe Shoklo Malaria Research Unit (SMRU) provides health services and conducts research of relevance to the local population of migrants and refugees on the border between Thailand and Myanmar (www.shoklo-unit.com). As part of the efforts to reduce malaria-related maternal mortality8, pregnant women are encouraged to attend SMRU antenatal clinics (ANC) as frequently as every fortnight. For refugees the service has been available since 1986, and for migrant communities, since 1998. Three SMRU clinics operate in border communities north and south of the town of Mae Sot: Maela (MLA,17°07′44″N 98°22′50″E) refugee camp (population circa 49,626) and in the migrant villages of Wang Pa (WPA,16°49'42\"N 98°32'25\"E) and Mawker Tai (MKT, 16°19'37\"N 98°40'12\"E) (population circa 30,000).\n\nDuring the first antenatal visit, a dating ultrasound, routine screening blood tests (malaria smear, haematocrit, syphilis, HIV, full blood count) are taken, and medical and obstetric examinations are performed. Pre-test counselling, using an “opt-out” system, is provided to all women at their first antenatal visit before any screening blood tests are performed. Malaria smears are read promptly and positive cases are treated immediately. At all visits tablets of ferrous sulphate (200 mg daily), folic acid (5 mg weekly) and thiamine (vitamin B1 100 mg daily)9 are supplied to all pregnant women. Anaemic patients receive 800 mg of ferrous sulfate and 5 mg of folic acid daily, and a tetanus vaccination is given to women who have not been previously immunized. The SMRU ANC program aims to provide integrated antenatal care for any medical or obstetric problem including treatment for HIV [life-long antiretroviral (ARV) triple therapy] or syphilis. ARV therapy was GPO-vir® (a combination of Stavudine (D4T) 30 mg, Lamivudine (3TC) 150 mg and Nevirapine (NVP) 200 mg) one tablet twice daily, for patients with low CD4 (<350/mm3) counts; and for late pregnancy presentation (34 weeks of more) or CD4 (≥350/mm3) then Zidovudine (AZT) 300 mg and Lamivudine (3TC) 150 mg as a combination tablet (ZilarVir) and Efvarinex (EFV) 600 mg taken in once dose once daily, is provided. Drug therapy for syphilis was benzathine penicillin G 2.4 million units by intramuscular injection.\n\nPoint of care HIV testing is done using an on-site rapid diagnostic test (Core™ HIV 1&2, Core Diagnostics, UK). At the first antenatal visit the results of the screening test are explained to the patient and the sera of positive patients is transported to Mae Sot Hospital laboratory (30–60 km from the sites) for confirmation using an immunoassay (HIV Combi PT, cobas®, Roche, Germany). Post-test counselling explaining the results of the confirmation test is provided the following week.\n\nSyphilis testing is conducted at Mae Sot Hospital on samples taken at SMRU ANCs. The hospital’s protocol10 uses the Venereal Disease Research Laboratory (VDRL) test (VDRL Carbon Particle Antigen Kit, Plasmatec, Lab21 part of Health Care Ltd, UK) and confirms positive VDRL results with Treponema pallidum haemagglutination (TPHA) assay (TPHA kit, Plasmatec, Lab21 part of Health Care Ltd, UK). If a screening using VDRL is negative, no further tests are performed. Counselling about the test results is provided by SMRU staff to all women at their next antenatal visit. A policy to treat all patients for whom both VDRL and TPHA were reactive with 2.4 million units penicillin IM weekly × 3 doses was employed. This simple regimen which should be effective for all stages of the disease and prevention of congenital syphilis was used due to the difficulties in determining the stage of infection in most of our patients who denied symptoms or exposure history. No further serological testing was carried out after treatment in line with current recommendations for resource-poor settings11.\n\nRetrospective review of anonymized data from antenatal records was approved by the local Tak Community Advisory Board and the Oxford Tropical Research Ethics Committee (OXTREC 28-09).\n\nData were analysed using SPSS for Windows™ (Version 20, SPSS Inc.) (Dataset 1). Continuous normally distributed data were described by their means and compared with the students’s t test, while non-normally distributed data were described by their median and compared with the Mann-Whitney U test. Percentages were calculated for categorical data, which were compared using the χ² test or Fisher’s exact test. Factors associated with a positive syphilis status or a positive HIV status, were compared by univariate analysis and odds ratios (OR) were calculated with a 95% confidence interval.\n\n\n\n\nResults\n\nFrom the 8th of August 2012, until the 7th of August 2013, there were 3,600 women who attended the SMRU antenatal clinics at least once. Most were regular attenders. The ethnic make-up of the refugee and migrant population was largely Karen and Burmese, and significant differences between baseline characteristics of refugee and migrant clinics and between the two migrant clinics were apparent (Table 1). Maela has the highest case load (1,477 ANC attenders), followed by Wang Pha (1,185) and then Maw Ker Thai (954). Age and gravidity were similarly matched at all clinics, but women attending the migrant sites (WPA and MKT) had a significantly higher number of marriages and a shorter duration of residence at their current address when compared with MLA. Duration of residence and literacy was lowest in WPA. In this border population, country of residence differed significantly between sites, with only 8% of ANC patients in MLA reporting an address in Myanmar, compared with 34% in Maw Ker Thai and 67% in WPA. Finally, significant differences were seen in the ethnic makeup of the patient populations. Karen ethnicities account for 82% of MLA patients, but only around 30% of the patients in MKT and WPA. Muslim patients make up 12% of MLA’s ANC attenders, but are less than 1% of the migrant populations. Around 40% of the migrant patients are ethnically Burmese, but less than 2% of the MLA patients report Burmese ethnicity. Other ethnic minorities comprise 10% of MKT’s patients, 5% of WPA and only 2% of MLA.\n\naEthnic group of the woman was derived from the ethnicity of the woman’s parents. Sgaw Karen implies both parents were Sgaw Karen and so on. Muslim is used locally to define people who originated from Bangladesh or Rhakine state.\n\nbOther as in not one of the leading 3 ethnic groups (Karen, Burmese or Muslim)\n\ncP<0.05 significantly different Wang Pha;\n\ndP<0.05 significantly different Maw Ker Thai;\n\neP<0.001 significantly different Wang Pha;\n\nfP<0.001 significantly different Maw Ker Thai\n\nSyphilis was tested in 3,592 of 3,600 women (99.78%). The remaining 8 patients were not tested due to interruption of the usual screening process such as a patient actively miscarrying. Off-site testing found 0.50% (18/3,592) VDRL reactive of whom 22.2% (4/18) were TPHA non-reactive indicating biological false positive reactions. Prevalence of serological syphilis (VDRL and TPHA reactive sera) was 0.39% (95% CI 0.23–0.65) (14/3,592). Of these, the majority 78.6% (11/14) were low VDRL titres < 1:8 (three were 1:2; eight were 1:4) and the remaining three were all 1:32. Only two women were symptomatic, both at a titre of 1:32 and one of these women was also HIV positive. The proportion of serological syphilis in MLA, MKT and WPA was 0.07% (1/1,469), 0.73% (7/954) and 0.51% (6/1,169) respectively. Syphilis prevalence was significantly lower in MLA compared to MKT P=0.008 and WPA P=0.049, but there was no difference between the two migrant sites (P=0.583). The overall prevalence of syphilis was lower in refugees 0.07% (1/1,469) (95% CI 0.01–0.38) compared to migrants 0.61% (13/2,123) (95% CI 0.36–1.04), P=0.011.\n\nAll active syphilis cases found in this audit (titre ≥ 1:8 and TPHA reactive) were in young migrant women who were also primigravidae and all were treated. Amongst their partners, one partner agreed to testing and treatment (HIV and syphilis positive case); one agreed to treatment but not to testing and one was not contactable. Amongst the 11 low titre couples: five husbands attended the clinic and all five were negative (VDRL titres in their wives were 1:4 (four cases) and 1:2 (one case)); the remaining six husbands did not get tested because they were away for work (five cases) and one woman never returned at all after the first consultation. Counselling couples with low VDRL titres who both reported to have one lifetime sexual partner was particularly challenging.\n\nTreatment with IM Penicillin was given to 71% (10/14) of patients with serological syphilis (positive VDRL and TPHA reactive) and four low titre (2 with 1:2 and 2 with 1:4) women remained untreated. Three of these women had low risk histories (no history of symptoms and reporting only one lifetime sexual partner for both the woman and her husband) and one history was unknown as the woman never returned to ANC after the first visit.\n\nA HIV test was performed on 3,599 of 3,600 women (99.9%) of whom 0.9% (34) were tested positive by a single on-site rapid diagnostic test (RDT). Off-site confirmation testing by double ELISA showed that 46.9% (95% CI 30.9–63.6%) (17/32) of these positive RDT results were false positives (including 2 cases for whom confirmation testing was initially indeterminate, but were ultimately negative on repeat samples). This high rate of RDT biological false positives is not unexpected in a low transmission setting as these tests are optimized for sensitivity at the expense of specificity12. The confirmed HIV-positive rate in pregnancy was 0.47% (95% CI 0.30–0.76) (17/3,599). Lowest HIV rates were again observed in the refugee camp MLA 0.27% (4/1,474) compared to the migrant sites, MKT 0.52% (5/594) and WPA 0.68% (8/1,171). While MLA was significantly lower than WPA (P=0.049) no other significant differences were observed: MLA vs. MKT P=0.329, and MKT vs. WPA P=0.783. The percentage of HIV cases in refugees was not significantly different from the combined percentage of the two migrant sites: 0.3% (95% CI 0.11–0.70) (4/1,474) vs. 0.61% (13/2125) (95% CI 0.36–1.0), P=0.215. There were 82.4% (14/17) of HIV-positive women treated with ARVs following guidelines based on WHO recommendations. The three untreated women were all migrants: one decided to return to Burma; one miscarried with a very high CD4 count and was followed up with 6-monthly CD4 counts and one woman did not return for the result.\n\nThere has been no significant change in the prevalence of HIV and syphilis from 19977 to 2013 in refugees, nor for syphilis7 in migrants 2005 to 2013. Data from Myanmar and Thailand (http://aidsdatahub.org/Country-Reviews) were included for comparison (Table 2).\n\naSerological syphilis positive using the same criteria, and the same hospital for confirmatory testing at each survey time point; n.a. not available\n\nbData from refugee and migrant populations in 1997 and 2005 previously published in Reference 7\n\ncData on Thailand and Myanmar’s infection rates provided for comparison from the Evidence To Action Website country profiles accessed Mar-2014 (http://aidsdatahub.org/Country-Reviews)\n\nFactors possibly associated with serological positive syphilis and HIV infection were examined by univariate analysis (Table 3). Further modelling was not done due to the low number of seropositive cases precluding meaningful conclusions.\n\naOnly 1 woman had both HIV and syphilis so these were not compared as a risk factor for each other\n\n\nConclusions\n\nThere is limited research on the prevalence of HIV and syphilis in migrant and refugee pregnant populations, despite the vulnerability of these populations. Here we describe low and stable HIV and syphilis prevalence in three displaced populations near Mae Sot, on the Thai-Myanmar border. No statistically significant difference in HIV rates between refugees and migrants was observed while syphilis was almost absent in the refugees but prevalent in the migrant population with rates comparable to regional and world averages1.\n\nUnivariate risk factor analysis demonstrated that age of 30 years or more, a history of remarriage, and non-Karen ethnicity, emerged as significant for both HIV and syphilis infection. However, unlike HIV, syphilis was significantly higher in the migrant population than the refugee population. Migration, and length of residence at the current address of < 6 months, was itself a risk factor. These findings suggest that the relatively stable social order in the refugee camp, compared with the more mobile migrant communities, may be protective against syphilis. Though the camp population is not fixed, local government, educational and religious institutions are well established, and largely controlled by a socially conservative Karen majority. Syphilis transmission differs from HIV transmission in that it is rarely transmitted during the chronic latent phase; the infection is passed most readily if the interval between partners is short13. HIV, on the other hand, can become increasingly infectious years after an untreated infection is established in one of the partners14. Karen culture, which does allow for remarriage in cases of divorce or death of a spouse, holds a strong taboo on multiple sexual partners or extramarital sex. This taboo may provide some protection in the Karen-dominated refugee camp and is supported by the ethnic trend shown in Table 1 and Table 3. Migrant communities contain more young men and women separated from their families and typical community support. High-risk sexual behaviours have been found to occur more frequently in migrant populations in Thailand than in non-migrants, possibly accounting for the higher prevalence of syphilis in this group.\n\nA significant potential confounder to our analysis is the fact that existing serologic tests cannot differentiate between syphilis and yaws or other non-venereal treponematoses15,16. Yaws is still included in Thailand’s program of neglected tropical diseases indicating that total eradication may not have been achieved yet17. The last reported outbreak of yaws in Thailand was published in 1994 (within our patients’ lifetime) and occurred in a remote village a few hundred kilometres south of the SMRU sites18. The last yaws-related publication from Myanmar was published in 196019 on the proposed yaws national programme. Myanmar has been amongst the world’s poorest 30 countries for decades, and populations on the borders, remote from central government, have had poor access to health services18,19. If a national program has been implemented to eradicate Yaws, it is unlikely it has reached these communities. More than 75% of TPHA-reactive patients in our cohort had low VDRL titres <1:817.\n\nAmongst these women, all of the husbands who came to the clinic were negative and the couples presented low risk histories. Latent syphilis (acquired via sexual contacts not disclosed by the patients) cannot be ruled out but the picture is suggestive of an unidentified treponemal infection (such as yaws) causing false-positive results. Publically available data of RPR or VDRL titres in pregnant women in Thailand (http://aidsdatahub.org/Country-Reviews) are similar to what was observed here and are in contrast to studies from Africa, where a greater proportion of high sera titres are reported20,21. Counselling VDRL discordant couples with low risk histories where the wife has a very low VDRL titre presents an ethical challenge in this conservative culture. The potential for serious social consequences, such as abuse or abandonment, of giving false positive results to these couples should be considered. While the stable HIV prevalence in this population supports continuation of routine HIV screening for all pregnant women, a risk-factor-based screening approach for syphilis could be considered for the refugee camp.\n\nAn additional factor limiting the analysis of risk factors in this cohort is the small number of positive results. These numbers become even smaller if the low-titre syphilis patients are excluded.\n\nReports from 2011 estimate over 7 million people live in protracted refugee situations, and over 27 million are internally displaced persons (http://www.prsproject.org/protracted-refugee-situations/). Contextual differences within such groups are highlighted here where differences were found in quite similar populations. While these results cannot be widely applied to other settings, the questions raised about unintended consequences of routine screening and the need for more conclusive syphilis testing strategies, have implications with global relevance. The overall cost-effectiveness and impact of syphilis serological testing in pregnancy in low prevalence areas requires more in-depth evaluation especially in settings where funding for the most basic health care needs remains precarious.\n\n\nData availability\n\nfigshare: HIV and syphilis antenatal screening data at SMRU 2012–13. Doi: 10.6084/m9.figshare.104412022", "appendix": "Author contributions\n\n\n\nRM and FH conceived the study. RM, METG, AMH, NWT, LK and FN designed the experiments. MEGT, AMH, NWT, LK and NC carried out the research. JK, IW, NC, and BM contributed to the design of experiments. IW, MBT, METG and RM prepared the first draft of the manuscript. RM, JK, IW, METG, MBT, BH, and FN contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a grant from the Wellcome Trust of Great Britain for the Thailand/Laos Major Overseas Programme 2010–2015 (Grant B9RTOZ2) jointly awarded to MORU and LOMRU. The Shoklo Malaria Research Unit is part of the Wellcome Trust Mahidol University Oxford Tropical Medicine (MORU) Research Programme.\n\n\nAcknowledgments\n\nWe would like to thank the women who attended the antenatal clinics and the midwifery, counsellors, laboratory, pharmacy, IT and logistic staff who supported the work.\n\n\nReferences\n\nNewman L, Kamb M, Hawkes S, et al.: Global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data. PLoS Med. 2013; 10(2): e1001396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhary M, Kashyap B, Bhalla P: Congenital syphilis, still a reality in 21st century: a case report. J Med Case Rep. 2007; 1: 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarballo M, Nerukar A: Migration, refugees, and health risks. Emerg Infect Dis. 2001; 7(3 Suppl): 556–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKusakabe K, Pearson R: Transborder Migration, Social Reproduction and Economic Development: A Case Study of Burmese Women Workers in Thailand. Int Migr. 2010; 48(6): 13–43. Publisher Full Text\n\nSalama P, Spiegel P, Brennan R: No less vulnerable: the internally displaced in humanitarian emergencies. Lancet. 2001; 357(9266): 1430–1. PubMed Abstract \| Publisher Full Text\n\nSpiegel PB: HIV/AIDS among conflict-affected and displaced populations: dispelling myths and taking action. Disasters. 2004; 28(3): 322–39. PubMed Abstract \| Publisher Full Text\n\nPlewes K, Lee T, Kajeechewa L, et al.: Low seroprevalence of HIV and syphilis in pregnant women in refugee camps on the Thai-Burma border. Int J STD AIDS. 2008; 19(12): 833–7. PubMed Abstract \| Publisher Full Text\n\nMcGready R, Boel M, Rijken MJ, et al.: Effect of early detection and treatment on malaria related maternal mortality on the north-Western border of Thailand 1986–2010. PLoS One. 2012; 7(7): e40244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGready R, Simpson JA, Cho T, et al.: Postpartum thiamine deficiency in a Karen displaced population. Am J Clin Nutr. 2001; 74(6): 808–13. PubMed Abstract\n\nWiwanitkit V: Screening for syphilis in pregnancy: which is the proper method? Arch Gynecol Obstet. 2007; 276(6): 629–31. PubMed Abstract \| Publisher Full Text\n\nSaloojee H, Velaphi S, Goga Y, et al.: The prevention and management of congenital syphilis: an overview and recommendations. Bull World Health Organ. 2004; 82(6): 424–30. PubMed Abstract \| Free Full Text\n\nShanks L, Klarkowski D, O’Brien DP: False positive HIV diagnoses in resource limited settings: operational lessons learned for HIV programmes. PloS One. 2013; 8(3): e59906. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo EL, Lukehart SA: Syphilis: using modern approaches to understand an old disease. J Clin Invest. 2011; 121(12): 4584–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWawer MJ, Gray RH, Sewankambo NK, et al.: Rates of HIV-1 transmission per coital act by stage of HIV-1 infection, in Rakai, Uganda. J Infect Dis. 2005; 191(9): 1403–9. PubMed Abstract \| Publisher Full Text\n\nNoordhoek GT, Cockayne A, Schouls LM, et al.: A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws. J Clin Microbiol. 1990; 28(7): 1600–7. PubMed Abstract \| Free Full Text\n\nNoordhoek GT, Wieles B, van der Sluis JJ, et al.: Polymerase chain reaction and synthetic DNA probes: a means of distinguishing the causative agents of syphilis and yaws? Infect Immun. 1990; 58(6): 2011–3. PubMed Abstract \| Free Full Text\n\nNarain JP, Dash AP, Parnell B, et al.: Elimination of neglected tropical diseases in the South-East Asia Region of the World Health Organization. Bull World Health Organ. 2010; 88(3): 206–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTharmaphornpilas P, Srivanichakorn S, Phraesrisakul N: Recurrence of yaws outbreak in Thailand, 1990. Southeast Asian J Trop Med Public Health. 1994; 25(1): 152–6. PubMed Abstract\n\nTha Hla U: The proposed national programme on the control of yaws in Burma. Burma Med J. 1960; 8: 96–7. PubMed Abstract\n\nTaiwo SS, Adesiji YO, Adekanle DA: Screening for syphilis during pregnancy in Nigeria: a practice that must continue. Sex Transm Infect. 2007; 83(5): 357–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatson-Jones D, Gumodoka B, Weiss H, et al.: Syphilis in pregnancy in Tanzania. II. The effectiveness of antenatal syphilis screening and single-dose benzathine penicillin treatment for the prevention of adverse pregnancy outcomes. J Infect Dis. 2002; 186(7): 948–57. PubMed Abstract \| Publisher Full Text\n\nMcGready R, Kang J, Watts I, et al.: HIV and syphilis antenatal screening data at SMRU 2012–13. figshare. 2014. Data Source" }	[ { "id": "5072", "date": "27 Jun 2014", "name": "R Matthew Chico", "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\nOverall comment:This is a well-written paper on an important topic, for which there is limited data: What is the prevalence below which routine screening of all pregnant women for HIV and, in particular, syphilis is no longer advisable? Specific comments:Syphilis is far more deleterious to birth outcomes than other bacterial sexually transmitted infections and, therefore, the potential risks associated with missed diagnoses should be made in clearer in the paper. Among unscreened pregnant women in Tanzania, 51% of stillbirths, 24% of preterm live births, and 17% of all adverse pregnancy outcomes were attributable to maternal syphilis 1. The authors should consider citing the comprehensive summary of the effect of curable STIs on birth outcomes by Chico et al. (Table 1. Effect of curable STIs/RTIs on pregnancy outcomes) 2. Clearly the rates of HIV and syphilis were low among the refugees and migrants tested along the Thai-Myanmar border. However, it is not clear in the manuscript (1) the proportion of pregnant women in these two populations who seek antenatal care services, and (2) the prevalence of syphilis and HIV in the two populations who do not seek ANC services. Are the pregnant women attending these health facilities healthier than the overall population? Pregnant women are encouraged to attend ANC clinics as frequently as every fortnight. This makes it more difficult to generalise results beyond the study population. The refugee and migrant populations in this setting are unique for cultural reasons described in the paper. There should be some discussion about other contexts in which women are at elevated risk of HIV and syphilis infection, and that women may enter into transient marital relationships that are based in personal security, but expose them to other risks. There should be some discussion about the use of rapid point of care (POC) tests for syphilis, in place of VDRL and THPA assays. POC tests for syphilis simplify the screening and treatment procedures. The World Health Organization currently recommends the use of POC tests for syphilis in the antenatal care setting 3. Using POC tests will expedite case finding and treatment because results will be available during the same consultation. Although published results are not yet available, a combination HIV and syphilis POC test has recently been validated. The paper would benefit from a table that delineates the demographic and birth-outcome data for the 14 women who were found to have syphilis. The risk profile in Table 3 suggests that (1) migrant women, (2) who have been married more than once, (3) who are above the age of 30, (4) whose parents are not Karen, and (5) who have lived at their present address less than six months, are most likely to have a syphilis infection. Considering these five risk factors, how many women had just one, or two, or three, or four, or all five?", "responses": [ { "c_id": "1335", "date": "02 Jul 2015", "name": "Rose McGready", "role": "Author Response", "response": "We appreciate and would like to sincerely thank the significant effort of both reviewers in regards to this manuscript. We apologize for the tardiness of the response. We have provided a point by point response to the comments below.This is a well-written paper on an important topic, for which there is limited data: What is the prevalence below which routine screening of all pregnant women for HIV and, in particular, syphilis is no longer advisable? Specific comments:1.Syphilis is far more deleterious to birth outcomes than other bacterial sexually transmitted infections and, therefore, the potential risks associated with missed diagnoses should be made in clearer in the paper. Among unscreened pregnant women in Tanzania, 51% of stillbirths, 24% of preterm live births, and 17% of all adverse pregnancy outcomes were attributable to maternal syphilis 1. The authors should consider citing the comprehensive summary of the effect of curable STIs on birth outcomes by Chico et al. (Table 1. Effect of curable STIs/RTIs on pregnancy outcomes) 2.We agree this is important and have amended the text as suggested in the 2nd paragraph of the introduction. 2.Clearly the rates of HIV and syphilis were low among the refugees and migrants tested along the Thai-Myanmar border. However, it is not clear in the manuscript (1) the proportion of pregnant women in these two populations who seek antenatal care services, and (2) the prevalence of syphilis and HIV in the two populations who do not seek ANC services. Are the pregnant women attending these health facilities healthier than the overall population? Pregnant women are encouraged to attend ANC clinics as frequently as every fortnight. This makes it more difficult to generalise results beyond the study population.Thank you for pointing this out and this information has been added to the methods section.We estimate the proportion at 90% - see 2nd paragraph of the methods section. The “prevalence of syphilis and HIV in the two populations who do not seek ANC services” is impossible to answer. Data of HIV rates from the border wide camp Health information system suggest similarly low prevalence and this has been added to the discussion first paragraph.The ANC has always encouraged fortnightly voluntary visits (and care during childbirth in the clinic) and 4 or less antenatal visits in this setting has been associated with an increased risk of maternal death: AOR 2.50 (95%CI 1.41–4.43) ( doi: 10.1371/journal.pone.0040244) and this detail has been been added to the methods first paragraph. 3.The refugee and migrant populations in this setting are unique for cultural reasons described in the paper. There should be some discussion about other contexts in which women are at elevated risk of HIV and syphilis infection, and that women may enter into transient marital relationships that are based in personal security, but expose them to other risks.This has been added into the discussion towards the end of the first paragraph in the conclusions.4.There should be some discussion about the use of rapid point of care (POC) tests for syphilis, in place of VDRL and THPA assays. POC tests for syphilis simplify the screening and treatment procedures. The World Health Organization currently recommends the use of POC tests for syphilis in the antenatal care setting 3. Using POC tests will expedite case finding and treatment because results will be available during the same consultation. Although published results are not yet available, a combination HIV and syphilis POC test has recently been validated.We have not got experience with POC for syphilis. Our experience with POC for HIV (higher positivity rate than syphilis) has not been good as explained in the results – nearly ½ were false positive. As the incidence goes down the false positive rate goes up and this is one of the reasons we have not moved to POC tests for syphilis in this population. Currently the turn around time with the local Thailand hospital where the POC positive results are sent for confirmation and the distance involved is a minimum of 3 days (usually a week). This is very stressful for women. This has been added to the discussion. We are not against POC tests and use malaria POC testing frequently. POC tests have limitations in their own right and the limitations and advantages need to be determined locally.On another note we have also found that hepatitis B POC tests also present us with a similar problem but to a lesser degree (the incidence of Hepatitis B in adults in this area approaches 10%). Approximately 10% of Hepatitis B Surface Ag POC positive tests turn out to be incorrect. This is not a problem when the point is to screen for healthy donors but it is a problem when you are telling an individual they have a chronic disease.We would be delighted to be a site for testing sensitivity and specificity of such tests.5.The paper would benefit from a table that delineates the demographic and birth-outcome data for the 14 women who were found to have syphilis. The risk profile in Table 3 suggests that (1) migrant women, (2) who have been married more than once, (3) who are above the age of 30, (4) whose parents are not Karen, and (5) who have lived at their present address less than six months, are most likely to have a syphilis infection. Considering these five risk factors, how many women had just one, or two, or three, or four, or all five?We appreciate this suggestion as a method to try to focus on at risk women. We have looked at this in 2 ways: scoring the number of risk factors a woman has (below)We appreciate this suggestion as a method to try to focus on at risk women. We have looked at this in 2 ways: scoring the number of risk factors a woman has (below)ScoreSyphilis n=14No syphilis n=357800 (0)646 (100%)12* (0.2%)1082 (99.8%)20 (0)1032 (100%)34 (0.7%)99.3%)45 (2.3%)216 (97.7%)53 (10.0%)27 (90.0%)*Scored: one woman scored 1 because of ‘migrant’ status; and one woman scored 1 because of age ≥ 30 y. A risk factor score of ≥ 3 includes 1.44% (12/831) and a score <3 includes 0.07% ( 2/2762). And trying to identify if a pattern of risk factors is common (below) Finally as suggested by the 2nd reviewer we have run a multivariate analysis which can be found at the request of the 2nd reviewer (see comments). The main message of the manuscript is not to identify risk factors (which also does not seem practical given the low number of positive case). References1. Watson-Jones D, Chanhalucha J, Gumodoka B, Weiss H, Rusizoka M, Ndeki L, Whitehouse A, Balira R, Todd J, Ngeleja D, Ross D, Buvé A, Hayes R, Mabey D: Syphilis in pregnancy in Tanzania. I. Impact of maternal syphilis on outcome of pregnancy. J Infect Dis. 2002; 186 (7): 940-947 PubMed Abstract \| Publisher Full Text \| Reference Source2. Chico RM, Hack BB, Newport MJ, Ngulube E, Chandramohan D: On the pathway to better birth outcomes? A systematic review of azithromycin and curable sexually transmitted infections. Expert Rev Anti Infect Ther. 2013; 11 (12): 1303-1332 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source3. World Health Organization: The use of rapid syphilis tests: Geneva:WHO. 2006. Reference Source" } ] }, { "id": "6142", "date": "16 Sep 2014", "name": "Nitika Pant Pai", "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\nOverall:This manuscript must be indexed subject to revisions/clarifications that are highlighted in specific sections mentioned below. Strengths:The strengths of the study lay in its design, recruitment of displaced, vulnerable populations, and evaluation of rapid testing interventions (antenatal screening for HIV and Syphilis) with downstream implications for mother-to-infant transmission. Data on this population are sparse. Little is known about effectiveness of screening in migrant populations and actions taken to link them to care and prevention.Limitations:A convenience sample is enrolled - leading to a biased sample of a migrant population whose primary study base is ill-defined. This limits generalizability of their findings. Confounding: a discussion is needed - especially because a multivariate analyses was not attempted. A small sample, and fewer infections detected in the small sample, limits a multivariate analysis. Two groups are compared with a null finding. A clear hypotheses, a clear primary objective and a clean sample size calculation based on that is needed. Implication section for policy and practice is missing. Detailed suggestions are provided below:Title:The title specifies the studied population, the location and the correct study design. A suggested alternative title could be: “Audit of the antenatal screening for syphilis and HIV in migrant and refugee women on the Thai-Myanmar border: a descriptive study” for the following reasons:The HIV and syphilis prevalence estimates obtained when screening the sampled population may not be a true reflection of the prevalence among the underlying source population (refer to the limitation in the discussion section below). The rates of infections are not the only focus of the study. The study also explores counselling challenges following screening and some consequential considerations for future testing strategies; however these are not covered in the title. Abstract:The abstract provides informative and balanced summary of the study. There is a discrepancy between the aim presented in the abstract and the one presented in the introduction. The aim of auditing the first year of the syphilis routine screening is mentioned in the abstract but not the one of reassessing the trends in HIV rates.Introduction:The scientific background is provided and the rationale for the investigation was well explained: the 2 studied STIs present major health risks on maternal and child health (it would be useful to add the health risk on the woman and not only on the pregnancy outcomes). The pregnant woman is especially vulnerable to infection when displaced (low income was mentioned as a risk factor – along with low education and lack of access to health services – and it would be useful to explain the correlation). There is a large displaced population between Thailand and Myanmar and data on prevalence of infections among this population is scarce. The objective of the study is stated. The aim of auditing a screening program is quite ambitious (many of its facets are discussed which may be overwhelming to the reader).Methods:The study design is overall well presented (including study site, data collection and laboratory testing, treatment plan and adequate statistical analysis plan). Regarding the study population, the eligibility criteria and the sample size considerations (sample should be large enough to reflect important variations) are not discussed. Training and expertise of the people executing the screening tests is not mentioned. Authors should discuss the comparability of the testing methods in the three SMRU clinics included in the study and the quality assurance and control measures applied.Results:The results section presents and adequately summarizes all relevant characteristics of the study participants (Table 1). However, it would be helpful to define the number of marriages that a woman has as the number of remarriages (which is explained in the discussion). Literacy should also be defined. Additionally, 2 MLA and 14 WPA patients are not segregated into the different ethnic groups and are therefore missing from this part of the table; this mismatch can be detected when computing the total number of participants from each of these two sites. Finally, it is advised to keep one of the two significance levels presented below the table (P<005 or P<0.001). The results on prevalence rates of syphilis and HIV are properly presented in Table 2. However, the data from other studies on prevalence rates in Myanmar and Thailand are not very informative given the fact that confidence intervals are not provided. The reasons for non-participation and missing data are provided for each of those who did not complete the screening. However, the authors should consider providing an explanation for the one instance where a partner was treated for syphilis without being tested first. A multivariate analysis would have been more informative than a univariate analysis in order to get a clearer understanding of the risk factors associated with contracting syphilis and HIV. But, as the authors mention, this type of analysis is not possible when having a small number of cases. The authors should consider adding the type of analysis in the title of Table 3. Also, the confidence interval of the odds ratio for syphilis in the migrant group (1.182 – 6.921 instead of 1.182- 69.214) A wide confidence interval could be due to small number of cases, and sampling variability issues - these could be acknowledged.Discussion:The discussion summarises the key results. But the conclusion on the HIV rates does not accurately encapsulate what is presented in the results section. It is advised to state that no statistically significant difference in HIV rates is observed between the refugees and the combined migrant population attending both MKT and WPA sites (since it is mentioned in the results section that there is a statistically significant difference in HIV rates between MLA and WPA). The discussion is contextualized in relation to the studied population. The authors present sound explanations and interpretations of the results based on a good understanding of the cultural traits and traditions of the studied population. The authors discuss the clinical applicability of the study findings. It would be useful to further clarify the ethical difficulties faced and the challenges created during post-test counselling when a false positive syphilis result is undetected. For example, the authors can explain that a positive syphilis screening test does not necessarily confirm syphilis when titres are low and that yaws – which is not sexually transmitted – may instead be suspected. This inability to determine the true cause of the positive result can lead to serious consequences when counselling a patient with low titres levels – as it may increase her risk of abuse and abandonment. It can also be specified that that yaws are transmitted through skin-to-skin contact and promoted by overcrowding and poor hygiene (which is very likely the case in the study population). The authors discuss limitations of the study. However, the convenience sampling of the study population is a limitation that is not mentioned in the discussion. There is a risk of selection bias since the study population consists of regular attendees of the SMRU antenatal clinics who may be very different from those who are not. For example, regular attendees of the clinic may be more health conscious – engage in less risky behaviors – and may have been more exposed to health recommendations and counselling than those who miss antenatal care visits. The generalizability of results (external validity) to the whole population of migrants and refugees in the studied location is difficult to establish given the risk of selection bias. In conclusion, the study is scientifically sound however some editing is advised. It presents interesting data on HIV and syphilis prevalence for regular antenatal clinic attendees coming from a disadvantaged and understudied population of migrant and refugee at the Thai- Myanmar border.It provides valuable ethical considerations and highlights a caveat pertaining to the currentsyphilis testing strategy and post-test counselling.This review could help public health officials better understand the situation in regard to HIVand syphilis among the studied population in order to revise the routine screening program.", "responses": [ { "c_id": "1336", "date": "02 Jul 2015", "name": "Rose McGready", "role": "Author Response", "response": "We appreciate and would like to sincerely thank the significant effort of both reviewers in regards to this manuscript. We apologize for the tardiness of the response. We have provided a point by point response to the comments below. Overall:This manuscript must be indexed subject to revisions/clarifications that are highlighted in specific sections mentioned below. Strengths:The strengths of the study lay in its design, recruitment of displaced, vulnerable populations, and evaluation of rapid testing interventions (antenatal screening for HIV and Syphilis) with downstream implications for mother-to-infant transmission. Data on this population are sparse. Little is known about effectiveness of screening in migrant populations and actions taken to link them to care and prevention.Limitations:A convenience sample is enrolled - leading to a biased sample of a migrant population whose primary study base is ill-defined. This limits generalizability of their findings. Thank you – we need to clarify. This is not a convenience sample but the whole population of pregnant women in well attended antenatal clinics. This point is similar to point 2 raised by reviewer one about the population. The methods (2nd paragraph) has been modified to better describe this population.Confounding: a discussion is needed - especially because a multivariate analyses was not attempted.The original submissions did a univariate analysis (Table 3) and in the results a sentence was added “Further modelling was not done due to the low number of seropositive cases precluding meaningful conclusions.” At this reviewer’s request a multivariate analysis has been added here (next page) and as you can see the confidence intervals of the AOR are very large, which means the level of uncertainty are so high that we cannot confidently interpret the result. If the reviewer insists we can include the multivariate analysis in the main body of the paper with the appropriate changes to the methods section as well (included here).Methods, statistical analysis: Factors associated with a diagnosis of syphilis or a diagnosis of HIV, were evaluated by univariate analysis; two logistic regression models were created using “syphilis (yes/no)” and “HIV (yes/no)” as dependent variables. All factors with a P < 0.10 in univariate analysis were entered in their respective stepwise forward logistic regression model, and were included in the relevant tables. Adjusted odds ratios (AOR) were given with their 95% confidence interval.[see table 3 in new version - resubmission] A small sample, and fewer infections detected in the small sample, limits a multivariate analysis. We have acknowledged this. The main reason to write this manuscript is not to explore risk factors but to question the prevalence at which syphilis testing is not viable for resource constrained settings.Two groups are compared with a null finding.Yes – the incidence if HIV and syphilis is very low in both populations which is what we are trying to illustrateA clear hypotheses, a clear primary objective and a clean sample size calculation based on that is needed.This is a non-selected inclusive cohort of all consecutively registered pregnant women so no sample size was provided. This has been clarified, 2nd paragraphs in the methods.Implication section for policy and practice is missing.These implications are clarified in the paragraph starting at line 293.Detailed suggestions are provided below:Title:The title specifies the studied population, the location and the correct study design.A suggested alternative title could be: “Audit of the antenatal screening for syphilis and HIV in migrant and refugee women on the Thai-Myanmar border: a descriptive study” for the following reasons:The HIV and syphilis prevalence estimates obtained when screening the sampled population may not be a true reflection of the prevalence among the underlying source population (refer to the limitation in the discussion section below). The rates of infections are not the only focus of the study. The study also explores counselling challenges following screening and some consequential considerations for future testing strategies; however these are not covered in the title.We have amended the title in line with what has been suggested although we are confident that we have good coverage of the population. We agree with the second point. Abstract:The abstract provides informative and balanced summary of the study. There is a discrepancy between the aim presented in the abstract and the one presented in the introduction. The aim of auditing the first year of the syphilis routine screening is mentioned in the abstract but not the one of reassessing the trends in HIV rates.Thank you for this comment and the aims are now aligned.Introduction:The scientific background is provided and the rationale for the investigation was well explained: the 2 studied STIs present major health risks on maternal and child health (it would be useful to add the health risk on the woman and not only on the pregnancy outcomes). We agree and a sentence added to the end of the first paragraph of the introduction.The pregnant woman is especially vulnerable to infection when displaced (low income was mentioned as a risk factor – along with low education and lack of access to health services – and it would be useful to explain the correlation). There is a large displaced population between Thailand and Myanmar and data on prevalence of infections among this population is scarce. Thank and this has been amended in the introduction (see end of the 2nd paragraph).The objective of the study is stated. The aim of auditing a screening program is quite ambitious (many of its facets are discussed which may be overwhelming to the reader).Given the challenges we encountered, we found that such an audit (ambitious and complex as it is) is necessary to shed light on the issues faced in this context. It is our hope that the overwhelming experience of the reader will be lessened by the changes we have made in response to the general reviewer comments. No specific changes made to change the objective. Methods:The study design is overall well presented (including study site, data collection and laboratory testing, treatment plan and adequate statistical analysis plan).OK Regarding the study population, the eligibility criteria and the sample size considerations (sample should be large enough to reflect important variations) are not discussedThank you. The sample is exhaustive and the study population is more clearly defined now in the methods section.Training and expertise of the people executing the screening tests is not mentioned. Authors should discuss the comparability of the testing methods in the three SMRU clinics included in the study and the quality assurance and control measures applied.Thank you for this and the information has been added to the first paragraph of laboratory sampling.Results:The results section presents and adequately summarizes all relevant characteristics of the study participants (Table 1). However, it would be helpful to define the number of marriages that a woman has as the number of remarriages (which is explained in the discussion). AmendedLiteracy should also be defined. Amended: this was self-reported.Additionally, 2 MLA and 14 WPA patients are not segregated into the different ethnic groups and are therefore missing from this part of the table; this mismatch can be detected when computing the total number of participants from each of these two sites. Thank you for detecting this. It is an error and the numbers in the manuscript has been carefully reviewed in line with this finding.Finally, it is advised to keep one of the two significance levels presented below the table (P<005 or P<0.001).Amended: only P<0.05 has been maintained.The results on prevalence rates of syphilis and HIV are properly presented in Table 2. However, the data from other studies on prevalence rates in Myanmar and Thailand are not very informative given the fact that confidence intervals are not providedWe think the comparative data compiled in one table is helpful to understand the situation of a population that is rarely provided with any type of sero-surveillance. We agree a better data source that provides confidence intervals would be useful but we are unable to find exact numbers for Thailand. We have expanded the paragraph in the results further and provided clearer reference material; including confidence intervals for the Myanmar data.The reasons for non-participation and missing data are provided for each of those who did not complete the screening. However, the authors should consider providing an explanation for the one instance where a partner was treated for syphilis without being tested first.High risk case and this has been added to the results section.A multivariate analysis would have been more informative than a univariate analysis in order to get a clearer understanding of the risk factors associated with contracting syphilis and HIV. But, as the authors mention, this type of analysis is not possible when having a small number of cases. The authors should consider adding the type of analysis in the title of Table 3. Also, the confidence interval of the odds ratio for syphilis in the migrant group (1.182 – 6.921 instead of 1.182- 69.214) A wide confidence interval could be due to small number of cases, and sampling variability issues - these could be acknowledged.Analysis of risk factors is not the main objective of this manuscript, and the limitation of such an analysis due to low positivity is acknowledged in the original submission. We have clarified in the laboratory sampling section that sampling variability is minimized by regular quality control and standardization, and this is not a significant contributor to the wide confidence interval, unlike the small number of positive cases. The comment about the confidence interval of the odds ratio for syphilis is a little unclear to us, but the correct CI is as included in the table (1.182-69.214) i.e. it really is 69 and no 6.9.Discussion:The discussion summarises the key results. But the conclusion on the HIV rates does not accurately encapsulate what is presented in the results section. It is advised to state that no statistically significant difference in HIV rates is observed between the refugees and the combined migrant population attending both MKT and WPA sites (since it is mentioned in the results section that there is a statistically significant difference in HIV rates between MLA and WPA).Thank you for this suggestion and it has been amended as suggested.The discussion is contextualized in relation to the studied population. The authors present sound explanations and interpretations of the results based on a good understanding of the cultural traits and traditions of the studied population.The authors discuss the clinical applicability of the study findings. It would be useful to further clarify the ethical difficulties faced and the challenges created during post-test counselling when a false positive syphilis result is undetected. For example, the authors can explain that a positive syphilis screening test does not necessarily confirm syphilis when titres are low and that yaws – which is not sexually transmitted – may instead be suspected. This inability to determine the true cause of the positive result can lead to serious consequences when counselling a patient with low titres levels – as it may increase her risk of abuse and abandonment. It can also be specified that that yaws are transmitted through skin-to-skin contact and promoted by overcrowding and poor hygiene (which is very likely the case in the study population). We have amended the text as suggested.The authors discuss limitations of the study. However, the convenience sampling of the study population is a limitation that is not mentioned in the discussion. There is a risk of selection bias since the study population consists of regular attendees of the SMRU antenatal clinics who may be very different from those who are not. For example, regular attendees of the clinic may be more health conscious – engage in less risky behaviors – and may have been more exposed to health recommendations and counselling than those who miss antenatal care visits.Sampling was exhaustive, it was not a convenience sample and we included women who came for only one ANC visit or arrived at the clinic for the first time while in labor.The generalizability of results (external validity) to the whole population of migrants and refugees in the studied location is difficult to establish given the risk of selection bias. The sampling was exhaustive amongst consecutively enrolled pregnant women. The data is similar to previous surveys and consistent with other reports from the area suggesting the risk of selection bias is low. This has been better clarified in relation to the methods (the study population) and relation to other data sources included in the first paragraph of the discussion. In conclusion, the study is scientifically sound however some editing is advised. It presents interesting data on HIV and syphilis prevalence for regular antenatal clinic attendees coming from a disadvantaged and understudied population of migrant and refugee at the Thai- Myanmar border.It provides valuable ethical considerations and highlights a caveat pertaining to the current syphilis testing strategy and post-test counselling.This review could help public health officials better understand the situation in regard to HIVand syphilis among the studied population in order to revise the routine screening program." } ] } ]	1	https://f1000research.com/articles/3-123
https://f1000research.com/articles/3-302/v1	11 Dec 14	{ "type": "Research Note", "title": "Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia", "authors": [ "Somary Nhem", "Joanne Letchford", "Chea Meas", "Sovanndeth Thann", "James C. McLaughlin", "Ellen Jo Baron", "T. Eoin West", "Somary Nhem", "Joanne Letchford", "Chea Meas", "Sovanndeth Thann", "James C. McLaughlin", "Ellen Jo Baron" ], "abstract": "Melioidosis infection, caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown’s medium to standard culture methods. Two specimens (0.8%) were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4%) was positive using standard methods. These findings demonstrate that B. pseudomallei is rarely detected in sputum at this hospital. The low frequency of B. pseudomallei in sputum specimens precludes drawing any conclusions about the relative benefits of an enhanced sputum testing protocol at this site. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are likely to be important factors in facilitating identification of melioidosis.", "keywords": [ "Burkholderia pseudomallei", "melioidosis", "pneumonia", "diagnostic microbiology", "lung", "sputum", "Cambodia" ], "content": "Introduction\n\nMelioidosis, infection with the Gram negative bacterium Burkholderia pseudomallei, has increasingly been described in Cambodia in recent years1–6. Infection is caused by inoculation, inhalation, or ingestion of B. pseudomallei, an environmental saprophyte7. In one single center Cambodian study, melioidosis (defined as growth of B. pseudomallei in any clinical specimen) was lethal in more than half of cases, many of whom had received inappropriate initial antibiotic therapy2. The clinical presentation of melioidosis is notoriously variable, but pneumonia and sepsis occur commonly7. Sub-acute and chronic forms of melioidosis may be confused with tuberculosis and it is important to distinguish these diseases8. B. pseudomallei is resistant to penicillin, ampicillin, first- and second-generation cephalosporins and aminoglycosides7. Use of quinolones or third-generation cephalosporins to treat melioidosis is associated with poorer outcomes9–11. Thus, many of the first line antimicrobial therapies administered for pneumonia and sepsis do not adequately treat melioidosis. The treatment course for melioidosis is prolonged, requiring initial intensive therapy for at least 10–14 days with intravenous ceftazidime, imipenem, or meropenem, followed by 3–6 months of oral eradication therapy with trimethoprim-sulfamethoxazole7. Moreover, ceftazidime and carbapenems are relatively expensive and may not be universally accessible in Cambodia. Therefore, rapid identification of the bacterium is essential to guide clinical management. In pulmonary melioidosis, culture of sputum may provide critical diagnostic information. In this report, we present our experience implementing an enhanced sputum testing protocol in a Cambodian provincial hospital diagnostic microbiology laboratory to increase detection of B. pseudomallei.\n\nKampong Cham Provincial Hospital is a 260 bed referral hospital approximately 120 kilometers by road northeast of Phnom Penh. The diagnostic microbiology laboratory at the hospital began offering culture and antibiotic susceptibility testing in 2009. Several cases of B. pseudomallei have been isolated at the laboratory since then. Given resource limitations, standard media used to culture sputum specimens in this laboratory are blood, chocolate, and MacConkey agars. In northeast Thailand, where melioidosis is highly endemic7, the use of selective enrichment broth and Ashdown’s medium has enhanced the detection of B. pseudomallei from sputum12,13. We hypothesized that the use of selective enrichment broth and Ashdown’s medium at Kampong Cham Provincial Hospital would similarly increase the identification of cases of pulmonary melioidosis.\n\n\nMethods\n\nTo compare the use of selective enrichment broth and Ashdown’s medium to standard sputum culture at Kampong Cham Provincial Hospital, we performed specific testing for B. pseudomallei on all sputum specimens submitted to the laboratory and accepted for bacterial culture between March 25 and September 30, 2013, following a previously described protocol12,14. We chose these dates to include much of the rainy season, when melioidosis is diagnosed more frequently2. Specimens accepted for culture had fewer than 10 epithelial cells per low power field or moderate to high numbers of polymorphonuclear cells on Gram stain. Specimens were inoculated into selective enrichment broth containing Tryptic Soy Broth 10g, glycerol 40ml, crystal violet 0.1% 5ml, and colistin 50mg per liter of distilled water for two days at 37°C in aerobic conditions. Specimens were then sub-cultured onto Ashdown’s medium (Tryptic Soy Broth 10g, agar 15g, glycerol 40ml, crystal violet 0.1% 5ml, neutral red 1% 5ml, and gentamicin 4mg per liter of distilled water) and incubated for four days at 37°C in aerobic conditions. Colonies that grew were tested using the oxidase test; oxidase-positive colonies were tested using a highly sensitive and specific monoclonal antibody-based latex agglutination test for B. pseudomallei15,16. In parallel, as per routine laboratory practice, all sputum specimens were also cultured onto sheep blood, chocolate, and MacConkey agars.\n\n\nResults\n\nTwo hundred and forty one sputum specimens deemed acceptable for culture were received by the laboratory during the study period and tested using the enhanced protocol. B. pseudomallei was isolated from one specimen (0.4%) using standard media and from two specimens (0.8%) using the enhanced protocol. The single specimen positive using standard media was also positive using the enhanced protocol; the enhanced technique therefore detected one additional isolate of B. pseudomallei compared to the standard protocol. In this case, Klebsiella pneumoniae grew on standard media, raising the possibility that overgrowth of K. pneumoniae precluded detection of B. pseudomallei in the absence of selective enrichment broth. Among the 241 sputum specimens collected during the study period, B. pseudomallei accounted for two (1.6%) of the 122 that were culture positive. Other bacteria isolated from the sputum samples during the study period were K. pneumoniae (54), Pseudomonas species (37), Enterobacter species (7), Acinetobacter species (5), Staphylococcus aureus (4), Stenotrophomonas maltophilia (3), Streptococcus pneumoniae (3), Haemophilus influenzae (3), Escherichia coli (3), K. ozaenae (3), unknown Enterobacteriaceae (2), Vibrio alginolyticus (1), and a non-fermenting Gram negative bacillus (1). On standard media, three other specimens were positive for B. pseudomallei during the study period: a pleural fluid specimen from one patient with a B. pseudomallei-positive sputum culture, and pus from two other individuals.\n\n\nConclusions/Discussion\n\nThese data confirm that respiratory melioidosis is detected in patients presenting to Kampong Cham Provincial Hospital although the overall number of sputum specimens positive for B. pseudomallei was low. Our rate of detection of B. pseudomallei in sputum samples submitted to the laboratory is similar to a previously published study evaluating the etiology of clinical respiratory infection at this hospital3. This study of patients with acute lower respiratory tract infection identified melioidosis in four of 422 (0.9%) patients from the hospital. The diagnosis was determined by culture of B. pseudomallei from sputum or blood at an off-site laboratory.\n\nIn light of the small number of specimens positive for B. pseudomallei we could not ascertain an advantage to the use of the enhanced sputum testing protocol. However, the ramifications of identification of B. pseudomallei are substantial. Although ceftazidime and imipenem may not be available in Cambodian hospital pharmacies and cost prevents the outside purchase by most patients, mortality from melioidosis is greatly diminished when appropriate antibiotic therapy is instituted17. Others have reported a benefit of a similar diagnostic testing strategy. For example, the use of selective enrichment broth and B. pseudomallei selective agar to test 154 respiratory samples in a referral center in Kuala Lumpur identified three cases of B. pseudomallei that were not detected using routine media18. Thus, the relative benefit of the enhanced sputum testing protocol may be greater in circumstances where B. pseudomallei is isolated more commonly.\n\nA limitation to our investigation is the relatively small total number of sputum specimens submitted to the laboratory during the six month period. As the hospital microbiology laboratory has only been operational since 2009, this may reflect the established clinical practice of not relying on microbiology data to make treatment decisions. We further observed that the majority of sputum specimens came from patients presenting to the hospital’s tuberculosis screening area and ward. This suggests that in many cases clinicians may have been considering the diagnosis of tuberculosis. Melioidosis and tuberculosis may be difficult to distinguish clinically8 and both infections should be considered in patients with chronic symptoms of respiratory infection in Cambodia. However, most respiratory melioidosis is associated with symptoms for less than two months and the mean incubation period in acute melioidosis is nine days19,20. We speculate that sputum from melioidosis patients presenting elsewhere in the hospital with acute clinical syndromes of pneumonia and sepsis may not have been submitted for culture. This may have reduced our rate of detection of melioidosis in this study. Furthermore, this highlights the importance of informing clinicians of the utility of diagnostic microbiology testing, preferably before initiation of treatment.\n\nSeveral other techniques should also be considered in the diagnosis of pulmonary melioidosis. Many cases of pulmonary melioidosis have concurrent bacteremia19 and B. pseudomallei bacteremia is reported in Cambodia. In a recent study, B. pseudomallei accounted for 12.6% of clinically significant positive blood cultures collected from adults between July 2007 and December 2010 at the Sihanouk Hospital Centre of HOPE, Phnom Penh21. From January to December 2013, B. pseudomallei was isolated in 18 of 2,230 (0.8%) blood cultures submitted to microbiology laboratories at five Cambodian government hospitals, with the majority of B. pseudomallei cultures (15/434, 3.5%) occurring at Takeo Provincial Hospital (unpublished data). Throat swabs cultured in selective enrichment broth, while insensitive, are easily obtained and highly specific for the diagnosis of melioidosis22,23. Thus, routinely culturing blood and throat swabs in patients with pneumonia and sepsis may enhance the diagnosis of respiratory melioidosis in Cambodia.\n\nIn conclusion, these data confirm that B. pseudomallei is a respiratory pathogen in Kampong Cham province but show that it is rarely detected in sputum samples submitted to the diagnostic microbiology laboratory at Kampong Cham Provincial Hospital. As a result, our study cannot determine the utility of an enhanced sputum testing protocol at this site. However, the benefit of the enhanced testing protocol may be more apparent elsewhere in Cambodia where melioidosis is more common. In addition, given the severity of melioidosis and importance of identifying the infection to guide treatment, several other factors should be considered in order to increase detection of the disease. In particular, clinician awareness of the diverse presentations of melioidosis and routine utilization of diagnostic microbiology services for patients presenting with pneumonia and sepsis should be promoted.\n\n\nData availability\n\nF1000Research: Dataset 1. Detection of B. pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium Compared to Standard Culture, 10.5256/f1000research.5935.d4042324", "appendix": "Author contributions\n\n\n\nTEW conceived the study. SN, JCMc, EJB, and TEW designed the protocol with the support of CM. SN, JL, and ST carried out the study. SN, JL, and TEW analyzed the data. All authors participated in the generation or revision of the manuscript, and approved the final version.\n\n\nCompeting interests\n\n\n\nNo completing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by support from the University of Washington to TEW.\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 Ms. Vanaporn Wuthiekanun for providing the sputum testing protocol and advice. We also thank Dr. Narisara Chantratita for providing latex agglutination tests and Dr. Michael Prouty for support. We are grateful to the Kampong Cham Provincial Hospital diagnostic microbiology laboratory staff for their dedication to the project.\n\n\nReferences\n\nOvertoom R, Khieu V, Hem S, et al.: A first report of pulmonary melioidosis in Cambodia. Trans R Soc Trop Med Hyg. 2008; 102(Suppl 1): S21–5. PubMed Abstract \| Publisher Full Text\n\nVlieghe E, Kruy L, De Smet B, et al.: Melioidosis, Phnom Penh, Cambodia. Emerg Infect Dis. 2011; 17(7): 1289–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRammaert B, Beaute J, Borand L, et al.: Pulmonary melioidosis in Cambodia: a prospective study. BMC Infect Dis. 2011; 11: 126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChheng K, Carter MJ, Emary K, et al.: A prospective study of the causes of febrile illness requiring hospitalization in children in Cambodia. PLoS One. 2013; 8(4): e60634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPagnarith Y, Kumar V, Thaipadungpanit J, et al.: Emergence of pediatric melioidosis in Siem Reap, Cambodia. Am J Trop Med Hyg. 2010; 82(6): 1106–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoesser N, Pocock J, Moore CE, et al.: Pediatric suppurative parotitis in Cambodia between 2007 and 2011. Pediatr Infect Dis J. 2012; 31(8): 865–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiersinga WJ, Currie BJ, Peacock SJ: Melioidosis. N Engl J Med. 2012; 367(11): 1035–44. PubMed Abstract \| Publisher Full Text\n\nSuntornsut P, Kasemsupat K, Silairatana S, et al.: Prevalence of melioidosis in patients with suspected pulmonary tuberculosis and sputum smear negative for acid-fast bacilli in northeast Thailand. Am J Trop Med Hyg. 2013; 89(5): 983–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaowagul W, Simpson AJ, Suputtamongkol Y, et al.: Empirical cephalosporin treatment of melioidosis. Clin Infect Dis. 1999; 28(6): 1328. PubMed Abstract \| Publisher Full Text\n\nChaowagul W, Suputtamongkul Y, Smith MD, et al.: Oral fluoroquinolones for maintenance treatment of melioidosis. Trans R Soc Trop Med Hyg. 1997; 91(5): 599–601. PubMed Abstract \| Publisher Full Text\n\nCheng AC, Currie BJ: Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev. 2005; 18(2): 383–416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWuthiekanun V, Dance DA, Wattanagoon Y, et al.: The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice. J Med Microbiol. 1990; 33(2): 121–6. PubMed Abstract \| Publisher Full Text\n\nWalsh AL, Wuthiekanun V, Smith MD, et al.: Selective broths for the isolation of Pseudomonas pseudomallei from clinical samples. Trans R Soc Trop Med Hyg. 1995; 89(1): 124. PubMed Abstract \| Publisher Full Text\n\nWuthiekanun V, Limmathurotsakul D, Wongsuvan G, et al.: Quantitation of B. Pseudomallei in clinical samples. Am J Trop Med Hyg. 2007; 77(5): 812–3. PubMed Abstract\n\nAnuntagool N, Naigowit P, Petkanchanapong V, et al.: Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J Med Microbiol. 2000; 49(12): 1075–8. PubMed Abstract\n\nWuthiekanun V, Anuntagool N, White NJ, et al.: Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am J Trop Med Hyg. 2002; 66(6): 759–61. PubMed Abstract\n\nWhite NJ, Dance DA, Chaowagul W, et al.: Halving of mortality of severe melioidosis by ceftazidime. Lancet. 1998; 2(5665): 697–701. PubMed Abstract \| Publisher Full Text\n\nRoesnita B, Tay ST, Puthucheary SD, et al.: Diagnostic use of Burkholderia pseudomallei selective media in a low prevalence setting. Trans R Soc Trop Med Hyg. 2002; 106(2): 131–3. PubMed Abstract \| Publisher Full Text\n\nMeumann EM, Cheng AC, Ward L, et al.: Clinical features and epidemiology of melioidosis pneumonia: results from a 21-year study and review of the literature. Clin Infect Dis. 2012; 54(3): 362–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurrie BJ, Fisher DA, Anstey NM, et al.: Melioidosis: acute and chronic disease, relapse and re-activation. Trans R Soc Trop Med Hyg. 2000; 94(3): 301–4. PubMed Abstract \| Publisher Full Text\n\nVlieghe ER, Phe T, De Smet B, et al.: Bloodstream infection among adults in Phnom Penh, Cambodia: key pathogens and resistance patterns. PLoS One. 2013; 8(3): e59775. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng AC, Wuthiekanun V, Limmathurosakul D, et al.: Role of selective and nonselective media for isolation of Burkholderia pseudomallei from throat swabs of patients with melioidosis. J Clin Microbiol. 2006; 44(6): 2316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWuthiekanun V, Suputtamongkol Y, Simpson AJ, et al.: Value of throat swab in diagnosis of melioidosis. J Clin Microbiol. 2001; 39(10): 3801–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNhem S, Letchford J, Meas C, et al.: Detection of B. pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium Compared to Standard Culture. F1000Research. 2014. Data Source" }	[ { "id": "7216", "date": "20 Jan 2015", "name": "David Dance", "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\nThis paper describes an evaluation of the use of a selective enrichment method to detect Burkholderia pseudomallei in sputum samples in a provincial hospital in Cambodia. The additional yield compared with conventional cultures was relatively low (only one of 241 samples tested). The paper is well written and presents the results clearly. It would have been improved by the inclusion of an assessment of the additional costs of using selective culture. Clearly the benefits in this hospital are relatively small (although potentially life-saving for the one additional patient with melioidosis who was detected), but cannot be extrapolated to other areas because the distribution of melioidosis may be remarkably focal (for example in Thailand the incidence is very low in the central region but high in the northeast). It is also unclear why a selective enrichment technique was used as opposed to direct culture onto solid media, since enrichment culture was not shown significantly to increase the B. pseudomallei isolation rate from sputum compared with plating directly onto Ashdown's medium in a previous study (Wuthiekanun et al.,1990). It might also have been possible to have been more selective about the samples that were cultured and to have analysed patient characteristics in greater detail, although the majority of samples were thought to have been from patients suspected of having TB, a group in whom it has previously been suggested that testing for melioidosis (using a similar enrichment technique) might be warranted (Suntornsut et al., 2013).Although this is a relatively small scale study with relatively unremarkable results, it warrants being in the public domain. Laboratories in melioidosis-endemic areas should still be encouraged to consider looking for B. pseudomallei in appropriate samples and should conduct their own assessment of the costs and benefits of doing so based on local epidemiology.", "responses": [ { "c_id": "1339", "date": "15 May 2015", "name": "T Eoin West", "role": "Author Response", "response": "Thank you for the helpful comments.We opted not to perform cost-benefit analyses in light of our small numbers of positive samples and wide confidence intervals around point estimates. We agree that these calculations are important to perform in the future in larger studies.We chose to use selective broth based on Wuthiekanun V et al, Am J Trop Med Hyg 77(5), 2007, 812-13. This study reported that “a total of 94 of 120 (78%) respiratory secretions were positive for B. pseudomallei, of which 33 (35%) were positive from enrichment broth alone.”Unfortunately we do not have any data on patient characteristics. Our future studies aim to investigate patient characteristics and indications for sampling." } ] }, { "id": "7217", "date": "06 Feb 2015", "name": "Jan Jacobs", "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\nThis is a well-designed and clearly written paper about describing the incremental yield of selective culture for Burkholderia pseudomallei (BPM) in sputum samples routinely submitted for culture in a Provincial hospital in Cambodia, where melioidosis is endemic.I have the following comments:Most readers will not be familiar with melioidosis. It may be useful to state more explicitly in the Introduction that culture for the time being is the only reliable way for diagnosis of melioidosis and that recovery of BPM from any sample and in any quantity always means infection: this is especially important as for most other bacteria grown from non-sterile samples (as respiratory secretions), colonization and oral flora contamination must be excluded, which may be done by, among other criteria, based on density of growth. Second, based on this observation, selective media have been recommended as part of routine sputum culture in endemic areas. The main weakness of this study is that there are no data about indications of sampling and selection of patients and from the Discussion can be learned that probably no well-defined indications have been used. So it is not clear whether all patients were suspected of lower respiratory tract infection and acutely ill, or if they selected, for instance, based upon initial treatment success and ability to pay for the culture etc.. The authors should emphasize this non-selection somewhat more, and, for instance, display data or at least an idea about the proportion of patients cultured in relation to the total (estimated) numbers of eligible patients with acute respiratory tract infections. Likewise, some data about the patients’ profile (age (median/range or IQR) and gender would be of interest, as well as the distribution between hospitalized/outpatients - the spectrum of organisms cultured as mentioned in the Results suggests a high proportion of hospitalized patients. In line with the above, I think the authors should reconsider amendingtheir conclusion: they are right by stating that selective enrichment culture media would be more rewarding in settings with high(er) incidence of melioidosis, but there is for the time being no sound evidence about the to assume that the incidence of melioidosis in their place will be lower than, for instance, in Thailand. Moreover, and as the authors state in the Discussion above, sputum and other microbiological cultures are probably not yet exploited to the best extent by the clinicians and probably also cost-prohibitive for many patients, reason why identification and selection of at-risk patients and pre-test probability may have been more important than overall incidence of melioidosis (as the authors state in the Discussion). In other words, if the enhanced testing protocol would have applied on for instance, middle-aged patients with diabetes presenting with overt signs of pneumonia/sepsis, incremental yield and cost-effectiveness may be much higher. So I suggest to consider rephrasing “the benefit …may be more apparent … in patients selected upon risk factors and clinical presentation…” Likewise, the Abstract (final sentence) may be rephrased to focus more on selected indications for sputum sampling. The authors may add some information about the referral population of Kampong Cham hospital: rural, agriculture...", "responses": [ { "c_id": "1338", "date": "15 May 2015", "name": "T Eoin West", "role": "Author Response", "response": "Thank you for this constructive review. We agree and have modified the introduction to clarify these points as suggested.This is indeed a shortcoming of this study; unfortunately we do not have any data on the patients who provided the sputum samples tested for these patients. Sputum samples are submitted to the laboratory from both clinic and hospitalized patients. Our future studies aim to investigate patient characteristics and indications for sampling.We agree with these very helpful comments, and have modified the abstract and discussion as suggested.We have now added this information as suggested." } ] } ]	1	https://f1000research.com/articles/3-302
https://f1000research.com/articles/4-119/v1	14 May 15	{ "type": "Opinion Article", "title": "A(a)LS: Ammonia-induced amyotrophic lateral sclerosis", "authors": [ "Bhavin Parekh" ], "abstract": "Amyotrophic lateral sclerosis (ALS) is a dreadful, devastating and incurable motor neuron disease. Aetiologically, it is a multigenic, multifactorial and multiorgan disease. Despite intense research, ALS pathology remains unexplained. Following extensive literature review, this paper posits a new integrative explanation. This framework proposes that ammonia neurotoxicity is a main player in ALS pathogenesis. According to this explanation, a combination of impaired ammonia removal— mainly because of impaired hepatic urea cycle dysfunction—and increased ammoniagenesis— mainly because of impaired glycolytic metabolism in fast twitch skeletal muscle—causes chronic hyperammonia in ALS. In the absence of neuroprotective calcium binding proteins (calbindin, calreticulin and parvalbumin), elevated ammonia—a neurotoxin—damages motor neurons. Ammonia-induced motor neuron damage occurs through multiple mechanisms such as macroautophagy-endolysosomal impairment, endoplasmic reticulum (ER) stress, CDK5 activation, oxidative/nitrosative stress, neuronal hyperexcitability and neuroinflammation. Furthermore, the regional pattern of calcium binding proteins’ loss, owing to either ER stress and/or impaired oxidative metabolism, determines clinical variability of ALS. Most importantly, this new framework can be generalised to explain other neurodegenerative disorders such as Huntington’s disease and Parkinsonism.", "keywords": [ "amyotrophic lateral sclerosis", "ammonia", "neurodegenerative disorders" ], "content": "Introduction\n\nAmyotrophic lateral sclerosis (ALS) is the most feared, frequent, flummoxing and fatal motor neuron disease1–3. It is a biphasic disease which starts insidiously, later followed by relentless progression once symptomatic4. Although rare, it is a grim and demeaning illness: it slowly cripples and confines its victims in their own body, ultimately killing them by breathing failure within 3–5 years after onset2,5. No cure exists1. Ever since Charcot’s description of ALS (1869), a classical view defines ALS as an adult-onset neurodegenerative disease of upper and lower motor neurons6,7. However, ALS is clinically characterised by variability about the type and degree of motor neuron and non-motor neuron involvement8.\n\nALS pathology involves an interaction of multiple genes and environmental factors. Indeed, ALS is mainly a polygenic disease (70%–90); and the heritable form (familial ALS) contributes to merely 30% of total ALS cases3,9. Remarkably, mutant C9ORF72, TARDBP, FUS, and SOD1 genes account for 70% of all familial ALS cases10. Evidence shows that environmental factors such as intense physical activity, cigarette smoking, viral infections, and the ingestion of non-protein amino acids (i.e. β-N-methylamino-L-alanine) play a role in ALS5,11,12.\n\n\nALS aetiology: an enduring enigma\n\nDespite nearly 150 years of research, ALS remains an enigma13, although, at the cellular, molecular and metabolic levels, a staggering and ever expanding list of pathogenic mechanisms have been linked to ALS13,14. These include protein aggregation, mitochondrial dysfunction, oxidative/nitrosative stress, endoplasmic reticulum (ER) stress, axonal transport defects, glutamate excitotoxicity, impaired macroautophagy, impaired glycolysis, neuroinflammation, and glucose and fat metabolism impairments13–19. However, hitherto no hypothesis exist that effectively links all these mechanisms to a singular central cause3. Hence, despite steadily accumulating knowledge about ALS, a key question still lingers: what cause ALS?\n\n\nALS: a multi organ disease\n\nSince ALS is manifestly a neurological disorder, researchers have long embraced an intuitive neurocentric view of ALS, assuming that intrinsic neuronal pathology causes ALS7,20. Against this view, however, growing evidence suggests that ALS pathology extends well beyond neuronal cells and involves multiple organs7,14,21. Unsurprisingly, ALS is now deemed as a systems disease20,22. Not only that, evidence increasingly shows that primary pathological events, inherited or acquired, within these organs may act as distal cause of ALS14,21,22. Such evidence is reviewed below.\n\n\nRole of skeletal muscle\n\nALS starts and spreads from skeletal muscle14,23. Indeed, some early symptoms of ALS involve the neuromuscular system: muscle atrophy, cachexia (wasting), weakness, and fasciculation (twitches)7,11,14,19,23. In fact, cachexia reduces survival of ALS patients19. Reinforcing such observations, data from animal models of ALS showed neuromuscular dysfunction precede motor neurons loss14. For instance, Frey et al. showed selective loss of fast-fatigable neuromuscular synapses of SOD1G93A mice by 6 weeks of age, 2 month before symptomatic phase24. Cogently, a study showed that the expression of mutant gene (SOD1G93A) exclusively in skeletal muscle of transgenic mice caused cachexia, neuromuscular denervation, paresis, and motor neuron degeneration25.\n\nSkeletal muscle possesses mainly two types of muscle fibres: fast twitch and slow twitch26. Lately, evidence suggests that in ALS fast twitch muscle motor units are selectively damaged before overt symptoms, whereas slow twitch motor units show damage after overt symptoms27,28. For example, a set of studies showed a rapid motor unit loss during the presymptomatic phase (5 weeks of age) in fast but not slow-twitch muscles of the SOD1G93A mouse27. Accordingly, fast twitch muscle appears to be more susceptible to damage in ALS patients28. Therefore, fast twitch muscle pathology appears to be the distal cause of ALS.\n\nTogether, those findings have led to the “dying-back” hypothesis14. This holds that ALS is a distal axonopathy in which pathological changes first arise distally at the neuromuscular junction and progress backward toward the spinal cord cell body14. That said, however, recent and prior research mandates refinement of this hypothesis. Recently, experiments in the SOD1G93A mice showed independent and parallel degeneration of both upper and lower motor neurons at early stage, hinting at a common pathological mechanism29. Consistent with this, recent neuroimaging studies showed early stage involvement of upper motor neuron (UMN) in ALS patients30. In fact, Gower (1886), Charcot’s contemporary, suggested simultaneous and independent degeneration of upper and lower motor neurons in ALS31. Thus, a common but hitherto unidentified pathological factor emanating from skeletal muscle appears to damages both upper and lower motor neurons.\n\n\nLiver: an emerging locus of ALS\n\nAside from skeletal muscle, mounting evidence suggests that liver dysfunction commonly occurs in ALS. Indeed, literature on the liver pathology in ALS has existed for over a half century32,33. Earlier, researchers showed a range of liver abnormalities in ALS patients including the disturbance of unconjugated bilirubin metabolism, mitochondrial defects, and copper accumulation in hepatic lysosomes32. More recently, clinical studies suggest that hepatic steatosis (fatty liver degeneration) is a common and unique phenomenon in motor neuron diseases including ALS22,34,35. Nodera et al. found that hepatic steatosis was present in 76% of ALS patients22. In line with this, studies showed reduced growth hormone/insulin-like growth factor-1 (GH/IGF-I) levels, which induce hepatic steatosis, in ALS21,36. In keeping with this, hyperhomocysteinemia, which is associated with hepatic fat accumulation, commonly occurs in ALS patients37,38. Moreover, research showed that ALS-associated environmental factors such as virus infection (i.e. retrovirus virus and HIV) and cigarette smoking cause hepatic steatosis5,12,39,40. Furthermore, viral hepatitis, which causes hepatic insufficiency and frequent fatty liver degeneration, has been linked to motor neuron disease41–44. Finally, Reye-like syndrome, associated with fatty liver degeneration, has been associated with spinal muscular atrophy (SMA), a lower motor neuron disease35.\n\nA number of genetic findings also support this notion. Iron dysregulation disorders such as HFE gene-related hemochromatosis and hyperferritinemia, which induces hepatic steatosis, frequently (30%) occurs in ALS45,46. Additionally, mutant cholesterol and lipid pathways genes such as TDP-43 ATXN2, paraoxonase and CYP7A1, implicated in hepatic steatosis, have been linked to ALS47–55. Moreover, an interaction between disturbances in hepatic mitochondrial function and ER homeostasis causes hepatic steatosis; and investigators discovered morphological changes in ER structure and mitochondria in the liver of ALS patients32,56. These findings support the evidence that mutant ER-stress regulating genes such as XBP1, SigR1, VCP, TDP-43, FUS, SOD1, and VAPB are linked to ALS57,58. Furthermore, SMN gene, implicated in ALS and SMA, have been shown to regulate the development and function of liver35.\n\nFinally, hepatic steatosisis is linked to the metabolic syndrome, characterised by hyperglycaemia, hyperglucagonemia, insulin resistance and altered serum triglycerides; and such findings have been reported in ALS59–65. In this regard, it is interesting to note that damage to fast twitch skeletal muscle, the main site of glucose disposal and the largest reservoir of glycogen in humans, leads to hepatic steatosis66.\n\nNotably, Li et al. showed exendin-4, which counteracts hepatic steatosis, ameliorated motor neuron degeneration partly by correcting this systemic metabolic alteration67,68. This clearly suggests that, much like skeletal muscle, liver pathology is not merely an innocent bystander, but rather a premorbid condition, which plays an active role in ALS pathogenesis.\n\n\nAims\n\nThus, (i) identifying skeletal–muscle produced unknown pathological factor, (ii) unravelling its nexus and synergism with hepatic steatosis, (iii) understanding the mechanisms by which this pathology factor causes motor neuron damage, and (iv) revealing the cause(s) of clinical heterogeneities would fully untie the Gordian knot of ALS pathology, allowing the development of predictive and prognostic biomarkers as well as potent drugs3,13. Hence, by taking a systems view, this paper aims to fill these knowledge gaps. Moreover, by fusing these separate pieces together, this paper presents a full picture of ALS pathology.\n\n\nImpaired glycolysis in fast twitch muscle: one of the pathological triggers of ALS\n\nEvidence suggests that defective energy deficit in skeletal muscle triggers ALS. Investigators found impaired skeletal muscle metabolism, characterised by low ATP levels and hypermetabolism, causes neuromuscular dysfunction in ALS mouse model69,70. Conversely, metabolic interventions such as high-calorie diets and reducing hypermetabolism improved survival and alleviated symptoms in ALS19,70. However, the functional link between skeletal muscle metabolic impairment and ALS remains nebulous. Instructively, since ALS begins from fast twitch muscle, which relies on anaerobic glycolysis for energy (i.e., ATP), this immediately suggests that impaired anaerobic glycolysis produces the unknown pathological trigger.\n\n\nImpaired glycolysis in ALS\n\nCompelling evidence suggests that muscle glycolysis is impaired in ALS. Valosin-containing protein (VCP), a gene linked to ALS, causes defective muscle glycolysis and reduced ATP levels. Dupis et al. linked upregulation of mitochondrial uncoupling proteins UCP1 and UCP3—which suppresses glycolysis and causes hypermetabolism—to muscle denervation in ALS71. Bernardini et al. showed low expression of glycolysis genes such as FBP2 and enolase 3 in the skeletal muscles of ALS patients72. Brockington et al. uncovered down regulation of glycolytic enzyme lactate dehydrogenase 1 in the VEGFδ/δ mouse model of ALS73. Moreover, experiments showed that the gain-of-interaction of the SOD1G93A mutant with cytosolic malate dehydrogenase induces glycolytic impairments74. Dunckley et al. linked variants of FLJ10986, a protein linked to glycolysis, with the susceptibility of sporadic ALS75. Collectively, these findings clearly show impaired glycolysis in skeletal muscle of ALS patients and mouse model.\n\n\nImpaired muscle glycogen and glucose homeostasis in ALS\n\nNotably, fast twitch skeletal muscle glycolysis depends on muscle glycogen storage and glucose transporter 4 (GLUT4)-mediated muscle glucose uptake26,76. Accumulating evidence suggests defective muscular glycogen metabolism and impaired GLUT4-mediated muscular glucose uptake in ALS. Derave et al. discovered diminished muscle ATP and glycogen accumulations in SOD1 G93A mice27. Smittkamp et al. revealed impaired insulin-stimulated glucose uptake exclusively in fast twitch skeletal muscle in middle-stage SOD1 G93A mice77. Accordingly, fast twitch skeletal muscle fibres of TDP-43 transgenic mice show defective insulin-induced GLUT4 translocation and glucose uptake77. Moreover, in the mutant TDP-43-linked ALS mice, Perera et al. reported decreased AMPK, which mediates muscle contraction-induced glucose entry and glycogen synthesis76,78,79. Conversely, AMPK activator drugs (i.e. latrepirdin) delayed ALS in SOD1G93A mice80. Furthermore, muscle contraction facilitated glucose uptake involving Ca2+/calmodulin-dependent GLUT4 translocation appears to be defective in ALS. For example, investigators linked mutant neuregulin-ERBB4 gene, involved in calcium-induced glucose uptake during muscle contraction, to ALS79,81. Thus, it is obvious that ALS involves impaired carbohydrate metabolism that supports muscle glycolysis.\n\n\nALS resistance of extraocular muscles (EOMs): role of glycolysis\n\nFinally, the metabolic characteristics of—ALS-resistant—extraocular muscles (EOMs) further consolidate this notion82. Two fundamental differences exist between EOMs and skeletal muscle metabolism83. First, compared to skeletal muscles, EOMs have high glycolysis capacity, evident by the overexpression of glycolytic enzymes (e.g. lactate dehydrogenase, enolase)83. Second, owing to their high vascularity, EOMs rely more on instantaneous glucose uptake—less on glycogen storage and GLUT4-mediated muscle glucose uptake83. All in all, these three sets of findings point that defective glycolysis causes ATP deficits in fast twitch skeletal muscle of ALS patients. Hence, the unknown pathological factor emanating from skeletal muscle appears to have direct connection with defective muscle glycolysis. How?\n\n\nAmmonia: the elusive pathological factor\n\nNotably, defective glycolysis, which reduces ATP levels, in fast twitch skeletal muscle activates catabolic reactions of adenine nucleotides (i.e. purine nucleotide cycle) and amino acids (Figure 1)84,85. Intriguingly, such catabolic reactions produce ammonia—a neurotoxin 1000 times more toxic than ethanol at equimolar concentrations85,86. Since ammonia is toxic, it is obligatorily removed mainly through hepatic urea cycle which transforms ammonia into urea87. Notably, when the urea cycle is impaired, as it occurs in fatty liver disease, increased ammonia production from skeletal muscle or from dietary sources can cause chronic hyperammonia (>35–50 µM) and consequent neurodegeneration and motor impairments (Figure 1 and Figure 2)33,88–92. Indeed, in many liver diseases, including fatty liver disease which commonly occurs in ALS, because of impaired urea cycle-mediated ammonia removal, hyperammonia frequently leads to corticospinal hyperexcitability, myelopathy and spasticity—features strikingly reminiscent of neurophysiological phenotypes of ALS symptoms93–97.\n\nMechanism of motor neuron degeneration in ALS involves two main factors: (i) ammonia neurotoxicity and (ii) down regulation of neuronal calcium binding proteins (CaBPs). Owing to imbalanced interorgan ammonia metabolism, ammonia, a well-known neurotoxin, accumulates in neurons. Among the five organs (brain, skeletal muscle, gut, liver and kidney) involved in ammonia metabolism, ALS appears to mainly involve the role of liver and skeletal muscle in that confluence of impaired ammonia removal—owing to impaired hepatic urea cycle—and increased muscular ammoniagenesis—owing to impaired glycolysis in fast twitch skeletal muscle—lead to chronic hyperammonia in ALS. In the brain, ammonia activates several neurodegenerative pathways such as (1) autophagy-endolysosomal dysfunction (2) neuroinflammation (3) oxidative stress (4) Golgi fragmentation and (5) neuronal hyperexcitability. In the absence of neuronal calcium binding proteins (CaBPs) such as parvalbumin, calbindin, calreticulin, activation of these degenerative pathways lead to motor neuron damage. Notably, decrease in calreticulin, because of increased ER stress, leads to lower motor neuron damage, whereas the down-regulation of parvalbumin and calbindin, because of defective mitochondrial respiration, leads to upper motor neuron damage.\n\n(Modified with permission from 232). Ammonia intoxication directly damages motor neurons through five mutifactorial pathological mechanisms: 1) alkalisation-induced impairment of macroautophagy-endolysosomal system, 2) Golgi impairment, 3) increased oxidative/nitrosative stress and MAPK up-regulation 4) neuronal hyperexcitability and 5) neuroinflammation. These mechanisms explain frequently found cellular, molecular and neurophysiological phenotypes of motor neuron damage in ALS. A. Owing to ammonia-induced alkalisation, impairment of macroautophagy-endolysosomal system induces several key molecular histopathological features of ALS including : (i) ubiquitinated (Lewy and skein body-like inclusions) and non-ubiquitinated inclusion bodies (i.e. bunia bodies) formation, (ii) amyloid precursor protein (APP), (iii) gangliosides accumulation (i.e. GM2), (iv) autophagy vacuoles, (v) neurofilament aggregation and axonal swelling. B. Ammonia activates CDK5 which in turn leads to frequently observed Golgi fragmentation. C. Ammonia-induced oxidative/nitrosative stress and MAPK-up-regulation lead to multiple cellular and molecular pathological features such as: (i) blood brain barrier (BBB) breakdown, and (ii) MMP-9-induced ER stress. D. Ammonia causes neuronal hyperexcitability by (i) down-regulating astrocyte glutamate transporters (GLAT-1 and GLAST) and (ii) lowering potassium-chloride co-transporter KCC2 level which suppresses GABA and Glycine-mediated inhibitory neurotransmission. E. Ammonia leads to neuroinflammation secondary to reactive microglial and astrogliosis. This occurs because of (i) quinolic acid release from microglia (ii) up-regulation of pro-inflammatory cytokines (TNF, IL-1β, NF-κB, and PGE2) in astrocytes (iii) TLR-4 activation and (iv) neutrophil burst derived NADPH oxidase (NOX)-induced oxidative stress.\n\n\nAmmonia neurotoxicity: hypothesis and evidence\n\nTogether, these findings provide a compelling rationale for a new hypothesis. ALS pathology might involve not only skeletal muscle-induced increased ammonia production, because of impaired glycolysis, but also impair ammonia removal, secondary to hepatic steatosis-induced faulty urea cycle, leading to chronic hyperammonia and consequent progressive motor neuron degeneration (Figure 1 and Figure 2). Astonishingly ammonia’s role has seldom been directly investigated. Nonetheless, diverse data obtained from clinical and animal studies support this hypothesis showing that hyperammonia increases ammoniagenesis and decreases ammonia removal in ALS.\n\nA clinical study showed elevated ammonia level in motor neuron disease patients—with ammonia levels inversely correlated to disease duration98. These investigators also found a causal relationship between ammonia and ALS by noting that infusion of amino acids, which causes ammoniagenesis, aggravates ALS98. Moreover, dietary supplements of branched chain amino acids was one of the factors associated with the early onset of ALS (45 years) in Italian soccer players99. Accordingly, other investigators reported accelerated skeletal muscle protein catabolism ALS100. This chimes with the fact that hepatic steatosis-induced glucagon secretion, which occurs in ALS, increases ammoniagenesis through protein degradation101,102. Consistent with this, as noted above, intense or prolonged physical exertion, an ammoniogenic activity, is an ALS risk factor11,103.\n\nBeside a link between hepatic steatosis and a faulty urea cycle, other lines of clinical evidence further implicate the impaired urea cycle in ALS48,91,104. For example, Iłzecka et al. showed decreased arginine levels, an amino acid required for liver urea cycle function, in ALS patients105. Additionally, research showed that metabolic acidosis, which impairs urea cycle, occurs in ALS60,106. Impaired hepatic urea cycle activates glutamine synthetase, an alternative ammonia detoxification pathway, and researcher also found increase in glutamine synthetase expression in blood platelets of ALS patients107,108.\n\nAnimal models of ALS further cement this ammonia hypothesis. Investigators showed hyperammonia and impaired urea cycle in 50 day old SOD1G93A mice compared to wild type mice of the same age109. Moreover, these investigators showed increased glutamine, a precursor of ammonia, in SOD1G93A mice109. Additionally, in the mutant SOD 1 G86R mice, de Aguilar et al. showed early (3 months of age) muscle denervation along with increased AMP deaminase-3 (AMPD3), an enzyme of purine nucleotide cycle involved in ammoniagenesis110. Furthermore, a set of studies showed increased arginine vasopressin release in the SOD1 mice, and independent research showed that arginine vasopressin causes muscle protein degradation and consequent ammoniagenesis111,112. Conversely, research showed that ammonia-counteracting compounds such as phenylbutyrate, ariginine, resveratrol and l-carnitine alleviated symptoms and enhanced survival in the ALS mouse model97,113–118.\n\nFurther supporting this hypothesis, experiments have shown that environmental neurotoxins implicated in ALS causes ammonia toxicity. Dietary intake of β-N-methylamino-L-alanine, a non-protein amino acid linked to Guam's ALS-PDS complex epidemic, causes liver damage and ammonia toxicity119,120. Similarly, the ingestion of Lathyrus sativus seeds, implicated in neurolathyrism (an upper motor neuron disease), causes liver dysfunction, urea cycle impairment, and chronic ammonia toxicity121,122. Finally, animal studies showed that the pesticide pyrethroid, which causes an ALS-mimicking syndrome, leads to protein catabolism ammonia toxicity123,124.\n\nYet another line of evidence bolsters the ammonia neurotoxicity hypothesis. Interestingly, reports showed that motor neuron disease could be one of Huntington's disease (HD)’s presenting features125,126. In fact, aside from genetic overlap with ALS, HD shares many pathophysiological characteristics with ALS: skeletal muscle atrophy, hepatic steatosis, hyperglycaemia and adipose tissue dysfunction127,128. Tellingly, although often regarded as curious findings rather than telltale observation, impaired urea cycle as well as hyperammonia occur in HD127. Strikingly, data from mouse models of HD showed that protein-restricted diets not only reduced hyperammonia but also prevented the motor deterioration90. This suggests that ammonia could be a common culprit in range of neurodegenerative conditions, especially affecting motor system.\n\nIn addition to muscles and the liver, ammonia metabolism involves other organs, including the gut, the kidneys and the brain (Figure 1)87,89. Hence, this hypothesis does not preclude a role of these organs. Although no evidence has yet emerged to implicate the gut and kidneys in ALS, some data at least suggest a role of cerebral ammoniagenesis in ALS. Studies showed increased deamination of catecholamine, which causes cerebral ammoniagenesis, in ALS, evident by the overactivity of catecholamine oxidising enzymes such as MAO-B and aldehyde oxidase89,129–131. Put together, these findings implicate ammonia neurotoxicty in ALS.\n\n\nMechanisms of ammonia’s neurotoxicity\n\nWhen hyperammonia occurs, ammonia enters into the brain, leading to neurotoxicity. Ammonia exerts pleiotropic neurotoxic effects by activating an array of cellular mechanisms, which are the proximal causes of ALS (Figure 1 and Figure 2). These mechanisms include: 1) alkalisation-induced impairment of macroautophagy-endolysosomal system, 2) Golgi impairment, 3) increased oxidative/nitrosative stress and mitogen-activated protein kinase (MAPK) up-regulation 4) neuronal hyperexcitability and 5) neuroinflammation89,132–136.\n\nAs described below, taken together, these five mechanisms not only explain several frequent cellular and molecular histopathological hallmarks of ALS but also neurophysiological features of ALS (Figure 1 and Figure 2). The cellular histological features, explained by ammonia’s toxicity, include axon swelling, blood brain barrier breakdown and astrogliosis and microgliosis137–139. The molecular pathological features, explained by ammonia’s toxicity, include formation of inclusion bodies such as bunia bodies and Lewy bodies, gangliosides accumulation, glycogen aggregation, neurofilament derangement, Golgi fragmentation, and reduced glutamate transporters60,140–145. Moreover, ammonia toxicity explains a key neurophysiological feature of ALS: neuronal hyperexcitability17. Finally and most importantly, ammonia toxicity explains why ALS is mainly a motor neuron disease.\n\n\nAlkalisation-induced impaired macroautophagy-endolysosomal system\n\nAmmonia-induced alkalisation impairs the macroautophagy-endolysosomal system, one of the main cellular garbage disposal systems. This occurs at least in two ways. First, ammonia, a weak base, preferentially accumulates in lysosomes because of their low acidity (PH~4.5)134. Consequently, intra-lysosomal alkalisation and lysosomal enzyme leakages occur, impairing the lysosomal hydrolysis of proteins, lipids and carbohydrates (Figure 2)134. Second, ammonia alkalises acidic membranous compartments of axon terminals, jamming membrane microtubules and thereby blocking the anterograde-to-retrograde transport of endosomes146. Consequently, impaired fusion of endocytic compartments with lysosomes occurs, causing defective autophagy of endocytosed material (Figure 2)89,146,147. As a result, toxic accumulation of protein aggregates, glycolipids, and carbohydrates occurs89. In turn, these toxic by-products activate the apoptosis programme, causing cell death148.\n\nAmmonia’s alkalisation-induced toxicity is especially relevant to ALS because macroautophagy-endolysosomal dysfunction causes motor neuron degeneration149. Indeed, mutant genes of this pathway such as SOD1, FIG4, CHMP2B, SQSTM1, DCTN1, DYNC1H1, and RAB7A have been linked to ALS149. Consistent with this interpretation, research showed impaired dynein-dependent retrograde axonal transport, required for autophagosome-lysosome fusion, causes motor neuron degeneration150,151. Furthermore, consistent with lysosomal enzyme leakage, investigators reported increased lysosomal enzyme levels (i.e. acid phosphatase, Cystatin C) in the cerebrospinal fluid (CSF) and plasma of ALS patients152,153.\n\n\nImpaired lysosomal proteolysis\n\nAmmonia-induced impaired lysosomal proteolysis explains key histopathological hallmarks of ALS including formation of inclusion bodies (Figure 2). Ammonia-induced alkalinisation in lysosomes impairs the activities of protease enzymes including cathepsin B and cathepsin D134,154. Strikingly, investigators showed downregulation of cathepsin B and cathepsin D in ALS155,156. Notably, defective lysosomal proteolysis causes swollen axonal dystrophy (spheroids), with histological features such as ubiquitinated and non-ubiquitinated inclusion bodies, amyloid precursor protein, and neurofilament aggregation157. In keeping with this, research revealed such findings in ALS144,145,156,158–161.\n\nIn regard to non-ubiquitinated inclusion bodies, Kikuchi et al. showed that decreased cathepsin B generates Bunina bodies (small eosinophilic intraneuronal lysosomal inclusion bodies) in motor neurons, a hallmark of ALS (Figure 2)140,156. Since cathepsin D mediates lipofuscin and α-synuclein clearance, and since downregulation of cathepsin D occurs in ALS, this explains frequently observed deposits of lipofuscin granules and α-synuclein aggregation in ALS patients148,162–164. Moreover, reduced cathepsin B activity induces amyloid precursor protein (APP) accumulation, and Bryson et al. showed increased APP level in the SOD1 G93A mouse, which contributed to motor neuron damage158,165. As for impaired proteolysis-induced ubiquitinated inclusion bodies, ubiquitin inclusion aggregates such as Lewy body-like inclusions’ and ‘skein-like inclusions’ have been found in ALS (Figure 2)145,166. This finding accords with the observations that inhibition of macroautophagy impairs the ubiquitin proteasome system (UPS)167. Finally, neurofilament aggregation and spheroid formations have been found in the ALS mouse model and in patients (Figure 2)161.\n\n\nImpaired lysosomal ganglioside clearance\n\nGangliosides are complex sialylated glycosphingolipids, particularly found in the CNS168. Notably, GM2 ganglioside is a main ganglioside in motor neurons169. Accumulation of GM2 ganglioside, owing to impaired lysosomal Hexosaminidase (Hex) enzymes, frequently causes motor neuron disease170–172. For example, Banerjee et al. reported slow accumulation of GM2 ganglioside, primarily in motor neurons, in patients with progressive motor neuron disease associated with partial Hex A and no Hex B activity172. By implication, this suggests that accumulation of gangliosides including that of GM2 occurs in ALS and that ammonia increases GM2 ganglioside levels. Indeed, although scantly investigated, some investigators reported increased ganglioside levels in ALS including GM2 ganglioside142,173,174. In line with ammonia’s role in ganglioside metabolism, Perez et al. showed that ammonia causes leakage of Hexosaminidase A (Hex A), indicating GM2 accumulation175,176. Thus, ammonia-induced GM2 accumulation could partly explains the heightened vulnerability of motor neurons in ALS (Figure 2).\n\n\nImpaired lysosomal carbohydrate clearance\n\nAnimal and clinical studies reported neuronal and glial glycogen accumulation and polyglucosan bodies (branched chained glycogen aggregates) in ALS (Figure 2)60,177,178. Notably, Dodge et al. showed that decreased level of α-glucosidase—a glycogen degrading lysosomal enzyme—partly causes glial and neuronal glycogen accumulation in ALS, and experiments showed that ammonia leaks α-glucosidase from lysosomes60,179. Thus, ammonia-mediated lysosomal dysfunction explains yet another histological feature of ALS. Of note, this fits with the observations that upper and lower motor neuron lesions frequently arise in polyglucosan body diseases180.\n\n\nImpaired Golgi function\n\nAmmonia toxicity could explain Golgi apparatus fragmentation in ALS, an early and frequently observed event141. Sun et al. showed that CDK5 activation fragments Golgi apparatus181. Interestingly, Cagnon and Braissant showed that ammonia activates CDK5. They also showed that CDK5 activation led to neuronal cell death and impairment of axonal outgrowth135. Apparently, p25-induced mislocalization and deregulation of CDK5 activity occurs in ALS (Figure 2)143,182. In fact, Nguyen et al. reported that an attempted re-entry of motor neurons into the G1-S phase of the cell cycle subsequent to CDK5 deregulation is a critical step of neurodegeneration in ALS182.\n\n\nIncreased oxidative/nitrosative stress and MAPK expression\n\nAdditionally, data suggested that ammonia induces oxidative/nitrosative stress and MAPK expression, frequently found pathological features of ALS (Figure 2)132. Research showed that oxidative/nitrosative stress and MAPK increases extracellular matrix degrading enzymes such as urokinase-type plasminogen activators and MMP-9183. Unsurprisingly, experiments found that increased levels of these extracellular matrix degrading enzymes occur in ALS184. Strikingly, Kaplan et al. observed overexpression of MMP-9 increased the vulnerability of fast fatigable limb-innervating motor neuron185. MMP-9 appears to exert neurotoxicity mainly through up-regulation of ER stress (Figure 2)185. Moreover, Skowrońska et al. showed that increase in MMP-9, which degrades the extracellular matrix, destroys the blood brain barrier (BBB)186. Predictably, Nicaise et al. showed impaired blood-brain and blood-spinal cord barriers in mutant SOD1-linked ALS rodents138. Additionally, since MAPK regulates cytoskeletal homeostasis, ammonia-induced MAPK activation explains why cytoskeleton abnormalities such as intermediate filaments accumulation occur in ALS160,187.\n\n\nNeuronal hyperexcitability\n\nFurthermore ammonia intoxication explains neuronal hyperexcitability in ALS—a cardinal characteristic of ALS16. By decreasing potassium-chloride cotransporter KCC2, located in the brain and spinal cord, ammonia increases chloride levels in neurons (Figure 2)188. Increased neuronal chloride levels in turn suppress GABA and Glycine-mediated inhibitory neurotransmission, causing neuronal hyperexcitability (Figure 2)189. In keeping with this, Fuchs et al. discovered decreased KCC2 expression in ALS-vulnerable motoneurons in spinal cord and hypoglossal nuclei of SOD1-G93A mice but not in EOMs190. Concordantly, researchers reported spinal motor neuron hyperexcitability and degeneration in ALS patients191. In fact, Hübner et al. showed that KCC2 knockout mice died after birth owing to motor deficits that caused respiratory failure, a feature similar to ALS189.\n\nFurthermore, ammonia causes glutamatergic excitotoxicty. By MAPK activation and increasing oxidative stress, ammonia decreases the glutamate transporter EAAT2 (GLT-1) and glutamate-aspartate transporter (GLAST) (EAAT-1) in astrocytes (Figure 2)133,192. Consequently, decreased transporters impair astrocyte-mediated high affinity glutamate uptake and clearance, leading to defective glutamatergic neurotransmission and excitotoxicity133,192,193. In line with this, decreased GLT-1 and GLAST have been found in the spinal cord of SOD1 G93A mice and ALS patients194–196. Interestingly, increased CSF glutamate was associated with a spinal onset of the disease and with severity of the symptoms in 41% of ALS patients196.\n\n\nNeuroinflammation\n\nAmmonia extensively affects the function of astrocytes and microglia (Figure 2). Through several mechanisms including (1) quinolinic acid (QUIN) production, (2) NADPH oxidase (NOX) activity-induced reactive oxygen species (ROS) generation, (3) Toll-like receptor 4 (TLR-4) activation, and (4) extracellular-signal-regulated kinase (ERK) pathway stimulation, ammonia induces a transition from a resting state into reactive astroglia and microglia phenotype (Figure 2)113,197–199. Consequently, reactive astroglia and microglia increase oxidative stress and stimulate the release of a range of proinflammatory cytokines including NF-κB, IL-1β, and PGE2, leading to neuroinflammation and degeneration (Figure 2)200.\n\nEmerging data indicate a role of QUIN in ALS201. Chen et al. detected overproduction of serum tryptophan, kynurenine and QUIN in the CSF of ALS patients compared to controls, concomitant with microglial activation and neuroinflammation (Figure 2)139. Similarly, experiments showed increased microglial and neutrophil-derived NOX activity correlated with fast ALS progression202. In keeping with increased ammonia-induced inflammation, investigators showed TLR-4 activation, and elevated levels of various pro-inflammatory cytokines in ALS203,204.\n\n\nClinical heterogeneities in ALS: the role of calcium-binding proteins (CaBPs)\n\nThe postulated ammonia neurotoxicity as the sole cause of ALS raises an awkward question. If ammonia damages both upper and lower motor neurons equally, then why does ALS often deviate from its classical pattern, manifesting as either the upper or lower motor neuron dominant subtype8? Moreover, why it is a relatively rare disorder? These questions clearly indicate that a protective factor exists that counteracts ammonia toxicity, and that anatomic-region specific loss of this factor causes the clinical heterogeneities in its presentation.\n\nOne such neuroprotective factor identified in ALS is the ER family of calcium binding proteins (CaBPs) (Figure 1 and Figure 2)205. By regulating voltage-gated calcium ion channels, CaBPs reduce calcium overload and cytotoxicity, thus protecting neurons from cell death205. The CaBPs involved in motor neuron protection include calreticulin, parvalbumin, and calbindin which are distributed in anatomic region specific manner within motor neurons206,207. Calreticulin expression mainly occurs in limb-innervating lower motor neuron regions such as the lumbar spinal cord area and fast-fatigable motoneuron, whereas calbindin and parvalbumin are expressed in both lower and upper motor neurons206,207.\n\nDifferential anatomic region-specific distribution of CaBPs in the CNS partly explains different patterns of motor neurodegeneration206,208. In the SOD1 G93A ALS mouse model, during the presymptomatic stage, fast-fatigable motoneuron denervation mainly accompanies calreticulin loss208,209. By contrast, investigators showed that loss of calbindin and parvalbumin correlated with both upper and lower motor neuron damage206.\n\n\nRegion specific regulation of CaBPs: ER stress and bioenergetics\n\nHow do neurons lose different CaBPs in different anatomic regions of the CNS? Research showed that ER stress downregulates calreticulin in limb-innervating lower motor neurons motor. In fact, calreticulin co-localises with the ER207. This accords with the finding that neuronal MMP-9—which enhances ER stress—selective damages fast fatigable lower motor neurons185. Additionally, since androgens modulate ER stress, this explains why sexual dimorphism occurs in lower motor neuron damage210. Interestingly, research revealed increased ER stress and reduced calreticulin in Alzheimer’s disease (AD)209. This explains why AD occasionally co-exists with motor neuron disease211.\n\nAs for the causes of reduced parvalbumin and calbindin expression in ALS, research implicates impaired oxidative metabolism secondary to defective mitochondrial electron transport (the respiratory chain) system212,213. Indeed, of the five protein complexes of the mitochondrial respiratory chain, research has frequently showed reduced respiratory chain complex I and IV activity in sporadic ALS patients205,212. Within these two complexes, complex IV appears to be particularly involved in ALS. This chimes well with the fact that 90% of all parvalbumin and calbindin-immunoreactive cells showed dense staining for respiratory complex IV (cytochrome c oxidase)214. Furthermore, hyperhomocysteinaemia, found to be highly prevalent in ALS, damages mitochondria and suppresses respiratory complex IV activity37,215. Revealingly, compared to skeletal muscle, the EOMs have slow metabolism characterised by low complexes I and IV activities (~50%) yet elevated mitochondria density with increased complex I and IV levels (30% to 2 times)—explaining why parvalbumin and calbindin levels remain relatively unaffected in EOMs205,216,217.\n\n\nThe role of respiratory chain complex subunits\n\nInterestingly, alterations in mitochondrial respiratory chain complex subunits also partly determine the spectrum of motor neuron damage. Investigators reported that altered Cytochrome c oxidase subunit Vb caused spinobulbar muscular atrophy, whereas Cytochrome c oxidase subunit I microdeletion induced upper motor dominant motor neuron damage218,219. Furthermore, deficiency of complex I involved lower motor neuron damage involving spinal and bulbar areas220. This fits with the findings that anatomic region-specific differences in mitochondrial respiration contribute to the localized neurodegeneration221.\n\nIn summary, these findings suggest that the regional loss of CaBPs expression, dependent on ER stress and defective mitochondrial respiration in the brain determines the anatomically variable manifestation of ALS. Collectively, it is also clear that motor neuron degeneration depends not only on postulated ammonia neurotoxicity but also on deficits of CaBPs within motor neuron.\n\n\nBiomarkers and therapeutics\n\nFrom this insight about ALS pathogenesis, diagnostic, disease monitoring and therapeutic measures emerge—fostering real hopes that ALS can be halted or even cured. Because ammonia is a volatile organic compound, excreted from breath and skin, an ammonia breath test would present a simple, reliable, robust, inexpensive and non-invasive tool for diagnosis and monitoring of ALS222. This ammonia breath test would prove invaluable in expediting drug discovery process. Aside from ammonia, gangliosides (e.g. Sialosylglobotetraosylceramide) and serum lysosomal enzymes could also serve as reliable adjuvant biomarkers of ALS142.\n\nAs for therapeutics, since ammonia toxicity appears to be a major player in ALS, ammonia-removal strategies seem to be the most effective strategy for ALS treatment223. Many existing ammonia-lowering agents including those that act on the hepatic urea cycle can be employed224,225. These could include salbutamol, conclevan, neomycin, sodium benzoate, ornithinephenyl acetate and L-ornithine aspartate223,226,227. Moreover, since impaired fast-twitch skeletal muscle glycolysis plays a role in ALS, improving muscle glycolysis through various existing drugs such as serotonin agonists and AMPK agonists (e.g. D-xylose) is another promising pharmacological strategy228,229. Additional therapeutic strategies could involve correcting system metabolic defects such as hyperglucagonemia and acidosis60,68. Moreover, other potent therapeutic targets could involve MAPK inhibitors, K-Cl co-transporters, and hexosaminidase agonists (e.g. Pyrimethamine)192,230,231. Finally, interventions that restore the levels of CaBPs should also be simultaneously applied for effective treatment.\n\n\nSummary\n\nALS is a ghastly and incurable disease. Despite increasing wealth of data, ALS remains poorly understood. By analysing existing literature, this paper has not only identified important knowledge gaps in ALS aetiopathology but also filled them and tied them together. In doing so, this paper postulates a new integrative explanation of ALS and suggests potent therapeutic measures to treat ALS. Central to this explanation is the notion that ALS is a neurological disease of metabolic origin—resembling hepatocerebral degeneration223. This explanation posits that ALS pathology involves the interplay of two critical factors: 1) chronic hyperammonia caused by imbalanced interogan ammonia metabolism, mainly due to muscle and liver pathology (Figure 1 and Figure 2) altered CaBPs homeostasis, mainly due to increased ER stress and impaired mitochondrial respiration (Figure 1).\n\nConsidering all these together in sequence, impaired fast twitch skeletal muscle carbohydrate metabolism activates purinergic and amino acid catabolism, leading to a release of ammonia, a neurotoxin. Alternatively, ammonia toxicity can also be induced or exacerbated by other endogenous (e.g. cerebral deamination, intestinal ammoniagenesis) and exogenous sources (i.e. neurotoxins). Owing to concurrent liver pathology (e.g. hepatic steatosis) in ALS, impaired hepatic ammonia detoxification occurs. Consequently, ammonia levels progressively builds up, leading to chronic hyperammonia. Since ALS pathology also involves loss of neuroprotective CaBPs (i.e. calbindin, calreticulin and parvalbumin), ammonia neurotoxicity in the absence of CaBPs leads to ALS. Ammonia damages motor neurons through a range of pathways. These pathways include impaired macroautophagy-endolysosomal impairment, Golgi fragmentation, oxidative/nitrosative stress and reactive microglial and astrogliosis. These mechanisms explain a range of histopathological and neurophysiological hallmarks of ALS such as bunia bodies and neuronal hyperexcitability. Finally, since ALS appears to be associated with HD, dementia and Parkinsonism this framework can be generalised to explain these disorders33,89,90.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI am very grateful to Prof. Rod Nicolson, at the University of Sheffield, for his kindness, constant support, and editorial guidance during this work. I am thankful to Prof. Paul Overton, at the University of Sheffield, for his incisive comments and criticisms. I am also thankful to Navin Shah and Nirav Parekh for their support.\n\n\nReferences\n\nMaessen M, Veldink JH, Onwuteaka-Philipsen BD, et al.: Euthanasia and physician-assisted suicide in amyotrophic lateral sclerosis: a prospective study. J Neurol. 2014; 261(10): 1894–901. PubMed Abstract \| Publisher Full Text\n\nCarus R: Motor neurone disease: a demeaning illness. 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J Neurosci. 2006; 26(18): 4774–84. PubMed Abstract \| Publisher Full Text\n\nZhou BG, Norenberg MD: Ammonia downregulates GLAST mRNA glutamate transporter in rat astrocyte cultures. Neurosci Lett. 1999; 276(3): 145–8. PubMed Abstract \| Publisher Full Text\n\nHowland DS, Liu J, She Y, et al.: Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proc Natl Acad Sci U S A. 2002; 99(3): 1604–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeath PR, Shaw PJ: Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis. Muscle Nerve. 2002; 26(4): 438–58. PubMed Abstract \| Publisher Full Text\n\nDunlop J, Beal McIlvain H, She Y, et al.: Impaired spinal cord glutamate transport capacity and reduced sensitivity to riluzole in a transgenic superoxide dismutase mutant rat model of amyotrophic lateral sclerosis. J Neurosci. 2003; 23(5): 1688–96. PubMed Abstract\n\nFelipo V, Butterworth RF: Neurobiology of ammonia. Prog Neurobiol. 2002; 67(4): 259–79. PubMed Abstract \| Publisher Full Text\n\nReinehr R, Görg B, Becker S, et al.: Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices. Glia. 2007; 55(7): 758–71. PubMed Abstract \| Publisher Full Text\n\nJayakumar AR, Tong XY, Curtis KM, et al.: Increased toll-like receptor 4 in cerebral endothelial cells contributes to the astrocyte swelling and brain edema in acute hepatic encephalopathy. J Neurochem. 2014; 128(6): 890–903. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodrigo R, Cauli O, Gomez-Pinedo U, et al.: Hyperammonemia induces neuroinflammation that contributes to cognitive impairment in rats with hepatic encephalopathy. Gastroenterology. 2010; 139(2): 675–84. PubMed Abstract \| Publisher Full Text\n\nGuillemin GJ, Meininger V, Brew BJ: Implications for the kynurenine pathway and quinolinic acid in amyotrophic lateral sclerosis. Neurodegener Dis. 2005; 2(3–4): 166–76. PubMed Abstract \| Publisher Full Text\n\nMarrali G, Casale F, Salamone P, et al.: NADPH oxidase (NOX2) activity is a modifier of survival in ALS. J Neurol. 2014; 261(11): 2178–83. PubMed Abstract \| Publisher Full Text\n\nCasula M, Iyer AM, Spliet WG, et al.: Toll-like receptor signaling in amyotrophic lateral sclerosis spinal cord tissue. Neuroscience. 2011; 179: 233–43. PubMed Abstract \| Publisher Full Text\n\nEvans MC, Couch Y, Sibson N, et al.: Inflammation and neurovascular changes in amyotrophic lateral sclerosis. Mol Cell Neurosci. 2013; 53: 34–41. PubMed Abstract \| Publisher Full Text\n\nAppel SH, Beers D, Siklos L, et al.: Calcium: the Darth Vader of ALS. Amyotroph Lateral Scler Other Motor Neuron Disord. 2001; 2(Suppl 1): S47–54. PubMed Abstract \| Publisher Full Text\n\nInce P, Stout N, Shaw P, et al.: Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease. Neuropathol Appl Neurobiol. 1993; 19(4): 291–9. PubMed Abstract \| Publisher Full Text\n\nCopray JC, Liem RS, Kernell D: Calreticulin expression in spinal motoneurons of the rat. J Chem Neuroanat. 1996; 11(1): 57–65. PubMed Abstract \| Publisher Full Text\n\nBernard-Marissal N, Sunyach C, Marissal T, et al.: Calreticulin levels determine onset of early muscle denervation by fast motoneurons of ALS model mice. Neurobiol Dis. 2015; 73: 130–6. PubMed Abstract \| Publisher Full Text\n\nBernard-Marissal N, Moumen A, Sunyach C, et al.: Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS. J Neurosci. 2012; 32(14): 4901–12. PubMed Abstract \| Publisher Full Text\n\nMontague K, Malik B, Gray AL, et al.: Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy. Brain. 2014; 137(Pt 7): 1894–906. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrake ME Jr: The association of motor neuron disease and Alzheimer-type dementia. Am J Med Sci. 1984; 287(3): 26–7. PubMed Abstract \| Publisher Full Text\n\nCrugnola V, Lamperti C, Lucchini V, et al.: Mitochondrial respiratory chain dysfunction in muscle from patients with amyotrophic lateral sclerosis. Arch Neurol. 2010; 67(7): 849–54. PubMed Abstract \| Publisher Full Text\n\nMenzies FM, Ince PG, Shaw PJ: Mitochondrial involvement in amyotrophic lateral sclerosis. Neurochem Int. 2002; 40(6): 543–51. PubMed Abstract \| Publisher Full Text\n\nCarr PA, Yamamoto T, Karmy G, et al.: Analysis of parvalbumin and calbindin D28k-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content. Neuroscience. 1989; 33(2): 363–71. PubMed Abstract \| Publisher Full Text\n\nLinnebank M, Lutz H, Jarre E, et al.: Binding of copper is a mechanism of homocysteine toxicity leading to COX deficiency and apoptosis in primary neurons, PC12 and SHSY-5Y cells. Neurobiol Dis. 2006; 23(3): 725–30. PubMed Abstract \| Publisher Full Text\n\nPatel SP, Gamboa JL, McMullen CA, et al.: Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function. Invest Ophthalmol Vis Sci. 2009; 50(1): 180–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrade FH, McMullen CA, Rumbaut RE: Mitochondria are fast Ca2+ sinks in rat extraocular muscles: a novel regulatory influence on contractile function and metabolism. Invest Ophthalmol Vis Sci. 2005; 46(12): 4541–7. PubMed Abstract \| Publisher Full Text\n\nBeauchemin AM, Gottlieb B, Beitel LK, et al.: Cytochrome c oxidase subunit Vb interacts with human androgen receptor: a potential mechanism for neurotoxicity in spinobulbar muscular atrophy. Brain Res Bull. 2001; 56(3–4): 285–97. PubMed Abstract \| Publisher Full Text\n\nComi GP, Bordoni A, Salani S, et al.: Cytochrome c oxidase subunit I microdeletion in a patient with motor neuron disease. Ann Neurol. 1998; 43(1): 110–6. PubMed Abstract \| Publisher Full Text\n\nRygiel KA, Grady JP, Turnbull DM: Respiratory chain deficiency in aged spinal motor neurons. Neurobiol Aging. 2014; 35(10): 2230–2238. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan PG, Rabchevsky AG, Keller JN, et al.: Intrinsic differences in brain and spinal cord mitochondria: Implication for therapeutic interventions. J Comp Neurol. 2004; 474(4): 524–34. PubMed Abstract \| Publisher Full Text\n\nTurner C, Parekh B, Walton C, et al.: An exploratory comparative study of volatile compounds in exhaled breath and emitted by skin using selected ion flow tube mass spectrometry. Rapid Commun Mass Spectrom. 2008; 22(4): 526–32. PubMed Abstract \| Publisher Full Text\n\nWright G, Noiret L, Olde Damink SW, et al.: Interorgan ammonia metabolism in liver failure: the basis of current and future therapies. Liver Int. 2011; 31(2): 163–75. PubMed Abstract \| Publisher Full Text\n\nDiaz-Herrero MM, del Campo JA, Carbonero-Aguilar P, et al.: THDP17 decreases ammonia production through glutaminase inhibition. A new drug for hepatic encephalopathy therapy. PLoS One. 2014; 9(10): e109787. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKristiansen RG, Rose CF, Fuskevåg OM, et al.: L-Ornithine phenylacetate reduces ammonia in pigs with acute liver failure through phenylacetylglycine formation: a novel ammonia-lowering pathway. Am J Physiol Gastrointest Liver Physiol. 2014; 307(10): G1024–31. PubMed Abstract \| Publisher Full Text\n\nIkarashi N, Fukazawa Y, Toda T, et al.: Effect of Conclevan on endurance capacity in mice. Biol Pharm Bull. 2012; 35(2): 231–8. PubMed Abstract \| Publisher Full Text\n\nMatthys D, Calders P, Pannier JL: Inhaled salbutamol decreases blood ammonia levels during exercise in normal subjects. Eur J Appl Physiol Occup Physiol. 1998; 79(1): 110–3. PubMed Abstract \| Publisher Full Text\n\nGruzman A, Shamni O, Ben Yakir M, et al.: Novel D-xylose derivatives stimulate muscle glucose uptake by activating AMP-activated protein kinase alpha. J Med Chem. 2008; 51(24): 8096–108. PubMed Abstract \| Publisher Full Text\n\nCoelho WS, Costa KC, Sola-Penna M: Serotonin stimulates mouse skeletal muscle 6-phosphofructo-1-kinase through tyrosine-phosphorylation of the enzyme altering its intracellular localization. Mol Genet Metab. 2007; 92(4): 364–70. PubMed Abstract \| Publisher Full Text\n\nRangroo Thrane V, Thrane AS, Wang F, et al.: Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nat Med. 2013; 19(12): 1643–1648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsher E, Fattal-Valevski A, Sagie L, et al.: Pyrimethamine increases beta-hexosaminidase A activity in patients with Late Onset Tay Sachs. Mol Genet Metab. 2011; 102(3): 356–63. PubMed Abstract \| Publisher Full Text\n\nKiernan MC, Vucic S, Cheah BC, et al.: Amyotrophic lateral sclerosis. The Lancet. 2011; 377(9769): 942–955. PubMed Abstract \| Publisher Full Text" }	[ { "id": "15775", "date": "01 Sep 2016", "name": "Viswanathan Krishnan", "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\nThis Opinion Article introduces an interesting hypothesis connecting ammonia with ALS. The article initially begins with a discussion about the variety of etiologies of ALS and how their disparate onset and pathology is an unexplained area in the field. His main argument is the liver may be a locus for the variety of etiologies and specifically, hepatic steatosis is a unique link to motor neuron diseases, including ALS.\n\nThis Opinion Article is replete with references supporting each of the statements from skeletal muscle to the various metabolic pathways (e.g. glycolysis and glycogen metabolism), to the deficits in vital organelles (e.g. lysosomes and Golgi). The latter part of the review introduces the role of calcium binding proteins and how ammonia dyshomeostasis contributes to neurodegeneration.\n\nThe only shortcoming is the discussion on the mechanism of ammonia which mainly focuses on the in vitro application and exposure to ammonia; no chemical mechanisms following ammonia’s path of chemical reaction using labeled material is cited. While it is not the role of the Opinion Article to present these types of experiments. The lack of these cited references does weaken the hypothesis of ammonia’s connection to ALS. However, this article provides an interesting area of reading that may open new avenues of experimentation for researchers that are focused on understanding the connection between ammonia and not only ALS, but other diseases such as HD and PD.", "responses": [ { "c_id": "3456", "date": "26 Feb 2018", "name": "Bhavin Parekh", "role": "Author Response", "response": "The hypothesis that ammonia plays a critical role in ALS is further bolstered by this recently study: http://www.manchester.ac.uk/discover/news/major-cause-of-dementia-discovered/." } ] }, { "id": "16323", "date": "07 Oct 2016", "name": "Smita Saxena", "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\nThis opinion article by Bhavin Parekh puts forward an interesting hypothesis about how ammonia neurotoxicity might influence and propagate ALS pathology. The article provides a concise introduction to the etiology of ALS with primary focus on the involvement of the skeletal muscle and liver in ALS pathogenesis. The author postulates a novel premise centering on the involvement of the liver and the distinctive occurrence of hepatic steatosis in motor neuron diseases including ALS. In depth literature is discussed for the existence of hepatic steatosis and hyperhomocysteinemia in ALS patients and other motor neuron diseases.\n\nThe opinion article then focuses on ammonia and imbalances in interorgan ammonia metabolism and further discusses how this specific ammonia imbalance induces functional deficits in organelles (Golgi, ER, lysosomes), thereby impairing critical ALS associated pathways such as macroautophagy, oxidative/nitrosative stress, neuroinflammation, and hyperexcitability of motor neurons. Lastly, the authors search for a conceptual framework to account for clinical heterogeneity observed with respect to upper and lower motor neurons in ALS and they suggest that calcium binding proteins (CaBPs) are key molecules involved in neutralizing ammonia toxicity. The author further discusses the role of calcium binding proteins such as Calreticulin in ER stress and ALS pathogenesis and proposes that ammonia neurotoxicity and the parallel loss of expression of CaBPs leads to ALS.\n\nOverall the review is informative, well written and postulates an interesting hypothesis which might be of general interest to the neurodegeneration field. One of the limitations of this opinion article is that the discussion about CaBPs is restricted to Calreticulin in motor neurons, while recent studies have implicated various other ER chaperones in the ALS pathogenesis. The lack of these recent citations in the discussion concerning the involvement of ER chaperones in ALS does weaken the hypothesis. Nevertheless, this article provides an interesting area of reading and brings forward new experimental ideas for the further understanding of ALS and other neurodegenerative disorders.", "responses": [] }, { "id": "18396", "date": "12 Dec 2016", "name": "Scott T Brady", "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\nALS is a devastating disease of uncertain etiology and no effective therapies are available. As a result, there is a temptation to speculate about underlying causes in the hopes of hitting on the right answer. In this case, the author without any record of studying either ALS pathology or ammonia metabolism has proposed a novel integrative explanation in which impaired glycolytic metabolism in fast twitch skeletal muscle and liver pathology leads to chronically elevated ammonia levels that leads to altered Ca2+ binding protein homeostasis due to ER stress and impaired mitochondrial respiration. Based on this idea, the author proposes that ammonia removal therapies would be effective treatments for ALS.\nThe first requirement of any disease model is that it be consistent with known facts about the disease. Unfortunately, this model fails immediately on two counts. First, there is no evidence that interorgan ammonia levels are chronically elevated in ALS. People have been looking for plasma biomarkers for early diagnosis and none have reported elevated ammonia as a candidate in either ALS patients or animal models. The author does not cite any relevant patient studies and the one animal model cited (Bame, et al. 2014) does not find a significant correlation between ammonia levels and SOD1 G93A pathology. Similarly, liver pathology is not a hallmark of the disease. While skeletal muscle wasting is seen, this is associated with lack of activity, rather than a primary defect in glycolysis.\n\nSecond, ammonia toxicity is a well-documented condition in the brain and other tissues. While there are neurotoxic effects that include cortical atrophy, demyelination and edema (see for example Braissant, et al. 2013), none of these changes are specific to motor neurons (upper or lower) nor is there any evidence of ALS-like pathology in either patients with hyperammonemia or animal models with chronically elevated ammonia. Thus, patients with ALS show no evidence of elevated ammonia levels and patients with hyperammonemia do not have ALS-like symptoms. Curiously, both the Bame and the Braissant references are listed in the bibliography, but the conclusions are misrepresented as being consistent with the thesis.\n\nAlthough the manuscript uses many buzzwords currently popular in the neurodegeneration field (autophagy, ER-stress, Ca2+ homeostasis, neuroinflammation, etc.), the logic that relates changes in these parameters to ammonia metabolism is never made clear. The diagrams are convoluted and have little explanatory power. In particular, Figure 2 manages to be so densely packed with symbols and labels that it is uninterpretable. In contrast, the source figure from Kiernan, et al. 2011 that was adapted is sparse and focused.\nIn sum, while the author has gathered a substantial bibliography, the main hypothesis is falsified by the literature and the utility of this opinion piece is therefore minimal. I would not consider this suitable for indexing in a rigorous journal.", "responses": [] } ]	1	https://f1000research.com/articles/4-119
https://f1000research.com/articles/4-117/v1	13 May 15	{ "type": "Research Article", "title": "A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.", "authors": [ "Reinier Gesto-Borroto", "Miriam Sánchez-Sánchez", "Raúl Arredondo-Peter", "Reinier Gesto-Borroto", "Miriam Sánchez-Sánchez" ], "abstract": "Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58.", "keywords": [ "Burkholderia", "Cupriavidus", "flavohemoglobin", "globin-coupled sensor", "Rhizobium", "single-domain globin", "truncated (2/2) hemoglobin" ], "content": "Introduction\n\nGlobins (Glbs) are proteins widely distributed in organisms from the three kingdoms of life, i.e. in Archaea, Eubacteria and Eukarya1. Structurally, Glbs fold into a tertiary structure known as the globin fold. This protein folding consists of six to eight α-helices (designated with letters A to H) that form a hydrophobic pocket where a heme prosthetic group is located2. Two structural types of the globin fold have been identified in Glbs: the 2/2- and 3/3-fold. In the 2/2-Glbs, helices B and E overlap to helices G and H3 and in the 3/3-Glbs helices A, E and F overlap to helices B, G and H4,5. Likewise, three evolutionary families have been identified in Glbs6,7: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs), the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs) (which are further classified into class 1, class 2 and class 3 tHbs). Canonical tHbs are ~20 to 40 amino acids shorter than the globin fold, resulting in an almost absent helix A and a helix F that is reduced to a single turn8,9. The M and S Glbs fold into the 3/3-fold whereas the T Glbs fold into the 2/2-fold.\n\nA variety of gaseous ligands bind to the heme Fe of Glbs, most notably O2 and nitric oxide (NO). The reversible binding of O2 is associated with the major function of Glbs in organisms: the transport of O2. Binding of NO by oxygenated Glbs is essential to NO-detoxification via NO-dioxygenase activity10,11. Several additional functions have been reported for Glbs, including dehaloperoxidase activity and reaction with free radicals, binding and transport of sulfide and lipids, and O2-sensing (reviewed by Giardina et al.12 and Vinogradov et al.13). This indicates that in vivo, Glbs might be multifunctional proteins.\n\nGlbs are widespread in bacteria. A comprehensive genomic analysis revealed that glb genes belonging to the M, S and T Glb families exist in the genomes of 1185 Eubacteria, including several rhizobial genomes7. However, only few rhizobial glb genes have been characterized. Characterizing rhizobial Glbs is of interest because rhizobia establish symbiotic relationships with leguminous plants. A result of this plant-microbe interaction is the symbiotic fixation of atmospheric N2, which occurs within specialized plant organs called nodules14. Symbiotic N2-fixation is a process modulated by a variety of factors, such as the O215 and NO16,17 levels in the surrounding environment. Glbs bind O2 and NO and thus may function in some aspects of the N2-fixation, e.g. by transporting O2 and detoxifying NO. Modulation of O2 levels in the plant cell cytoplasm from nodules is well characterized18,19. A plant Glb (leghemoglobin (Lb)) that is synthesized at high (~3 to 5 mM) concentrations in nodules apparently facilitates O2-diffusion to the symbiotic rhizobia and maintains low (submicromolar) concentrations of O2 within nodules. This is essential for sustaining the (micro) aerobic respiration of symbiotic rhizobia and preventing the inactivation of nitrogenase (which fixes the atmospheric N2 into NH4+) by O2. The binding and metabolizing of NO by Lb and other Glbs is also well documented11,20. Thus, a likely function for Lb in nodules is to detoxify the NO that is generated during the plant infection by rhizobia21. However, little is known about the properties and functions of Glbs either within the symbiotic or free-living rhizobia.\n\nForty-six years ago Appleby22 was the first to propose the existence of Glbs in rhizobia. This author detected absorption peaks and troughs that are characteristic of Glbs in differential (dithionite reduced + CO minus dithionite reduced) spectra of soluble extracts from Bradyrhizobium japonicum 505 (Wisconsin). Subsequent spectroscopic analyses suggested the existence of soluble Glbs in Rhizobium leguminosarum bv. viciae23, B. japonicum NPK6324 and R. etli CE325. The first rhizobial glb gene was identified in the pSymA megaplasmid of Sinorhizobium meliloti 102126. BLAST analysis revealed that this gene corresponded to an fhb gene and thus was named smfhb. A bioinformatics analysis showed that smfhb is flanked by nos and fix genes (which code for denitrification enzymes and high O2-affinity terminal oxidases and an O2-sensor, respectively) and that apparently it is regulated by an Fnr-like promoter. These observations suggested that smfhb is regulated by the concentration of O2 and that SmfHb functions in some aspects of nitrogen metabolism. A transcriptomic analysis of the S. meliloti response to NO in culture showed that smfhb (also designated as a S. meliloti hmp) is upregulated by NO and the analysis of a smfhb- mutant exhibited a high sensitivity to NO in culture and led to a reduced N2-fixation efficiency in planta. These observations suggested that SmfHb functions in some aspects of NO metabolism, possibly by detoxifying NO27.\n\nGenomic analysis reported by Vinogradov et al.7 revealed that Glb sequences exist in several rhizobia. However, in spite of the above reports knowledge on the rhizobial Glbs is quite limited. Hence, in order to obtain information on the properties of rhizobial Glbs we characterized Glb sequences from selected rhizobial genomes by using bioinformatics methods. These included gene characterization, polypeptide sequence and phenetic analysis, as well as protein modeling. Also, we analyzed soluble extracts from B. japonicum USDA38 and USDA58 by differential spectroscopy. Our main results showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work; that several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr-like promoters; that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively; that rhizobial Glbs segregate into the M, S and T Glb families; that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-fold, respectively, and that spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibit peaks and troughs characteristic of bacterial and vertebrate Glbs.\n\n\nMethods\n\nPutative Glb sequences and Glb domains were identified in databases (Table S1) containing the genomes of rhizobial species and strains using the query sequences S. meliloti fHb; Vitreoscilla SDgb; Agrobacterium tumefaciens GCS; Methanosarcina acetivorans protoglobin; Methylacidiphilum infernorum sensor single domain globin; Mycobacterium tuberculosis tHb class 1; A. tumefaciens tHb class 2, and M. avium tHb class 3 (Genbank accession numbers AY328026, AAA75506, NP_354049, 2VEB_A, YP_001939425, NP_216058, WP_020813663 and BAN32501, respectively) and the SUPERFAMILY database (http://supfam.mrc-lmb.cam.ac.uk)28. Resulting sequences were subjected to a FUGUE analysis (http://tardis.nibio.go.jp/fugue/prfsearch.html)29 to determine the most similar Glb structure and presence of proximal H at the myoglobin-fold position F8. Putative Glbs had to satisfy the following criteria: length higher than or ~100 amino acids, a FUGUE Z score higher than 6 (which corresponds to 99% specificity29) with known Glb structures, and the presence of proximal H at position F8.\n\nScaffolds containing copies of the glb gene were used for mapping glbs. This included the detection of open reading frames (ORFs) ~5 kb up- and downstream to glbs and ORF length, transcription direction and localization in the +/- strand. Canonical (-10 and -35) and Fnr30 promoter sequences and Shine-Dalgarno sequences were searched within 130 nucleotides upstream to the rhizobial glb genes either by using the search tool of MS Word® or by pairwise sequence alignments using the ClustalX program (http://www.clustal.org/clustal2/)31.\n\nPairwise and multiple sequence alignments were performed using the ClustalX program31. Multiple sequence alignment was manually verified using the procedure described by Kapp et al.32 based on the myoglobin-fold33. A phenogram was constructed from the aligned sequences using the UPGMA method from the ClustalX program. The resulting phenogram was edited using the iTOL program (http://itol.embl.de/)34.\n\nThe tertiary structure of rhizobial Glbs was modeled using the automated mode of the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/)35–37, which also provided the best structural homologs to the query sequences. Models were edited using the VMD program (http://www.ks.uiuc.edu/Research/vmd/)38 and Adobe Photoshop® software. Distance and dihedral angles of amino acids at the heme prosthetic group were calculated using the distance and dihedral tools of the SwissPDBViewer program (http://spdbv.vital-it.ch/) as described by Gopalasubramaniam et al.39 and Sáenz-Rivera et al.40, respectively.\n\nBradyrhizobium japonicum USDA38 and USDA58 were kindly provided by Drs. Donald Keister and Douglas Jones (United States Department of Agriculture, USA). All reagents were purchased from Sigma-Aldrich (St. Louis MO, USA). B. japonicum cells were grown in YM (Yeast Mannitol) broth (per 100 ml: KH2PO4, 50 mg; MgSO4, 20 mg; NaCl, 10 mg; mannitol, 1 g; yeast extract, 50 mg, pH 7.0) for 3 to 5 days at 30°C with shaking at 200 rpm. Cells were harvested by centrifugation at 11,000 × g, pellets were resuspended in 50 mM Na-phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by sonication at maximum power (three cycles of 1 min each in ice) and incubation at 4°C overnight with gentle agitation after the addition of DNAse I (40 U/ml), RNAse A (3 U/ml) and lysozyme (2 mg/ml). The resulting solution was cleared by centrifugation at 22,000 × g for 40 min at 4°C, and the supernatant was fractionated with solid ammonium sulphate between 35 and 65% saturation. The resulting pellet was resuspended in 5 ml of 50 mM Na-phosphate buffer (pH 7.2) containing 1 mM EDTA and 1 mM PMSF and dialyzed for 18 h against the same buffer to remove the excess of salts. 0.5 to 1 ml aliquots of the dialyzed solution were used to obtain the dithionite reduced + CO minus dithionite reduced differential spectra in a Beckman DU6 spectrophotometer. Control spectra were obtained from commercial (Sigma-Aldrich) preparations of the sperm whale myoglobin and bovine blood hemoglobin.\n\n\nResults and discussion\n\nRecently, Vinogradov et al.7 reported that Glb sequences exist in the genomes of 96 rhizobia. However, this report did not provide the rhizobial Glb sequences or links to rhizobial scaffolds containing the Glb sequences. Hence, we searched in databases (see the Methods section and Table S1) in order to obtain rhizobial Glb sequences for analysis. We selected 62 out of the 96 rhizobial genomes reported by the above authors representing the major rhizobial genera, species and strains, which included α- and β-rhizobia (i.e. those classified within the α- and β-proteobacteria, respectively). A total of 197 glb sequences were detected in the 62 rhizobial genomes, corresponding to 7 fhbs, 47 sdgbs, 40 gcss and 103 thbs (4 thbs class 1, 56 thbs class 2 and 43 thbs class 3). Individual Glb nucleotide and polypeptide sequences and links to rhizobial scaffolds containing the Glb sequences are provided in Dataset 1 and Dataset 2, respectively. All the rhizobial genomes analyzed in this work contained glb sequences, thus indicating that glbs are widespread in rhizobia. However, protoglobin and sensor single domain globin sequences were not detected in the rhizobial genomes. This observation indicates that apparently only the fhb, sdgb, gcs and thb lineages evolved within rhizobia.\n\nA distribution analysis showed that most (61) of the rhizobial genomes analyzed in this work contain thbs, either as single thbs (13) or in combination with fhbs, sdgbs and/or gcss (48). Furthermore, one rhizobial genome contained only a gcs and none contained only fhbs and sdgbs and the combinations fhbs + sdgbs, fhbs + gcss and sdgbs + gcss (Figure 1). These observations indicate that in the rhizobia analyzed in this work thbs predominate over other glbs and that in these bacteria fhbs, sdgbs and gcss mostly exist in combination with thbs. Also, analysis of the glb copy number showed that in the rhizobia analyzed in this work fhbs mostly exist as single copy (ranging from one to two copies), sdgbs mostly exist as two copies (ranging from one to four copies), gcss exist as either single or two copies (ranging from one to two copies) and thbs mostly exist as two copies (ranging from one to three copies) although quite a few thbs exist as single copy (Table 1). Thus, apparently rhizobial glbs mostly exist as either single or two copies.\n\nNumbers correspond to rhizobial genomes containing glbs.\n\nThe glb genes detected in this work were mapped within the rhizobial genomes in order to identify genes that flank nearby to and could coexpress with glbs. Mapping analysis showed that rhizobial glb copies are located in different scaffolds and that they are not tandemly arrayed. Figure S1A shows that either no ORFs or ORFs coding for hypothetical or non-identified proteins are located nearby most of the rhizobial fhb genes. However, genes coding for the transcriptional regulator NsrR, 2-nitropropane dioxygenase and NosR, Z, D, F, Y and X are located nearby cupnecN1fhb1, rhilegUPM1137fhb and sinmel1021fhb, respectively. Figure S1B shows that B. elkanii and B. japonicum sdgbs are mostly flanked by genes coding for proteins that function in nitrate/nitrite metabolism and sugar transport. Figure S1C shows that genes coding for proteins that function in chemotaxis are located nearby several rhizobial gcss, although genes coding for a peptide deformylase, sugar and nitrate transport proteins and NAD(P)H nitrate reductase are located nearby some other rhizobial gcss. Figure S1D shows that genes flanking the rhizobial thbs are rather variable. However, B. japonicum thbs are often flanked by genes coding for the transcriptional regulator Rieske Fe-S, shikimate kinase and alcohol dehydrogenase; mesorhizobia thbs are often flanked by genes coding for permeases and tRNA-Trp, and R. leguminosarum thbs are often flanked by genes coding for membrane proteins. Thus, if glb and flanking genes coexpress in rhizobia, and proteins coded by these genes function within the same metabolic pathways, the above observations suggest that rhizobial Glbs could play a variety of roles in rhizobial physiology, including nitrate/nitrite metabolism, transport processes, gene regulation and chemotaxis. Interestingly, with the exception of sinmel1021fhb which is flanked by nos and fix genes (Figure S1A)26, nif and fix genes coding for proteins that function in N2-fixation were not detected nearby the rhizobial glb genes. This observation suggests that rhizobial Glbs might not directly function in N2-fixation.\n\nIdentification of promoter sequences is crucial to an understanding of gene regulation and ultimately protein function within the cell's physiology. Hence, we searched for canonical (-10 and -35) promoters and the O2- and NO-regulated Fnr promoter30,41,42 within 130 nucleotides upstream to 44 selected rhizobial glb genes (i.e. those representative of major rhizobial Glb clades identified in this work (see Figure 2)). Also, we searched for Shine-Dalgarno sequences within the same region, which indicate that Glb transcripts could be translated into proteins. Results showed that, with the exception of burphySTM815thb1, burphySTM815thb2 and rhilupHPC(L)thb1, a -10 promoter is absent upstream of the selected rhizobial glbs. In contrast, with the exception of cupnecN1thb1 and rhilupHPC(L)thb2, a -35 promoter exists upstream of the selected rhizobial glbs. Searching for Fnr promoter sequences revealed that Fnr-like promoters exist upstream to 30 out of the 44 selected rhizobial glbs, including fhb, sdgb, gcs and thb genes. A Shine-Dalgarno sequence was detected upstream to most of the selected rhizobial glbs (Table 2). These observations suggest that the -35 promoter is a major canonical promoter that regulates most of the rhizobial glbs, that it is likely that several rhizobial glbs are regulated by levels of O2 and NO throughout an FNR mechanism41–44 and that rhizobial Glb transcripts are translated into proteins.\n\nConsensus sequences are indicated in parenthesis. Identical and non-identical nucleotides into the Fnr-like promoter sequences to the consensus Fnr promoter sequence are indicated with upper- and lowercase letters, respectively. N.D., non-detected.\n\nPairwise sequence alignments showed that the rhizobial fHbs, SDgbs, GCSs and tHbs analyzed in this work are 34.6 to 85.4%, 6.7 to 100%, 10.9 to 100% and 3.5 to 100% identical, respectively. This indicates that variability among the rhizobial Glb sequences is high. Moreover, identity values for the fHbs globin and flavin domains were 39.1 to 93.7% and 26.5 to 81.1%, respectively, and identity values for the GCSs globin and transmitter domains were 17.5 to 100% and 5.9 to 100%, respectively. Thus, apparently in the rhizobial fHbs and GCSs analyzed in this work the globin domain is more conserved than the flavin and transmitter domains.\n\nThe average length and molecular mass for the rhizobial fHbs, SDgbs, GCSs and tHbs analyzed in this work are 400 amino acids and 44 kDa, 141 amino acids and 15 kDa, 510 amino acids and 55 kDa and 149 amino acids and 17 kDa, respectively. However, sequence analysis revealed that globin domain from BraelkUSDA76tHb1, BraelkUSDA94tHb1 and Braelk587tHb2 contains 119 to 237 extra amino acids at the N-terminal and 131 extra amino acids at the C-terminal, and that the globin domain from BrajapUSDA123tHb1, BrajapUSDA135tHb1, BraelkWSM1741SDgb2, RhietlCFN42GCS1, BrajapUSDA4tHb2 and BrajapWSM2793tHb3 contains 27 to 73 extra amino acids at the N-terminal. In contrast, a large deletion comprising helices A and B, CD loop and part of helix E was detected in the BraelkUSDA94SDgb2 sequence indicating that BraelkUSDA94SDgb2 is 89 amino acids in length (Figure S2).\n\nMultiple sequence alignment showed that, with the exception of 21 GCSs, in the rhizobial Glbs analyzed in this work, the proximal (F8, located at position 322/323 in Figure S2) amino acid to the heme Fe is H. Apparently, in the above rhizobial GCSs, F8 is E. Amino acids other than H occupying the F8 position in bacterial Glbs were previously reported by Vinogradov et al.7. However, because H F8 is absolutely conserved in Glbs (i.e. from bacteria to mammals)1,32,45–47, assigning E F8 to rhizobial (and other bacterial) GCSs should be taken with caution as this assignment might result from a sequence alignment artifact. Ideally, F8 from rhizobial GCSs should be identified by experimental methods, such as x-ray crystallography. Multiple sequence alignment also showed that in the rhizobial Glbs analyzed in this work, the distal (E7, located at position 285/289/290 in Figure S2) amino acid to the heme Fe is Q in fHbs, can be Q/R/K/M/L in SDgbs, Q in GCSs and can be H/F/L/V/R in tHbs. This indicates that distal Q is conserved in rhizobial fHbs and GCSs and that amino acids occupying the distal position in rhizobial SDgbs and tHbs are variable. The B10 and CD1 amino acids (located at positions 257 and 270/271/273 in Figure S2, respectively), which also participate in binding of ligands to the heme Fe48–50, are Y and F in most of the rhizobial Glbs analyzed in this work followed by (in order of abundance) F, S and V and H, I, S and Y, respectively.\n\nPhenogram was obtained from the Glbs sequence alignment shown in Figure S2. The fHb, SDgb, GCS, tHb class 1, tHb class 2 and tHb class 3 clusters are indicated with light blue, dark blue, red, light green, bright green and dark green, respectively. Stars indicate Glbs selected for the detection of promoter sequences upstream to the glb genes and Glb protein modeling.\n\nA phenogram was constructed from the above multiple sequence alignment. Figure 2 shows that the rhizobial Glbs analyzed in this work segregate into two main lineages: one containing fHbs, SDgbs and GCSs, and the other containing tHbs (the fHb/SDgb/GCS and tHb lineages, respectively). This is consistent with the main evolutionary lineages identified in bacterial Glbs1,51,52 thus indicating that major evolutionary patterns for rhizobial Glbs were identical to those for other bacterial Glbs. Rhizobial fHbs and GCSs cluster with rhizobial SDgbs within the fHb/SDgb/GCS lineage owing to the similarity between the fHb and GCS globin domains and SDgbs. This has been postulated to be the result of an early divergence from a common ancestor to the bacterial fHb and GCS globin domains and SDgbs1,6. The tHb lineage segregates into rhizobial tHbs class 1, tHbs class 2 and tHbs class 3. Within this lineage the rhizobial tHbs class 3 segregate in ancestral position to the rhizobial tHbs class 1 and tHbs class 2. Also, the bradyrhizobial, azorhizobial, mesorhizobial, rhizobial and burkholderial tHbs class 3 segregate from each other; the segregation within rhizobial, sinorhizobial, mesorhizobial and β-rhizobial tHbs class 2 is rather conserved, and bradyrhizobial tHbs class 2 and class 3 segregate into the B. elkanii and B. japonicum tHb sublineages. These observations indicate that rhizobial tHbs evolved similarly to other bacterial tHbs7,8,52 and that evolution of rhizobial tHb sublineages was rather conserved.\n\nStructure elucidation is essential to a full understand of a protein´s function within the cell´s physiology. The structure of a considerable number of bacterial and non-bacterial Glbs has been elucidated by x-ray crystallography. However, with the exception of a S. meliloti fHb whose tertiary structure was predicted using bioinformatics methods26, the structure of rhizobial Glbs is not known. Hence, we used bioinformatics methods to predict and analyze the tertiary structure of 44 selected rhizobial Glbs (i.e. those representative of major rhizobial Glb clades identified in this work (see Figure 2 and Table S2)) using the best structural homologs as templates (Dataset 3).\n\nPredicted structures for selected rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-globin fold, respectively (Figure 3 to Figure 8). Figure 3 shows that structures among the predicted rhizobial fHbs are highly similar. Yet major differences were detected in the BurphySTM815fHb, CupnecHPC(L)fHb and RhilegUMP1137fHb flavin domains, which exhibited two additional helices. Dataset 3 shows that among globin domains from predicted rhizobial fHbs the distance of the proximal H and distal Q to the heme Fe is 1.44 to 2.47 Å and 6.71 to 15.35 Å, respectively. This observation suggests that the heme Fe in rhizobial fHbs is pentacoordinate.\n\nStructural homologues are indicated in Dataset 3. Distal and proximal amino acids to the heme Fe and amino acids that interact with the FAD cofactor are shown in brown. Heme and FAD are shown in red and yellow, respectively. Helices within the globin domain are indicated with letters A to H. All structures are displayed in the same orientation.\n\nStructural homologues are indicated in Dataset 3. Distal and proximal amino acids to the heme Fe are shown in brown. Heme is shown in red. Helices are indicated with letters A to H. All structures are displayed in the same orientation.\n\nStructural homologues are indicated in Dataset 3. Distal and proximal amino acids to the heme Fe are shown in brown. Heme is shown in red. Helices are indicated with letters A to H. All structures are displayed in the same orientation.\n\nDistal and proximal amino acids to the heme Fe are shown in brown; only potential distal E11 is shown in the CupnecN1tHb1 structure. Heme is shown in red. Helices are indicated with letters A to H.\n\nStructural homologues are indicated in Dataset 3. Distal and proximal amino acids to the heme Fe are shown in brown; only potential distal E11 is shown in the tHbs structure. Heme is shown in red. Helices are indicated with letters A to H. Pre-helix F is indicated with the Greek letter φ. All structures are displayed in the same orientation.\n\nStructural homologues are indicated in Dataset 3. Distal and proximal amino acids to the heme Fe are shown in brown; only potential distal E11 is shown in the tHbs structure. Heme is shown in red. Helices are indicated with letters A to H. All structures are displayed in the same orientation.\n\nFigure 4 shows that 3/3-globin folding is highly conserved in the predicted structure of the rhizobial SDgbs AzodoeUFLA1-100SDgb, BraelkUSDA3254SDgb2, BraelkUSDA3259SDgb1 and BrajapUSDA38SDgb2. Major variations to 3/3-globin folding from predicted rhizobial SDgbs consisted of the existence of an unusually short helix E in BraelkUSDA94SDgb2, a long helix H in BraelkUSDA3254SDgb1 and BrajapUSDA124SDgb1, and the existence of a pre-helix A followed by a long loop at the N-terminal of BraelkWSM1741SDgb2. Dataset 3 shows that among the predicted rhizobial SDgbs the distance of proximal H and distal Q/R/K/M to the heme Fe is 2.11 to 4.44 Å and 5.08 to 6.63 Å, respectively. This observation suggests that the heme Fe in rhizobial SDgbs is either penta- or hexacoordinate.\n\nOnly the globin domain from bacterial GCSs has been crystalized and analyzed by x-ray crystallography53,54 (Dataset 3). Crystal structure for the bacterial GCSs transmitter domain has not been elucidated. Hence, we only predicted and analyzed the tertiary structure of globin domains from the selected rhizobial GCSs. Figure 5 shows that the predicted rhizobial GCSs globin domain exhibits a 1.5- to 3-turn pre-helix A, that (with the exception of SinfreGR64GCS) no loop exists between helices A and B, and that helix H is unusually long in Rhietl8C3GCS, RhietlCIAT652GCS2 and RhilegGB30GCS2. Dataset 3 shows that among the predicted rhizobial GCSs globin domain distance of proximal H/E and distal Q to the heme Fe is 1.77 to 5.56 Å and 4.09 to 9.04 Å, respectively. This observation suggests that the heme Fe in the rhizobial GCSs globin domain is either penta- or hexacoordinate.\n\nFigure 6 to Figure 8 show that 2/2-globin folding is highly conserved in the predicted rhizobial tHbs class 1, class 2 and class 3. Major variations to 2/2-globin folding from predicted rhizobial tHbs consisted of the existence of a 2.5-turn pre-helix A followed by a long loop at the N-terminal of (class 1) CupnecN1tHb1 (Figure 6); the existence of a one-turn pre-helix F (designated as φ in Figure 78) in the rhizobial tHbs class 2; the existence of a long and extended C-terminal region in (class 2) BraelkUSDA94tHb1 (Figure 7), and the substitution of helix A by a long loop that connects to helix B through a 1- to 2.5-turn pre-helix B in (class 3) BraelkUSDA76tHb2, BrajapUSDA123tHb1, BurphySTM815tHb1, MeslotNZP2037tHb2 and Sinmel1021tHb2 (Figure 8). Dataset 3 shows that among the predicted rhizobial tHbs, the distance of proximal H and distal H/L/F to the heme Fe is 1.77 to 7.51 Å and 4.09 to 8.25 Å, respectively. This observation suggests that the heme Fe in the rhizobial tHbs is either penta- or hexacoordinate.\n\nThe above observations suggest that in spite of sequence variability (see the Sequence alignments and phenetic analysis of rhizobial Glbs subsection) the structure of rhizobial Glbs is similar to the canonical 3/3- or 2/2-globin folding of bacterial and non-bacterial Glbs. However, a number of predicted rhizobial Glbs exhibited variations at the N- and C-terminal regions suggesting that their structural properties could be different to those of canonical Glbs.\n\nData also shows that (with few exceptions) in addition to proximal and distal amino acids the distance of B10 and CD1 amino acids to the heme Fe and the orientation of proximal, distal, B10 and CD1 amino acids are similar within and among the predicted rhizobial SDgbs, fHbs and GCSs globin domain and tHbs. These amino acids participate in the binding of ligands to the heme Fe. Thus, these observations suggest that the mechanisms and chemistry for ligand binding are similar among the rhizobial Glbs.\n\nThe prerequisites for being able to infer a protein’s function are isolating and characterizing either native or recombinant proteins and detecting protein synthesis in vivo. No rhizobial Glb has been isolated and characterized thus far. However, spectroscopic evidence indicates that putative Glbs exist in soluble extracts from B. japonicum 505 (Wisconsin), R. leguminosarum bv. viciae, B. japonicum NPK63 and R. etli CE3 (see the Introduction section). In order to extend these analyses to other rhizobia, we analyzed soluble extracts from B. japonicum USDA38 and USDA58 by (dithionite reduced + CO minus dithionite reduced) differential spectroscopy using as controls the sperm whale myoglobin and bovine blood hemoglobin. Table 3 shows that absorption peaks and troughs in the Soret and Q regions for the B. japonicum USDA38 and USDA58, B. japonicum 505 (Wisconsin), R. leguminosarum bv. viciae, B. japonicum NPK63 and R. etli CE3 soluble extracts, Vitreoscilla VHb, E. coli K12 Hmp, sperm whale myoglobin and bovine blood hemoglobin are nearly identical. This preliminary evidence indicates that putative soluble Glbs are synthesized in B. japonicum USDA38 and USDA58. Interestingly, genes coding for SDgbs (brajapUSDA38SDgb1 and brajapUSDA38SDgb2) and tHbs (brajapUSDA38tHb1 and brajapUSDA38tHb2) were identified in the B. japonicum USDA38 genome (Dataset 1). Thus, it is likely that putative B. japonicum USDA38 Glbs corresponds to a combination of SDgbs and tHbs. Inferences from the preliminary results reported here should be confirmed by Glb detection, isolation and unequivocal identification after protein sequencing. This may open the possibility to carry out further experimental analyses on rhizobial Glbs.\n\nn.i., non-identified\n\n\nConclusions\n\nRhizobial Glbs have been poorly studied. However, results reported in this work provide molecular and biochemical data from a bioinformatics perspective that contribute to a better understanding of these proteins. For example, the distribution and outline for the evolution of glb genes and Glb proteins among rhizobia was clarified, genes that could coexpress with the rhizobial glbs were identified and the predicted tertiary structure for rhizobial Glbs was elucidated. Also, spectroscopic analysis suggested that soluble Glbs are synthesized in free-living B. japonicum USDA38 and USDA58. This information will be useful in designing future experimental work focused on clarifying Glb functions within the physiology of free-living and symbiotic rhizobia.\n\n\nData availability\n\nF1000Research: Dataset 1. Globin genes detected in the genomes of rhizobial bacteria. 10.5256/f1000research.6392.d46189\n\nF1000Research: Dataset 2. Predicted Glb polypeptides detected in the genomes of rhizobial bacteria. 10.5256/f1000research.6392.d46190\n\nF1000Research: Dataset 3. Distance to the heme Fe and orientation of distal, proximal, B10 and CD1 amino acids in the predicted structure of selected rhizobial Glbs (Table S2). 10.5256/f1000research.6392.d46191", "appendix": "Author contributions\n\n\n\nRGB, MSS and RAP conceived the study. RGB and MSS executed the experiments. RAP prepared the first draft of the manuscript. RGB, MSS and RAP revised the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was partially financed by SEP-PROMEP (grant number UAEMor-PTC-01-01/PTC23) and Consejo Nacional de Ciencia y Tecnología (CoNaCyT grant numbers 25229N and 42873Q), México. R. Gesto-Borroto is a graduate student financially supported by CoNaCyT (registration no. 293307).\n\nI confirm that the 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 Drs. Donald Keister and Douglas Jones (United States Department of Agriculture, USA) for kindly providing the Bradyrhizobium japonicum USDA38 and USDA58 strains, and Dr. Serge N. Vinogradov (Wayne State University, Detroit MI, USA) for critical reading of this article and providing constructive comments.\n\n\nSupplementary materials\n\nSupplementary File S1. Mapping of the fhb (A), sdgb (B), gcs (C) and thb (D) genes in the genomes of rhizobial bacteria.\n\nDNA fragments correspond to ~5 kb up- and downstream to glb genes. Arrows indicate the transcription orientation. The glb genes are shown in red, predicted polypeptides functioning in nitrogen metabolism are shown in blue and predicted polypeptides functioning in chemotaxis are shown in green. The ORF sizes and distances between ORFs are shown at an approximate scale. Abbreviations for the predicted polypeptides are indicated at the end of the figure. Location of DNA fragments in the rhizobial genome: Chr, chromosome; Pmd, plasmid; n.i., non-identified.\n\nSupplementary File S2. Sequence alignment of Glbs detected in the genomes of rhizobial bacteria.\n\nThe tHb, fHb, SDgb and GCS sequences are shown in green, light blue, dark blue and red, respectively. Distal and proximal amino acids located in helices E and F, respectively, are indicated within black boxes; potential distal E7 and E11 are indicated in the tHb sequences. Amino acids that interact with FAD and NAD(P)+ cofactors in the fHb flavin domain are indicated within gray boxes. Limits for the globin domain are indicated with right- and left-oriented arrows within black circles. Helices are indicated with letters A to H within the 2/2- and 3/3-fold of the tHb and SDgb and fHb and GCS globin domains, respectively. Outgroups for fHbs correspond to BacsubfHb, EsccolfHb and SaccerfHb (Genbank accession number YP_003865693, NP_289108 and NP_011750, respectively); outgroup for SDgbs corresponds to VitSDgb (Genbank accession number AAA75506); outgroups for GCSs correspond to AgrtumGCS and BacsubGCS (Genbank accession number NP_354049 and NP_388919, respectively); outgroups for tHbs correspond to MyctubtHb class1, MyctubtHb class 2, AgrtumtHb class 2 and MycavitHb class 3 (Genbank accession number NP_216058, NP_216986, WP_020813663 and BAN32501, respectively).\n\n*Formerly classified as Alcaligenes eutrophus and Ralstonia eutropha.\n\n\nReferences\n\nVinogradov SN, Hoogewijs D, Bailly X, et al.: A phylogenomic profile of globins. BMC Evol Biol. 2006; 6: 31–47. 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Mol Gen Genet. 1991; 226(1–2): 49–58. PubMed Abstract \| Publisher Full Text\n\nGesto-Borroto R, Sánchez-Sánchez M, Arredondo-Peter R: Dataset 1 in: A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling. F1000Research. 2015. Data Source\n\nGesto-Borroto R, Sánchez-Sánchez M, Arredondo-Peter R: Dataset 2 in: A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling. F1000Research. 2015. Data Source\n\nGesto-Borroto R, Sánchez-Sánchez M, Arredondo-Peter R: Dataset 3 in: A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling. F1000Research. 2015. Data Source" }	[ { "id": "8649", "date": "26 May 2015", "name": "Manuel Becana", "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\nThis is a well-written paper on a subject of great interest. There is very little information of rhizobial globins and the authors have done a good job by systematically analyzing the composition of globin genes of 62 genomes in various genera, species and biovars of rhizobia. The authors are experts in the phylogeny and evolution of plant hemoglobins, and I have no major comments to improve this work. It will nevertheless be of interest for future work to address the issue of why several types of hemoglobins coexist in rhizobia. For example, both truncated hemoglobins and flavohemoglobins seem to be present within the same species and strain, although this would have to be verified by identifying the proteins themselves rather than by only gene sequencing or by analyzing differential spectra (reduced + CO vs reduced) in bacterial extracts. Both classes of hemoglobins have been proposed to act as modulators of NO concentration, but they are unlikely to have redundant functions. An interesting, additional aspect of the work is the mapping analysis, including the report of flanking sequences. This hints to a role of at least some rhizobial globins in nitrogen metabolism. This observation is very timing because of the recent discovery that truncated hemoglobins of Chlamydomonas regulate nitrate reductase.", "responses": [ { "c_id": "1388", "date": "26 May 2015", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Becana for evaluating this article and constructive comments." } ] }, { "id": "9023", "date": "08 Jul 2015", "name": "Paul Twigg", "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 find this paper by Gesto-Borroto et al. to be well written and analyzed. This paper fills a gap in the knowledge base for what is known about bacterial or more specifically rhizobial globins. The authors seem to have taken great care to analyze all available globins from various rhizobial species and biovars. I have no major revisions for this work. It is comprehensive and demonstrates some interesting points about globins in rhizobia. The coexistence of various globins in the bacteria begs questions about their control and functions that undoubtedly other researchers will address. The work with the promoter analysis was particularly interesting to me indicating more than once that the globin expression is likely tied to nitrate/nitrogen metabolism. The absence of the -10 promoter area was also unexpected. The amino acid sequence alignment data also shows interesting information about the conserved positions in the sequence. The phenetic relationships are also interesting and appropriately analyzed. The structural modeling is also well done and reveals interesting points about the structure and function of the various globins. Lastly, the authors back up some of their proposals with spectroscopic data from globing extracts. Again, overall I thought that the work was well done and needs no major revisions.", "responses": [ { "c_id": "1456", "date": "08 Jul 2015", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Twigg for evaluating this article and constructive comments." } ] } ]	1	https://f1000research.com/articles/4-117
https://f1000research.com/articles/4-116/v1	13 May 15	{ "type": "Research Article", "title": "Effect of rice variety and blending proportion on the proximate compositions, minerals and phytic acid contents of bread from rice-teff blend", "authors": [ "Sintayehu Legesse", "Solomon Worku", "Geremew Bultosa", "Solomon Worku", "Geremew Bultosa" ], "abstract": "Development of bakery products containing rice (Oryza sativa, Linn.) and teff (Eragrostis tef) could have potential health benefits due to their gluten free nature. Nine experimental runs were generated using custom design by JMP 8 software. The effect of two factors, rice variety (Edeget, X-jigna and Nerica-4) and blending proportions of rice and teff (0.5:0.5, 0.7:0.3 and 0.9:0.1) were studied. The data analysis was conducted using SAS software package for the mean comparison and custom design by JMP 8 software. Response surface methodology was applied to study the interaction effect of the main factors and to generate the predictive equations. An optimal value (1.60%) of fiber was obtained when the proportion of the blend was 50% Edeget and 50% teff because teff grain is high in fiber. A maximum value (10.75%) of protein was obtained when the proportion of the blend was 70% Nerica-4 and 30% teff. Carbohydrate was optimal (81.37%) when 90% Edeget and 10% teff were blended because rice grain is high in carbohydrate. Optimal iron content (12.97 mg/100g) was obtained when the proportion of the blend was 50% Nerica-4 and 50% teff because teff grain is high in iron. Optimal zinc content (4.14 mg/100g) was obtained when the proportion of the blend was 50% X-jigna and 50% teff. The optimal value (61.25 mg/100g) of calcium was obtained when the proportion of the blend was 50% Edeget and 50% teff. Optimum (lower) value (0.31mg/g) of phytic acid was obtained when the proportion of the blend was 90% Nerica-4 and 10% teff because rice grain is lower in phytic acid content. It was concluded that rice variety and rice-teff blending proportion had a significant effect on the physico-chemical properties of rice-teff blend bread. An optimal nutrient blend (high in nutrients, low in anti-nutrients) was obtained when 70% Edeget rice variety was blended with 30% teff. All the derived mathematical models for the various responses were found to fit significantly to the predicted data.", "keywords": [ "Blending proportions", "Gluten-free bread", "Response surface methodology" ], "content": "Introduction\n\nRice (Oryza sativa, Linn.) is the most important cereal in terms of the numbers of people it nourishes. Traditionally, it has been the staple food and main source of income for hundreds of millions of people throughout the world. It holds the 2nd place next to wheat in its importance as a food cereal in the human diet, and produces more food energy than other cereal grains (Aklilu et al., 2002). Rice can play a very important role in human nutrition especially for developing countries like Ethiopia because it is a popular, gluten free source of carbohydrates, B-vitamins and is non-allergenic to celiac patients (Dziezak, 1991). It also contains about 6–7% of high quality protein (Choudhury & Gautam, 1998).\n\nIn Ethiopia, rice is consumed in different forms; as a substitute for other major cereals mainly for injera (by mixing with millet and teff, Eragrostisteff), in bread (alone), as cooked rice, in brewing local drinks (“Farssoo” and “Araqee”), porridge and Kinche (splatted and cooked oats). Thus it fits well into the food habits of Ethiopians. Moreover, rice is a good source of income for farmers and has a higher yield and price than that of teff in the local market. However, it is claimed that consuming rice brings constipation especially in children due to its low fiber content. It is also poor in mineral and fat content. Mixing rice with other cereal crops can improve these problems (Aklilu et al., 2002).\n\nOn the other hand, rice flour has become an attractive ingredient in the processing industry due to its unique attributes such as white color, hypoallergenicity and ease of digestion (Kadan et al., 2003). It has also an excellent expansion property because of its high starch content and is well suited to thermal processing to produce a variety of food products (Ibanoglu et al., 2006).\n\nTeff is a staple cereal crop indigenous to Ethiopia that supplies a large proportion of the daily calorie intake for the majority of the Ethiopian population (Seyfu, 1997; Bultosa & Taylor, 2004). According to a Central Statistical Authority (CSA) (2010/11) report, teff cultivation takes up the largest amount of land under cereal cultivation (27.49%, 2.72 million hectares) and is the third largest crop (after maize and wheat) in terms of grain production (19.92%, 34.34 million quintals) in Ethiopia. As it is in high demand and it has a high market value, farmers earn more from growing teff than growing other staple crops. At present, teff is produced predominantly by smallholders who rely on a rainfall. Teff cultivation as a cereal food grain is restricted to Ethiopia, except in very small quantities in Eritrea and recently, in Israel, the Netherlands and U.S.A. Teff is also gaining popularity as health food (Spaenij-Dekking et al., 2005).\n\nTeff is as nutritious as major cereals like barley, oats, rice and wheat and even better in some aspects (Seyfu, 1993). It is a rich source of B-vitamins and minerals and is considered to be an excellent source of essential amino acids with higher levels than wheat and barley (Seyfu, 1993). The grain is small, with an average length and width of 1.00 to 1.20 and 0.59 to 0.75 mm, respectively (Bultosa & Taylor, 2004; Zewdu & Solomon, 2007). This makes it inconvenient to separate the germ from the bran and so the germ and the entire seed are consumed. This results in better nutrient provision and higher fiber content.\n\nStudies made on the utilization of teff are few and have been limited to the biological and biochemical changes taking place during the fermentation process (Asrat & Frew, 2001). However recently there has been a growing interest to develop new products from teff using modern processing techniques, like extrusion cooking to harness its potential (Laike, 2006). Research into these techniques has been limited in Ethiopia.\n\nDue to the many functional as well as nutritional properties of rice, it can be used for gluten free bread-making in combination with teff, which serves to compensate its limitations. Teff is gluten free in nature and has the potential to increase fiber, fat, B-vitamins and minerals consumption in the products. This paper seeks to characterize rice-teff bread by reporting its proximate compositions, including minerals and phytic acid content.\n\n\nMaterials and methods\n\nThree varieties of rice (Oryza sativa, Linn.) and teff (Eragrostis teff) grain were obtained from Adet Agricultural Research Center (AARC) and Debre Zeit Agricultural Research Center (DZARC), Ethiopia respectively. The rice was manually cleaned, milled so as to be able to pass through 710 µm sieves (Laike, 2006) and blended with the required ratios (defined below).\n\nBread was baked using straight-dough method as described in the AACC (2000) method № 10 - 10B. Fresh baked bread was dried for 24h at 65°C in an oven (Model: 101-1A; Tianjin Taisite Instrument Co., Ltd, Tianjin, China) and ground by mortar and pestle to pass through a 750 µm sieve. This sample was kept in sealed plastic bag at refrigeration temperature (5°C) and was used for proximate composition, minerals and phytic acid analysis.\n\nThe effect of rice variety and blending proportions of rice and teff on bread composition were studied using a custom design. The proportions of rice to teff ranged from 50 to 90% whereas teff from 10 to 50%. Rice bread (100% rice) was used as a control. Each formulation had nine runs and was done in triplicate.\n\nIn building the model, a regression equation was established to describe the relationship between the response Y and variable X. A second order model was generated for the two mixture components as follows:\n\nY = β1X1 + β2X2 + β12X1X2 + β3X3 + β4X4 + β5X5 (1)\n\nWhere: Y is the predicted response; β1 and β2 are linear coefficients; β12 is the interaction coefficient; β3, β4 and β5 are varietal coefficients and X1, X2, X3, X4 and X5 are independent variables.\n\nTotal ash was determined according to AOAC (1995). Approximately 3g dried bread sample was carbonized on hot plate and transferred to a muffle furnace (MF 120, Ankara TURKEY) and combusted at 550°C until ashing was completed (over 12 hrs). The residue was cooled to ambient temperature in desiccators (Nalgene Model 5317-0120) and then, the total ash was calculated.\n\nThe crude fiber was analyzed according to AOAC (1995). Approximately 3g dried bread sample was digested with 1.25% sulfuric acid and washed with distilled water and further digested with 1.25% sodium hydroxide, filtered through coarse porosity (75μm) crucible in apparatus at a vacuum of about 25 mm. The residue left after refluxing was washed again with 1.25% sulfuric acid at near boiling point. The residue was dried at 100°C for 2 hrs, cooled in a desiccator. After being dried the sample was ashed at 550°C for 2 hrs; after ashing the sample was cooled in a desiccator. Total crude fiber was then calculated.\n\nThe crude fat analysis was determined by Soxhlet extraction method in accordance with AOAC (1995), method 920 - 85. A thimble with approximately 2g dried bread sample was placed in a 50 ml beaker and dried in an oven for 2 hrs at 110°C. The sample contained in the thimble was extracted with petroleum ether in a Soxhlet extraction apparatus for 8 hrs. After the extraction was completed, the extracted fat was placed in a fume hood to evaporate the solvent on a steam bath until no odor of the solvent was detectable. The extracted fat was then dried in an oven for 30 minutes at 100°C. Finally, it was removed and cooled in a desiccator. Crude fat content was calculated.\n\nThe total nitrogen content of the sample was analyzed by micro-Kjeldahl method as described in AACC (2000) Method № 46 – 11. Approximately 0.3g dried bread sample was digested in a flask containing 2.5 mL of a mixture of H2SO4 + Se (100 mL) and salicylic acid (7.2g) and three pieces of boiling chips. The content of the flask was digested at a temperature of 350°C on the digestion apparatus until the digestion was completed (the digest becomes clear). The acid digest was allowed to cool at room temperature. The digested sample was transferred to a distillation unit (Model UDK-142, Europe) and distillation was under taken by adding 30 mL of distilled water followed by 25 mL of 40% NaOH and connecting it to distillation apparatus whose outlet tube was immersed in 25 mL of 4% boric acid solution. The distillate (about 150 mL) was collected and titrated by standard acid (0.1N HCl). Urea was used as a control in the analysis.\n\nThese were determined using an atomic absorption spectrophotometer (Model: 210 VGP spectrophotometer, Buck Scientific, East Norwalk, CT, USA) after digestion of approximately 3.0g dried bread using air-acetylene as a source of energy for atomization (AACC, 2000). For iron content determination absorbance was measured at 248.3nm and iron was estimated from a standard calibration curve (3–8µg Fe/mL) prepared from analytical grade iron wire. For zinc content determination, absorbance was measured at 213.8nm and zinc level was estimated from a standard calibration curve (0.1–1.0µg Zn/mL) prepared from ZnO. For calcium content determination, absorbance was measured at 422.7nm after addition of 1% lanthanum (i.e., 1mL La solution/5mL) to sample and standard to suppress interferences. Calcium content was then estimated from standard solution (0.1–1.0 µg Ca/mL) prepared from CaCO3.\n\nPhytic acid was determined after 0.25 g of flour sample was extracted with 12.5 mL of 3% Trichloroacetic acid (TCA), precipitation of phytate as ferric phytate with addition of 4 mL of FeCl3 (2mg/mL) (Poiana et al., 2009) followed by phytate phosphorus (Ph-P) analysis (Morrison, 1964) using a conversion factor i.e., phytate = P × 3.55 (Poiana et al., 2009).\n\nAt least triplicate data were analyzed by ANOVA and modeled using the statistical software JMPTM 8, 2008 (by SAS Institute Inc., Cary, NC, USA). Response surface methodology was applied to the experimental data using JMP version 8 to study the interaction effect of the main factors and to generate the prediction equations. Mean comparison has been done by Duncan’s Multiple Range Test (DMRT) by SAS 9.1.3. Mean values were considered at 95% significance level.\n\n\nResults and discussion\n\nThe ash content of the product ranged from 2.71–3.74% (Table 1). The ash content of the control was 2.7%, which was significantly (P<0.05) increased on blending with teff at different proportions. This is mainly due to the higher ash content of teff flour as compared to rice flour. Bultosa (2007) reported that teff grain ash content ranged from 1.99 to 3.16% with mean of 2.45%. This is due to teff grain’s proportionally high bran content (Bultosa & Taylor, 2004). Ash content indicates milling performance by indirectly revealing the amounts of bran removed. The highest ash content (3.74%) was obtained when 50% X-jigna rice variety was blended with 50% teff and lowest ash content was obtained when 90% Edeget rice variety and 10% teff were blended.\n\nValues are in means ± standard deviation on dry matter basis. Means within a column with the same letter are not significantly different at 95% probability levels. Where: V=rice variety, E=Edeget, X=X-jigna and N=Nerica-4.\n\nThe combined effects of rice variety and blending proportion on ash content was significant (P<0.0001). All the linear terms, interaction term (RT) and Edeget and X-jigna rice variety were significant for ash content (Appendix table 1.4). The following model (Equation 2) was developed to predict the crude ash content of the rice-teff blend bread.\n\nAC = 2.69R + 5.22T–1.45(RT)–0.085E + 0.026N + 0.06X [R2=0.97] (2)\n\nWhere: AC is predicted ash content (%), R is rice, T is teff, E, N and X were Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed value versus predicted plot of ash content (Figure 1a) was randomly distributed along the diagonal line with the regression coefficient of R2 = 0.97. Figure 1b shows the values of the residuals based on the fitted model. The points were randomly distributed about the zero value line on the vertical axis which indicates the fitted model was adequate to describe the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of ash (%).\n\nThe crude fiber content of the blended products ranged from 0.63 to 1.60% (Table 1) which is higher than 100% wheat flour bread (0.29%) (Mongi et al., 2011). The crude fiber content of the control was 0.46%. Blending rice with teff significantly (P<0.05) increased the crude fiber content of the product (Table 1). This is due to the high fiber content of teff grain. The highest value of crude fiber was obtained when 50% Edeget rice variety and 50% teff were blended. The lowest value was obtained when 90% Edeget rice variety and 10% teff were blended.\n\nThe combined effect of rice variety and blending proportion on crude fiber were significant (P<0.0001). The linear terms of rice and the interaction term (RT) were significant (P<0.05) on crude fiber content. The effect of the linear terms of teff and rice varieties was not significant (P>0.05) on the fiber content of the product (Appendix table 2.4). The following model (Equation 3) was developed to predict crude fiber.\n\nCF = 0.34R – 0.27T + 5.87(RT) – 0.02E + 0.06N – 0.04X [R2 = 0.85] (3)\n\nWhere: CF is crude fiber (%) predicted, R is rice, T is teff, and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed value versus predicted plot to crude fiber (Figure 2a) was well modeled at regression coefficient of (R2 = 0.85). The points were randomly distributed around the diagonal line which indicates the good fits of the model to the results. The residual versus predicted plot to crude fiber is shown in Figure 2b. The points were scattered on the zero value of the horizontal line indicating that the model is adequate to describe the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of fiber (%).\n\nThe crude fat content of the products ranged from 0.85 to 1.90% which is higher than 30% cocoyam-wheat composite bread (0.54%) and lower than 100% wheat bread (2.02%) (Mongi et al., 2011). The crude fat content of the control (0.63%) was significantly (P<0.05) increased on blending with teff; because the crude fat content of teff is higher than rice (Table 1). High crude fat content was obtained when 50% Edeget rice variety and 50% teff were blended and low value was obtained when 90% Edeget rice variety and 10% teff were blended.\n\nThe combined effect of the rice variety and blending proportion on crude fat content was significant (P<0.0001). The estimated parameters of linear terms and interaction (RT) term were significant for crude fat content (P<0.0001) (Appendix table 3.4). Rice varieties Edeget, Nerica-4 and X-jigna had no significant effect on crude fat content (P<0.05). The following model (Equation 4) was developed to predict crude fat content of the product.\n\nF = 0.81R + 4.41T – 3.28 (RT) + 0.004E – 0.015N + 0.01X [R2 = 0.95] (4)\n\nWhere: F is crude fat (%), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed value versus predicted plot (Figure 3a) was randomly distributed nearby the diagonal line which indicates the goodness of fit of the model. The residual versus predicted plot (Figure 3b) shows data points randomly distributed over the zero valued horizontal line which indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of fat (%).\n\nThe analysis shows that the protein content is significantly (P<0.05) increased as the proportion of teff increased. The protein content of the rice-teff blend bread had ranged from 9.71–10.75% (Table 1). All blended products were found to have higher crude protein contents than the control (9.74%) except Edeget rice variety (9.71%) which was blended at 50% with 50% teff (Table 1). This study shows that the crude protein content of rice-teff blend bread is lower than 100% wheat bread (12.54%) and higher than 30% cocoyam-wheat composite bread (9.04%) (Mongi et al., 2011).\n\nThe combined effect of the rice variety and blending proportion on crude protein was insignificant (P>0.05) (Appendix table 4.2). The linear terms of rice and teff had a significant effect on crude protein content (P<0.0001). Inclusion of the Edeget rice variety had a significant effect on crude protein (P<0.05) content. The following model (Equation 5) was developed to predict the crude protein content of the product.\n\nP = 9.98R + 8.43T + 4.82(RT) – 0.19E + 0.13N + 0.09X [R2 = 0.60] (5)\n\nWhere: P is crude protein (%), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed value versus predicted plot to protein is presented in Figure 4a. The determinant coefficient (R2) was 60%. The residual versus predicted plot to protein was presented in Figure 4b. The points were randomly distributed about the zero value horizontal line.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of protein (%).\n\nThe carbohydrate content of rice-teff blend bread ranged from 77.84–81.37% (Table 1). This is higher than the carbohydrate content of 100% wheat bread which is 63.25% (Mongi et al., 2011). A significant (P<0.05) decrease in carbohydrate content was observed with an increase in teff proportion (Table 1). This may be due to the fact that rice flour is higher in carbohydrate as compared to teff flour (Edeogu et al. 2007). The carbohydrate content of the control (X-jigna rice variety) was 81.78% which was significantly (P<0.05) decreased when blended with teff (Table 1). The lowest carbohydrate content (77.84%) was obtained when 50% Edeget rice was blended with 50% teff. Odetokun (2000) had reported that the increase in carbohydrate content during fermentation might be due to a reduction in the fiber content and an increase in both reducing sugars and total soluble sugars. These observations may also be attributed to the fact that during fermentation carbohydrate including cellulose, pectin, lignocellulose and starch are broken down by fermenting microorganisms thereby reducing the fiber content of such food (Raimbault & Tewe, 2001).\n\nThe combined effect of the rice variety and blending proportion on carbohydrate content was significant (P<0.0001) (Appendix table 5.2). The linear terms of rice and teff were significant (P<0.0001). The interaction term (RT) and Edeget rice variety did not differ significantly on carbohydrate content (P>0.05). The following model (Equation 6) was developed to predict carbohydrate content of the product.\n\nCHO = 81.77R + 78.39T – 7(RT) + 0.15E + 0.31N – 0.46X [R2 = 0.84] (6)\n\nWhere: CHO is predicted carbohydrate (%), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nFigure 5a shows the points were randomly distributed near to the diagonal line with regression coefficient of R2 = 0.84 which indicates the goodness of fit of the model to the data. The residual by predicted plot is presented in Figure 5b. The points were randomly distributed about the zero value horizontal line which indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of carbohydrate (%).\n\nThe iron content of rice-teff blended bread is shown in Table 2. The values ranged from 2.73–12.97 mg/100g. The iron content of the product was significantly (P<0.05) increased on blending with teff. The increase in iron content is due to the high iron content of teff compared to rice (Abebe et al., 2007). A maximum value of iron was obtained when 50% Nerica-4 and 50% teff were blended and a minimum value was obtained at 90% Edeget and 10% teff. An increase in iron after fermentation may be due to a reduction of phytate during fermentation. This is because fermentation is known to reduce phytate that forms complexes with different minerals and in part contributed by high iron contents of grain teff (Abebe et al., 2007).\n\nValues are in means ± standard deviation on dry matter basis. Means within a column with the same letter are not significantly different at 95% probability levels. Where: V=rice variety, E=Edeget, X=X-jigna, N=Nerica-4, Ca=calcium, Fe=iron and Zn=zinc.\n\nThe combined effect of the rice variety and blending proportion was significant (P<0.0001) (Appendix table 6.2). The linear terms of rice (P<0.05) and teff (P<0.0001) had a significant effect on iron content (Appendix table 6.4). The interaction term (RT) and X-jigna rice variety had a significant effect (P<0.05) on the iron content of the product. The inclusion of Edeget and Nerica-4 rice varieties had no significant effect (P>0.05) on the iron content of the product. The iron prediction model was developed as shown in Equation 7 below.\n\nFe = 4.78R + 41.9T – 45.44 (RT) – 0.28E – 0.63N + 0.91X [R2 = 0.84] (7)\n\nWhere: Fe is predicted iron (mg/100g), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed and predicted values of iron (Figure 6a) were closely correlated with the data as demonstrated by regression coefficient (R2 = 0.84). The majority of the points were randomly distributed nearby the diagonal line which indicates the goodness of fit of the model. The residual versus predicted plots for iron (Figure 6b) were randomly distributed about the zero value horizontal line on the vertical axis. This indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of iron (%).\n\nIron carries oxygen to the cells and it is necessary for the production of energy, the synthesis of collagen and the functioning of the immune system. Iron deficiency is a global problem with children and pre-menopausal women are highly affected. However, great care must also be taken not to take too much iron, as excess amounts are stored in the body’s tissues and adversely affect the body’s immune function, cell growth and heart health (Halliday, 1998; Rebouche et al., 1999).\n\nIron absorption can be influenced by calcium, magnesium, manganese, zinc, anti-acids and tetracycline (a common antibiotic) (Agency of Toxic Substances and Disease Registry, 2004). Iron deficiency deprives body tissues of oxygen and results in anemia which is characterized by low blood iron level, small red blood cells and low blood hemoglobin values. Outward effects of anemia include; fatigue, paleness, dizziness, sensitivity to cold, irritability, poor concentration and heart palpitation (Agency of Toxic Substances and Disease Registry, 2004). Recommended daily allowance of iron depending on age level and health condition is 10 to 30 mg and the recommended daily intake is 15 mg. The iron content of rice-teff blend bread was within the recommended range (Table 2).\n\nThe zinc content of the blended products ranged from 2.70 to 4.14mg/100g (Table 2). The analysis illustrated that there was a significant (P<0.05) difference in zinc content of the product between blends of rice and teff. The zinc content of the control was 3.46mg/100g. The highest value (4.14mg/100g) was obtained when 50% X-jigna rice variety and 50% teff were blended and the lowest value was obtained when 70% Edeget rice variety and 30% teff were blended. Fermentation has been reported to significantly increase zinc solubility (2 to 28%) and zinc uptake by intestinal segment from 1 to 16% (Agte et al., 1997). This may be due to the microbial fermentation, which enhances zinc bioavailability through hydrolysis induced by microbial phytase enzymes (Walingo, 2009).\n\nThe combined effect of the rice variety and blending proportion was significant (P<0.0001). The linear terms of rice and teff and the interaction term (RT) had a significant effect on zinc content (P<0.0001) (Appendix table 7.4). The difference between rice varieties was insignificant (P>0.05) for zinc content. The model used to predict zinc content was presented by Equation 8 as follows.\n\nZn = 4.45R + 14.21T – 21.83(RT) + 0.06E + 0.052N – 0.112X [R2 = 0.76] (8)\n\nWhere: Zn is zinc (mg/100g), R is rice, T is teff and E, N and X were Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nThe analyzed value versus predicted plot for zinc is shown in Figure 7a. Both values were closely correlated (R2 = 0.76). This indicates the majority of the points were randomly distributed near the diagonal line which indicates the goodness of fit of the model. The residual versus predicted plot for zinc (Figure 7b) was randomly distributed about the zero value horizontal line on the vertical axis. This indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of zinc (%).\n\nZinc is an essential micronutrient for animals, plants and microorganisms. Organisms can accumulate a considerable amount of zinc in their system without any damaging effect (Agency for Toxic Substances and Disease Registry, 1995). It is essential for carbohydrate metabolism, protein synthesis and inter-nodal elongation (stem growth). Zinc participates in all major biochemical pathways and plays multiple roles in the perpetuation of genetic material, and ultimately cell division. When the supply of dietary zinc is insufficient to support these functions, biochemical abnormalities and clinical signs of zinc mal-absorption occur. Zinc deficiency leads to iron deficiency causing similar symptoms to anemia; loss of appetite, growth retardation and immunological abnormalities (Agency of Toxic Substances and Disease Registry, 2004; Craig, 1994; Kimura & Itokawa, 1990).\n\nThe recommended daily allowance of zinc is 15 mg/day for men and 12 mg/day for women. Recent research suggests that men have a higher need for zinc than do women. Thus, it is appropriate that the recommended daily allowance is sex-specific for zinc (Kimura & Itokawa, 1990).\n\nThe calcium content of the blended products ranged from 25.31 to 61.25mg/100g (Table 2). The analysis indicated that means with in a column were significantly (P<0.05) different and this shows that the calcium content is significantly different between products. All the blends had higher calcium content than the control (17.97mg/100g). The highest value (61.25mg/100g) was obtained when 50% Edeget rice variety and 50% teff were blended and the lowest value was obtained when 90% Nerica-4 rice variety and 10% teff were blended. The observed high calcium content may be contributed by high calcium content of teff (Umeta et al., 2005).\n\nThe combined effect of the rice variety and blending proportion on calcium content was significant (P<0.0001) (Appendix table 8.2). The linear terms of rice and teff and the interaction term (RT) had a significant effect on calcium content (P<0.0001). The inclusion of the Nerica-4 rice variety had a significant effect (P<0.05) while the inclusion of Edeget and X-jigna rice varieties were insignificant (P>0.05) on the calcium content of the product. The model used to predict calcium content was presented by Equation 9 as follows.\n\nCa = 14.49R + 70.32T + 70.84(RT) + 0.43E – 0.98N + 0.54X [R2 = 0.98] (9)\n\nWhere: Ca is calcium (mg/100g), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice varieties, respectively.\n\nCalcium forms a vital part of bone and tooth structure and it is important as a positive ion (Ca2+) in blood clotting, muscle contraction and nerve impulse transmission. It also participates in glycogen metabolism (Krebs-Smith et al., 1997; WHO, 1996). Inadequate intake of calcium increases the risk of osteoporosis (bone loss with no apparent cause). Excess intake of calcium may cause kidney stones and reduces mineral absorption in general. The recommended dietary allowance of Calcium for adult is 800 mg; for pregnant women and young adults it is 1200 mg (Tortora, 1997).\n\nThe analyzed value versus predicted plot to calcium is given in Figure 8a. The values were strongly correlated (R2 = 0.98). The residual versus predicted plots for calcium (Figure 8b) were randomly distributed about the zero value horizontal line on the vertical axis. This indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of calcium (%).\n\nThe phytic acid content of the control and blend is presented in Table 2. There was a significant (P<0.05) differences in phytic acid content among the blended products. Values ranged from 0.31–0.62mg/g. The phytic acid content of the control was 0.21mg/g. Natural fermentation can achieve a large reduction in phytic acid by the action of bacterial as well as grain phytases. These reduce the hexa form of phytic acid into lower forms, which have a lower binding capacity for metals like iron and zinc (Agte et al., 1999). Results of fermentation on wheat bread showed that it could significantly improve in vivo bioavailability of minerals (Turk et al., 2000).\n\nThe decrease in phytate content could be attributed to possible secretion of the hydrolytic enzyme (phytase) by microorganisms. This enzyme is capable of hydrolyzing phytate content in the fermented foods (Ojokoh et al., 2005).\n\nThe combined effect of rice variety and blending proportion on phytic acid content was significant (P<0.0001) (Appendix table 9.2). The linear terms of rice and X-jigna rice variety had a significant effect on phytic acid (P<0.0001). The linear term of teff, Edeget and Nerica-4 rice varieties had significant effect on phytic acid (P<0.05). Fermentation of grains significantly decreased the phytic acid content of the blended product. The most marked reduction of phytic acid in the product was obtained at proportion of 90% Nerica-4 rice variety to 10% teff. The following model was developed to predict the phytic acid content of the product as shown by Equation 10.\n\nPA = 0.25R + 0.56T + 0.6(RT) – 0.026E – 0.027N + 0.053X [R2 = 0.92] (10)\n\nWhere: PA is phytic acid (mg/g), R is rice, T is teff and E, N and X are Edeget, Nerica-4 and X-jigna rice variety, respectively.\n\nThe analyzed versus predicted value plots for phytic acid (Figure 9a) were closely correlated by the regression coefficient (R2 = 0.92) and the points were randomly distributed near by the diagonal line which indicates the goodness of fit of the model. The residual versus predicted value plot for phytic acid (Figure 9b) were randomly distributed about the zero value horizontal line which indicates that the model was adequate in describing the data.\n\nAnalyzed value versus predicted (a) and residual versus predicted (b) plot of phytic acid (%).\n\n\nConclusions\n\nThis study revealed that rice varieties and blending proportion leads to significant difference in the proximate compositions, minerals and phytic acid contents of rice-teff blend bread. Therefore, blending of rice and teff in different proportions when making bread can compensate for the limitation of whole rice bread and whole teff bread. The combined effect of rice variety and blending proportions were significant (P<0.0001) in all the responses analyzed except protein. Carbohydrate values were significantly decreased with an increasing proportion of teff (P<0.05) for all varieties of rice, as rice has a higher carbohydrate content than teff. Ash, crude fiber and fat content significantly increased (P<0.05) with the increased proportion of teff blend. Addition of teff to rice significantly increased the iron, zinc, calcium and phytic acid contents of the product. The protein content of the bread product was not significantly influenced by the rice variety and blending proportion of rice and teff (P>0.05).\n\nThe regression coefficient (R2) values of ash content, fat, calcium and phytic acid were greater than 0.90. All the derived mathematical models for the various responses were found to be fit significantly to predicted data.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for JMP analysis, 10.5256/f1000research.6201.d45224 (Legesse et al., 2015a).\n\nF1000Research: Dataset 2. Data for SAS analysis, 10.5256/f1000research.6201.d45225 (Legesse et al., 2015b).\n\nF1000Research: Dataset 3. Proximate compositions, minerals, and phytic acid contents of rice-teff blend bread, 10.5256/f1000research.6201.d45226 (Legesse et al., 2015c).", "appendix": "Author contributions\n\n\n\nSintayehu Legesse designed the experiment, carried out the research and prepared the manuscript under the supervision of Solomon Worku and Geremew Bultosa. Solomon Worku contributed to the design of experiments and review of the manuscript. He was the major advisor. Geremew Bultosa contributed to the methods of sample analysis, review of the manuscript and provided expertise in cereal chemistry. He was the co-advisor. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was fully funded by Ethiopian Ministry of Education.\n\n\nAcknowledgements\n\nThe authors thank Ethiopian Ministry of Education (EMoE) for financial support; Adet Agricultural Research Center (AARC) for three varies of rice and Debre Zeit Agricultural Research Center (DZARC) for grain teff offers. Centre of Research on Grain Quality, Processing and Technology Transfer (CRGQPTT) at Food Science and Postharvest Technology (FSPT) department, Haramaya University, Ethiopia is acknowledged for supporting with facilities.\n\n\nAppendix\n\nLeast squares fit to proximate compositions, minerals and phytic acid contents\n\nAppendix table 1. Model fitting for ash content.\n\nAppendix table 2. Model fitting for crude fiber content.\n\nAppendix table 3. Model fitting for crude fat content.\n\nAppendix table 4. Model fitting for crude protein content.\n\nAppendix table 5. Model fitting for carbohydrate.\n\nAppendix table 6. Model fitting for iron content.\n\nAppendix table 7. Model fitting for zinc content.\n\nAppendix table 8. Model fitting for calcium.\n\nAppendix table 9. Model fitting for phytic acid.\n\n\nReferences\n\nAACC (American Association of Cereal Chemists): Approved methods of the American association of cereal chemists. AACC: International, 10th ed., Minnesota, U S A. 2000. Reference Source\n\nAbebe YA, Bogale KM, Hambidge BJ, et al.: Phytate, zinc, iron and calcium content of selected raw and prepared foods consumed in rural Sidama, Southern Ethiopia, and implications for bioavailability. J Food Compost Anal. 2007; 20(3–4): 161–168. Publisher Full Text\n\nAgency of Toxic Substances and Disease Registry (ATSDR): Toxicological profile for copper. U.S. Department of Health and Human Service. Atlanta, U.S. 2004; 6553–6561. 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Publisher Full Text\n\nTortora GJ: Introduction to human body: The essential of anatomy and physiology, 4th ed., John Wiley and Sons: New York. 1997; 472–474. Reference Source\n\nTurk M, Sandberg AS, Carlsson NG, et al.: Inositol hexaphosphate hydrolysis by baker’s yeast. Capacity, kinetics and degradation products. J Agric Food Chem. 2000; 48(1): 100–104. PubMed Abstract \| Publisher Full Text\n\nUmeta M, West CE, Fufa H: Content of zinc, iron, calcium and their absorption inhibitors in foods commonly consumed in Ethiopia. J Food Composition Anal. 2005; 18(8): 803–817. Publisher Full Text\n\nWalingo MK: Indigenous food processing methods that improve zinc absorption and bioavailability of plant diets consumed by the Kenyan population. African J Food Agriculture Nutrition Dev. 2009; 9(1): 523–535. Publisher Full Text\n\nWHO (World Health Organization): Trace elements in human nutrition and health, WHO Geneva. 1996. Reference Source\n\nZewdu AD, Solomon WK: Moisture dependent physical properties of teff seed. Biosystems Eng. 2007; 96(1): 57–63. Publisher Full Text" }	[ { "id": "43046", "date": "12 Feb 2019", "name": "Afifa Jahan", "expertise": [ "Reviewer Expertise PhD in food and nutrition", "MSc in food and Nutrition and BSc in food and Nutrition" ], "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 research is clearly and accurately presented and the current literature is appropriately cited. The study design is appropriate and the work is technically sound. It has sufficient details of methods and analysis provided to allow replication by others. The statistical analysis and its interpretation used in the study is appropriate. The rationale for developing the products of rice usually consumed in Ethiopia is clearly explained and description of the method is clearly presented by the author. The conclusions drawn adequately support the results.\n\nThe research is good and the paper is very elaborate. The results are confined to Ethiopia and the products developed are commonly consumed there. Hence this paper can be accepted for indexing, provided the content is modified a little and tables can be made into a single format as too much content cannot be read by any researcher. The length of paper can also be reduced. All the mineral content and analysis can be explained under one sub head. The acceptance of product developed can be mentioned in the methodology as it was replaced with rice (oryza sativa). The statistical analysis is appropriate and the content is very interesting too. Overall, a positive feedback from my side.\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": "53369", "date": "23 Sep 2019", "name": "Joseph Adubofuor", "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\nThe presentation of the research work is satisfactory with citations of the literature. Authors should consider citing some recent references starting from 2010 onwards. The study design is technically sound. The design of experiment used is appropriate in establishing the model for the variables involved. The methods used in data collection of proximate composition, mineral and phytic acid contents were outlined and explained in detail for replication by others. The statistical analysis was done appropriately to establish the interaction effect of the main factors and generate prediction equations for the responses. All the data have been provided in tables and figures. Superscripts have been indicated to show the significant differences among the samples. The conclusions drawn have been summarized clearly and are adequately supported by the results.\nThe following should be addressed:\nDiscussion on crude fat: The sentence: “Rice varieties Edeget, Nerica-4 and X-jigna had no signifcant effect on crude fat content (P<0.05)”. Comment: the less than sign should be changed to greater than (>).\n\nDiscussion on protein content: The sentence: \"The analysis shows that the protein content is signifcantly (P<0.05) increased as the proportion of teff increased”. Comment: The sentence should be changed because from Table 1 the protein content increased and decreased as the proportion of teff increased. An appropriate reason should be given to explain this trend.\n\nDiscussion on Phytic acid: The sentence: \"There was a signifcant (P<0.05) differences in phytic acid content among the blended products” Comment 1: The sentence should be changed to read as follows: \"There were significant differences (p<0.05) in phytic acid content among the blended products\". Comment 2: In all three rice varieties, decrease in teff content of the blends resulted in decrease in phytic acid content. The reason(s) which could account for this trend should 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": [] } ]	1	https://f1000research.com/articles/4-116
https://f1000research.com/articles/4-114/v1	12 May 15	{ "type": "Research Note", "title": "Epidemiology and patterns of musculoskeletal motorcycle injuries in the USA", "authors": [ "Sean T. Burns", "Zbigniew Gugala", "Carlos J. Jimenez", "William J. Mileski", "Ronald W. Lindsey", "Sean T. Burns", "Carlos J. Jimenez", "William J. Mileski", "Ronald W. Lindsey" ], "abstract": "Introduction: Motorcycles have become an increasingly popular mode of transportation despite their association with a greater risk for injury compared with automobiles. Whereas the recent incidence of annual passenger vehicle fatalities in the United States of America (USA) has progressively declined, motorcycle fatalities have steadily increased in the past 11 years. Although motorcycle injuries (MIs) have been studied, to the author’s knowledge there are no published reports on MIs in the USA during this 11-year period.Methods: Study data were derived from a prospectively collected Level I trauma center database. Data sampling included motorcycle crash injury evaluations for the 10-year period ending on 31 August 2008. This retrospective analysis included patient demographic and medical data, helmet use, Glasgow coma scale (GCS) score, injury severity score (ISS), length of hospital stay (LOS), specific injury diagnosis, and death. Data statistics were analyzed using the Spearman correlation coefficient, Kruskal-Wallis tests, and logistic regression.Results: The study identified 1252 motorcycle crash injuries. Helmets were worn by 40.7% of patients for which helmet data were available. The rates of the most common orthopedic injuries were tibia/fibula (19.01%), spine (16.21%), and forearm (10.14%) fractures. The most common non-orthopedic motorcycle crash injuries were concussions (21.09%), skull fractures (8.23%), face fractures (13.66%), and hemo- and pneumothorax (8.79%). There was a significant correlation between greater age and higher ISS (r=0.21, P<0.0001) and longer LOS (r=0.22, P<0.0001). Older patients were also less likely to wear a helmet (OR=0.99, 95% CI: 0.98, 0.997), associated with a significantly higher risk for death (after adjustment for helmet use OR=1.03, 95% CI: 1.00, 1.05). All patients without helmets had a significantly lower GCS score (P=0.0001) and a higher mortality rate (after adjustment for patient demographic data OR=2.28, 95% CI: 1.13, 4.58). Conclusion: Compared with historical reports, the prevalence of skull, face, spine, and pelvis fractures have increased in American motorcycle crashes. Compared to recent European studies, the incidence of USA skull and face fractures is much higher, while the incidence of USA spine and pelvis fractures is more comparable; however, this is not associated with increased in-hospital mortality.", "keywords": [ "musculoskeletal motorcycle injuries", "epidemiology", "fractures" ], "content": "Introduction\n\nInjury secondary to motorcycle crashes is a major problem in the United States of America (USA). In 2007, the number of registered motorcycles in the USA totaled 7,138,476, of which 387,915 were registered in Texas19,21. That same year, 103,000 Americans were injured in motorcycle crashes, with 5,154 of them killed. Texas ranked third in terms of fatalities behind Florida and California18.\n\nIn recent years, the annual incidence of automobile-related fatalities in the USA has progressively decreased, while the annual incidence of motorcycle crash fatalities has progressively increased19. However, despite the expanding use of motorcycles, there has been a paucity of orthopedic epidemiological studies related to motorcycle crash in recent years. Our institution has been the primary Level I trauma facility for Galveston County and the surrounding region. The area is one of the most popular motorcycle destinations in Texas, and Galveston is home to the state’s largest motorcycle rally1. The purpose of this study was to review a single-institution experience with MIs in regard to the epidemiology and patterns of both musculoskeletal and non-musculoskeletal injuries. In addition, this study will compare our modern USA motorcycle injury data with those of earlier international reviews2,27.\n\n\nMaterials and methods\n\nThe study was conducted in compliance with the University of Texas Medical Branch policies and regulations regarding human subject research following study protocol review and approval by the Institutional Review Board (approval IRB #09-259). Study data were derived from a prospectively collected injury database at our Level I trauma center. The data sampling included 1,221 patients injured in motorcycle crashes for the 10-year period ending August 31, 2008. Each motorcycle trauma event was considered separately and evaluated independently; patients with more than one motorcycle trauma event evaluation during the study time period had each event considered as a separate incident.\n\nThis retrospective analysis included data on patient age, gender, race/ethnicity, documentation of helmet use, Glasgow Coma Scale (GCS) score at the time of presentation in the emergency department, injury severity score (ISS), length of hospital stay (LOS), specific injury diagnosis, and mortality.\n\nDescriptive statistics were used to describe the sample characteristics (age, gender, race) and distribution of the outcome variables (GCS, ISS, LOS, type of injury, and death). Non-parametric methods (Spearman correlation coefficient and Kruskal-Wallis tests) were used to test the association between the general patient demographic data, helmet use, and the outcome variables ISS and LOS. Logistic regression analyses were used to test the association of the general patient demographic data and helmet use with patient GCS classification (mild vs. moderate/severe), vital status at discharge and type of injuries (with/without central nervous system injury, dislocation, fracture, and thorax injury). All tests were two-sided with alpha of 0.05 and were performed using SAS 9.3 (SAS Institute Inc, Cary, NC, USA).\n\n\nResults\n\nThe study identified 1,252 motorcycle trauma event evaluations performed at our institution within a 10 year period. Of these trauma event evaluations, 31 were for a patient’s second or third motorcycle injury during the study period. These multiple-event evaluations involved 28 patients and constituted 59 of all 1,252 evaluations. Twenty-five patients were evaluated twice and three patients were evaluated three times. The interval between subsequent trauma event evaluations among these 28 patients ranged from 2 days to 8.5 years.\n\nMales made up 83% of the motorcycle injury evaluations. The average patient age was 36.0 years (range 4–83 years). There was an 8:1:1 White:Black:Hispanic ratio (Table I). Helmet use could be established for 1093 (87.3%) of motorcycle event evaluations. Within that subset, helmets were worn in 445 cases (40.7%) and not worn in 648 cases (59.3%).\n\na All values are for number of evaluations (N = 1252).\n\nThe average GCS score at presentation for the 1,244 trauma event evaluations with available data was 13.9. The diagnosis was a minor brain injury (GCS 13–15) in 1127 (90.6%), a moderate brain injury (GCS 9–12) in 13 (1%), and a severe brain injury (GCS 3–8) in 104 (8.4%).\n\nThe average ISS for all evaluations was 9.4 (median 5, range 0–75). The mortality rate (at arrival and/or post-admission) was 4.4% (55 evaluations), with 40% (22) of those patients dead on arrival (DOA). Excluding the DOA cases, the mortality rate in all other (in-hospital) evaluations fell to 2.7% (33/1230). The average LOS was 4.4 days (median 1 day, range 0–121 days).\n\nThe incidence and specific types of motorcycle injuries are listed in Table II–Table V. The most common orthopedic motorcycle injuries were tibia/fibula, spine, and forearm fractures, which occurred in 238 (19%) evaluations, 203 (16.2%), and 127 (10.1%) respectively (Table II and Table III). The highest rates of non-orthopedic motorcycle injuries were for concussions (264 evaluations, 21.09%), skull fractures (103, 8.23%), face fractures (171, 13.66%), and hemo- and pneumothorax injuries (110, 8.79%) (Table IV). Abdominal injuries (excluding vessel injuries) were seen in 192 evaluations (15.3%) (Table V).\n\na For patients with bilateral fractures only one side was counted (i.e., bilateral fractures were counted as one fracture not two).\n\nb Open fractures are included in the overall fractures column.\n\nc The percentages listed in this column are the percentages of patients with each fracture, as opposed to the percentages of fractures that are of a particular type (e.g., there was a skull fracture in 8.23% of evaluations, where skull fractures were 6.09% of all fractures).\n\nd The percentages listed in this column are the percentages of fractures that were open (e.g., 40% of patella fractures were open, but open patella fractures occurred in only 0.32% of evaluations).\n\na Evaluations in which there were bilateral dislocations were counted as having one.\n\nb The percentages listed in this column are the percentages of evaluations with each dislocation, as opposed to the percentage of dislocations that are of a particular type.\n\nc The percentages listed in this column are the percentages of dislocations that were open.\n\na The percentages listed are for the rates of particular injuries in the motorcycle injury evaluations.\n\na The percentages listed are for the rates of particular injuries in the motorcycle injury evaluations.\n\nb Vessel injuries are not included.\n\nOlder patients were less likely to use a helmet (OR=0.99, 95% CI: 0.98, 0.997), which was associated with a significantly higher risk for death (after adjustment for helmet use OR=1.03, 95% CI: 1.00, 1.05). Older patients without helmets had a significantly lower GCS score (P=0.0001) and higher mortality rate (after adjustment for patient demographic characteristics OR=2.28, 95% CI: 1.13, 4.58).\n\nLogistic regression analysis demonstrated that greater age was significantly associated with more severe injury (Spearman correlation coefficient r=0.21, P<0.0001) and a longer hospital stay (Spearman correlation coefficient r=0.22, P<0.0001). Greater age was also associated with significantly higher risks for death following injury, for fracture, and for thorax injury.\n\nWhites had significantly higher ISS and LOS and higher risk for fracture.\n\nAmong all patients who did not wear a helmet, the risk was significantly higher for a more severe GCS score (adjusted odds ratio 2.98) and for death (adjusted odds ratio 2.95). These patients were also more likely to have a fracture of the skull (P<0.001) or face (P<0.001).\n\nFracture results from our current study and from earlier studies from England, Sweden, and the USA are presented in Table VI. The rates of skull and face fractures are higher in our current study than in England or Sweden. The incidence of skull, spine, and rib fractures has nearly doubled in the USA over the last 30 years, and pelvis fractures are much more prevalent.\n\n\nDiscussion\n\nMany epidemiological studies have addressed motorcycle crash injuries, in particular the effect of helmet use3,5,6,9,25. There has been a relative paucity, however, of new information regarding orthopedic motorcycle crash injuries in USA over the last 30 years. Unfortunately, during this period motorcycle crashes continued to be a major cause of accident-related morbidity and mortality. It has been reported that in 2007 motorcyclists were 37 times more likely to die in a motor vehicle traffic crash than passenger car occupants, and nine times more likely to be injured20. Whereas car and truck fatality rates have decreased for the past 6 and 3 years, respectively, 2008 marked the eleventh consecutive year in which the annual motorcycle fatality rate increased19. Yet, to our knowledge, the most recent epidemiological studies on orthopedic motorcycle crash injuries in the USA were by Bried et al.6 in 1987 and Peek et al.22 in 1994.\n\nAccording to our data, the rates of skull, face, spine, rib, and pelvis fractures have increased in the USA motorcycle crashes compared with historical reports9,11,28. The rates of skull and face fractures in our study are substantially higher than in recent European studies (Table VI), whereas the rates of spine and pelvis fractures have remained similar. The higher incidence of some of the injuries documented in our study may be attributed to better injury detection. In the USA, whole-body computed tomography has become a routine trauma screening tool24. Moreover, European riders are more likely to wear a helmet2, and their lower incidence of skull and face fractures and death might be attributed to this protective device. The protective nature of helmets has been described in a number of studies13,16.\n\nIn contrast to Ankarath et al’s2 and Robertson et al’s23 studies from 2002 that analyzed similar patient populations from the Yorkshire region of the United Kingdom from the mid to late 1900s and Kupferschmid et al.15 study from 1989, we found lumbar spine fractures to be more common than thoracic. This is in agreement with Goslar et al.’s12 study (2008) that looked at spine fractures in motorcycle accident victims in Arizona. However, the incidence of facial fractures in the present study was nearly twice that reported by Kraus et al. (13.66% vs. 7.10%) in a study using early 1990s data14, when routine computed tomography was not as common a procedure.\n\nInjuries and death among older motorcyclists has recently become a focus topic of motorcycle crash studies. It has been reported that the age of motorcyclists has been increasing7. The Motorcycle Industry Council found the mean age of motorcycle ownership to rise from 33.1 years in 1998 to 40.2 years in 200317. Our study of patients aged 4 to 83 years (mean 36 years) joins other studies of motorcycle crash victims that have shown increased risk for a longer hospital stay, more severe injuries, and death among older patients. Brown et al., using 1996–2005 National Trauma Databank statistics on 61,689 motorcycle operators involved in crashes, found the average age to increase steadily over the study period, from 33.9 years to 39.1 years7. The fastest growing age group in relative frequency was that of 50 to 59 years, whereas the most rapidly declining was that of 20 to 29 years. Operators 40 years of age or older had higher ISS, LOS, intensive care unit LOS, and death rate compared with operators younger than 40 years. Brown et al. found significantly higher rates of severe thoracic and head injuries and rib fractures, as well as more complications and more than twice the rate of pre-existing comorbidities among motorcyclists aged 40 years or older7. Whereas their data indicated no difference in helmet use between younger and older injured operators, we found older patients significantly less likely to use a helmet, with lack of helmeting linked to skull fracture and death3,25. In contrast, in a 1981 NHTSA treatise Hurt et al. described younger patients as less likely to wear a helmet13.\n\nDischinger et al. found that older riders (40 years or older) have significantly higher incidence of thoracic injury, specifically multiple rib fractures10. Talving et al. investigated the relation of age to injury type, distribution, and the severity of motorcycle crash patients in a Los Angeles County-wide trauma registry study (N = 6,530) including data from 1995 to 199726. Age was classified as 18 years or younger, 19 to 55 years, or older than 55 years. Older patients were significantly more likely to suffer severe trauma, severe head and chest injuries, and spinal fractures. Death was 3-fold higher in the oldest age group compared with the youngest.\n\nThe limitations of our study include its retrospective nature and the fact that it relies upon a trauma database. Although the data were collected prospectively, they were not specifically designed for this study. Orthopedic injuries were grouped by anatomic location as opposed to specific fracture type to control for small errors of injury classification (e.g., injury was classified as a radial shaft fracture as opposed to a Galeazzi or simple distal radius fracture). However, these injury classifications are similar to those employed in prior studies2,6,8,9,11,22,27,28, and many of those study data were also derived from existing databases2,4,12.\n\nAlthough the motorcycle fatality rate continues to increase, we did not see a similar increase in in-hospital mortality rate, indicating that more of the involved participants are likely being declared dead at the scene. The increase in the incidence of skull, spine, and face injuries noted in the study may be attributed to better injury detection (routine CT and/or MRI). The higher incidence of USA skull and facial fractures, as compared to other countries, may also be attributed to differences in compulsory helmet wearing laws. Older MI patients are a higher risk for serious injury and mortality, and this is further potentiated by not wearing a helmet.\n\n\nData availability\n\nFigshare: Raw data used for the analysis in the study on epidemiology and patterns of musculoskeletal injuries. Doi: http://dx.doi.org/10.6084/m9.figshare.140218629", "appendix": "Author contributions\n\n\n\nSTB - data collection, data analysis, manuscript preparation.\n\nZG - study design, data analysis, manuscript preparation.\n\nCJJ - data collection, data analysis.\n\nWJM - data collection, manuscript preparation.\n\nRWL - data analysis, manuscript preparation.\n\nAll authors read and approved the revised manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors thank Alai Tan, PhD, for support with statistical analysis and Suzanne Simpson, BA for editorial assistance.\n\n\nReferences\n\nABC News: Lone Star Motorcycle Rally, Houston, TX. Reference Source\n\nAnkarath S, Giannoudis PV, Barlow I, et al.: Injury patterns associated with mortality following motorcycle crashes. Injury. 2002; 33(6): 473–7. PubMed Abstract \| Publisher Full Text\n\nBachulis BL, Sangster W, Gorrell GW, et al.: Patterns of injury in helmeted and nonhelmeted motorcyclists. Am J Surg. 1988; 155(5): 708–11. PubMed Abstract \| Publisher Full Text\n\nBledsoe GH, Schexnayder SM, Carey MJ, et al.: The negative impact of the repeal of the Arkansas motorcycle helmet law. J Trauma. 2002; 53(6): 1078–87; discussion 1086–7. PubMed Abstract\n\nBraddock M, Schwartz R, Lapidus G, et al.: A population-based study of motorcycle injury and costs. Ann Emerg Med. 1992; 21(3): 273–8. PubMed Abstract \| Publisher Full Text\n\nBried JM, Cordasco FA, Volz RG: Medical and economic parameters of motorcycle-induced trauma. Clin Orthop Relat Res. 1987; 223: 252–6. PubMed Abstract \| Publisher Full Text\n\nBrown JB, Bankey PE, Gorczyca JT, et al.: The aging road warrior: national trend toward older riders impacts outcome after motorcycle injury. Am Surg. 2010; 76(3): 279–86. PubMed Abstract\n\nClark DW, Morton JH: The motorcycle accident: a growing problem. J Trauma. 1971; 11(3): 230–7. PubMed Abstract\n\nDenaner RM, Fitchett VH: Motorcycle trauma. J Trauma. 1975; 15(8): 678–81. PubMed Abstract \| Publisher Full Text\n\nDischinger PC, Ryb GE, Ho SM, et al.: Injury patterns and severity among hospitalized motorcyclists: a comparison of younger and older riders. Annu Proc Assoc Adv Automot Med. 2006; 50: 237–49. PubMed Abstract \| Free Full Text\n\nDrysdale WF, Kraus JF, Franti CE, et al.: Injury patterns in motorcycle collisions. J Trauma. 1975; 15(2): 99–115. PubMed Abstract\n\nGoslar PW, Crawford NR, Petersen SR, et al.: Helmet use and associated spinal fractures in motorcycle crash victims. J Trauma. 2008; 64(1): 190–6. PubMed Abstract \| Publisher Full Text\n\nHurt HH, Quellet JV, Thom DR: Motorcycle Accident Cause Factors and Identification of Countermeasures. Vol 1. Technical Report. Washington, DC: US Department of Transportation, National Highway Traffic Safety Administration. 1981. Reference Source\n\nKraus JF, Rice TM, Peek-Asa C, et al.: Facial trauma and the risk of intracranial injury in motorcycle riders. Ann Emerg Med. 2003; 41(1): 18–26. PubMed Abstract \| Publisher Full Text\n\nKupferschmid JP, Weaver ML, Raves JJ, et al.: Thoracic spine injuries in victims of motorcycle accidents. J Trauma. 1989; 29(5): 593–6. PubMed Abstract \| Publisher Full Text\n\nLin MR, Kraus JF: A review of risk factors and patterns of motorcycle injuries. Accid Anal Prev. 2009; 41(4): 710–722. PubMed Abstract \| Publisher Full Text\n\nMotorcycle Industry Council. Statistical Annual, 2004, Irvine, CA. 2004.\n\nNational Highway Traffic Safety Administration. Traffic Safety Facts 2007: Motorcycles. Washington, DC: National Center for Statistics and Analysis. 2008. Reference Source\n\nNational Highway Traffic Safety Administration. Traffic Safety Facts 2008 Data. Washington, DC: National Center for Statistics and Analysis. 2008. Reference Source\n\nNational Highway Traffic Safety Administration. Traffic Safety Facts 2008: Motorcycles. Washington, DC: National Center for Statistics and Analysis. 2008. Reference Source\n\nNational Highway Traffic Safety Administration. Traffic Safety Facts Texas 2004–2008. Washington, DC: National Center for Statistics and Analysis. 2008. Reference Source\n\nPeek C, Braver ER, Shen H, et al.: Lower extremity injuries from motorcycle crashes: a common cause of preventable injury. J Trauma. 1994; 37(3): 358–64. PubMed Abstract \| Publisher Full Text\n\nRobertson A, Giannoudis PV, Branfoot T, et al.: Spinal injuries in motorcycle crashes: patterns and outcomes. J Trauma. 2002; 53(1): 5–8. PubMed Abstract \| Publisher Full Text\n\nSalim A, Sangthong B, Martin M, et al.: Whole body imaging in blunt multisystem trauma patients without obvious signs of injury: results of a prospective study. Arch Surg. 2006; 141(5): 468–75; discussion 473–5. PubMed Abstract \| Publisher Full Text\n\nShankar BS, Ramzy AI, Soderstrom CA, et al.: Helmet use, patterns of injury, medical outcome, and costs among motorcycle drivers in Maryland. Accid Anal Prev. 1992; 24(4): 385–96. PubMed Abstract \| Publisher Full Text\n\nTalving P, Teixeira PG, Barmparas G, et al.: Motorcycle-related injuries: effect of age on type and severity of injuries and mortality. J Trauma. 2010; 68(2): 441–6. PubMed Abstract\n\nWladis A, Boström L, Nilsson B: Injuries in 8927 patients admitted after motor-cycle crashes in Sweden 1987-1994 inclusive. Eur J Surg. 2002; 168(3): 187–92. PubMed Abstract \| Publisher Full Text\n\nZettas JP, Zettas P, Thanasophon B: Injury patterns in motorcycle accidents. J Trauma. 1979; 19(11): 833–6. PubMed Abstract\n\nBurns ST, Gugala Z, Jimenez CJ, et al.: Raw data used for the analysis in the study on epidemiology and patterns of musculoskeletal injuries. Figshare. 2015. Data Source" }	[ { "id": "8630", "date": "21 May 2015", "name": "Charles A. Reitman", "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\nWell written study on relevant topic.Weaknesses are discussed by the authors. Conclusions supported by the data.I support indexation.", "responses": [] }, { "id": "8631", "date": "28 May 2015", "name": "Arvind D. Nana", "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\nGood paper. Injury details are well captured. For instance, this study did a better job of capturing acetabulum, pelvis, patella, and scapula fractures compared to previous studies. The authors did identify dislocations in this patient population, and I wish they had discussed that further in the paper.", "responses": [] } ]	1	https://f1000research.com/articles/4-114
https://f1000research.com/articles/3-311/v2	10 Apr 15	{ "type": "Software Tool Article", "title": "Ant-App-DB: a smart solution for monitoring arthropods activities, experimental data management and solar calculations without GPS in behavioral field studies", "authors": [ "Zeeshan Ahmed", "Saman Zeeshan", "Pauline Fleischmann", "Wolfgang Rössler", "Thomas Dandekar", "Saman Zeeshan", "Pauline Fleischmann", "Wolfgang Rössler", "Thomas Dandekar" ], "abstract": "Field studies on arthropod ecology and behaviour require simple and robust monitoring tools, preferably with direct access to an integrated database. We have developed and here present a database tool allowing smart-phone based monitoring of arthropods. This smart phone application provides an easy solution to collect, manage and process the data in the field which has been a very difficult task for field biologists using traditional methods. To monitor our example species, the desert ant Cataglyphis fortis, we considered behavior, nest search runs, feeding habits and path segmentations including detailed information on solar position and Azimuth calculation, ant orientation and time of day. For this we established a user friendly database system integrating the Ant-App-DB with a smart phone and tablet application, combining experimental data manipulation with data management and providing solar position and timing estimations without any GPS or GIS system. Moreover, the new desktop application Dataplus allows efficient data extraction and conversion from smart phone application to personal computers, for further ecological data analysis and sharing. All features, software code and database as well as Dataplus application are made available completely free of charge and sufficiently generic to be easily adapted to other field monitoring studies on arthropods or other migratory organisms. The software applications Ant-App-DB and Dataplus described here are developed using the Android SDK, Java, XML, C# and SQLite Database.", "keywords": [ "Arthropod behaviour", "behavioural ecology", "Data collection", "GPS/GIS", "Android", "Tracking" ], "content": "Introduction\n\nThe traditional way of collecting and managing the data in behavioral field studies has been a tedious and laborious task. It requires the marking and monitoring of arthropods in the field along with the manual entry and management of the data about marked insects, feeders and experiments. Moreover, it becomes extremely complex, when the ecologists have to estimate solar positions and timings without any GPS system in remote and wild regions. In field studies monitoring of arthropods requires an easy to handle application, monitoring movement as well as behavioral parameters. A desktop application, installed in a laptop may not be a reliable solution, due to humid, warm and uncertain weather conditions, especially in deserts. For this, coupling modern database technology with a smart phone application can provide a strong, user-friendly tool to adopt1.\n\nSeveral beneficial applications have already been developed to improve the field of ecology e.g. animal and plant georeference phenological recording2, crowd-sourcing3,4, gearing community developmental research5 with scientific approach6, collecting data in the field with a GPS system7 or a GIS system8. Despite some existing useful technological solutions in the field, we found some gaps that still need to be addressed. For instance, there is no specific smart phone or tablet application available for optimized monitoring of desert ant species. Effective desert ant monitoring requires an application with an efficient data management system and the ability to estimate solar positions and timings without a GPS or GIS system.\n\nWe offer a thoroughly developed generic solution which can easily be adapted to investigate behavioral parameters in other organisms such as honey bees and fruit flies and is hence made freely available for such efforts. The application was originally developed and optimized for monitoring a desert ant species, Cataglyphis fortis, a social insect, which mainly uses a polarized skylight based compass and path integration for orientation and homing9–11. In addition, olfactory cues are used close to the nest entrance12.\n\nIn areas inhabited by Cataglyphis fortis (salt flats in North African deserts) –the lack of prominent visual landmarks means that ants mostly rely on celestial cues. High temperatures and an unpredictable distribution of food force the ants to make long-winded search runs to then return in a straight path back to the nest13. The high level of complexity in orientation and extreme environmental conditions requires novel tools to monitor the ant’s behavior in any easy to use fashion that allow production of accurate data on foraging runs and homing with relation to solar time, solar azimuth, time of the day, identification of individual ants and other parameters.\n\nA large amount of computational research has been performed with regard to behavior studies, for instance in artificial intelligence14 and different approaches have been proposed e.g.15–27. However, the specific field experimental paradigm related to skylight compass orientation leads to different kinds of information where an optimal, easy-to-handle tool and database has not yet been established. Without any swift and effectual technological solution, the experimentation process may become very complex and time consuming, as the observer has to do many tasks at one time e.g. managing information about the running experiment, food at feeders, marking of the ants, separating registered and unregistered ants and observing the continuous change in the current time of the location, solar time, solar zenith and azimuth angle.\n\nTo cope with this, we propose a new product line architecture (PLA) based scientific solution, the Ant-App-DB; a user friendly, smart phone and tablet application, helpful in efficient management of experimental data including location, date, time, geographical measurements, feeders, registered and unregistered ants28. We also present another new multi document interface (MDI) desktop application Dataplus; that enables quick data transfer from the smart phones exported database file and conversion into the Microsoft excel format for further data analysis.\n\nThe major reason for developing the smart phone application is to have a user friendly way of managing experimental processes along with data sharing. Moreover, it is also worthy to take advantage of the advanced mobile computing and service provision of this era, which offers small sized devices (easy to carry, usable worldwide and affordable), embedded with extra durable (rechargeable and replaceable) batteries, internal and external memory cards and most of all temperature resistance with the ability to withstand extreme conditions such as those found in deserts where laptops or other computational devices can experience problems.\n\nThe following sections of the manuscript explain the methodology, architected software and database designs, and implementation with modular description of the application.\n\n\nMethods\n\nAnt-App-DB is a well-developed application, following the principles of three layered Butterfly29,30 software development model towards scientific software engineering (SSE), integrating formal Unified Modelling Language (UML)31,32 perspectives and incorporating Human Computer Interaction (HCI) design patterns.\n\nThe overall software engineering process of the Ant-App-DB is well planned, as initially the possible number of requirements were gathered and discussed, abstract application designs were architected, and mockup designs of graphical user interface (GUI) were constructed following a brain storming session by the authors and other colleagues. Implementable designs (use case, database, dataflow, work flow, system sequence, class and components) were then drawn based on the finalized functional requirements, the most suitable technologies (both software and hardware) were chosen, comprehensive prototype development was performed and the end product was successfully deployed and tested in-house.\n\nThe conceptual architecture of Ant-App-DB (Figure 1) is divided into five different modules: Mobile System, Database, NOAA, Personal Computer and Export Excel Format. ‘Mobile system’ is the smart phone application to be used in the experiments on the field, ‘Database’ is the embedded data management system in the smart phone, ‘NOAA’ is an integrated module in smart phones to estimate solar timing and angles using different astronomical algorithms recommended by the National Oceanic and Atmospheric Administration.\n\nThis figure shows the conceptual architecture of the Ant-App-DB application, which consists of five main components: mobile system, database, solar estimations using recommended algorithms by the National Oceanic and Atmospheric Administration (NOAA), personal computer and exporting data in Microsoft Excel format.\n\nThe personal computer module uses the desktop application (Dataplus) to extract data from the smart phone database and then converts into the Microsoft Excel format.\n\n\nImplementation\n\nThe designed and implemented methodology is explained in the following UML notation and semantics for use case, activity, dataflow, system sequence, class, component and database (entity relationship) diagrams (Please see details in the Supplementary material).\n\nThe activity work flow (Figure 2) starts with the main GUI of the application (describing the available options provided to the observers), which is further categorized as three different processes: administration, experimentation, and solar estimation. Administration offers a secured access to the authorized users/observers for deleting or creating backups of the existing records in the internal or external storage locations, which then can be exported, reused and shared. Experimentation allows users to manipulate and manage information related to experiments, feeders (optional), registration of ants and ants to be used during experiments. Moreover, it also offers an additional interface (Quick Ant) to fasten the experimentation process and presents stored results in tabular form. The solar estimation process allows users to approximate the solar time and azimuth angle using any given (valid) date, time, UTC time zone, longitude and latitude. Finally, the data (SQLite database) can be exported from the smart phone application to a personal computer and then using Dataplus can convert data into the Microsoft Excel sheet format.\n\nThis figure shows the application’s activity workflow, which starts with the Main interface with four categorized options: Close, Administration, Solar Estimation and Experimentation. Choosing Close (default return option in almost all Android based smart phones), user can exit from the application. Using Administration an authorized user can delete or backup the data and via Solar Estimation user can calculate different solar timings and angles. Experimentation option allows user to enter and manage the information related to the Experiments, Feeders and Ants. All application’s data is stored in internal storage with sequential access, which then can be exported to the personal computer and using Dataplus can be converted in Microsoft Excel format.\n\nAs shown in the component diagram (Figure 3), Ant-App-DB is an Android operating system based application (tested using a Sony Xperia Z1 smart phone and the Android SDK based emulator). Eclipse Integrated Development Environment (IDE) was used for the entire smart phone application development using Java programming language, XML, Android SDK and SQLite database for embedded database scripting. The Dataplus module was developed in C-Sharp programming language in Microsoft dot net framework.\n\nThis figure shows wiring of the component of the Ant-App-DB. It consists of four major components: IDE (with five sub-components: Android SDK, Java, XML, SQLite DB, Eclipse), Personal Computer (PC), Smart Phone, Tablet PC and Microsoft Dot Net Framework (including C# programming language).\n\nFollowing the designed sequence of the application, the implemented source code is divided into two sections: GUI and the logic of the program. The designed GUI (8 horizontal and 8 vertical pages) are implemented in XML and the main logic of the application is implemented in Java programming language.\n\nData management. To manage the application’s data, we designed a normalized entity relationship model and implemented this in SQLite database management (please see Supplementary material for details).\n\nThe Ant-App-DB is divided in to six major interlinked GUIs: Main, Experiments, Ant Feeder, Registration, Ant and Quick Ant. The main GUI of the application can be accessed via a white image (an ant on a white background) marked by a red line (Figure 4a). It has six important options leading to six different GUIs. The green computer button navigates to the Experiment’s interface, the yellow bell button directs users to the Feeder’s interface, the orange pyramid button routes to the Ant interface, the red twisted button provides a connection to the Quick Ant interface, the blue earth button proceeds to the Approximate Solar Calculations and the button with the image of a man in a suit is linked to the Admin interface.\n\nThe Experiment’s interface (Figure 4b), is the first and the most important module of the application, where experiment-related information needs to be entered and managed. This module asks the user to provide information about the name of the experiment, the date and time of the experimentation and any additional notes. Furthermore, it asks the user to provide geographical information about the location of the experimentation which includes the latitude, longitude and UTC time zone. It allows the user to give positioning information in degree and/or minutes. The user can update existing information by editing, delete with reference to the automatically generated ID and view the stored information in tabular form.\n\nThe Ant Feeder’s interface (Figure 4b) manages information about the used feeders during experimentation. It is important, but optional. In the GUI ‘Experiment’, the user can update Feeder’s existing information by editing, delete with reference to the automatically generated ID and view the stored information in tabular form. The stored data using Experiment and Feeder GUIs is presented in Figure 4(c).\n\nThe ‘Registration’ interface (Figure 4d) is another very important module of the application. It can only be accessed from the Ant’s interface and is used to register the ants before experimentation. It asks the user to give information (names, numbers) about used (marked) ants and to select the experiment (from the list of the experiments). Furthermore the user can update existing information by editing, delete with reference to the automatically generated ID and view the stored information in tabular form.\n\nThe Ant’s interface is divided into two modules: Ant and Quick Ant (Figure 4d). The major difference is the availability of the options, as the Ant interface allows the user to select registered ants with feeder, as well as provides options to perform data manipulation. However the Quick Ant allows the user to only select the name of the Ant from the registered ant’s list. Both interfaces have in common the provision of an additional notes field and the automatic extraction of the information (from database) about associated experiments. Note helps the user to save any additional information and experiment information to help in getting the geographical details (latitude, longitude and time zone) to calculate, save and manage the solar time and azimuth angle. The main reason for dividing Ant section into two different modules is to help speed up the experimentation process. The stored Ant and Quick Ant’s results are shown in Figure 4(d).\n\nFigure 4(a) is the Android based smart phone’s graphical user interface (Sony Xperia Z1). It also presents the main graphical user interface of the application with 6 important buttons leading to 6 different interfaces. The green computer button navigates to the Experiment’s interface, yellow bell button directs to the Feeder’s interface, orange pyramid button routes to the Ant interface, red twisted button leads to the Quick Ant interface, blue earth button proceeds to the Approximate Solar Calculations and button with a man in a suit image goes to the Admin interface. Figure 2(b) is the Experiment and Feeder’s interface, where experiment and feeder-related information is entered, managed, deleted and viewed. Figure 2(c) shows the successfully inputted experiment and feeder’s data in the database. Figure 2(d) presents the Registration, Ant and Quick Ant graphical interfaces, where unregistered Ants can be registered and their visit related information can be managed into the system. Moreover it also successfully inputs Ant’s data in the database.\n\nSolar estimations. The solar estimation is a very important section of this application, as it increases the practical value of experiments in extreme conditions, especially since there is no internet available in deserts, and the use of printed tables is time consuming. Therefore, it is nearly impossible to compute solar time and azimuth angles manually (by use of tables) in the field at each marked insect’s visit to the feeder. These calculations normally have to be done afterwards in time consuming sessions.\n\nThe solar estimation module works independently, as well as in integration with the experimental data management system. Using different astronomical algorithms33–35 it estimates approximate Gregorian Day Number, Decimal Day, Decimal Day of the Year, Fractional Year, Equation of the Time, Declination, Solar Time Offset, Solar Time Solar Zenith Angle, Solar Hour Angle, Solar Azimuth Angle and Solar noon. For any registered ant and its visits to the feeder, it will automatically extract the information about latitude and longitude from the associated experiment. Current date and time is entered automatically and saved with the information about solar timing and angles.\n\nThe user gives information about latitude and longitude, adding manual or automatic date and time information. The current Gregorian calendar day number is calculated:\n\nDay of the year = 365 * year + year/4 – year/100 + year/400 + ((month+1) * 306)/10 + (day – 62)\n\nNext fractions of a full day are considered:\n\nDecimal day = (dhour/24) + (dminutes/1440)\n\nBoth are combined for the decimal day of the year:\n\nDecimal day of the year = Day of the year + Decimal day\n\nThe Fractional Year uses PI (3.14) and hour (current time in hours):\n\nFractional Year = (2 * PI/365) * (Decimal day of the year – 1 + ((hour – 12)/24))\n\nNow the equation of time and declination are calculated:\n\nEquation of Time = 229.18 * (0.000075 + 0.001868 * cos (Fractional Year) – 0.032077 * sin (Fractional Year) – 0.014615 * cos (2 * Fractional Year) – 0.040849 * sin (2 * Fractional Year))\n\nDeclination = 0.006918 – 0.399912 * cos (Fractional Year) + 0.070257 * sin (Fractional Year) – 0.006758 * cos (2 * Fractional Year) + 0.000907 * sin (2 * Fractional Year) – 0.002697 * cos (3 * Fractional Year) + 0.00148 * sin (3 * Fractional Year)\n\nSolar time offset and solar time are estimated:\n\nSolar Time offset = 4 * (longitude – (15 * Time zone)) + Equation of Time\n\nSolar Time = hour * 60 + min + sec/60 + Solar Time Offset\n\nUsing solar zenith angle and solar hour angle (ha) the azimuth angle is estimated:\n\nSolar Zenith Angle = (sin (Latitude) * sin (Declination)) + (cos (Latitude * cos (Declination) * cos (ha))\n\nSolar Hour Angle = Solar Time * 60\n\nAzimuth Angle = atan2 (sin (ha)), cos (ha) * sin (lat) – tan (Declination) * cos (Latitude))\n\nFinally, the solar noon is calculated:\n\nSolar noon = 720 + (4 * Longitude) – Equation of Time\n\nThe workflow of the solar estimation module starts with the user given information about the latitude and longitude with manual (by the user) or automatic (from system) date and time information. At first the day of the year is estimated, then decimal day of the year, then Fractional Year, then Equation of Time, then Solar Noon Time, then Declination, then Solar Time Offset, then Solar Time, then Solar Hour Angle, then Solar Zenith and Solar Azimuth Angle. At the end all results are presented in textual format (Figure 5).\n\nThis Figure presents a sequential process for the solar calculations, where almost each process’s estimated output directly or indirectly used as the input in the calculations of the following process.\n\nThe obtained results match well with the results produced by the calculators provided by or linked with the National Oceanic & Atmospheric Administration (NOAA) (99.99% accurate Solar Time and 99.8% accurate Azimuth Angle, please see attached supplement material for more details).\n\nThe online NOAA solar calculation is however not always accessible (as is the case in desert ant observation) and time-consuming to implement (not all necessary steps are readily apparent from the NOAA web site) and consult afterwards. Our main aim was to have an easy-to-use, stand-alone application to monitor accurately the behavior of the ant together with positional and behavioral data and directly import all calculations and observations in a custom-made database.\n\nData administration. The administration module of the application provides two major options: clearing or deleting records and creating backup of data. Only authorized users can delete the records of Ants, Registrations, Feeder and Experiments (individually or all at once), by entering a security key into the system. The generated backup of the data is stored in the external (e.g. SD card) or internal storage location of the smart phone or tablet, which can be later copied, exported and reused. The exported file’s name is based on the following structure: Ant-App-DB then current date and time in the mobile system, which helps in preventing duplication or replacement of data that has already been backed up (please see Supplementary material for more details).\n\nDataplus. Dataplus is another important module of the application which helps observers in transferring the data from the smart phone application’s generated SQLite database file into Microsoft Excel format, for future use, analysis, sharing and backup using a personal computer.\n\nDataplus (Figure 6) is a desktop MDI application, designed and developed following the concepts of the Butterfly Model29 in C-sharp programming language. The application is very simple to use and install, but can only be configured using a Microsoft Windows platform.\n\nFigure 3 show that the data is exported from Ant-App-DB in SQLite database file, which is loaded in to the Dataplus module by clicking the small Ant icon button, and can then be converted into Microsoft Excel format by pressing the Excel icon button. The button with the snowflake icon is to remove the data.\n\n\nOperation\n\nAnt-App-DB is very simple to use and install but can only be configured on Android based smart phones and tablets, while Dataplus is a desktop application that can only be configured on a Microsoft Windows platform (preferred OS version: 7). Installation of Ant-App-DB is a two step process. The application can be configured and installed on smart phones and personal computers, following the instructions in the Supplementary material.\n\nAn example operational workflow of Ant-App-DB and Dataplus is presented and briefly explained in Figure 7. As shown in the Figure 7a, the observer is required to first run the application and access the different modules of the application using the main GUI.\n\nThis figure presents the real time work flow of the different modules of the application. It shows the Main (Figure 7a), experiments (Figure 7b), feeders (Figure 7c), registration of Ants (Figure 7d), approximate solar calculations (Figure 7e), Quick Ant (Figure 7f), Ant (Figure 7g, Figure 7h), Admin (Figure 7i), Dataplus (Figure 7j) and the Microsoft Excel file format “.xlsx” (Figure 7k).\n\nThe most important steps are to give details about the experiment (Figure 7b), registered ants Figure 7c and feeders Figure 7d. Before starting the experiment, the observer can also estimate the solar position and timings using Approximate Solar Calculation module (Figure 7f).\n\nLater, during the experiment process, the observer is only required to run the module Quick Ant (Figure 7f), select each marked ant at its visit and press the button ‘Plus’ sign. The module Ant (Figure 7g), also offers a similar option to Quick Ant but it is recommended to use Quick Ant to avoid any unnecessary clicks etc. The results are stored in the created database, which the observer can view those (e.g. Figure 7h).\n\nThe results data can be deleted or backed up as well using module Admin (S-Figure 21i), which can be copied to the personal computer and converted in to the Microsoft Excel format (Figure 7k) using Dataplus (Figure 7j).\n\n\nDiscussion\n\nWe have tested and validated the Ant-App-DB application by successfully executing and performing available tasks e.g. entering and storing data using the Experiment, Feeder and Registration Interfaces modules. We have also tested and validated the deleting and backing up of data using the Admin module, as well as different solar estimations using different input values (date, time, latitude, longitude and time zone). Moreover, we have compared the estimated solar results with NOAA.\n\nThe app has the capability to handle multiple users and to synchronize in real time between those users. A real time backup is another important feature. It is possible to extract, share and combine the experimental data generated during one or multiple experiments by one of the multiple users using different smart phones. The data can then be exported into Microsoft Excel format for further editing and analysis.\n\nIn future, this application can also be enhanced by adding more computation and data management features to assist the observers during experiments e.g. linking to the service (if available) of GPS to get highly accurate geographical positions, sharing data using internet service (if available), and searching data using natural language based queries. Based on the observer’s feedback we can also improve the GUI and other features.\n\nSeveral of Ant-App-DB’s features compare favorably with other currently available solutions (e.g. Etholog36, JWatcher37, Nolduss EthoVison38, Cybertracker & Animal Behavior39) for effective and efficient insect monitoring such as2–8. The most significant advantages are that it does not require any GPS and/or GIS systems, can be used on any Android based device without internet service, allows Subscriber Identity Module (SIM) card and external SD card to be used to manage the experiment’s data and enables estimates of solar positions and timings. Additionally, unlike other applications, it provides a desktop application which helps in extracting the data from the smart phone’s database and converting it to Microsoft Excel formats for further analysis and sharing. Moreover, Ant-App-DB is more user friendly that other applications as it offers a ‘One Click’ operation during the experiments at the field. Only a few steps are needed to adapt the software for other arthropods or migratory animals. For more extensive changes to the configuration a software engineer is needed. For instance if one wants to monitor flight time the application needs the integration of a suitable tracking module. This is easily integrated into the modular system (see Supplementary material) and the system is configured such that other observational modules can be easily integrated once properly programmed and tested.\n\n\nConclusions\n\nAnt-App-DB couples a database and database conversion tool with direct access and data input using a smart phone application. We have used the application in the field and have found it to be a user friendly database tool developed for behavioral research on Cataglyphis fortis, managing experimental data and calculating observation data such as solar timing and position monitoring. However, all features, software code and database as well as Dataplus application are sufficiently generic to be easily adapted to other field monitoring studies on arthropods (e.g. on honey bees, fruit fly etc.) or other migratory animals. The Ant-App-DB is available to interested non-commercial users free of charge.\n\n\nSoftware availability\n\nThe software executables are freely available at the following web link: http://www.neurogenetics.biozentrum.uni-wuerzburg.de/en/project/services/ant_app_db/\n\nThe software download section provides three files in total: Ant-App-DB’s APK file to be installed in the Android based smart phones, Dataplus’s executable setup to be installed on the Microsoft Windows platform and an example dataset (SQLite database file, generated by the Ant-App-DB application).\n\nApp-Ant-Database (DOI: 10.5281/zenodo.13223)40; Dataset Ant-App-DB (DOI: 10.5281/zenodo.13225)41; Dataplus Application (DOI: 10.5281/zenodo.13226)42.\n\nAll associated files are licensed under the Academic Free License 3.0 (AFL 3.0).", "appendix": "Author contributions\n\n\n\nZA: developed the complete solution (including database designing, software designing, programming, testing, deployment and technical documentation). SZ assisted ZA. PF tested in-house and successfully evaluated the application in fields. WR lead and TD guided the study.\n\nAll authors participated in writing of the manuscript and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDeutsche Forschungsgemeinschaft (DFG), collaborative research center SFB1047 “Insect timing”, for funding this research (to Zeeshan Ahmed, Pauline Fleischmann, Wolfgang Rössler) and TR34/Z1 for support (to Saman Zeeshan and Thomas Dandekar).\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\nWe would like to give special thanks to Dr. Ulrike Rapp-Galmiche for stylistic and native speaker corrections.\n\nWe would like to thank all our interested colleagues for critical community input on the approach. We thank anonymous reviewers for helpful comments on the manuscript, University of Wuerzburg and the State of Bavaria, Germany.\n\n\nSupplementary materials\n\nAnt-App-DB: Work Flow, UML Designs, Configuration and Calculation Steps. Click here to access the file. http://dx.doi.org/10.5256/f1000research.5931.s45712\n\n\nReferences\n\nDufau S, Duñabeitia JA, Moret-Tatay C, et al.: Smart phone, smart science: how the use of smartphones can revolutionize research in cognitive science. PLoS One. 2011; 206(9): e24974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeacher AG, Griffiths DJ, Hodgson DJ, et al.: Smartphones in ecology and evolution: a guide for the app-rehensive. Ecol Evol. 2013; 3(16): 5268–5278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilvertown J: A new dawn for citizen science. Trends Ecol Evol. 2009; 24(9): 467–471. 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Publisher Full Text\n\nSpink AJ, Tegelenbosch RA, Buma MO, et al.: The EthoVision video tracking system--a tool for behavioral phenotyping of transgenic mice. Physiol Behav. 2011; 73(5): 731–44. PubMed Abstract \| Publisher Full Text\n\nAnsell S, Koening J: CyberTracker: An integral management tool used by rangers in the Djelk Indigenous Protected Area, central Arnhem Land, Australia. Ecol Manag Restor. 2011; 12(1): 13–25. Publisher Full Text\n\nZeeshan A: App-Ant-Database. Zenodo. 2014. Data Source\n\nZeeshan A: Dataset Ant-App-DB. Zenodo. 2014. Data Source\n\nZeeshan A: Dataplus. Zenodo. 2014. Data Source" }	[ { "id": "8266", "date": "20 Apr 2015", "name": "Charles Bargeron", "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\nArticle was very good and provided the needed technical and biological information to describe the problem and solutions. However, it was unclear to me why GPS could not be used, since it does not require the need for cellular service with most devices. Also why was the data not stored on the smartphone and then uploaded to a web server when internet access was available? This would allow for data from multiple users to be combined and could be outputted to Excel without the creation of another desktop application. There may be good reasons for why both of these solutions were not considered but I didn't feel they were addressed as part of the article.", "responses": [ { "c_id": "1334", "date": "12 May 2015", "name": "Zeeshan Ahmed", "role": "Author Response", "response": "Authors > Thank you so much for your time in reviewing our manuscript and giving valuable suggestions, which all helped to further improve our manuscript. Reviewer > Article was very good and provided the needed technical and biological information to describe the problem and solutions.Authors > Thank you so much for appreciating our work and we agree with you. Reviewer > However, it was unclear to me why GPS could not be used, since it does not require the need for cellular service with most devices.Authors > You are absolutely right in your point. In general, the known GPS coordinates of the location where the insects are studied can be used as an input to the system. However, our point was that this application can be used without online GPS service. We have tried to contribute for such cases, where scientists/observers/users do not have a GPS facility with them due to any reason, e.g. if it is costly, not compatible to the in use devices, not providing the spatial resolution needed, etc. Following your suggestion, we have highlighted this in our revision. Reviewer > Also why was the data not stored on the smartphone and then uploaded to a web server when internet access was available? This would allow for data from multiple users to be combined and could be outputted to Excel without the creation of another desktop application. There may be good reasons for why both of these solutions were not considered but I didn't feel they were addressed as part of the article.Authors > You are absolutely right and in fact it is possible. Data is stored inside the Smartphone memory and, potentially, can be shared via internet access, USB connection, Bluetooth etc. Moreover, it is also possible to combine multiple data files. We have not implemented direct data sharing options in our smart phone application because our data, which is stored in the internal or external memory card, can easily be transferred or shared using existing Android smart phone features. Following your suggestion, we have highlighted this in our revision. Authors > Thank you so much for your suggestions, we are keeping these for our future development tasks." } ] }, { "id": "8452", "date": "24 Apr 2015", "name": "Harald Wolf", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents an interesting and potentially powerful new tool for monitoring behavioral data on small animals, particularly arthropods in field experiments. This includes remote areas without internet or situations without GPS access. The basic idea and the present implementation appear useful indeed for field studies on animal navigation and related topics. It is a major advantage that the modular structure of the application should allow expansion towards related applications and the implementation of additional features, depending on the particular user requirements.There are, however, a few shortcoming, most of them minor, but also a couple of more serious ones. I shall list them below, roughly arranged in the sequence of their importance: Data input: While the text explains in some detail the modular structure of Ant-App-DB and the basic function of the different modules, it remains mostly unclear to me what kind of input data the application would accept. Perhaps the most important features when studying arthropod activity are track recordings, or the monitoring of locomotor paths. Can such data be recorded with Ant-App-DB? Or is it just single coordinates in space and time? Or is it simply numbers that represent something the researcher may be interested in, such as walking distance, walking times etc.? Considerable effort is spent to explain, for example, the solar estimations. This is good, particularly for the uninitiated reader and naturally for research topics where solar azimuth and similar data are important. It would be highly desirable to spend similar effort on the explanation of data recording itself and the different options that exist and possible simple expansions that may be possible. Parts of the text are written in good English, quite crisp and altogether clear. Other parts of the text, by contrast, suffer from a dense style with long-winding sentences and some unintelligible sentence structure. Together with the occasional lab jargon or computer speak, this makes parts of the text somewhat difficult to read and understand. The English language is not always correct, despite native English speaker correction at some point in manuscript evolution. Hyphenation is just one example here. Typographic errors may also fall into this category; e.g. figure numbers in legend of fig.4 are consistently wrong.", "responses": [ { "c_id": "1333", "date": "12 May 2015", "name": "Zeeshan Ahmed", "role": "Author Response", "response": "Authors > Thank you so much for your time in reviewing our manuscript and giving valuable suggestions, which all helped to further improve our manuscript. Reviewer > This manuscript presents an interesting and potentially powerful new tool for monitoring behavioral data on small animals, particularly arthropods in field experiments. This includes remote areas without internet or situations without GPS access. The basic idea and the present implementation appear useful indeed for field studies on animal navigation and related topics. It is a major advantage that the modular structure of the application should allow expansion towards related applications and the implementation of additional features, depending on the particular user requirements. Authors > Thank you so much for appreciating our work and we agree with you.Reviewer > There are, however, a few shortcoming, most of them minor, but also a couple of more serious ones. I shall list them below, roughly arranged in the sequence of their importance:Authors > Thank you so much for raising minor and major points. Reviewer > Data input: While the text explains in some detail the modular structure of Ant-App-DB and the basic function of the different modules, it remains mostly unclear to me what kind of input data the application would accept. Perhaps the most important features when studying arthropod activity are track recordings, or the monitoring of locomotor paths.Authors > Our application helps in recording the information about individually color-marked ants (three dot color code), their visits to the feeders and the respective solar estimations. It does not help in the monitoring of locomotor paths. However, it can also be used with the placement of multiple feeders, for example to track at which locations the ant was. Reviewer > Can such data be recorded with Ant-App-DB? Or is it just single coordinates in space and time? Or is it simply numbers that represent something the researcher may be interested in, such as walking distance, walking times etc.?Authors > You are right, it records only coordinates and numbers of individually registered ants, but we are keeping this as a very valuable suggestion by you for our future work. Due to the time and resource constrains, we cannot implement sophisticated locomotion path tracking features now. This certainly is an interesting aspect for future developments as we point out in the discussion. Reviewer > Considerable effort is spent to explain, for example, the solar estimations. This is good, particularly for the uninitiated reader and naturally for research topics where solar azimuth and similar data are important.Authors > Thanks and we agree with you. Reviewer > It would be highly desirable to spend similar effort on the explanation of data recording itself and the different options that exist and possible simple expansions that may be possible.Authors > We totally agree with you. As the scope of this paper was limited to the software/tool presentation (category: Software Tool Article), we did not provide detailed explanations of biological experimental procedures. We appreciate your suggestion and certainly will implement this in future reports on biological applications. Reviewer > Parts of the text are written in good English, quite crisp and altogether clear. Other parts of the text, by contrast, suffer from a dense style with long-winding sentences and some unintelligible sentence structure. Together with the occasional lab jargon or computer speak, this makes parts of the text somewhat difficult to read and understand.Authors > Thanks for the nice suggestion and we agree with you. We have revised and tried to improve the content presentation. Reviewer > The English language is not always correct, despite native English speaker correction at some point in manuscript evolution. Hyphenation is just one example here. Typographic errors may also fall into this category; e.g. figure numbers in legend of fig.4 are consistently wrong. Authors > Thanks for the suggestion and we agree with you. We have revised and improved the content presentation as well as corrected Figure 4 legend." } ] }, { "id": "8339", "date": "27 Apr 2015", "name": "David White", "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\nThe overall technical implementation of the software appears to be sound. I am not familiar with arthropod marking and tracking methods, thus the article could be improved by briefly outlining these methods in the introduction as to better align with the purpose/design of the Ant-App-DB. The paper does adequately explain methods and provides description of the underlying software framework through the use of figures and descriptions.There are some issues that the authors need to address before indexing:\"dot Net framework\" = .NET Framework Figure 4 caption needs to be reviewed as it has several typos \"Figure 2(b), Figure 2(c) etc...\" Figure 7 is difficult to read. It may be better to print on an entire page. What is \"(S-Figure 2li)\" pg. 10 The discussion reads as a rough draft as there are several run-on sentences and typos. For example, \"Several of Ant-App-DB's features compare favorably with other currently available solutions........ efficient insect monitoring such as2-8.\" Is not a good style. Providing some examples of how these systems all compare would strengthen this discussion. see pg. 12. The authors need to be careful with their subjective statements such as \"Moreover, Ant-App-DB is more user friendly that (typo) other applications.....\" See critique #5. A more thorough review of existing systems is really needed if you are going to make these statements. pg. 12.", "responses": [ { "c_id": "1332", "date": "12 May 2015", "name": "Zeeshan Ahmed", "role": "Author Response", "response": "Authors > Thank you so much for your time in reviewing and approving our manuscript, and giving valuable suggestions, which all helped to further improve our manuscript. Reviewer > The overall technical implementation of the software appears to be sound. Authors > Thank you so much for appreciating our work. Reviewer > I am not familiar with arthropod marking and tracking methods, thus the article could be improved by briefly outlining these methods in the introduction as to better align with the purpose/design of the Ant-App-DB.Authors > Thanks for the nice suggestion and we agree with you. We have tried to revise it accordingly. The desert ants that we describe as an example for the use of the applications were individually marked with a three dot color code to be able to register individual ants at different locations and solar times. This information now is included at various positions in the text. Reviewer > The paper does adequately explain methods and provides description of the underlying software framework through the use of figures and descriptions.Authors > Thanks and we agree with you. Reviewer > There are some issues that the authors need to address before indexing:Authors > Thanks for pointing these issues and we have tried to clarify these in revision. Reviewer > \"dot Net framework\" = .NET FrameworkAuthors > You are right, and we have corrected it.Reviewer > Figure 4 caption needs to be reviewed as it has several typos \"Figure 2(b), Figure 2(c) etc...\"Authors > You are right, and we have corrected it.Reviewer > Figure 7 is difficult to read. It may be better to print on an entire page.Authors > You are right, and we have provided new Figure with better resolution.Reviewer > What is \"(S-Figure 2li)\" pg. 10Authors > You are right, it’s a mistake and we have corrected it by “Figure 7i”.Reviewer > The discussion reads as a rough draft as there are several run-on sentences and typos. For example, \"Several of Ant-App-DB's features compare favorably with other currently available solutions........ efficient insect monitoring such as2-8.\" Is not a good style. Providing some examples of how these systems all compare would strengthen this discussion. see pg. 12.Reviewer > The authors need to be careful with their subjective statements such as \"Moreover, Ant-App-DB is more user friendly that (typo) other applications.....\" See critique #5. A more thorough review of existing systems is really needed if you are going to make these statements. pg. 12. Authors > You are right and we have revised it." } ] } ]	2	https://f1000research.com/articles/3-311
https://f1000research.com/articles/4-113/v1	12 May 15	{ "type": "Research Article", "title": "Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.", "authors": [ "Benjamin N. Rollo", "Dongcheng Zhang", "Johanna E. Simkin", "Trevelyan R. Menheniott", "Donald F. Newgreen", "Benjamin N. Rollo", "Dongcheng Zhang", "Johanna E. Simkin", "Trevelyan R. Menheniott" ], "abstract": "The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates. This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface.", "keywords": [ "Neural crest", "intestine", "enteric nervous system", "cell adhesion", "gangliogenesis", "quail", "chick" ], "content": "Introduction\n\nThe enteric nervous system (ENS) is derived from the neural crest (NC), a population of migratory mesenchyme cells originating in the dorsal neural tube. Most of the ENS arises from the caudal hindbrain (vagal) NC (Yntema & Hammond, 1954), chiefly from the level of somites s3 to s5 (Epstein et al., 1994). These cells migrate to the nearby foregut, changing en route (Simkin et al., 2013) to become enteric NC (ENC) cells which are capable of exploiting the gut mesoderm. ENC cells migrate in the mesoderm along the midgut and hindgut to colonise the entire gastro-intestinal tract. This migration takes the form of intersecting narrow chains of motile ENC cells (Druckenbrod & Epstein, 2005; Epstein et al., 1991; Young et al., 2004; Young et al., 2014). Later the ENS comprises a network of numerous small, closely-spaced ganglia with many types of neurons and glia, with each ganglion connected via neurites and glial cells to other ganglia and to the smooth muscle and the mucosa (Conner et al., 2003; Epstein et al., 1991; Fairman et al., 1995). This distributed ENS network controls peristalsis as well as other gut activities (Furness, 2012).\n\nDevelopmental disorders of the structure, size and organization of the ENS ganglia have consequences for ENS function. Hypoganglionosis (fewer, smaller ganglia) is associated with persistent constipation, and mice heterozygous for the neurotrophic factor GDNF are hypoganglionic (Flynn et al., 2007). Defects involving an over-abundance of ENS cells and disturbance of their distribution are also known; hyperganglionosis in the form of enteric ganglioneuromas occurs in multiple endocrine neoplasia type 2 (MEN2B) syndrome as a result of constitutive activation of RET (the receptor for GDNF on ENS cells) and is accompanied by dysfunction of the ENS (Takahashi et al., 1999). Mouse Zic2 mutants show an increased number of ENS neurons (Zhang & Niswander, 2013). Other ENS disorganizations have also been described. In mice where HAND2 is knocked out in ENS cells, the migration of ENC cells is not impaired but segregation into ganglia is abnormal (D'Autreaux et al., 2007; Lei & Howard, 2011). Mice with NC-specific knockout of β1-integrin show ENS ganglia which are morphologically different from normal (Breau et al., 2006).\n\nHow ENC cell chain migration evolves into a ganglionated network, and how this is disturbed in some pathologies, is not well understood. NC derivatives elsewhere form relatively large ganglia, such as the dorsal root ganglia (DRG) and sympathetic ganglia. In the forming DRG and sympathetic ganglia, early differentiating neurons occupy the centre of cell aggregates with NC cells or glioblasts surrounding this core. This segregation is maintained in part by Notch signalling which suppresses neuronal differentiation in the peripheral cells (Tsarovina et al., 2008; Wakamatsu et al., 2000). DRG and sympathetic ganglia are in stereotyped positions which are clearly related to and dependent on segmentally spaced cues from their mesodermal microenvironment (Teillet et al., 1987). Formation of each the NC-derived sympathetic ganglia, for example, relies on the scattered NC cells self-aggregating, driven innately by increased N-cadherin homophilic adhesion. This is combined with growth factor and cytokine attraction and repulsion from a patterned microenvironment to provide the positioning of each of the ganglia (Kasemeier-Kulesa et al., 2006; Kasemeier-Kulesa et al., 2010). The size of the ganglia is also regulated. In the DRG, experimental NC overload and NC ablation suggest that the initial size of the ganglia reflects both the number of NC cells that give rise to them, together with early proliferation. The final size of the ganglia is adjusted in normal and NC cell overload conditions by population reduction by programmed cell death, due at least in part to logistic competition for survival factors. Conversely in NC cell under-supply conditions, compensatory mitosis occurs to increase the DRG cell population (Barde, 1994; Kalcheim et al., 1987; Zarzosa et al., 2014).\n\nThe final ENS cell population is enormous but ENS ganglia are each very small and are located mainly in two narrow layers associated with the intestinal smooth muscle layers, the myenteric ganglia between the longitudinal and circular muscle layers and the sub-mucosal ganglia internal to the circular muscle. Typically migratory ENC cells occupy the myenteric layer early, prior to visible structural or molecular correlations including smooth muscle differentiation (Newgreen & Hartley, 1995). A spatial association of the early ENS with the pre-existing intestinal vascular layer has been suggested but this is controversial (Delalande et al., 2014; Hackett-Jones et al., 2011; Hatch & Mukouyama, 2015; Nagy et al., 2009; Schrenk et al., 2015; Young et al., 2004). The sub-mucosal ENS cells originate later from this outer layer by centripetal migration (except in the avian hindgut), a process involving the response of DCC-expressing ENC cells to netrin produced by the endoderm (Jiang et al., 2003).\n\nCues governing the size, shape and location of each ENS ganglion are not well understood, but are clearly reliant on molecules from their local mesoderm microenvironment. With different-sized starting populations of ENC progenitors, resultant ENS ganglia achieve a similar, small size (compared to DRG) and a similar density of distribution (Allan & Newgreen, 1980; Zhang et al., 2010), suggesting some regulatory ability. Cell death is of great importance in most of the nervous system but is slight in the ENS (Chalazonitis et al., 2012). However, it does occur normally in the early ENS-fated population, at and before gut colonisation, well before the ganglia form. Inhibition of this early cell death leads later to an increase in the ENS cell population in those regions of the gut that are first colonised, and this increase takes the form of more densely distributed ganglia, but significantly the ganglia are of normal size and shape (Wallace et al., 2009).\n\nThe early ENS is strongly active mitotically (Simpson et al., 2007) in response to growth factors from the local environment. In particular the gut mesoderm cells produce GDNF (glial cell line-derived neurotrophic factor). GDNF is a survival factor and also initially a mitogen (Hearn et al., 1998) and chemotactic factor (Young et al., 2001) via its receptor RET on ENC cells. Later however, its role changes to an inducer of neuronal differentiation (which reduces proliferation) and axon growth. Down-regulation of GDNF during migration not only reduces ENC proliferation but also triggers premature neuronal differentiation (Mwizerwa et al., 2011). Endothelin-3 (ET-3) is also important at early stages, via its receptor EDNR-B on ENC cells. ET-3 is thought to dampen the differentiation response to RET activation, thereby prolonging the proliferative phase of ENC cells (Hearn et al., 1998; Wu et al., 1999) and aiding distal intestinal colonisation (Nagy & Goldstein, 2006). The mesodermal factor BMP2/4 is also important in ENS morphogenesis and differentiation. However, the ENS cell proliferation and neuronal differentiation response to BMP2/4 signalling by ENS lineage cells is complex, and may be influenced by factor concentration and by the time of exposure to alter proliferation and differentiation (Chalazonitis et al., 2004). Early BMP inhibition impairs aggregation of ENC cells into ganglia and leads to hypoganglionosis (Goldstein et al., 2005). BMP also has an effect on enteric gliogenesis, probably by priming ENC cells to respond to factors including Glial Growth Factor-2 (Chalazonitis et al., 2011). Furthermore, BMP increases the number of smooth muscle cells which are a major source of GDNF. In addition, BMP promotes polysialylation of NCAM on ENS cells which likely down-modulates cell-cell adhesivity (Faure et al., 2007); this may favour morphogenetic movement of ENS cells to allow structure changes such as establishment of the sub-mucosal plexuses.\n\nThe morphogenesis of the ENS is affected not only by mesoderm-derived soluble factors but also by structural elements such as extracellular matrix (ECM) (Newgreen & Hartley, 1995). The shape, size and pattern of ganglia can be altered by manipulation of ECM adhesion properties. NC-restricted loss of β1-integrin receptor for ECM adhesion leads to larger, rounder, sparser and abnormally patterned ENS ganglia (Breau et al., 2006). It is assumed that a major β1-integrin ligand is the ECM adhesive molecule fibronectin. Interestingly, the morphogenetic disturbance of ENS gangliogenesis caused by genetic ablation of β1-integrin can be partially corrected by simultaneous deletion of the cell-cell adhesion molecule N-cadherin (Broders-Bondon et al., 2012).\n\nENS neurons, glia and ENC cells show differential labelling for various cell-cell adhesion molecules, and also differ from their surrounding mesodermal cells (Hackett-Jones et al., 2011; Nagy et al., 2012). Here we describe cell-cell adhesion molecules in the ENS, and the roles of cell-cell adhesion in aggregation tests in vitro. In particular we wished to explore why the ENS ganglia are similar and small in size and why the initial cell disposition in ganglia has the pattern of central neurons surrounded by ENC progenitor cells.\n\n\nMaterials and methods\n\nFertilised quail (Coturnix japonica) and White Leghorn/Black Australorp cross chicken (Gallus gallus domesticus) eggs were obtained respectively from Lago Game Supplies and Research Poultry Farm, Vic., Australia. Eggs were incubated at 38°C in a 60% humidity incubator. Embryos were staged according to the number of embryonic days (E) and Hamburger and Hamilton stages (HH) (Hamburger & Hamilton, 1951). Animal ethics permission was obtained for the Royal Children’s Hospital Animal Ethics Committee, AEC677.\n\nThe midgut (defined as the intestine caudal to the stomach to rostral to about half way along the caecum) was removed from quail (Q) embryos at half-day intervals from QE4.5 (HH25) to QE8 (HH34) and then at intervals to QE14 (HH43). These were fixed from times varying from 1 h to overnight in 4% PFA. Antigen retrieval of fixed specimens employed 10 mM citrate buffer pH 6 for 20 minutes at 95°C. Specimens were washed in phosphate-buffered saline (PBS) for 10 minutes, then blocked and permeabilised overnight with 1% horse serum (CSL, Melb., Aust.) and Triton X-100 (Sigma-Aldrich, USA) at 0.1% in PBS. These were then incubated for 1–2 days at 4°C sequentially in primary and secondary antibodies (see Supplemental Table T1) prepared in blocking/permeabilising solution. Between treatments, the specimens were washed extensively in PBS. Gut tissue and aggregate specimens were mounted in Vectashield antifade reagent (Vector Laboratories, Inc., CA, USA) between two coverslips with coverslip spacers.\n\nFour to eight areas of 100×100 μm were selected along the midguts from QE4.5 to QE8.5 and cell counts of Hu+ve and SoxE+ve cells were made from optical sections of the myenteric plexus. Cells outside this layer, such as submucosal ENS cells, were not counted.\n\nThe midgut was removed from QE5 (HH27) and from QE8 embryos and 9-day chick embryos (ChE9), about HH34-35 (Supplemental Figure S1). The intestinal tissue pooled from 15–60 embryos was digested for 35 minutes at 37oC in Ham’s F12 media (Gibco Cell Culture, Invitrogen, USA) with 0.5% w/v Dispase II (Roche, USA) and 0.05% w/v CLSAFA Collaganase (Worthington, USA). To disrupt cadherin-based cell interactions ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, USA)) was added to a concentration of 1 mM for a further 10 minutes. The tissue was mechanically triturated and the cell suspension was washed in F12 media with 5% BSA. Cells from mid-trunk dorsal root ganglia (DRG) from the QE8 embryos were dissociated in the same way.\n\nIntestinal and DRG cells were labelled in suspension with mouse anti-HNK-1 IgM antibody (1/50 volume of supernatant; hybridoma maintained at MCRI) followed by secondary labelling with goat anti-mouse IgMμ Alexafluor 488 antibody (Supplemental Table 1). In some cases mouse anti-NCAM was also included followed by goat anti-mouse IgG Alexafluor 647 (see Supplemental Figure S2). Cells were filtered through a 30 μm strainer (BD-Falcon, USA) and propidium iodide (Sigma-Aldrich, USA) was added (final concentration 10 μg/ml) to detect dead cells. Cells positive for Alexa 488 fluorescence which also excluded propidium iodide were sorted using a MoFlo cell sorter (MoFlo, USA). About 2–3% of the dissociated QE5 and 5–10% of QE8/ChE9 midgut cells were selected by this process (Supplemental Figure S2). Each QE5 midgut segment provided 1900–2900 HNK-1+ve cells (range from 4 runs, total 268 midgut segments). Each QE8 gut segment yielded about 40–50,000 HNK-1+ cells (range from 4 runs, total 128 midgut segments), and the ChE9 about 50–60,000 HNK-1+ cells (range from 2 runs, total 33 midgut segments). HNK-1 FACS of QE8 DRG as expected produced a yield of >70% of the dissociated cells being HNK-1+ve.\n\nAfter selection by FACS, HNK-1+ve and HNK-1-ve cells (as well as unselected cells) were examined by q-PCR for the NC marker Sox-10 and the neuronal marker Hu-D, and by cell culture.\n\nTotal RNA was isolated from cells using the RNeasy mini kit (Qiagen, USA) and contaminant genomic DNA removed with DNA-free reagents (Ambion, USA). Primer sequences were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) and are listed below.\n\nPrimer sequences\n\nGAPDH: 5’-TTATCATCTCAGCTCCCTCAGC-3’, 3’-AAGTTGTCATGGATGACCTTGG-5’;\n\nSOX10: 5’-AGGAAATTGGCTGACCAGTACC-3’, 3’-GTCCTTCTTGTGCTGCATCC-5’;\n\nHU-D: 5’-ACAGATGACAGCAAAACCAACC, 3’-ATTTTGTCTCTCACGAGCTTGC-5’.\n\nFor quantitative reverse transcription and polymerase chain reaction qRT-PCR, oligo-dT primed cDNA was synthesised from 200 ng total RNA using Murine Moloney Leukaemia Virus reverse transcriptase (Promega, USA). qRT-PCR was performed on an ABI Prism® 7500 Real Time PCR System using SYBR green master mix (Applied Biosystems, USA) according to the manufacturer’s protocols. Relative gene expression values were obtained by normalization to the reference gene GAPDH using the −2ΔΔCt method, where −2ΔΔCt = ΔCt sample−ΔCt calibrator as described (Peterson et al., 2010). All fold changes were calibrated to the negative sort population. Results are shown in Supplemental Figure S3.\n\nFor cell culture substrates, HLA Terasaki-plates (10 μl wells; Greiner Bio-One, Sigma M6062) were coated with human plasma fibronectin (FN; 20 μg/ml in PBS, 2 h; Roche 11051407001) or rat laminin-1 (LN; 50 μg/ml in PBS, 2 h; Roche 1124321700). HNK-1 and NCAM FACS-selected quail E8 midgut ENS cells were plated at 3000 cells/well into the above wells in Ham’s F12 with 1–10% heat inactivated fetal calf serum (FCS; Thermo-Fisher, USA) plus 0.5% BSA, and penicillin/streptomycin (pen/strep; Sigma-Aldrich, USA). In addition, the HNK-1-ve cells were also plated in the same way. Cells were fixed and immunolabelled at 18 h to 66h in vitro, as for gut wholemounts, except that Terasaki cultures were not antigen retrieved (see Supplemental Figure S4).\n\nBoth q-PCR and culturing of FACS sorted cells indicated that the HNK-1-based sorting accurately selected for virtually all cells in the gut that expressed NC and neuronal markers.\n\nAfter cell dissociation and FACS analysis, the HNK-1+ve cells remained for 1 h at 37°C in cell culture medium of F12 with 2% heat inactivated FCS, 0.5% BSA and pen/strep, to recover cell-cell adhesive potential (Steinberg et al., 1973; Takeichi, 1977). A low degree of aggregation occurred in the recovery period, indicated by a 6–15% reduction in particle number from the count recorded at the time of FACS. In three dissociation runs Calcein AM (1/4000; Invitrogen/Molecular Probes) was added for 20 min to reveal live cells, with the cells then centrifuged into fresh medium. This indicated that about 90% of cells were alive at this stage. The cell suspension was then aliquoted into Eppendorf tubes (100 μl cell suspension/tube) with particle density adjusted to 0.3–0.5×106 cellular particles/ml. A minimum of three replicate tubes were prepared for each assay, and each assay was repeated at least twice. In parallel tubes ethylene glycol tetraacetic acid (EGTA) was added to 1 mM; this chelates Ca2+ and therefore prevents cadherin-dependent cell-cell adhesions. The tubes were then incubated on a rotating platform (120 rpm, radius 1 cm) at 37°C. At t=0, 15, 30, 60 and 120 min., 10 μl samples were withdrawn from each tube and the particles were counted (see below). A particle was defined as a single cell or group of contacting cells of any size. Cell aggregation was indicated by a decreasing particle number.\n\nCells were allowed to aggregate as above, but with aggregation time of 2h, 4h, 6h, 18h and 48h. Rotation rates of 150, 120, 100, 75 and 0 rpm were tested. Starting cell densities were varied from 0.167×106 to 1.0×106 particles/ml. Aggregation assays were also performed with the inclusion of BrdU (Amersham-GE Healthcare, USA) for 4 h prior to fixation. At the end of the incubation period, aggregates were collected for imaging and image measurement by allowing them to settle in the Eppendorf tube for 5 min then removing the bottom 20 μl of medium plus aggregates. This was placed as a standing drop on a non-TC Petri dish which was oscillated at 80 rpm for 5 minutes to centralise the aggregates, which were then imaged. For fixation, 200 μl of 4% PFA in PBS was added to each Eppendorf tube. After overnight fixation, the fixative was washed out with PBS prior to immunolabelling as for midgut wholemounts.\n\nSamples were screened using an Olympus IX70 microscope (Olympus Optical Co., Tokyo, Japan), under selective Texas Red, FITC and AMCA filters, and by phase contrast. Images were recorded using a Spot Monochrome camera model 2.1.1 with Image-Pro Plus 4.5 (MediaCybernetics, Silver Spring, MD, USA). Confocal imaging was prepared on a Leica TCS SP2 with image processing via Leicalite and Image-Pro–Analyser 6.1 (MediaCybernetics). For short-term aggregation assays, particles (cells and cell groups) at each time point were counted in a haemocytometer chamber with 10–20 microscope field images each of 1.14 mm2 recorded using an Olympus IX70 microscope (Olympus Optical Co., Tokyo, Japan) with 10× objective. Particle counts were made from these images by operators blinded to the assay conditions. For the long term aggregation assays, aggregate diameters were measured from phase contrast images (×20 objective) of at least 50 aggregates per treatment and time. Aggregates were chosen for measurement on the basis of roundness and defined edges, and very small and or loose cell clusters and single cells were ignored.\n\nUnless specified, data were expressed as mean± standard error of mean (SEM). All statistical tests were performed using GraphPad Prism version 6. A difference between two groups was determined using a two-tailed Student’s t-test and for nonparametric data Mann-Whitney test was used. For differences among multiple groups, statistical comparisons were performed using one-way analyses of variance (one-way ANOVA) followed with Fisher’s LSD post-test. A p-value of <0.05 was considered significant.\n\n\nResults and discussion\n\nThe sparse ENS cell population in the nascent myenteric plexus of the midgut was dominated by SoxE+ve ENC cells. The total ENS cell density (cells/unit plexus area) continued to increase over the period QE4.5 to QE8 (Figure 1) and the plexus area increased by exponential gut growth (Binder et al., 2008). From about QE6, correlating with the assembly of ENS cells into coherent groups, the proportion of Hu+ve neurons increased to reach a ratio of 1.2:1 Hu+ve: SoxE+ve cells. This ratio was maintained until at least QE8 (Figure 1). The constancy of the ratio of Sox+ve cells to Hu+ve cells while the population number and density increased suggests an effective co-ordinate control between neurons and ENC cells.\n\nA. The density (number per 100×100 μm area) of neurons (Hu; red), ENC cells (SoxE; blue) and total ENS cells (Hu plus SoxE; purple) increases from QE4.5 to QE8. B. The ratio of neurons to ENC cells stabilises by E6. Error bar=SEM.\n\nInhibition of Notch activity in mice by NC cell-specific knockout of the Pofut1 gene, and Notch inhibition by DAPT in mouse and human enteric neurospheres in vitro (Okamura & Saga, 2008; Theocharatos et al., 2013) led to loss of Sox10+ve ENC cells and a bias towards enteric neuron differentiation. This strongly suggests that after cell aggregation has been achieved by morphogenetic cell re-arrangements, the Notch system forms part of the intercellular signaling agency maintaining the ENC cell/neuron balance in the developing ENS.\n\nIn the quail embryonic midgut at HH27 (QE4.5 to 5), shortly after arrival of vagal ENC cells at HH25/6 (QE4.25), the relatively sparse ENS cell population was mainly SoxE+ve ENC cells (Hu-ve) distributed in chains (Figure 2A). The number of Hu+ve neurons (SoxE-ve) increased in the nascent myenteric plexus and they commenced forming clusters by QE6 (Figure 2B). Later (QE8), the Hu+ve cells and the SoxE+ve cells formed co-aggregates with almost all the SoxE+ve cells segregated to the periphery of each neuronal cluster (Figure 2C). The ENS cell aggregates increased in size and developed increasingly smooth borders and by QE14 the SoxE+ve cells were found not only surrounding the neuron groups but also between the individual neurons, as well as along axon tracts (Figure 2D). In other NC-derived ganglia like the DRG a similar sequence of events, but chronologically earlier, has been observed, with the late-appearing intraganglionic cells being differentiated ganglionic glia (Henion et al., 2000).\n\nA. QE5 midgut with chains of SoxE+ve ENC cells and a smaller number of scattered Hu+ve neurons. B. QE6 midgut with relatively more neurons in small groups, with adjacent ENC cells. C. QE8 midgut with coherent ENS neuron groups surrounded by SoxE+ve cells. D. QE14 midgut with large ENS ganglia with SoxE+ve cells both around the ganglia and in the ganglia mixed with the neurons, and also distributed along interganglionic tracts. Images are single confocal optical sections through the myenteric plexus.\n\nWe sought an explanation in differential cell-cell adhesion (Steinberg, 2007) for the progressive aggregation of ENS cells into ganglia, with internal neurons and a shell of ENC cells. Time-lapse microscopy in mouse intestine has revealed that ENS cells (both neurons and ENC cells) are motile for a considerable period after the initial colonization phase (Hao et al., 2009; Young et al., 2014). It can be imagined that such motile ENS cells might, by differential adhesion, finally collect together to form few very large ganglia; we therefore also sought reasons for the ENS forming only small ganglia.\n\nUnsorted dissociated midgut cells showed modest NC (Sox10) and neuronal markers (Hu) by q-PCR. The HNK-1+ve sorted moiety showed high levels of these neural sequences whereas the HNK-1-ve moiety had very low expression (Supplemental Figure S3). HNK-1+ve sorted cells plated on fibronectin or laminin surfaces showed neural immunoreactivity, including SoxE (recognizes Sox9 and Sox10), HNK-1, HuC/D, Tuj1 and E-C8. A few cells (<2.5% at 18 h in vitro) were negative for neural markers but fibroblast-like in appearance and smooth muscle actin (SMA)+ve. We regard these as contaminating gut mesoderm cells. In contrast HNK-1-ve cells when plated were virtually entirely fibroblast-like in appearance and SMA+ve (Supplemental Figure S4). This cell sorting procedure therefore provides highly enriched ENS cells for performance of cell aggregation assays.\n\nDissociated HNK-1+ve midgut ENS cells in low serum aggregation assays rapidly formed clusters which were relatively small and uniform. We examined ENS cell aggregates (N=7) at 22 h with confocal microscopy after 4 h bromodeoxyuridine (BrdU) exposure, and examined 18 h aggregates (N=6) with phosphohistone-H3 antibody and detected no cells labeled by these markers of proliferation. We conclude that there was little or no cell proliferation in vitro, and therefore the cell clusters under these conditions are due to cell aggregation.\n\nAvian ENS cells in previous studies showed immunoreactivity for N-cadherin and NCAM and also for Ng-CAM (L1CAM) at the stages equivalent to the early stage shown above (Hackett-Jones et al., 2011; Nagy et al., 2012). Ng-CAM soon became almost undetectable while N-cadherin and NCAM labelling became more intense on both SoxE+ve and Hu+ve cells. By QE8 in the midgut NCAM was clearly more strongly labelled on the Hu+ve neurons compared to the SoxE+ve cells (Hackett-Jones et al., 2011). This suggests that there may be a general increase in cell-cell adhesion in the ENS and a further increase in adhesion between neurons.\n\nThe short-term rotating cell aggregation assays indicated that dissociated QE5 and QE8 midgut ENS cells adhered progressively (Figure 3A, Figure 4A–D), but aggregation was faster and more complete in cells from the older embryos. This confirms a developmental increase in ENS cell cohesion. QE5 and QE8 ENS cell aggregation occurred over the first 30 minutes at the same rate with Ca2+ chelation (i.e. 1 mM EGTA) as with normal medium but later the level of cell aggregation was impaired (Figure 3B, C, Figure 4E). The sensitivity to EGTA showed involvement of Ca2+-dependent (i.e. cadherin) mechanisms, but the residual aggregation indicated Ca2+-independent mechanisms (such as NCAM) were operative as well, and suggests that the initial phase of adhesion in these conditions was largely due to Ca2+-independent adhesion. This is in accord with the immunoreactivity for both N-cadherin and NCAM in situ noted above and in these cells in vitro (Figure 6A, B).\n\nAggregation was indicated by decrease in particle count and is expressed as % of time t=0 min. A. QE5 and QE8 ENS cells aggregated continuously, with particle count at each time point significantly less than at the previous time point (0 min vs 15 min, 15 min vs 30 min, 30 min vs 60 min and 60 min vs 120 min) (◊◊◊ p<0.001 for QE5; # p=0.0455, ### p<0.001 for QE8), except for QE5 at 60 min vs 120 min, where the particle counts were not significantly different. In addition, aggregation was greater for QE8 ENS cells compared to QE5 ENS cells (* p<0.001 QE8 vs QE5 at each time point), consistent with a developmentally increasing adhesive capacity. B. With EGTA, early aggregation (0 min, 15 min and 30 min) of QE8 ENS cells proceeded rapidly and was not significantly different from particle counts in control medium at the same time points. At later time points (60 min and 120 min) particle counts with EGTA were significantly greater than from the same time points in control medium (θθθ p<0.001 EGTA vs Ctrl). This strongly indicates that early aggregation events in these assays are largely calcium-independent but after about 30 min aggregation is dependent on cadherin function. C. QE5 ENS cells showed a similar early rate of decline in particle number with EGTA medium at matched time points of 0 min, 15 min and 30 min. Later, particle number decline decreased in EGTA (φφφ, p<0.001 EGTA vs Ctrl at 60 min; φ p=0.0225, EGTA vs Ctrl at 120 min;). This indicates that for QE5 ENS cells early aggregation is largely calcium-independent but further aggregation requires cadherin function. Error bar=SEM.\n\nThe earliest stage of aggregation for HNK-1+ve E8 quail (and E9 chick) midgut ENS cells was as small clumps and strings, at about 2 h in rotating culture. They formed spheres by 4 h, and initially cells bulged from the surface of spheres (resembling a “bunch of grapes”) but the spheres became more smooth-surfaced, and maintained this from 18 h to 48 h (Figure 4A–D). The diameter of the aggregates increased between 4 h and 48 h (Figure 4D, Figure 5A) and the range of diameters recorded became wider (Supplemental Figure S5) but aggregation into a few huge cellular masses did not occur. ENS cells from quail and chick behaved identically in these assays (Figure 5B).\n\nENS cells at 0 h (A), 4 h (B), 18 (C) and 48 h (D) show rapid aggregation without the later formation of super-aggregates. The importance of cadherins is shown by Ca2+-chelation with 1 mM EGTA, which reduced aggregate formation at 18 h (E). F–H. Altering the rotation speed (0 rpm (F), 75 rpm (G) and 150 rpm (H)) had only slight effect on aggregation at 18 h. I–L. Increasing the initial ENS cell density (0.167×106 cells/ml (I), 0.33×106 cells/ml (J), 0.67×106 cells/ml (K), 1.0×106 cells/ml (L)) resulted in a decrease in the aggregate size by 18 h.\n\nThe spherical form of the aggregates suggests isotropic adhesion forces while the evolution of the aggregates from a “bunch of grapes” appearance to smooth-surfaced spheres indicates an increase in cell-cell adhesion strength in vitro with time after initial cell-cell adhesion. A time-dependent increase in adhesive bond energy has been observed in direct measurement of cadherin-mediated adhesion maturation in biophysical tests (Chu et al., 2004).\n\nSoxE+ve/Hu-ve (ENC cells) and SoxE-ve/Hu+ve cells (neurons) occurred first in tiny clumps, with SoxE+ve/Hu-ve cells predominating in the strings; these ENC cells displayed both N-cadherin and NCAM immunoreactivity (Figure 6A’, A”). The transient presence of cell-strings has also been described in cells with only cadherin adhesive mechanisms operative (Takeichi, 1977). Cadherins move on the plane of the membrane and cluster in cis, via intercadherin bonds extracellularly and via binding to the cytoskeleton intracellularly (Hong et al., 2013). The generation of cell strings by SoxE+ve ENS cells suggests that the ENC cells have a limited number of adhesive molecules on their surface, this cis-clustering may restrict cadherin to a few patches capable of mediating adhesion in trans.\n\nA. QE8 cell aggregates gradually increased in diameter from 0–48 h in vitro. (# p=0.0229, 4h vs 2h; p<0.001 for all other times). Starting cell density 0.3×106 cells/ml. B. ENS cells from QE8, ChE9 and mixed populations (Q+Ch) showed identical aggregation at 18 h in vitro, with no significant difference (Q:Ch p=0.29; Q:Q+Ch p=0.23; Ch:Q+Ch p=0.93;). Starting cell density 0.3 ×106 cells/ml. C. Aggregate diameter attained at 18 h in vitro was slightly larger at the highest rotational speed ( p<0.001, 150 rpm vs 0 and 75 rpm). Starting cell density 0.5×106 cells/ml. D. Aggregate diameter at 18 h in vitro was starting cell density-related. Starting cell density at 1×106 cells/ml produced aggregates of least diameter. With the gradually reduced starting cell density, the aggregate diameter increased (relative to 1×106 cells/ml: * p<0.001; relative to 0.67×106 cells/ml: # p=0.0186, ### p<0.001). However, this increase plateaued, with no significant difference in aggregate diameter at 0.167, 0.33 and 0.5×106 cells/ml. Error bar=SEM.\n\nA’ A’’. At 2 h QE8 N-cadherin and NCAM+ve ENS cells formed small aggregates (mixed Hu+ve neurons and SoxE+ve ENC cells) and chains (mostly ENC cells). B. At 6 h spherical aggregates were formed but neurons (HuC/D+ve) and ENC cells (SoxE+ve) were only partially segregated. C. At 18 h most HuC/D+ve neurons formed the centre of each spherical aggregate surrounded by SoxE+ve ENC cells. D. NCAM immunoreactivity was low on the external face of SoxE+ve ENC cells (indicated by dotted line), but high around HuC/D+ve neurons, whether these were located centrally or on the periphery (arrows). E. N-cadherin immunoreactivity was associated with all cells in the aggregate, but was less distinct on the external surface (indicated by dotted line).\n\nVarying the speed of rotation (0 and 75 rpm) had little effect on aggregate form or size at 18 h, while aggregates at 150 rpm were somewhat larger in diameter (Figure 4F–H, Figure 5C). Since increasing rotation rates did not prevent aggregation, the intercellular cell-cell adhesions of these ENS cells must display a rapid “catch” to initiate adhesion, with initial adhesion strength sufficiently high to resist external distractive forces in the highest shear used here. On the other hand, the similar size and shape of the aggregates even down to zero rpm (Figure 4F, Figure 5C) suggests an overriding intrinsic adhesive mechanism that regulates not only accumulation of cells into aggregates but also governs the preferred size of the aggregates under these conditions.\n\nVarying the initial density of ENS cells in suspension over a 6-fold range (0.167×106 to 1.0×106 cells/ml) led, counter-intuitively, to smaller aggregates in much larger numbers at higher starting cell densities (Figure 4I–L, Figure 5D).\n\nConfocal examination of 4–6 h spherical aggregates showed mixed Sox10+ve and Hu+ve cells (Figure 6B), but by 18 h cells in the aggregates showed most of the Hu+ve cells located in the centre, with the SoxE+ve cells forming the outer part of the spheres (Figure 6C). Labelling for NCAM showed that this adhesion molecule was most strongly expressed on the internal Hu+ve cells with lower immunoreactivity on the SoxE+ve cells, and especially low on the outer surface (Figure 6D). Such differential labelling was also present with labelling for N-cadherin (Figure 6E).\n\nConfocal examination of four aggregates (diameter range: 63.8–98.7 μm; cell number range 276–861) revealed a remarkably uniform average cell density of 0.24 ± 0.02 cells/(10 μm) 3. Likewise the neuron/ENC cell ratio of 1.19 ± 0.06 (Hu+ve/SoxE+ve cells) was identical to that in the ENS in vivo (Figure 1). The spatial order of central neurons and peripheral ENC cells in the aggregates in vitro strikingly resembled that in the ENS ganglia in vivo (Figure 2C) (Hackett-Jones et al., 2011).\n\nIn assays of this kind, cells move within aggregates (“sort out”) with their final equilibrium positions determined by the most favoured adhesive balance, that is, with the least surface free energy of adhesion (Foty & Steinberg, 2005). To satisfy this, the external position of the ENC cells relative to neurons indicates that the ENC cells must have lower overall adhesive capacity than the neurons, and the lower levels of NCAM immunoreactivity in SoxE+ve cells is in accord with this. Cell variants with a step difference in adhesiveness segregate especially rapidly in co-aggregates (Zhang et al., 2011), and this is likely to be represented by the two ENS cell types -neurons and ENC cells- used here.\n\nIn vivo the neurons form recognisable groups first (Figure 2B), at a time when the ENC cells still appear randomly placed, and the ENC cells co-assemble around neurons later (Fairman et al., 1995; Hackett-Jones et al., 2011). In contrast, the final internal/external distribution observed here would be attained from any starting distribution of the cell types (Steinberg, 2007). Indeed, when we followed aggregation in vitro, the neurons and ENC cells were initially mixed (Figure 6B) and only later did the relative positions of neurons and ENC cells develop (Figure 6C).\n\nTo test whether cells were able to sort out within aggregates we combined pre-formed (E9 chick) ENS cell aggregates with freshly dissociated QE8 ENS cells. A few quail cells adhered to the surface of pre-formed ENS cell aggregates when combined for 4 h in rotating culture (Figure 7A. By 18 h some of these quail ENS cells had penetrated deep into the chick ENS cell aggregates (Figure 7B). This confirms that cells can move within the aggregates. This is consistent with the SoxE+ve/Hu-ve ENC cells and SoxE-ve/Hu+ve neurons physically sorting out, although it does not preclude an additional spatial differentiation whereby internally placed ENC cells differentiate mostly into neurons.\n\nA. Few freshly dissociated E8 quail ENS cells (arrows, labelled QCPN+ve in green) at 3 h in vitro attached to the periphery of pre-formed (21 h) E9 chick ENS QCPN-ve cell aggregates. B. At 18 h in vitro some quail ENS cells (arrows) relocated deep into the chick ENS 36 h aggregate. C. Most quail ENS cells formed entirely quail cell aggregates after 18 h, rather than adhering to pre-formed chick ENS cell aggregates. D. QE8 and ChE9 ENS cells when combined as freshly dissociated cells formed mixed aggregates at 6 h, showing that there is no species-related adhesive incompatibility. All specimens were labelled with HuC/D, QCPN and SoxE. Scale bar applies to all images.\n\nWhen freshly dissociated QE8 HNK-1+ve cells were added to pre-formed (18 h) chick E9 ENS cell aggregates, QCPN-labelling showed that only a few quail cells adhered to the outer surface of pre-formed chick cell aggregates (Figure 7A), and most quail ENS cells formed separate aggregates entirely of quail cells (Figure 7C). This separation was not caused by species-specific adhesive differences, because when freshly dissociated E9 chick and QE8 ENS cells were mixed at the time of dissociation, all cell aggregates were a mixture of both chick and quail cells (Figures 5B, Figure 7D).\n\nThis shows that the outer surface of pre-formed aggregates is relatively non-adhesive, and is consistent with the observation that the outer surface of the peripheral cells of aggregates showed low immunoreactivity for CAMs (Figures 6D, E). We therefore propose that the outer SoxE+ve ENC cells segregate a limited number of CAMs mainly to the internal face to bind to similar but more numerous molecules on the neurons, leaving the external surface relatively non-adhesive. This process would automatically restrict the size of ENS cell aggregates by preventing new cells from binding to the surface.\n\nIf the peripherally located Sox10+ve ENC cells in the aggregates form an insulating coating preventing ever-larger aggregates from forming, then reducing the number of these cells should allow larger aggregates to form. By combining NCAM with HNK-1 FACS, we produced HNK-1+ve populations of higher and lower NCAM levels (Figure 6D, Supplemental Figure S2C). Previous immunolabelling in vivo (Hackett-Jones et al., 2011) and here in aggregates in vitro shows that the NCAM-high sub-population will be enriched for neurons, and the NCAM-low sub-population depleted in neurons. Culturing these cells in Terasaki wells confirmed this was the case, both populations had SoxE+ve cells but HuC/D+ve cells with E-C8+ve, Tuj1+ve neurites occurred only in the NCAM high fraction. Aggregation of NCAM-high and NCAM-low sub-populations produced respectively larger and smaller irregularly shaped aggregates than was usual for unsorted HNK-1+ve cells (Figure 8A–C).\n\nA. NCAM-high FACS fraction of HNK-1+ve ENS cells form aggregates with a wide range of diameters including very large aggregates. B. NCAM-low fraction ENS cells form small misshapen aggregates. C. Histogram of aggregate diameters formed at 18h in vitro by NCAM-high and NCAM-low ENS cell fractions. Difference is highly significant (*** p<0.001). Starting cell density 0.67 ×106 cells/ml. Error bar=SEM.\n\nQuail E8 trunk DRG were dissociated as for the ENS cells and placed in aggregation assays. Like the ENS cells, DRG cells rapidly formed aggregates with similar average size although the range of size of DRG aggregates was larger (Figure 9A). Interestingly, the internal structure of the aggregates was strikingly different: SoxE+/Hu-ve cells and SoxE-/Hu+ve neurons were mixed not segregated (Figure 9B inset). In addition, although all Hu+ve neurons were strongly NCAM+ve and all outer SoxE+ve cell membranes were deficient in NCAM labelling (as in equivalent ENS cells), the internal SoxE+ve cells were strongly NCAM-labelled (Figure 9B, C).\n\nA. QE8 DRG and ENS HNK-1+ve cell aggregates were similar in average size and size distribution. Box plots show the first quartile to inter quartile range and whiskers show minimum and maximum range of data, while the median is represented by a vertical line. There was no difference between DRG and ENS in aggregate size when analysed using Mann-Whitney test for nonparametric data. Starting cell density was 0.5 ×106 cells/ml. B. Unlike QE8 ENS cell aggregates, Hu+ve neurons and SoxE+ve cells are not segregated in DRG aggregates (inset), but like ENS aggregates, peripheral SoxE+ve cells show little outer NCAM labelling (arrows). C. In contrast, internal SoxE+ve cells (stars) as well as surface and internal Hu+ve cells show strong NCAM labelling in DRG cell aggregates.\n\nIn vivo, NC-derived cells coalesce to form DRG as early as about E4 (in chick; equivalent to E3.5 in quail), and at these early stages the DRG display NC cell/neuron segregation as seen for ENS cells, with all neurons placed centrally (Wakamatsu et al., 2000). As early as E5 in chick, however, early DRG glial cells identified by transitin expression (which are also SoxE+ve) spread centrally between the DRG neurons (Henion et al., 2000). We propose that the different sorting behavior of QE8 ENS cells and QE8 DRG cells represents a difference in developmental stage of the two ganglion types of the same chronological age, with DRG being much further advanced in ganglion cell differentiation, marked by the appearance of highly NCAM+ve glial cells. The morphogenetic consequence of this drives the internal relocalisation of glial cells among the neurons they support by E8 in trunk DRG. We propose that a similar process evolves later, by E14, in midgut ENS (Figure 2D).\n\nA mechanism of separation by sorting of more and less adhesive cells would produce the observed core/shell spatial distribution of SoxE+ve and Hu+ve cells (Figure 10A). Translocation of a numerically limited number of CAMs in the plane of the cell membrane to the internal-facing side of the less adhesive cells (i.e. the Sox10+ve cells) to engage the more numerous homophilic CAMs on the more adhesive cells (i.e. Hu+ve neurons) (Figure 10B) would automatically limit the size of the aggregates by denuding the external surface of CAMs. This mechanism would predict that the size of the aggregate depends at least in part on the ratio of more adhesive neurons to less adhesive ENC cells. We suggest that this operates also in vivo and is of importance for ensuring that the ENS consists of relatively small and uniform ganglia. However, alteration of adhesion of ENS cells to ECM leads to ENS ganglia of abnormal size, shape and pattern of distribution in vivo (Breau et al., 2009; Broders-Bondon et al., 2012). This shows that the balance of cell-ECM adhesion as well as cell-cell adhesion, needs to be taken into account to achieve correct ENS ganglionic morphogenesis.\n\nA. Initially ENC cells and neurons cohere in a disorderly fashion (2–4 h) but gradually the neurons sort out to a central position (18 h), indicating neurons have a greater adhesive bond energy. B. Neurons and ENC cells have adhesive molecules (coloured circles) which are free to move in the cell surface, but the neurons have more adhesive molecules. Cell-cell adhesion requires juxtaposition in trans of two adhesive molecules (red-red, blue-blue or red-blue). To minimise adhesive free energy, adhesive molecules on ENC cells move (pink arrows) in order to form an adhesion, thereby denuding the outer face of adhesion molecules. This insulates the group from further adhesion.\n\nF1000Research: Dataset 1. Raw data for Figure 1, 10.5256/f1000research.6370.d45928\n\nF1000Research: Dataset 2. Raw data for Figure 3a, b and c, 10.5256/f1000research.6370.d45929\n\nF1000Research: Dataset 3. Raw data for Figure 5a and Supplemental Figure S5, 10.5256/f1000research.6370.d45930\n\nF1000Research: Dataset 4. Raw data for Figure 5b, 10.5256/f1000research.6370.d45931\n\nF1000Research: Dataset 5. Raw data for Figure 5c, 10.5256/f1000research.6370.d45932\n\nF1000Research: Dataset 6. Raw data for Figure 5d, 10.5256/f1000research.6370.d45933\n\nF1000Research: Dataset 7. Raw data for Figure 8c, 10.5256/f1000research.6370.d45934\n\nF1000Research: Dataset 8. Raw data for Figure 9a, 10.5256/f1000research.6370.d45935\n\nF1000Research: Dataset 9. Raw data for Neuron: ENC ratio (page 12), 10.5256/f1000research.6370.d45939\n\nF1000Research: Dataset 10. Raw data for Supplemental Figure S3, 10.5256/f1000research.6370.d45936", "appendix": "Author contributions\n\n\n\nBR and DN conceived the study. DZ, BR and DN designed the experiments. BR, DZ and JS carried out the research and DZ performed statistical analysis. TM contributed to the design of experiments and provided expertise in q-PCR. DN and DZ prepared the initial manuscript draft, and all authors contributed to the preparation of the manuscript. All authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Health and Medical Research Council grants 436971 and 607379. MCRI facilities are supported by the Victorian Government's Operational Infrastructure Support Program.\n\nI confirm that the 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 Vanda Lennon, Mayo MN for the human Hu antibody and Craig Smith, MCRI, for the SoxE antibody. QCPN (B. and J. Carlson), E/C8 (G. Ciment) antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. Matt Burton, MCRI, assisted with confocal microscopy and FACS. Dr. Lincon Stamp and Ms. Sophie McConnell contributed to blinded aggregate counting. Alexander Luisetto, a local school student from Brunswick Secondary College, performed some of the cell culture of ENS cells.\n\n\nSupplementary information\n\nIntestine (midgut) caudal to the gizzard (giz.) to about half way along the cacum (cec.) was obtained. This was removed from E5 (about HH27) and E8 quail embryos (about HH34-35). HH34-35 midgut was also obtained from 9-day chick embryos. The intestine was cut at the dorsal border of the mesentery to exclude cells of the Nerve of Remak. The tissue used did not include the cecal root because here the Nerve of Remak is applied to the gut so closely that contamination is unavoidable. C-r = colo-rectum, prov. = proventriculus\n\nFollowing tissue digestion, single cells were fluorescently labelled with HNK1 and HNK1/NCAM and cells sorted by FACS. (A) Scatter plots show typical results of sorting embryonic tissue from quail embryonic day 5 (QE5), QE8, and chicken embryonic day 9 (ChE9). The typical percentage yields of HNK1+ve cells are shown following FACS of single cells from the midgut and dorsal root ganglia (DRG). (B) QE5 MG HNK1+ve cells plated on laminin show the characteristic multipolar morphology of ENC cells. (C) QE8 midguts were digested and single cells fluorescently labelled with HNK1 (Alexa 488) and NCAM (Alexa 647). The scatter plot shows that the majority of NCAM+ve cells also express HNK1. NCAM+ve/HNK1+ve are able to be differentially sorted from the NCAM-ve/HNK1+ve population.\n\nTo assess the enrichment for ENC markers following FACS for HNK1, QE8 midgut-derived cells were assessed for expression of SOX10 (a NC marker) and Hu-D (a neuronal marker). Cell populations analysed by qPCR were cells which were not sorted (pre-sort), sorted cells which were not HNK1+ve (-ve sort) and HNK1+ve sorted cells (HNK1). HNK1+ve cells showed a significant increase in expression of the markers tested when compared with pre-sorted cells indicating successful enrichment of ENC cells by FACS.\n\nA. HNK-1+ve cells plated for 18 h on laminin (phase contrast). B. HNK-1+ve cells at 66 h in vitro showed NC markers: HuC/D (neurons) and SoxE (ENC cells). C. HNK-1–ve cells plated for 66 h had flat fibroblastic morphology (phase contrast). D. Nearly all HNK-1–ve cells plated for 66 h were highly flattened and exhibited smooth muscle actin (SMA) labelling.\n\nAggregate diameter increased on average and the range of diameters increased over the period 2 h to 18 h in rotation aggregation assays. Starting cell density was 0.3×106 cells/ml.\n\n\nReferences\n\nAllan IJ, Newgreen DF: The origin and differentiation of enteric neurons of the intestine of the fowl embryo. Am J Anat. 1980; 157(2): 137–154. PubMed Abstract \| Publisher Full Text\n\nBarde YA: Neurotrophins: a family of proteins supporting the survival of neurons. Prog Clin Biol Res. 1994; 390: 45–56. PubMed Abstract\n\nBinder BJ, Landman KA, Simpson MJ, et al.: Modeling proliferative tissue growth: a general approach and an avian case study. Phys Rev E Stat Nonlin Soft Matter Phys. 2008; 78(3 Pt 1): 031912. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8623", "date": "26 May 2015", "name": "Miles L. Epstein", "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\nThis paper represents a vanguard analytical investigation on the role of adhesion molecules in regulating the size of enteric ganglia. This paper demonstrates that both in vitro and in vivo, Hu+ve enteric neural crest cells showing a neuronal phenotype tend to “sort out” within the centre of ganglia and adhere together while Hu-ve enteric neural crest cells show less adhesion and are located more in the periphery.I have a few comments that I would like to be addressed in order to increase the clarity and impact of this manuscript for the reader.In the Introduction the authors state “: Hypoganglionosis (fewer, smaller ganglia) is associated with persistent constipation…” Could they please provide a citation for this statement? On page 2 the authors wrote. “Formation of each the NC-derived sympathetic ganglia, for example, relies on the scattered NC cells self-aggregating…” Please delete “the” from this statement. On page 4 “The tubes were then incubated on a rotating platform (120 rpm, radius 1 cm) at 37oC. At t=0, 15, 30, 60 and 120 min.,” Please clarify the position that the tubes were incubated, vertical or horizontal? On page 6. “HNK-1+ve sorted cells plated on fibronectin or laminin surfaces showed neural immunoreactivity, including SoxE (recognizes Sox9 and Sox10). In the description of the antibodies it states that this antibody also recognizes Sox8. Therefore, could Sox8 please be added within the parentheses. In Fig. 1 . The y axis label reads cells per 100mm2, I think that this might be error and should be cells per 100 microns squared. Could the authors please amend this? On Page 6. The authors wrote, “The sensitivity to EGTA showed involvement of Ca2+-dependent (i.e. cadherin) mechanisms, but the residual aggregation indicated Ca2+-independent mechanisms (such as NCAM) were operative as well, suggests that the initial phase of adhesion in these conditions was largely due to Ca2+-independent adhesion.” This section could be made more clear with some revision. As the words residual aggregation suggest that it is a later time point and so maybe the removal of the word residual would make this sentence easier to read and understand what the authors are trying to say. Figure 6 needs to be modified so that each of the fluorophores are shown separately, especially 6D. It would be useful for the reader to be informed where the optical section was taken from. Are we looking at a section from the surface or deeper within the aggregate as this changes how we can interpret what we are looking at. Figure 6. It is not clear from visual inspection that the levels of NCAM are really different as stated “Labelling for NCAM showed that this adhesion molecule was most strongly expressed on the internal Hu+ve cells with lower immunoreactivity on the SoxE+ve cells,…”. The level is quite low between some Hu cells. Some quantification of the intensity of the NCAM staining would greatly improve the validity of the authors statements here. Fig 7B. This image doesn’t support the assertion from the authors that the Hu cells are found in the center of the aggregates as many Hu+ve cells are found on outside in this image. Do the authors have an alternative image that could be used to demonstrate their conclusions? Alternatively could they make sure that they state the proportion of aggregates that had Hu+ve cells on their surface. On page 10 the authors wrote, \"This shows that the outer surface of pre-formed aggregates is relatively non-adhesive, and is consistent with the observation that the outer surface of the peripheral cells of aggregates showed low immunoreactivity for CAMs (Figures 6D, E).” The outer surface of the aggregates must be sticky at initial stages or the aggregates would not have increased in size during these stages since the authors have already shown us that aggregate formation at the early stages is not being regulated by cell proliferation. Could the authors please amend this statement to ensure that it is a true reflection of what is occurring within their experiments? On page 11. “Culturing these cells in Terasaki wells confirmed this was the case, both populations had SoxE+ve cells but HuC/D+ve cells with E-C8+ve, Tuj1+ve neurites occurred only in the NCAM high fraction.” It would be really good if the authors could show this data. If this is not possible then could they please state, data not shown here. Fig 9A. “Box plots show the first quartile to inter quartile range and whiskers show minimum and maximum range of data, while the median is represented by a vertical line.” The authors are very imaginative but some readers don’t shave. Please change to vertical lines show minimum and maximum range of data respectively, while the median is represented by a horizontal line. On page 11 the authors state, “Interestingly, the internal structure of the aggregates was strikingly different: SoxE+/Hu-ve cells and SoxE-/Hu+ve neurons were mixed not segregated (Figure 9B inset)”. Please add “in the DRG” at the end of this sentence.", "responses": [] }, { "id": "8815", "date": "29 May 2015", "name": "Hans-Henning Epperlein", "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\nRollo et al. studied ganglion formation of ENS cells in vitro by using disaggregated cells from midguts of E5 and E8 quail and E9 chick embryos. Aggregation was investigated in cell-cell adhesion assays with HNK-1 fluorescence-activated cell sorting (FACS). While the developing neurons (Hu-positive) sorted out to the centre of the aggregates, NC cells (SoxE-positive) stayed more peripherally. This sorting behaviour was consistent with a higher adhesion centrally and a parallely higher N-cadherin- and NCAM-labelling. This main result suggests that a differential adhesion mechanism of enteric neurons and NC cells controls the inner/outer layering of the ganglia with the ENC cell fraction limiting the size of aggregates. The merit of this careful and detailed study is that it tries to visualize quantitatively at a cellular level what cannot be observed in vivo in this way or only exemplarily with severe difficulties via 3D-reconstructions. I recommend indexation. I have a small “major” concern and give a list of several minor comments below. Major concernResults and discussion p 5, section “ENS ganglia undergo progressive morphogenesis in vivo”: The size of the aggregates forming in vivo (see Fig. 2D) should be indicated here in this section, i.e. as much as one can recognize with the confocal studies used (diameter/cell number). Then this size and the in vitro size can be compared. Obviously the in vitro aggregates are much larger, similar to DRG size in vivo. This should be explained. The in vitro size of aggregates is indicated in detail on p 9. (see also minor comments 4, 8, 12). Minor commentsIntroduction: The authors might consider to move paragraph 4 starting with “The final ENS cell population is enormous…” after paragraph 1. Introduction, paragraph 3: citation Teillet et al. 87; here 1-2 papers from R. Mayor could be added (e.g. Theveneau and Mayor, 2012; Kuriyama and Mayor, 2008).Introduction, paragraph 4 What is “DCC”? Last paragraph of Introduction: “In particular we wished to explore why the ENS ganglia are similar and small in size……” If this is your wish you should add here 2-3 sentences about how “real” your results are. How much bigger/smaller are your ganglia obtained in vitro compared to those on the slices cut from an in vivo specimen (Fig. 2)? Think also of and compare the in vitro-sizes of enteric ganglia to your previous work! Mat.+Meth.: “Counts of myenteric ENS cells in wholemounts”. One does not know what “Hu” stands for and why “SoxE” is mentioned here but Sox-10 further down on the page (last line). Explanation of abbreviations here or before the paper starts? “Cell dissociation”. While a non-expert might imagine how a “midgut” is removed, the skill that is needed to harvest DRG may be beyond his/her imagination. Add perhaps 1-2 sentences for explanation?“The intestinal tissue pooled from 15–60 embryos”: why 15-60? Is this one of many similar preparations using between 15 and 60 guts? Otherwise you should indicate a fixed number.One gets no information about the proportion of ENS cells obtained from the myenteric and submucosal plexuses. Are the ganglia in both places equal in size and structure? At certain developmental stages there could be many more Hu-positive or SoxE-positive ganglia in the myenteric than in the submucosal plexuses because the latter are settled earlier. Section: “Fluorescent labelling and fluorescence-activated cell sorting (FACS)”: It is hardly imaginable that from such a broth obtained by filtering disaggregated tissue or single cells through a 30 µm strainer cells were obtained that could form such beautiful aggregates as shown in Fig. 6. May be you can add a sentence about the consistency of the filtered cells? Results: “Figure 2”: Why are myenteric plexuses chosen and not submucosal ones? What is the final size of a ganglion in Fig. 2D (in vivo) compared to Fig. 6C (in vitro)? Is the diameter range (63.8-98.7 µm) and the cell number range (276-861) indicated on p. 9 (right column) comparable to the diameters/sizes of the in vivo aggregates ? “ENS cell clusters in vitro are due to cell aggregation, not proliferation”. This section is nearly too short to merit section status – could it be subsumed somewhere else? Fig. 6D: “NCAM” is hardly readable. “ENS cells are mobile within aggregates, allowing sorting out”. I only wonder whether this initial mobility of ENS and ENC cells and their later sorting out and finally the outer placement of the ENC cells with restricting ENS cells is not all a little contradictory? Fig. 9: ..DRG…..cell aggregates were similar in average size… But the DRG would be definitely much bigger than the enteric ganglia ? If not, then your in vitro aggregates are much bigger than the in vivo ones! See again point 8 (above) and compare ENS aggregate sizes in Fig. 2d and Fig. 6C with DRG size in Fig. 9. May be you could discuss that at the very end of the paper?", "responses": [] } ]	1	https://f1000research.com/articles/4-113
https://f1000research.com/articles/4-24/v1	26 Jan 15	{ "type": "Opinion Article", "title": "The endothelial deprotection hypothesis for lupus pathogenesis: the dual role of C1q as a mediator of clearance and regulator of endothelial permeability", "authors": [ "József Prechl", "László Czirják", "László Czirják" ], "abstract": "Systemic lupus erythematosus (SLE) is a heterogeneous multifactorial systemic autoimmune disease affecting several organs. SLE can start relatively early in life and results in impaired quality of life and shortened life expectancy because of a gradual disease progression leading to cardiovascular, renal and neoplastic disease. The basic mechanisms of the pathogenesis of the disease still remain to be clarified. It is clear that complement proteins play a key and complex role in the development of SLE. Complement component C1q has been known to be a fundamental component of lupus development, but most explanations focus on its role in apoptotic debris removal. Importantly, C1q was recently found to play a key role in the maintenance of vascular endothelial integrity.We suggest that apoptotic products, endothelial cells and extracellular matrix components, which display negatively charged moieties, compete for binding to molecules of the innate humoral immune response, like C1q. Genetic or acquired factors leading to an increased load of apoptotic cell debris and decrease or absence of C1q therefore interfere with the regulation of endothelial permeability and integrity. Furthermore, we suggest that lupus is the net result of an imbalance between the two functions of immune clearance and vascular endothelial integrity maintenance, an imbalance triggered and sustained by autoimmunity, which skews C1q consumption by IgG-mediated complement classical pathway activation on autoantigens. In this triangle of innate clearance, autoimmunity and endothelial integrity, C1q plays a central role.Hence, we interpret the pathogenesis of lupus by identifying three key components, namely innate immune clearance, autoimmunity and endothelial integrity and we establish a link between these components based on the protective role that innate clearance molecules play in endothelial renewal. By including the vasoprotective role of C1q in the interpretation of SLE development we attempt to provide novel explanations for the symptoms, organ damage, diagnostic and therapeutic difficulties of the disease.", "keywords": [ "lupus", "SLE", "systemic lupus erythematosus", "pathogenesis", "complement", "C1q", "autoimmunity", "autoantibody", "natural", "IgM", "endothelium", "clearance", "apoptosis" ], "content": "Introduction\n\nA database search for the word “lupus” in the title of biomedical publications brings up 39,306 papers as of the writing of this manuscript. Thus, there is an abundance of experimental and clinical research data on systemic lupus erythematosus (SLE), yet the comprehensive etiopathogenesis of this group of heterogeneous diseases with multifactorial origin is still unknown (Figure 1). SLE is an enigmatic disease, with a range of manifestations. Indeed, currently systemic lupus erythematosus is classified, but not diagnosed, on the basis of the coexistence of several alterations from a list of criteria1,2. Immune complexes containing IgG and complement are found deposited in various tissues and are responsible for inflammatory processes causing skin rash, mucosal ulcers, arthritis, nephritis, and serositis. Hematological changes include diverse cytopenias, while immunological tests show antibodies against nuclear material, dsDNA, Sm antigen and phospholipids3.\n\nThe development of lupus is influenced by genetic factors, controlling individual variability especially with regard to the immune system and also with regard to physiology. Anatomical features may determine organ damage and source of autoantigens. Environmental factors can act upon all these elements, and may be the component we could try to modulate with the aim of preventing disease development. SLE, systemic lupus erythematosus; BCR, B-cell antigen receptor; TCR, T-cell antigen receptor; ECM, extracellular matrix; FcγR, Fc receptor for IgG.\n\nIn an attempt to simultaneously monitor antibody and complement binding to various autoantigens, we have developed a functional immunomics approach4,5 allowing a complex analysis of serological events in SLE. Interestingly, we observed that copious amounts of complement products are fixed by nucleic acids but not other negatively charged molecules in SLE patients with decreased complement C4 levels6 (and manuscript in preparation), which finally led us to formulate the hypothesis presented below. In this paper, we attempt to collect all the pieces of knowledge of the lupus puzzle and place them next to each other in a way that a novel picture emerges. We hope that this hypothesis will stimulate discussions along a novel course and finally will result in a better understanding of the lupus syndrome.\n\n\nThe pieces of the puzzle we already have\n\nWe can arrange most of the currently accepted mechanisms of lupus pathology in three main sets of factors: impaired clearance, autoimmunity and vascular injury. The elements of these particular processes are highly interconnected but for didactic reasons we will discuss them within one of these categories.\n\nIncreased load of cellular debris. The initiation of pathological autoimmune responses requires autoantigens to reach and trigger lymphocytes in the lymphoid organs. Cellular debris released from dying and dead cells is thought to be the most important source of self-antigen in lupus. One of the most widely used animal models of human SLE is the MRL/lpr mouse strain. These animals carry multiple susceptibility genes, which control lymphoproliferation and apoptosis7, and spontaneously develop a lupus-like disease with antinuclear antibody production and nephritis. Interestingly, in human autoimmune lymphoproliferative syndrome (ALPS), where apoptotic signaling is impaired, some of the symptoms observed in SLE also appear8, underlining the role of this factor. Direct evidence for the role of apoptotic load is also available, since increased apoptosis of monocytes9, neutrophils10, lymphocytes11 and endothelial cells (EC)12 has been described in SLE patients.\n\nImpairment of apoptotic debris removal. Cells undergoing programmed cell death are cleared from the body without inducing inflammation. This is part of the physiological tissue maintenance and regeneration events continuously occurring in the body. Dead cells, apoptotic blebs and debris are recognized by several soluble molecules and cell surface receptors, all promoting uptake by tissue macrophages and dendritic cells. This silent removal locally prevents inflammation and systemically the development of autoreactive lymphocytes13. Inefficient removal of apoptotic cell debris in lupus14 leads to the clonal expansion of autoreactive lymphocytes, with both B cells and T cells involved. Nucleosomes become accessible on the cell surface15, exposing negatively charged nucleic acid containing complexes. NETosis, a form of programmed cell death recently described in neutrophil granulocytes, has also been implicated as a source of cellular debris that contributes to lupus pathogenesis16.\n\nThe complement system plays an important role in apoptotic cell removal: C1q binds to negatively charged molecules like phosphatidyl serine and cardiolipin17 and polyanionic targets, like DNA18,19. Various cells display receptors for C1q and help silent phagocytosis of apoptotic cells opsonized by C1q13,20,21. The classical pathway of complement is activated; deposited C4 and C3 fragments are then recognized by the CR3 of myeloid cells, a receptor encoded by lupus susceptibility gene ITGAM. The allelic variant of CR3 associated with lupus shows impaired phagocytotic and adhesion function22.\n\nHereditary complement deficiencies. Early complement components have long been known to play a key role in lupus development. Genetic deficiency of C1q is the strongest susceptibility factor for lupus23, with close to 100% of the deficient subjects showing signs of the syndrome. Deficiency in the components involved in the later steps of classical pathway activation, C2 and C4, also predisposes to lupus development, albeit with lower probability. Interestingly, people with lupus show a secondary deficiency of these particular complement components suggesting the consumption of these proteins by factors playing a role in disease pathogenesis. These intriguing relations between complement and lupus have been discussed in depth by excellent reviews24,25.\n\nComplement deficiency due to consumption. Immunoglobulins undergo a conformational change upon antigen binding. This event coupled with immobilization and provision of affixed C1q binding sites promotes the binding of the C1 complex. The attachment of C1 will activate C1r and C1s, initiating the complement cascade26. The fact that DNA specific immunoglobulins trigger complement activation to such an extent that the systemic consumption is measurable as decreased C4, C3 and CH50 levels has been known for decades27. The measurement of these parameters forms part of the diagnostic routine even today, because secondary complement deficiency in lupus is associated with disease activity28. Complement is also consumed by being deposited on blood cells in the circulation of lupus patients, a fact that is beginning to be exploited for diagnostic purposes29.\n\nBreaking of tolerance. Lymphocytes go through several checkpoints during their development, ensuring that self-reactivity is kept within a rational range30. The process results in immunological tolerance to self-antigens. Tolerance can be broken by increased load of apoptotic cell debris reaching the secondary lymphoid organs31, increased propensity for positive selection of B cells32, the presence of molecular patterns in autoantigen that activates Toll-like receptors33, the production of cytokines regulating B-cell development34. The breaking of tolerance is characterized by the production of immunoglobulins specific for the autoantigens which induce autoimmunity, therefore these autoantibodies can be used as disease markers and also to identify the source of the autoantigens.\n\nDevelopment of autoantibodies. Once tolerance against self is broken, antibodies against various nuclear components appear, including various forms of DNA, RNA, nucleosome complexes and nuclear proteins35. Autoantibodies are detectable before the clinical onset of SLE36 and with the development of organ damage various specific autoantibodies appear in the circulation37. These can involve various extractable nuclear antigens38, phospholipids39, complement proteins40,41 and even cytokines like BAFF42. The composition of the immune complexes has important consequences regarding its cell activating properties: DNA in the immune complexes that are formed upon the production of IgG antibodies stimulates plasmocytoid dendritic cells, which in turn release type I interferons, promoting tissue injury43. The antigen-driven development and appearance of high affinity double-stranded DNA specific IgG is considered a hallmark of systemic lupus erythematosus44.\n\nCirculating immune complexes bind to vessel wall. In lupus, immune complexes are found in the circulation, attached to vessel walls and deposited perivascularly. Binding to capillary wall endothelium was shown to be dependent on the presence of C1q in the immune complexes C1q45–47. Thus, once immunity is triggered against autoantigens, the presence of both autoreactive IgG and autoantigen in the circulation will lead to the formation of C1q containing immune complexes. Circulating immune complexes with characteristic components are found in various autoimmune diseases48.\n\nNeutrophil granulocytes and FcγRs as effectors of inflammatory injury. Neutrophil granulocytes rolling along the vessel wall bind to the IgG component of deposited ICs via Fc gamma receptors. This binding, as modelled in our functional antibody assay49, will trigger adhesion and activation of the cell. Neutrophil granulocytes secrete type I interferon and play important roles in the initiation and perpetuation of the disease50. Fc gamma receptors displayed by the granulocyte play intricate roles in the recognition and uptake of IgG immune complexes and the induction of NETosis51. Dysregulation of NET formation itself has also been suggested to play a role in lupus pathogenesis52.\n\nEndothelial cell (EC) dysfunction in lupus. Lupus patients have a high risk of developing cardiovascular disease. Endothelial dysfunction, one of the key factors of atherogenesis, can be triggered by various endothelium damaging factors present in lupus53. The effects of cytokines, inflammatory cells and immune complexes are combined with compromised endothelial functions resulting in increased atherogenicity in lupus53. Endothelial repair is also compromised by a decrease in the number of bone marrow derived endothelial cell progenitors54. Interestingly, C1q and mannose binding lectin (MBL), recognition molecules of the classical and lectin pathways of complement activation, respectively, help remove atherogenic lipoproteins55, establishing a link between C1q deficiency and cardiovascular disease development in lupus. Increased vascular endothelial permeability resulting in edema, infiltration of inflammatory cells and deposition of immune complexes are the commonly observed histological features of the disease.\n\nCoagulation and thrombosis defects in lupus. SLE patients are susceptible to cardiovascular morbidity and mortality56. Abnormal coagulation and thrombus formation is associated with the presence of anti-phospholipid antibodies, exemplified by anti-cardiolipin, anti-β2-glycoprotein and lupus anticoagulant antibodies57. These autoantibodies have been suggested to directly cause endothelial injury or promote atherogenesis by altering lipoprotein metabolism58.\n\n\nThe missing piece: molecules of innate immunity contribute to endothelial integrity\n\nOne important question that has been left largely unanswered so far is why and how exactly are the vascular wall and surrounding tissues damaged? How do the above named three main categories, impaired clearance, autoimmunity and vascular injury interact with each other? We propose that the answer lies in the protective role that innate clearance molecules play in endothelial renewal.\n\nThe globular head of complement C1q binds to negatively charged molecules like DNA59 and cardiolipin17, and also to immunoglobulins mainly via ionic interactions60. C1q has also been shown to bind heparan sulfate61, a component of the extracellular matrix (ECM) and of the surface structures of adherent cells62. Thus, C1q beyond its role in the clearance of apoptotic cell debris could also be involved in endothelial cell interactions with the ECM. Indeed, C1q has been shown to play a role in vascular regeneration by binding to endothelial cells and promoting endothelial adhesion and spreading63 and exert proangiogenic effects like stimulation of endothelial proliferation, migration and permeability64. With these multiple roles of C1q in mind, we propose the following two scenarios, one describing events occurring in the presence of intact endothelium and another for leaky endothelium.\n\nIn a healthy adult male, the endothelium is quiescent65. Significant amounts of C1q bind to EC only in tissues with discontinuous endothelium. Discontinuous endothelium is found at sites of transendothelial trafficking and is characterized by the fusion of the luminal and abluminal plasma membranes, the presence of pores of various diameters and high heparan sulfate content of the glycocalyx66,67. Discontinuous endothelium is divided into sinusoidal and fenestrated types. Sinusoidal endothelium lines bone marrow sinuses, splenic and liver sinuses; fenestrated endothelium covers capillary walls in kidney glomeruli, in the gastrointestinal tract, in endocrine glands and in the choroid plexus65. In healthy adult females of the childbearing age the endothelium is subject to the effects of factors that regulate cyclic renewal of the endometrium, an event accompanied by vascular regeneration68.\n\nThe glycocalyx is a layer of macromolecules, mostly glycosaminoglycans, decorating the surface of ECs. Heparan sulfate constitutes more than 50% of the glycosaminoglycan pool in EC, localizing especially in cave-like structures (caveolae)69 and the fenestrae66, both areas playing primary roles in transendothelial transport and filtration, respectively. We envisage that C1q binds to heparan sulfate-rich regions on the luminal surface of EC and is also efficiently transported to the abluminal side into the tissues.\n\nIf the integrity of the endothelium is disrupted, the subendothelial lamina becomes exposed to the blood plasma. When ECs are in the process of cell division or death, large pores with diameters reaching 1 micrometer are formed in the endothelium, as a result of cellular discontinuity70. Upon exposure, subendothelial collagen immediately binds to several molecules from the blood plasma, triggering repair, coagulation and thrombocyte binding. This event remains silent as long as the endothelium is only modestly damaged. Even though the physiological turnover of EC is low71 subendothelial collagen can be exposed whenever and wherever endothelial cells die. The renewal process is restricted in time and space, unless massive endothelial cell apoptosis is triggered by external factors, such as UV radiation. C1q or C1q containing IC can pass through these leaky junctions and deposit in the ECM, where heparan sulfate is an essential proteoglycan component72. Since C1q is an eat-me-silently signal for myeloid cells, as discussed above, C1q deposition protects these areas of endothelial regeneration from myeloid cell-mediated damage, until integrity is reconstituted.\n\nBased on the above observations we hypothesize that sufficient amounts of free C1q should be available in the blood in order to maintain endothelial trafficking, integrity and renewal. Free serum C1q protects exposed collagen from triggering attachment, activation and extravasation of monocytes and neutrophil granulocytes during endothelial renewal.\n\nHowever, C1q is actually not the only multivalent molecule which binds negatively charged moieties. In addition there is – at least – one other molecule with the ability to bind to apoptotic cells multivalently. Natural IgM (nIgM) molecules, which are produced without a clearly identifiable antigenic stimulus, have been shown to promote the clearance of apoptotic cells in mice73 and enhance phagocytic clearance of host cells74. IgM against dsDNA was shown to be protective in a murine autoimmune model75. The general immunological protective properties of natural IgM were recently reviewed by Grönwall et al.76,77. Anti-apoptotic cell IgM antibodies bind C1q and promote clearance by phagocytes78. An interesting aspect of IgM is that it can bypass the classical activation pathway by binding MBL and induce C4b deposition via MASPs. MBL deficient mice displayed impaired apoptotic cell clearance, without overt signs of autoimmunity, suggesting an alternative role for clearance by the lectin pathway79. Natural IgM binding and the ensuing events lead to the recognition and removal of apoptotic cells without activating the phagocytic cells74. The fact that antibodies of the IgM class do bind to collagen and this binding is decreased in lupus patients has been reported6,80. The question that remains to be answered is whether it is nIgM that binds to collagen. It is also intriguing whether other molecules of the innate humoral immune system, such as pentraxins and collectins may play similar roles.\n\nIn summary, multivalent molecules with a propensity to bind negatively charged targets and the ability to initiate complement activation are consumed by apoptotic cells, immune complexes, EC and the ECM. As long as these molecules are available in excess this competition will go unnoticed. Once this balance is tipped pathological events start to take place.\n\n\nPutting the pieces together: the endothelial deprotection hypothesis\n\nIf C1q binds negatively charged molecules then those molecules compete for C1q binding. Competition for C1q binding by deoxyribose and heparan sulfate has been experimentally confirmed61. Thus, negatively charged components of the exposed nuclear content of dying cells and of the EC fenestrae or the exposed subendothelial matrix can all bind C1 complex from the circulation. Physiologically there is sufficient amount of C1 available and a balance exists between the vessel wall and cellular debris. This binding results in tightly controlled classical complement pathway activation, leading to the production of C4b. Other factors controlling complement activation also bind to negatively charged molecules; these include complement factor H, C4bBP81 amongst several other recognition molecules of the innate humoral immune system, like pentraxins82 and surfactant proteins83,84. These molecules regulate further activation of the complement cascade, the production of anaphylatoxins and the activation of surrounding and recruited cells. Opsonized apoptotic debris will therefore be silently removed by macrophages (Figure 2A). The fact that not only the absence of C1q but also functional C1q deficiency leads to lupus development85 suggests that initiation of the classical pathway activation is required for the removal of cellular debris and prevention of development of immune complex disease.\n\nMaintenance of endothelial integrity and clearance of cellular debris both requires multimeric innate molecules (C1q, nIgM) with the ability to bind anionic surfaces (A). C1q binds to fenestrated regions of the endothelial cell and to collagen exposed due to leaky endothel junctions. Increased use of these innate molecules by the clearance mechanism, or deficiency of these molecules shifts this balance, which results in cellular debris reaching the lymphoid organs (B). This triggers the production of IgM against the autoantigens. Induced autoreactive IgM binds to apoptotic autoantigens, activates the complement system and promotes clearance. Sustained autoantigenic stimulus and genetically determined clearance deficiency coupled with tendency of mounting inflammatory immune responses will result in the production of IgG against the tissue derived autoantigens (C). Immune complexes containing IgG and C1q will bind to the vascular wall, since free C1q levels are low and circulating immune complexes will outcompete them. Deposited immune complexes containing IgG recruit white blood cells with Fcγ receptors, which can trigger cell activation, release of inflammatory cytokines, frustrated phagocytosis, NETosis (D). Immune complexes and autoantibodies can penetrate the tissues via the damaged endothelium, causing organ specific damage. Sustained inflammation leads to irreversible organ damage.\n\nThis balanced binding to EC, subendothelial collagen and cellular debris can be tipped basically by three main factors: decrease in C1q levels, increased load of apoptotic cells and increased collagen exposure. Genetic deficiency in C1q is accompanied by highly increased (more than 90%) likelihood of developing SLE86. Increased cell death can be triggered by ionizing radiation or by drugs with cytotoxic effects87. Inefficient phagocytic capacity of CR3 polymorphic variant r77h88, a lupus susceptibility factor, may also increase apoptotic load. Increased collagen exposure can be the result of endothelial apoptosis induced by sunlight but physiological turnover of the endothelium is also accompanied by macromolecular permeability and access to the subendothelial lamina89.\n\nAs a result of one or more of these factors, cellular debris will reach secondary lymphoid organs, triggering immunity against self-molecules, including DNA. IgM is first produced, which may help restore the balance by enhancing apoptotic debris removal via complement activation (Figure 2B). This is in agreement with the findings of Li et al., who reported increased IgM reactivity to several autoantigens in patients with incomplete lupus erythematosus syndromes35. The generation of DNA specific IgG further tips the balance towards opsonization of cellular debris, taking away more C1q, leaving exposed subendothelial collagen unprotected (Figure 2C). Furthermore, circulating complexes of nuclear material, IgG and C1q will bind to exposed collagen by nature of the multivalent C1q molecule and diffuse into the tissues. Alternatively, in organs with discontinuous endothelium, immune complexes will bind to the fenestrae and be transported into the tissue. Deposition of immune complexes containing IgG will trigger activation of monocytes and neutrophil granulocytes, attracted by C3 and C5 derived anaphylatoxins of the alternative pathway. This results in damage to the vessel wall itself, to increased permeability and to IgG-mediated damage to the tissues (Figure 2D).\n\nTo summarize, impaired clearance of cell debris and immune complexes together with pathological anti-nuclear antibodies consume C1q, an important vascular regeneration factor, from the circulation, by directing it to immune complexes and apoptotic cells. In turn, not free but immune complex-bound C1q will attach to EC and exposed subendothelial collagen. IgG will trigger inflammation instead of regeneration. According to this scenario, dsDNA IgG triggers inflammation, while dsDNA IgM can act against it, by competing with IgG for dsDNA binding. Indeed, the ratio of dsDNA IgG to IgM has been shown to be a good indicator of renal damage in SLE90,91.\n\n\nInterpreting the lupus syndrome in light of the hypothesis\n\nEven though lupus can develop in both men and women, 90% of patients diagnosed with the disease are women, most of them being in the childbearing age92. How hormones contribute to this skewed susceptibility is not defined. Our hypothesis emphasizes the role of vascular endothelial renewal in the pathogenesis of SLE, pointing to sex differences in angiogenesis.\n\nIn fact, angiogenesis is a critical component of endometrial renewal. Various hormones and growth factors interact during the formation of new vessels, including vascular endothelial growth factor (VEGF)68,93. VEGF is produced in ovarian tissues during the menstrual cycle and regulates vascular remodeling and repair. VEGF is a permeability factor as well, its topical administration can induce the development of fenestrations in the endothelium of small venules and capillaries94. Estradiol itself can also directly increase permeability95 and act indirectly96 by modulating VEGF production in endothelial cells. As highlighted above and further discussed below, fenestration, accessibility of the subendothelial lamina promotes the deposition of both bare C1q and nIgM and C1q containing immune complexes. This would render women of the childbearing age, with functioning ovaries, more susceptible to immune complex deposition and vascular damage in SLE.\n\nOur hypothesis suggests that anatomical sites with discontinuous endothelium and tissues where collagen in the subendothelial lamina is exposed will be more vulnerable to immune complex induced damage. There is one more important aspect we have to consider: what is the source of the autoantigen that will induce autoimmunity? Keeping with the notion that in lupus it is the material from dead cells that induces autoimmune response we need to locate the source of apoptotic cells. Here we consider two main categories: cell death within the circulation and outside of the circulation. Within the circulation it is the corpuscles in the blood97 and endothelial cells12 that are sources of cellular debris. Actually we will also consider the bone marrow as a source of apoptotic debris within the circulation because of the high rate of apoptotic death during lymphocyte development and the sinusoidal structure of the endothelium in the tissue. Apoptotic antigens within the circulation will be distributed by the blood flow throughout the body. Immune complexes formed from this material will be deposited anywhere where there is blood flow, and preferentially where blood flow is high, the endothelium is discontinuous and where subendothelial collagen is more accessible. We suggest that renal, bone marrow, joint, serosal and synovial, and partly skin damage in lupus is mediated by this route and constitutes the core components of SLE.\n\nApoptotic cells that come into contact with blood also initiate coagulation events98. Therefore in addition to lipids that become exposed on apoptotic cells, phospholipid-binding proteins (β2-glycoprotein) and components of the coagulation cascade will serve as autoantigens targeted by the immune response. Anti-cardiolipin antibodies and lupus anticoagulant could be produced as the result of these pathological events99, and are responsible for the secondary anti-phospholipid antibody syndrome in SLE.\n\nOutside of the circulation, basically meaning in the tissues, apoptotic cells and their fragments and antigen presenting cells carrying and processing those will reach the secondary lymphoid organs first. Antibodies generated in the lymphoid organs will enter the circulation and immune complexes may or may not be formed, depending on the presence or absence of antigen. Once these autoantibodies appear, they could sustain organ specific damage and disease course by binding to their targets thanks to increased permeability by general impairment in endothelial regeneration.\n\nPhotosensitivity. Cutaneous manifestations of SLE are often linked to exposure to sunlight or artificial sources of ultraviolet (UV) light. Malar rash, the butterfly shaped erythematous lesion on the face is a classical sign of lupus. It may be present in about 50% of SLE patients at the time of the diagnosis100 and is part of the general photosensitivity observed in SLE. UV-light induced apoptosis of keratinocytes is thought to be a source of cellular debris that promotes the induction of rheumatic diseases101. Actually endothelial cells are quite sensitive to radiation-induced, ceramide-mediated apoptotic cell death102–104. We speculate that UV light penetrating the epidermis may cause endothelial damage in dermal capillaries. Dead endothelial cells will be removed inefficiently in the relative absence of C1q and nIgM, while the exposed subendothelial lamina will be less protected by these molecules. Increased paracellular leakage and transcellular trafficking in activated EC would result in increased deposition of IC, edema and extravasation of myeloid cells and the appearance of rash. Additionally, the entry of apoptotic endothelial cells12 or their products containing nuclear material and proinflammatory mediators105 into the circulation may contribute to disease flares triggered by UV exposure.\n\nRenal involvement. SLE can lead to the development of lupus nephritis, which is one of the most disabling complications106. The kidneys are prone to immune complex mediated damage for at least two reasons. It is the organ with the second highest blood flow rate107 and the endothelium is fenestrated, leaving access to C1q bearing immune complexes. Apoptotic debris that is generated within the circulation or enters the circulation will have a very high chance of ending up in the glomeruli. Should these complexes contain IgG, the necessary component for triggering inflammation will be present. Other factors, such as DNAse activity may modulate the severity and prognosis of nephritis108.\n\nSynovitis and serositis. Besides ionizing radiation mechanical injuries may also negatively influence endothelial integrity. Synovial and serosal membranes are rich with blood circulation and are continuously exposed to micromechanical injuries due to the movement of the joints and inner organs, respectively. We suggest that the healing of these microinjured sites would be slower and accompanied by edema and cellular infiltration, because of the deposition of circulating immune complexes. These microinjuries may therefore be responsible for arthritis, pleuritis and pericarditis in SLE.\n\nBone marrow involvement. The bone marrow is a site of intensive cell proliferation and cell death. Lymphocyte development involves selection steps when useless or harmful clones are deleted by programmed cell death. Any defect in the clearance of apoptotic cells is therefore expected to influence homeostasis in this tissue. Additionally, it is a site where there is intensive migration via the endothelial layer in both directions, facilitated by a special endothelial structure: the sinusoidal endothelium. We suggest that this tissue would be vulnerable to immune complex deposition and inflammation. In SLE patients abnormal bone marrow histology is observed109. Comparative analysis of gene expression revealed upregulation of genes involved in cell death and granulopoesis in active SLE patients, confirming the role of apoptosis and granulocytes in the pathogenesis of the disease110. We suggest that cytopenias, the detection of which constitute pillars of the diagnostic algorithm of SLE2, are the consequence of abnormal bone marrow function, in addition to specific antibody mediated direct damage.\n\nClassification of systemic lupus erythematosus relies on clinical examination, hematological tests and serological test. The presence of dsDNA specific IgG is quite specific but less sensitive for the identification of SLE patients6. Decreased complement levels and complement activity are also used but not specific for the disease. Indeed our hypothesis suggests that there is no single protein marker, which could be used alone, because a state of imbalance can only be assessed by the measurement of the different components characterizing the degree of imbalance. The measurement of free C1q or C1 complex and also of nIgM could be assessed for incorporation into the set of laboratory tests characterizing autoimmune conditions. Alternatively, instead of measuring the levels of individual proteins, a functional test that is capable of gauging the degree of endothelial damage, the propensity of immune complex deposition and inflammatory cell reactions all together, could be used for assisting diagnosis.\n\nThe causal therapy for lupus, based on the endothelial deprotection hypothesis, would be the restoration of endothelial integrity by the introduction or induction of C1q or nIgM. In the case of genetic C1q deficiency replacement therapy seems to be the logical solution111. Indeed, hematopoetic stem cell transplantation was recently shown to be successful treatment for hereditary C1q immunodeficiency112. For replacement therapy the administration of C1q or natural IgM is a potential solution. Immunoglobulin preparations for high dose intravenous immunoglobulin therapy contain mainly IgG and exert their effects via pathways related to IgG. However, successful management of SLE with IgM enriched intravenous immunoglobulin (IVIG) has also been reported113, providing support for the beneficial role of nIgM.\n\nIt is tempting to speculate that the production of natural IgM, endothelial turnover and integrity and innate clearance are influenced by environmental factors such as nutrition and lifestyle114,115 or factors produced by the mucosal microbiome, like vitamin K116. If so, we could even look for prevention strategies in light of this hypothesis. The relatively low concordance of SLE in monozygotic twins117 implies that there is ample room for the modulation of environmental effects.\n\n\nConcluding remarks and future directions\n\nWe propose an interaction scheme for SLE pathogenesis with three key components, each of these contributing to disease development by their mutual interactions (Figure 3). These components are innate immune clearance, adaptive immune response quality and endothelial integrity. The endothelial deprotection hypothesis assumes that the mechanisms of innate clearance and endothelial integrity share molecules like C1q and nIgM, therefore the two systems can interfere. We suggest that SLE is the net result of an imbalance between these two systems, which is aggravated by the development of autoreactive antibodies, leading to the leakage of immune complexes from the circulation and the triggering of inflammation in the vessel walls and in the tissues.\n\nC1q and natural IgM are gatekeepers that ensure innate immune clearance of apoptotic cellular debris and immune complexes, and also maintain endothelial integrity (A). Under physiological conditions these processes do not interfere with each other. Major abnormality in one or more of these processes, or combinations of minor abnormalities lead to imbalance, the breakdown of these gates and the development of lupus erythematosus (B). Depending on the contribution of these factors lupus will have different colors and shades, which define distinct disease entities or subtypes within such entities.\n\nIt will be imperative to create sets and networks of genetic factors that underlie these events and upon that superimpose the protein interactions to create a framework for further interpretation of cellular and immunological processes leading to various forms and manifestations of lupus. We speculate that other systemic autoimmune diseases will share some of these components while also possessing distinct other susceptibility factors to create a continuum of diseases with overlaps. We hope that this hypothesis will serve the further understanding of lupus and these other related diseases as well, leading to novel medical approaches and improvement in the quality of life of all those suffering from these conditions.", "appendix": "Author contributions\n\n\n\nJP developed the theoretical part of the hypothesis, LC supervised clinical aspects of the studies leading to this hypothesis and also clinical aspects of the hypothesis itself. Both authors contributed to the writing and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nDiagnosticum and MTA-TKI, the employers of JP, are the licenser/owner of a patent on measuring complement activation on microarrays and owners of a patent application on the assessment of immunological reactivity using cell-based functional assays.\n\n\nGrant information\n\nThe project that helped the development of this hypothesis was funded by the European Union Seventh Framework Programme FP7/2007–2013 under grant agreement n° 314971 (GAPAID-314971, FP7-SME-2012), entitled ‘Genes and proteins for autoimmunity diagnostics’ and by support from the National Science Fund to JP, grant number K109683 and to LC, grant number K 112939.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis theoretical work is dedicated to János Gergely who dreamed about finding a treatment for systemic autoimmune diseases. I wish to thank Anna Erdei who established the complement study group at the department, which I joined 17 years ago, for planting the seeds of complement science in me. Krisztián Papp helped me in setting up the protein microarray group and the development of functional antibody profiling technology, which played key role in developing this hypothesis. I thank my wife for her patience and critical comments on this work. Last but not least, I wish to express my gratitude to Ferenc Péterfy and Klára Rásky, whose trust and experience helped our work ever since we started studying autoimmunity.\n\nWhile we strived to introduce to the reader most of the aspects, theories and thereby research groups who contributed to the understanding of lupus, considering the breadth of this field and the huge number of publications it is practically impossible to provide a complete overview within the frame of this hypothesis. 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PubMed Abstract \| Publisher Full Text\n\nArkwright PD, Riley P, Hughes SM, et al.: Successful cure of C1q deficiency in human subjects treated with hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2014; 133(1): 265–7. PubMed Abstract \| Publisher Full Text\n\nEnk AH, Knop J: [Successful management of systemic lupus erythematosus with IgM enriched immunoglobulins]. Hautarzt. 2000; 51(6): 416–8. PubMed Abstract \| Publisher Full Text\n\nSakthiswary R, Raymond AA: The clinical significance of vitamin D in systemic lupus erythematosus: a systematic review. PLoS One. 2013; 8(1): e55275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWong MS, Leisegang MS, Kruse C, et al.: Vitamin D promotes vascular regeneration. Circulation. 2014; 130(12): 976–86. PubMed Abstract \| Publisher Full Text\n\nWebb JH, Blom AM, Dahlback B: Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells. J Immunol. 2002; 169(5): 2580–6. PubMed Abstract \| Publisher Full Text\n\nDeapen D, Escalante A, Weinrib L, et al.: A revised estimate of twin concordance in systemic lupus erythematosus. Arthritis Rheum. 1992; 35(3): 311–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7494", "date": "23 Feb 2015", "name": "Marko Radic", "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\nPrechl and Czirjak present an attempt to synthesize several aspects of lupus pathogenesis. In many respects, the opinion article reviews well known territory and the novel aspect is that the authors seek to include a role for complement C1q in the processes of transendothelial transport and paracellular leakage. These events could be compromised by the reduced levels of C1q that are associated with lupus or with DNA-anti-DNA immune complexes (IC). The proposed hypothesis is appealing because it may account for the early steps in pathogenesis. It may also provide a convincing connection to sites of vascular disease and IC deposition. One aspect that is not thoroughly covered is the deposition of C1q at sites of endothelial discontinuity. These are presented as areas that arise from the apoptotic death of endothelial cells which could expose underlying ECM. However, previous reports of apoptosis in a continuous endothelium revealed the rapid displacement of the dead cell by surrounding cells in the cell layer. This needs to be discussed more fully. In addition, the primary references describing the role of C1q in endothelial healing and renewal are sparse and incomplete. Possibly, the involvement of C1q in angiogenesis could be related to the proposed process but the connection is not clearly developed. Overall, the opinion piece could be streamlined by focusing on the innovative concept of C1q's role in regenerating endothelial gaps. Minor comments:Figure 1 shows brackets that do not make sense mathematically. Why are genetic factors shown in purple? \"Endothel\" should be endothelium. Figure 2 is a bit busy and difficult to deconstruct. Also the color of arrows is not explained. Figure 3 is confusing as to what factors make the three colored ovals merge and overlap.", "responses": [ { "c_id": "1330", "date": "11 May 2015", "name": "József Prechl", "role": "Author Response", "response": "Thank you for your thorough analysis of our hypothesis and its description. We do agree that the data supporting the role of C1q in regulating endothelial permeability, healing and renewal is sparse. This is main the reason for calling this work a hypothesis and not yet a theory. The reports of C1q influencing endothelial renewal however do fit into the overall emerging big picture of the complement system playing a general role in morphogenesis and tissue repair. We hope and expect to see more experimental data on these aspects of complement in the near future. To highlight the interactions of C1q with the endothelium and the consequences of C1q-containing immune complexes binding to the endothelium we have inserted two new figures into the second revised version of the paper.The speed of dead endothelial cell replacement and also the turnover rate of these cells are difficult issues. Data are not consistent on these values but because of the huge surface the endothelium covers we expect that even minor and accidental discontinuities count and serve as sites of damage initiation. This may well contribute to the randomness of the development of damage sites and of flares.We accept that brackets and parentheses do not change the result of a multiplication when numbers are multiplied. If we consider these factors not as numbers but something more complex, like matrices, then brackets might make sense. Here, using the brackets and blue/purple color, we only intended to highlight that genetic variability mainly affects proteins contributing to physiology and immunity but not anatomy. We explained this in the legends now.Syntax errors were corrected as suggested.We explained the colors of the arrows in figure 2. To make this figure somewhat less busy we also decreased the size of the arrows in between the boxes.We think that it is one major abnormality (e.g. C1q deficiency) or the combination of several abnormalities (multiple genetic factors affecting e.g. apoptotic debris clearance via CR3, adaptive response e.g. via HLA alleles, together with environmental effects, like an infection) that trigger the imbalance and the merging of these different pathways. The consumption and relative depletion of C1q and natural IgM is key to this overlap and lupus development.To help the reader follow this line of thinking we added „consumption of the gatekeepers,” to the legend.We hope that our responses and corrections are acceptable and improve understandability." } ] }, { "id": "7993", "date": "17 Mar 2015", "name": "Gregg Silverman", "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\nThe manuscript of Prechl and Czirjak provides a thoughtful review of the interface of endothelial cells and soluble factors of complement and antibodies in the pathogenesis of SLE and related diseases. I would recommend to the authors that they also consider the work of two independent groups on the effects of autoantigen specific IgM in the development of post ischemic injury to the vasculature 1,2. Candidate auoantigens have been identified (i.e., annexin IV, non-muscle myosin heavy chain II)3. Overt apoptosis of the endothelial cells may not be an absolute requirement for the expression of modified self antigens that then become accessible to IgM molecules. Furthermore the authors comment that products of complement activation are involved in the clearance of injured and apoptotic cells, but there is extensive evidence that deposition of C1q or MBL are themselves sufficient to serve as “eat me “ signals 4,5. Complexes of (auto) antigen and IgM can therefore enhance phagocytic clearance as they can further enhance MBL and C1q recruitment 6.", "responses": [ { "c_id": "1329", "date": "11 May 2015", "name": "József Prechl", "role": "Author Response", "response": "Thank you for taking the time to read and constructively comment on our hypothesis. We have incorporated some of the suggested papers as references. Actually we tried to build our hypothesis mostly on human data, because of the known differences in complement regulation between human and mouse. It was of course unaviodable to utilize murine experimental observations, but we kept it to a minimum. Nevertheless, some of the references are murine experimental data and biochemical observations probably hold for the human as well. More caution is probably needed when biological phenomemon and specific immune mechanisms, as specificity of natural antibodies, are considered. A careful comparison and analyis of human and murine data is probably required but this exceeded the scope of this hypothesis paper." } ] }, { "id": "8022", "date": "18 Mar 2015", "name": "Fleur Bossi", "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\nThe title and Abstract are appropriate.The article is well written, and the hypothesis is consistent and supported by a good number of references.The conclusions are balanced and well documented. Minor comments:Page 3 Figure 1 endothel should be endothelialPage 7 Figure 2 suffient should be sufficientPage 7 Figure 2 Legend (A) endothel should be endothelial", "responses": [ { "c_id": "1328", "date": "11 May 2015", "name": "József Prechl", "role": "Author Response", "response": "Thank you for reading our paper with scrutiny and helping to improve the quality of the paper. We have made the corrections you suggested in the second version." } ] } ]	1	https://f1000research.com/articles/4-24
https://f1000research.com/articles/4-111/v1	11 May 15	{ "type": "Case Report", "title": "Case Report: Pulmonary Alveolar Calcification as a Result of Severe Hypercalcemia due to Acute Lymphoblatic Leukemia.", "authors": [ "Jose Colleti Junior", "Eliana Carla Armelin Benites", "Gustavo Spadaccia dos Santos Fernandes", "Norberto Antonio Freddi", "Walter Koga", "Werther Brunow de Carvalho", "Eliana Carla Armelin Benites", "Gustavo Spadaccia dos Santos Fernandes", "Norberto Antonio Freddi", "Walter Koga", "Werther Brunow de Carvalho" ], "abstract": "Severe hypercalcemia is a rare metabolic disorder in pediatric medicine. This report describes a rare case of severe hypercalcemia and its clinical manifestations in a 2-year-old toddler. The radiological findings caused by hypercalcemia and osteolysis were emblematic of the osteolytic lesions. Hypercalcemia led to massive pulmonary alveolar calcification. The hypercalcemia was successfully treated with pamidronate, a bisphosphonate drug class. Further investigation resulted in a diagnosis of acute lymphoblastic leukemia (ALL). The patient is currently on chemotherapy and has a favorable prognosis. Although severe hypercalcemia alone is an unusual finding as the first sign for ALL, this should be considered, not to mention the radiological images resulted from calcium deposits.", "keywords": [ "hypercalcemia", "acute lymphoblastic leukemia", "pulmonary alveolar calcification", "osteolytic lesions", "paediatric case report" ], "content": "Introduction\n\nSevere hypercalcemia is unusual in children and can be either caused by elevated parathyroid hormone (PTH) or a PTH-independent mechanism. Primary hyperparathyroidism, familial hypocalciuric hypercalcemia, familial hyperparathyroidism and secondary hyperparathyroidism are examples of PTH-mediated causes of hypercalcemia. In patients with these conditions, in the early stages of the disease, there is a rise in PTH (or inappropriately normal PTH - not suppressed at the beginning of the hypercalcemia), which can confound the differential diagnosis1,2.\n\nThe most common PTH-independent mechanisms for hypercalcemia are related to malignancies1–3, with many different contributing mechanisms. One such mechanism is local metastatic osteolysis (also known as local osteolytic hypercalcemia or malign humoral hypercalcemia (MHH)), which contributes to hypercalcemia through the production of humoral factors by the tumor. This particular mechanism accounts for 80% of the hypercalcemias related to malignancies1,2. Most MHH are due to PTHrP and, less frequently, by the production of Vitamin D (1, 25(OH) 2D3). Tumors that secrete PTHrP can induce an increase in bone resorption and calcium reabsorption from the distal kidney tubule, raising the plasmatic calcium from both mechanisms1–3.\n\nWe report a case of alveolar pulmonary calcification after severe hypercalcemia in a 2-year-old toddler with a diagnosis of acute lymphoblastic leukemia (ALL); a rare condition in the pediatric age group. The radiological images of the osteolytic lesions and the alveolar calcification prompt this report, in the hope that they might help to alert clinicians to this unusual condition.\n\n\nCase report\n\nA 2-year-old Brazilian white male toddler weighing 11.8kg - previously healthy - presented with vomiting after meals, muscular weakness, generalized pain, abdominal cramps, intestinal constipation and inappetence that had begun 25 days prior to his first examination. There was no relevant information about family history.\n\nHe was admitted to the pediatric intensive care unit dehydrated, pallid, referring generalized pain, mainly on legs, arms and in abdomen, and was almost unable to walk. At first, the staff thought it could be sepsis or some endocrinological disorder. However, the initial laboratory tests showed heightened ionic calcium levels in the blood (2.95 mmol/L; normal: 1.11 to 1.40 mmol/L). Other laboratory analyses showed hemoglobin: 9.5 g/mL, white blood cell count: 9,460 cells/mm³, platelet count: 206,000/mm³. Urinalysis showed an elevated leukocyte presence (100 leukocytes/field; normal: < 10/field).\n\nAs soon as we received the results of the calcium analysis we submitted the patient to a pelvic X-ray to check calcification status. The X-ray revealed substantial osteolysis (Figure 1).\n\nWe started treatment for hypercalcemia with hydration and low doses of furosemide (1mg/kg/day divided into 3 doses, duration 2 days) in order to raise the calcium excretion by the kidneys2. We decided to start pamidronate (0.5mg/kg/day, duration 3 days) – a second-generation bisphosphonate class drug – to stop the osteolysis by the inhibition of calcium resorption2. The symptoms of hypercalcemia subsided and the patient improved.\n\nSubsequent laboratory analysis showed that PTH was low at 8 pg/mL (normal: 10 to 65 pg/mL), calcitonin was normal at 8 pg/mL (normal: less than 12 pg/mL) and 1, 25(OH) 2D3 was low at 21 ng/mL (normal: 30 to 60 ng/mL).\n\nA myelogram was performed and was compatible with acute leukemia. The immunophenotype showed the presence of immature T-type cells that expressed intracytoplasmic CD3 antigens, CD7, CD5, CD1a and partial terminal deoxynucleotidyl transferase (TdT). The presence of CD45 at moderately high levels and a lack of CD2 expression in the studied cells were also observed.\n\nThe patient underwent chemotherapy based on the standard Berlin-Frankfurt-Munich (BMF) protocol for pediatric ALL. Complete clinical remission occurred after the first cycle of chemotherapy. As a result, calcium levels returned to normal (ionic calcium: 1.2 mmol/L).\n\nSeven months after starting treatment, fever and bacteremia occured, with no associated neutropenia. An infectious disease screen was performed (blood and urine cultures included), and chest x-ray revealed multiple dense nodular structures. A CT scan confirmed structures resembling calcium nodules not exceeding 1 cm in diameter with peribronchovascular distribution, affecting both lungs mainly in the inferior lobes (Figure 2). Plasma calcium levels (ionized calcium: 1.2 mmol/L) were normal at that time. Tests for fungal infection, and specifically for Aspergillus spp., gave negative results. We also tested for Cryptococcus neoformans (agglutination test), Cytomegalovirus spp. (antigenemia), tuberculosis (three gastric lavages) and respiratory viruses (nasal secretion tests), with all negative results. The patient had no respiratory symtoms or hypoxemia. Despite the negative results, while the search for an infectious agent was ongoing, he received empiric antibiotic therapy, cefepime (150mg/kg/day) and vancomycin (60mg/kg/day) for 10 days, and lipossomal amphotericin B (5mg/kg/day) for 7 days.\n\nThe patient was free from infectious or respiratory disorders. Therefore we attributed the nodules observed in the CT images (Figure 2) to previous hypercalcemia which possibly led to a process of pulmonary alveolar calcification. We did not perform a biopsy, since we saw no benefit to the patient of doing so.\n\nPresently the patient is on maintenance chemotherapy with methotrexate (20mg/m²/once a week) and 6-mercaptopurine (50mg/m²/day) completing 106 weeks of treatment.\n\n\nDiscussion\n\nHypercalcemia in children is rare, especially when it is associated with signs and symptoms that precede a malignant disease. The etiology of hypercalcemia in children is different from adults3,4. Primary hyperparathyroidism and malignant diseases accounts for 90% of hypercalcemia in adults, but both these conditions are rare in children. The first association between hypercalcemia and malignancy was demonstrated by Myers5. The incidence of hypercalcemia in pediatric malignant diseases has been reported to be from 0.4% to 1.3%.\n\nAlveolar calcification in itself is a rare condition, often associated with hypercalcemia6,7 and has been reported only rarely in literature6,7 in association with acute leukemia, making this an unusual finding.\n\nAt first, the atypical clinical presentation of this case – the signals and symptoms associated with hypercalcemia - led to a wrong turn in the diagnostic path, seeming to indicate sepsis or endocrine disorders, before we knew about the calcium status. Thereafter, once the cause of the hypercalcemia was detected, our team of specialists acted in harmony and quickly came to a diagnosis.\n\nThe treatment of hypercalcemia depends on the primary cause3. Hydration and bone resorption inhibition with bisphosphonate agents are the most important interventions. Bisphosphonate treatment forms the basis of therapy for malignancy-associated-hypercalcemia, and may provide the necessary time for other antitumor therapies to act. Bisphosphonates (in the same mechanism of action used to treat osteoporosis), prevent the bones from losing calcium in primary hyperparathyroidism, but do not decrease the calcium levels. However, when the hypercalcemia is PTH-mediated, surgery is the standard therapy when possible.\n\nThe atypical radiological images, if they had not been associated with severe hypercalcemia could have led to unnecessary procedures like biopsies, or the wasteful use of other therapies, like antibiotics and antifungals.\n\nFor the clinician it is important to consider a diagnosis of pulmonary calcium alveolar deposits when faced with images resembling what we present in this report, and when other clinical issues are compatible so that a rapid diagnosis is possible, with the hope of sparing the patient unnecessary therapies.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the parent of the patient.", "appendix": "Author contributions\n\n\n\nJCJ prepared the first draft of the manuscript, EB was from the oncologist team, GSSF prepared the images, NAF and WBC contributed with their expertise for the therapeutic plan. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTrehan A, Cheetham T, Bailey S: Hypercalcemia in acute lymphoblastic leukemia: an overview. J Pediatric Hematol Oncol. 2009; 31(6): 424–7. PubMed Abstract \| Publisher Full Text\n\nShane E, Irani D: Hypercalcaemia: Pathogenesis, Clinical Manifestations, Differential Diagnosis and Management. In: Favus MJ (ed). Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. American Society for Bone and Mineral Research, USA. 2006; p. 176–80. Reference Source\n\nHamdy NA, Papapoulos SE: Management of malignancy-associated hypercalcaemia. Clin Rev Bone Min Metabol. 2002; 1(1): 65–76. Publisher Full Text\n\nJacobs TP, Bilezikian JP: Clinical review: Rare causes of hypercalcemia. J Clin Endocrinol Metab. 2005; 90(11): 6316–22. PubMed Abstract \| Publisher Full Text\n\nMyers WP: Clinical aspects and management of hypercalcemia. Med Clin North Am. 1956; 40(3): 871–85. PubMed Abstract\n\nIzadyar M, Mahjoub F, Ardakani SN, et al.: Pulmonary metastatic calcification in a leukemic patient: a case report. J Pediatr Hematol Oncol. 2010; 32(3): e108–e110. PubMed Abstract \| Publisher Full Text\n\nNorthcutt AD, Tio FO, Chamblin SA Jr, et al.: Massive metastatic pulmonary calcification in an infant with aleukemic monocytic leukemia. Pediatr Pathol. 1985; 4(3–4): 219–29. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8865", "date": "02 Jun 2015", "name": "Jerry Zimmerman", "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\nThis article submitted by Colleti and colleagues from Santa Catarina Hospital, São Paulo, Brazil, provides an interesting case report of an unusual entity, namely pulmonary alveolar calcification. The report is well documented and the conclusions not overstated. However, additional data, if available would be of interest. Specific comments regarding the manuscript are noted below:‘Acute lymphoblastic leukemia’, not ‘acute lymphoblatic leukemia’. At the time of initial presentation, what was the calcium x phosphate product? If the tumor burden was high and ongoing lysis of leukemia cells was occurring, serum phosphate might also be expected to be elevated and contributing to the calcification of soft tissue. Did the patient present with hypertension? Was a shortened QT interval noted on the admission EKG? Did the medical team specifically inquire about a family history of multiple endocrine neoplasia? At the time of initial presentation, did radiographs demonstrate any evidence of soft tissue calcification? Was calcification of the lungs present on admission; was nephrolithiasis present? The CT scan reproduced in Figure 2, also appears to demonstrate calcification of the heart and great vessels, as well as severe osteopenia of the lower thoracic vertebrae. Did the patient exhibit any structural cardiac problems, such as calcified valve leaflets by echocardiography? Did the patient exhibit any neurological findings attributable to thoracic vertebral osteolyis? Although biphosphonates represent the safest approach to therapy for this patient, is there any role for calcitonin, mithramycin, and indomethacin? It is probably worth mentioning pulmonary alveolar microlithiasis as well as the SLC34A2 gene mutation in the Na/PO4 co-transporter, as other causes of pulmonary alveolar calcification. Given the appearance of thoracic CT imaging, longitudinal pulmonary function testing is probably warranted for the patient presented.", "responses": [ { "c_id": "1426", "date": "22 Jun 2015", "name": "Jose Colleti Junior", "role": "Author Response", "response": "The comments of Dr Zimmerman about this article are relevant and helpful.I will try to elucidate some questions as follows:The phosphate was really high at the initial presentation, contributing to the calcification of soft tissue; The patient did not have hypertension; The initial EKG was normal (we did not expect that); There was no evidence of family neoplasias; We considered other drugs, besides bisphosphonates, to treat the hypercalcemia. We decided on pamidronate because of the availability, and our personal experience; Although the thoracic CT imaging is striking, the patient did not present any clinical signal of respiratory disorder. The patient is still undergoing treatment and is doing well." } ] }, { "id": "9334", "date": "08 Jul 2015", "name": "Ana Paula de Carvalho Panzeri Carlotti", "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\nThe authors report a rare case of severe symptomatic hypercalcemia as the initial manifestation of acute lymphoblastic leukemia associated with massive pulmonary alveolar calcification which was diagnosed several months later. The case is well documented and image findings are striking. This case report may be useful to alert pediatricians for the diagnosis of malignancy and also favor an early investigation of metastatic calcification of soft tissues in patients with such a severe degree of hypercalcemia.", "responses": [] } ]	1	https://f1000research.com/articles/4-111
https://f1000research.com/articles/4-110/v1	08 May 15	{ "type": "Software Tool Article", "title": "AGRIS: providing access to agricultural research data exploiting open data on the web", "authors": [ "Fabrizio Celli", "Thembani Malapela", "Karna Wegner", "Imma Subirats", "Elena Kokoliou", "Johannes Keizer", "Fabrizio Celli", "Karna Wegner", "Imma Subirats", "Elena Kokoliou", "Johannes Keizer" ], "abstract": "AGRIS is the International System for Agricultural Science and Technology. It is supported by a large community of data providers, partners and users. AGRIS is a database that aggregates bibliographic data, and through this core data, related content across online information systems is retrieved by taking advantage of Semantic Web capabilities. AGRIS is a global public good and its vision is to be a responsive service to its user needs by facilitating contributions and feedback regarding the AGRIS core knowledgebase, AGRIS’s future and its continuous development. Periodic AGRIS e-consultations, partner meetings and user feedback are assimilated to the development of the AGRIS application and content coverage. This paper outlines the current AGRIS technical set-up, its network of partners, data providers and users as well as how AGRIS’s responsiveness to clients’ needs inspires the continuous technical development of the application. The paper concludes by providing a use case of how the AGRIS stakeholder input and the subsequent AGRIS e-consultation results influence the development of the AGRIS application, knowledgebase and service delivery.", "keywords": [ "Semantic Web", "Linked Open Data", "AGRIS community", "agriculture", "agricultural data" ], "content": "1.0 Introduction\n\nIn the last decade Semantic Web technologies have introduced changes into the way structured data is published, shared and consumed on the Web. The Web has become a powerful bedrock where emerging online applications use it as an infrastructure to exchange, query and link semantically related data and information1. In order to take advantage of the prowess of the emerging Web, many repositories have adopted linked data principles making the vision of a semantic Web of data a reality2. Overtime, two important roles of linked open data (LOD) have emerged: consuming and publishing data, thereby facilitating innovation and wider knowledge creation and sharing3. The principle of linked data has been extensively described in publications and books4,5. There are still challenges faced in browsing, analyzing, reusing and consuming linked data by the research community, Semantic Web community and policy makers. The major fallacy1 of these emerging technologies is that they assume that connectivity to data repositories and entity resolution services are always online and available.\n\nIn the agricultural domain, the Agricultural Information Management Standards (AIMS) Team of the Food and Agriculture Organization of the United Nations (FAO) has taken advantage of the possibilities of LOD in making agricultural data, information and knowledge accessible. Often-cited examples include the publication of AGROVOC (http://aims.fao.org/vest-registry/vocabularies/agrovoc-multilingual-agricultural-thesaurus) as a linked data set6, and the AGRIS database and application7. AGRIS (the International System for Agricultural Science and Technology) is an initiative that was set up in 1974 by the FAO to make agricultural research information discoverable and globally available. Since then AGRIS has been collecting from more than 150 data providers located in more than 65 countries. AGRIS collects and disseminates bibliographic information on scholarly and scientific publications in agriculture and related subjects.\n\nAGRIS today is a ‘global public good’8, built and maintained by a big community of data providers, partners and users. This is based on two overarching principles. Firstly, that AGRIS grants complete core access to data where users are allowed to download and use the content subject to an acceptable use policy (http://agris.fao.org/content/acceptable-use-policy). Secondly, users are invited to give ideas on the development of AGRIS and its vision through e-consultations, stakeholder meetings, user surveys and feedback. This paper will briefly overview the recent developments in AGRIS and outline the latest technical implementations. The objective of this paper is to show the responsiveness of AGRIS to the community (clients’) needs and review the steps leading to the technical development and future direction of AGRIS.\n\n\n2.0 The AGRIS mashup\n\nSince December 2013, AGRIS has exposed its database as LOD, defining uniform resource identifiers (URIs) for bibliographic publications and allowing anyone to reuse the database also through a SPARQL endpoint. After an initial period where LOD opportunities were tested in the OpenAGRIS system9, the AGRIS team decided to adopt LOD standards into the deployed system. The goal was to take advantage of the latent knowledge available in the AGRIS data, in order to automatically discover and display related and relevant information from the Internet. AGRIS seeks to become the prime information service for agricultural research, where domain experts, agricultural extentionists, students, researchers, librarians/ information managers and decision makers can discover needed information with precision and recall it in a short response time. When the user is searching for a publication, the AGRIS system is able to enrich the user’s query by displaying a mashup page with results of related information available on the same topic. To achieve this, AGRIS adopted a dual approach that allows users to access agricultural information through:\n\n- Bibliographic metadata in the domain of agricultural science and technology are stored in a central database, currently storing nearly 8 million bibliographic references of scholarly and scientific publications.\n\n- Other types of information (distribution maps, passport data, pictures, other bibliography, etc.) that are interlinked to the AGRIS central database.\n\nTwo things are crucial to build a useful mashup page: the selection of the data sources and the precision of the automatic extracted resources. Precision in this context means the relevance of displayed resources10 must be of the same subject coverage as that of the article selected by the user. In AGRIS this is possible through AGROVOC6, which is a Simple Knowledge Organization System (SKOS) concept scheme used to index the AGRIS database. AGROVOC brings additional value as a thesaurus consisting of more than 32,000 concepts and is available in 21 languages, covering all areas of interest to the AGRIS database. Therefore, AGROVOC is the backbone of the resource discovery process where AGRIS records (which are indexed with AGROVOC concepts) are used to query external Web services (e.g. by scientific names) and SPARQL endpoints by using AGROVOC URIs or alignments with other thesauri related to agriculture. External data sources are identified based on the content, the relevancy to the AGRIS domain, and after evaluating, the information provider11.\n\nFigure 1 shows a mashup page which displays an AGRIS record selected by the user with some AGROVOC descriptors and URIs (left). Once AGRIS loads the mashup page, it reads the list of AGROVOC URIs available within the AGRIS record, and run asynchronous queries to external Web services and SPARQL endpoints to get information related to the content of the selected AGRIS record. In the screenshot below for the AGROVOC concept “Oryza sativa”, AGRIS displays a distribution map from GBIF (http://www.gbif.org; the Global Biodiversity Information Facility), as well as some germplasm collecting missions from Bioversity International (http://www.bioversityinternational.org/). AGRIS pulls and visualizes data from World Bank, CGRIS germplasm database, and International Food Policy Research Institute (IFPRI). A full listing of external data sources AGRIS pulls from is available on AGRIS website (http://agris.fao.org/content/how-it-works).\n\nThis allows users to tell the data source of the main bibliographic data of the AGRIS article.\n\n\n3.0 AGRIS recent developments\n\nAGRIS is constantly evolving to provide its users with new valuable services and many new different sources of information to be explored. The development of AGRIS, both on the service and data sides, is mainly driven by AGRIS users, who can provide feedback, ideas and needs in different ways: using the “feedback” form available in the AGRIS Web site; responding to periodic surveys designed by the AGRIS team; sending emails to the AGRIS Team or joining online events like AIMS Webinars [http://aims.fao.org/capacity-development/webinars] or AGRIS e-consultations. All this feedback is collected and analyzed to define priorities and provide new services to the community. For instance, after the adoption of a linked data infrastructure (when AGRIS and OpenAGRIS were merged at the beginning of 2014), the AGRIS team prepared an online survey and Webinars to collect feedback about the new AGRIS Web application. Two main activities were considered as top priorities to improve the service:\n\n1. Inclusion of all the available bibliographic metadata in the AGRIS mashup page;\n\n2. Multilingual search with the possibility to get results in several languages when searching with keywords in a specific language.\n\nThe first activity was carried out because, even though the AGRIS Web site mashes-up many sources of information to provide its users with a good browsing experience, for many AGRIS users access to the complete bibliographic metadata set is a valuable piece of information. Thus, the mashup view was extended to include all bibliographic metadata available, and advanced search functionality (namely, “classical view”) was re-introduced to allow filtering results according to specific metadata elements.\n\nIn the second activity, the multilingual search, the objective was to allow users to query the AGRIS database in their own native language, as well as retrieving results in different languages. This is exemplified by the following use case:\n\nXian is a Chinese researcher and he wants to discover some knowledge from the AGRIS database. He wants to know something more about “rice” and recent research activities surrounding it, but he prefers to query the database using his own native language. So he starts querying AGRIS using the keyword “稻米”. The AGRIS system discovers only 14 documents: they are not enough to add additional filters and they refer only to documents indexed with a Chinese keyword. Xian wants to access the international literature, so he also wants English articles. On the right side of the AGRIS interface, Xian enables the multilingual search and clicks on “GO”: 150,000 results! Maybe now Xian has too many articles to examine, but he can use other keywords to restrict the number of the results…\n\nThe multilingual search is very important to facilitate access to literature in different languages: a future improvement of this feature will be offering the possibility of selecting sub-sets of languages to be included in the output of a query. The implementation of this feature relies on AGROVOC and on the AGRIS-linked open data infrastructure. In fact, while AGRIS records are indexed with AGROVOC keywords in a specific language, the translation to resource description framework (RDF) makes AGROVOC URIs usable. From an AGROVOC URI there is a possibility to extract labels of a concept in all the languages available in AGROVOC: those labels can be considered as “translations” of a query term, so that they can be used to expand the user’s query to include the translation of terms in different languages. To be more precise, the implementation of the multilingual search feature required two activities:\n\n- Indexing AGROVOC URIs in Apache Solr (http://lucene.apache.org/solr)\n\n- Implementation of a software component that expands the user’s query to match results in all languages available in AGROVOC. The query expansion is transparent to the end user, who does not need to know technical details of this feature.\n\nAnother improvement was the inclusion of Chinese research content in the AGRIS database where a large amount of Chinese metadata were directly interlinked to the AGRIS database and displayed in the mashup pages. In the context of AgINFRA (http://aginfra.eu/) and the collaboration between AGRIS and the Chinese Academy of Agricultural Sciences [http://www.caas.cn/en/administration/research_institutes/research_institutes_beijing/77772.shtml], 500,000 resources from the Chinese Agricultural Sci-tech Documents Database (CASDD) and 410,000 resources from the CGRIS germplasm database were exposed as Web services and exploited as AGRIS external data sources, relying on the AGROVOC formal alignment with the Chinese Agricultural Thesaurus (CAT). The outcome of this activity was the inclusion of a large batch of Chinese research in agriculture in the AGRIS system, together with a unique collection of all types of plant genetic resources information from China, enriching the AGRIS knowledge base.\n\n\n4.0 AGRIS data ingestion\n\nAGRIS is supported by a community of data providers, partners and users. AGRIS ingests bibliographic metadata provided by the community and publishes it as open data; the metadata is captured through either (i) pulling data through harvesting from clients or (ii) by data being pushed to the AGRIS from clients9. AGRIS uses various tools and technologies to consume metadata from content providers and accepts any metadata records that meet the Meaning Bibliographic Metadata (M2B) standards. AGRIS’s data providers come from an international audience, with users often at varying stages of technological development. Figure 4 below summarizes the AGRIS data workflow, ingestion and processing.\n\nThe resultant AGRIS content is exposed via the AGRIS Web application – which is a mashup application that allows users to query the AGRIS content, interlinking all records to external sources of information. (See Figure 1 above and section 2.0 for more details).\n\n\n5.0 AGRIS community needs\n\nAGRIS’ vision is to be a responsive service to its global users’ needs by facilitating their contribution to the AGRIS core knowledgebase, AGRIS’s future and continuous development. Since 2014, FAO, Agro-Know (http://www.agroknow.gr/agroknow/) and the Agricultural Information Institute of Chinese Academy of Agricultural Sciences (http://www.caas.cn/en/administration/research_institutes/research_institutes_beijing/77772.shtml) (CAAS) have established a collaboration for the maintenance and centralization of AGRIS data processing. The collaboration is keen to keep AGRIS a community-driven product responding to the needs of the clients. The AGRIS team has a commitment to see AGRIS visitors and data providers as clients who contribute to the continuous development of AGRIS. In pursuit of this goal, periodic AGRIS stakeholder meetings, AGRIS e-consultations in the form of online surveys and user feedback are carried out to inform the development of the AGRIS application and coverage of the knowledgebase. Four thematic areas of focus have emerged since the initial discussions: 1.) AGRIS subject coverage, 2.) geographical accessibility of the system, 3.) improvement in user interactions and multilingualism and 4.) Strengthening the infrastructural backbone of AGRIS.\n\nIn considering these thematic areas, AGRIS partners agreed to map a strategy in each respective thematic area where the resultant output will drive technical developments, new functionalities and usability features. The involvement of the AGRIS community of users and further collaboration on technical developments will be invaluable in strengthening and developing new functionalities for the AGRIS portal. Feedback received from the community of data providers, partners and users is important for the possible improvements to the AGRIS portal and the knowledgebase. Furthermore, as stated earlier AGRIS has also been involved in a number of projects with the European Commission. For example, within the SemaGrow (http://www.semagrow.eu/) project, AGRIS served as a demonstrator of a technical infrastructure based on the federation of many triple stores; relying on the two backend components Agro Tagger (http://aims.fao.org/vest-registry/tools/agrotagger) and Web Crawler, AGRIS will be able to crawl the Web and to index discovered resources with AGROVOC URIs.\n\nThe AGRIS maintenance partners sought the engagement of the broader community into the further technical developments of AGRIS in the key thematic areas outlined above. The following issues emerged in the aforementioned four key areas:\n\nIn terms of subject coverage, the AGRIS database collects bibliographic references in agriculture as defined by the FAO which includes nutrition, forestry, and fisheries. Since the nomenclature of AGRIS defines it as an international system for Agricultural Science and Technology, technology could also be included. The full list of subject categories can be downloaded online (http://www.fao.org/scripts/agris/c-categ.htm): they will be revised in the coming months to enable the list to cope with the increased subject coverage requirement.\n\nIn terms of content, AGRIS core data initially focused on grey literature and later came to include papers, reports and other content types. The partners felt that AGRIS backbone data should continue to be bibliographic metadata, but felt that linked data technologies should be fully exploited to allow the inclusion of other relevant content types. To further develop the coverage of AGRIS content and to prevent stagnation, the AGRIS team aims to work out a new adequate subject scope for the AGRIS knowledgebase and discover new sources of information and data in collaboration with community partners. There are possibilities of linking AGRIS with science blogs and automatically updated feeds, and of further strengthening the relationship between AGRIS and AgriFeeds (http://www.agrifeeds.org/) (for example, http://esciencenews.com and other feeds from scientific presses and universities).\n\nUsing data mining, the AGRIS database could be a way to access already existing information. In the AGRIS e-consultation users expressed their demand for more additional data like statistics, multimedia, price data, daily crops prices etc. The user survey additionally underlined the high demand for accessing full text resources. The AGRIS team has already responded with the implementation of the mashup page that allows linking to full text resources in the internet. The AGRIS Team is aware of the potential in identifying relevant content to interlink with AGRIS core data (http://aims.fao.org/activity/blog/aginfra-promotes-integration-biodiversity-information-agris). The work on providing even more full-text links and resources will be continued, with the possibility of enriching AGRIS metadata with newly discovered full-text links and of setting up a link-checking mechanism to remove broken links. There will be a need for AGRIS’s authors’ disambiguation and the initial option could be to use unique author identifiers, for example AGRIS intends to use the AgriVIVO’s (http://aims.fao.org/vest-registry/tools/agrivivo) scientific profiles. Another interesting activity will be the analysis of AGRIS full-text links to extract relevant information, such as a database of pictures indexed with AGROVOC, which will help enrich the content of a specific paper and to allow the re-use of pictures for personal reports or research activities, subject to copyright.\n\nAlthough AGRIS can be accessed from anywhere in the world, it has been noted that there is a lack of good performance in some regions – especially in China and East Asia. This might be due to the fact that the front-end Web application is hosted only in Rome. The AGRIS team is aware of this challenge and is trying to look for a solution to geographical accessibility by collaborating with community partners. In the meantime, the possibility of having two replicas of the database in two continents to minimize this challenge is being considered. This solution will require resources (such as a system administrator in each replica), a synchronization mechanism and a networking mechanism to geographically serve users seamlessly from different places in the world.\n\nThe user survey which registered 279 respondents who confirmed the lack of performance in some geographic regions. The overall feedback on performance was positive: around 80% of the users rated the performance of the web portal as extremely good or moderately good. Half of the users that are not satisfied with the AGRIS performance come from Asian countries. Several options to improve performance especially for countries in East Asia are currently being discussed and need testing. The AGRIS team must ensure that a better connection to Asia will not put other regions at a disadvantage. Recently, the multilingual search is an example of a client-demand service that has already been implemented. (see recent developments in section 3.0 above).\n\nThe creation of a user registration facility is one of the main goals of the AGRIS team and was demanded by the community. Both AGRIS partners and users expressed their interest for a private area that allows the creation of personal profiles and the customization of the AGRIS interface (a step towards “social AGRIS”). The implementation of functions to define the portal design and select preferred datasets in the mashup page are possible as well as the addition of social functions like comments, ratings and quoting. The AGRIS team sees potential in having AGRIS sparking debates and collaborations based on AGRIS content. Support for mobile devices (e.g. smartphones and tablets) is another possible improvement that has been demanded in the surveys and discussed with individual community partners. In the survey users regard mobile device accessibility as very important with 40% of respondents wanting AGRIS to be read on a Tablet and 24% wanting access to AGRIS on their Smartphone.\n\nThe strengthening of the AGRIS backbone is important to provide a sustainable system. Currently, the AGRIS database is replicated in two types of models – (i) the AGRIS AP file system XML database and (ii) the AGRIS RDF triplestore. The AGRIS team will cease to maintain the AGRIS AP XML database and will design a new streamlined data model (most probably based on AGRIS RDF and linked open data-enabled bibliographic data (LODE-BD) (http://aims.fao.org/lode/bd) that allows ingestion of data directly into the triple store. One of the goals is to design a more scalable and stable backend solution, as a system of load balancing of different instances of the AGRIS triplestore. In order to involve the community, the AGRIS team will have experiments as part of ‘Hackathons’ where participants can try different triplestore solutions simulating AGRIS queries to the database.\n\nThe above summarized feedback represents the many suggestions of the clients’ needs and expectations from AGRIS. The value of the user-driven and responsive service is a core part of the AGRIS Vision and its continuous development. The move to a ‘social AGRIS’ will ensure that the AGRIS community of partners, data providers and users shape AGRIS service into the future. The collection of feedback from AGRIS spurs a number of potential enhancements now and in the future, with experiments made possible by the community (in the form of Hackathons). Redesign of workflows and AGRIS architecture, alongside strategic collaboration with global partners are some of the evident processes and activities within the AGRIS future vision.\n\n\n6.0 Conclusion\n\nAGRIS seeks to be a technological service that embraces the linked open data technologies while continuing to be a service relevant to its clients. This paper establishes that AGRIS is a global good, in that it is truly global in terms of data contribution and access, and also a public good in that it is built, maintained and responds to its community of partners, data providers, and users. AGRIS has been a bedrock for a number of semantic tools (as exhibited by the SemaGrow project) yet also provides a gateway to scientific research in Agriculture, Science and Technology. Semantic Web features have afforded AGRIS the ability to continue to be ‘the’ portal in Agriculture, Science and Technology for students, researchers and policy makers while at the same time constantly providing new and valuable services to the community in a dynamic and changing world.", "appendix": "Author contributions\n\n\n\nFC acted as the AGRIS technical lead and decided on the conceptual content, was responsible for technical parts of this paper, contributed section 2.0 and section 3.0 and was responsible for production of all the images.\n\nTM acted as corresponding author and coordinated inputs from all sections as well as contributing section 1.0 and part of section 5.0 and section 6.0, including the literature search. TM also fine-tuned the references and organization of the paper.\n\nKW contributed to section 5.0, interpreted the survey results and related materials and also contributed the whole AGRIS community section.\n\nIS provided editorial guidance and edited the paper for appropriateness regarding F1000 guidelines and correctness in discussing metadata related aspects.\n\nEK is responsible for AGRIS data processing and contributed to section 5.0 by providing the analysis of the survey.\n\nJK made provided guidance as to the title of the paper and direction it took, as well as taking responsibility for this content from FAO’s perspective. JK also reviewed the article and cleared it for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe work described in this article was partly funded by the EC project “agInfra: A data infrastructure to support agricultural scientific Communities”, Grant agreement no: 283770.\n\n\nReferences\n\nGueret C, Boyera S, Powell M, et al.: The Semantic Web for all. 2014. Reference Source\n\nGayo JEL, Kontokostas D, Auer S: Multilingual Linked Data Patterns. 2012. Reference Source\n\nBauer F, Kaltenböck M: Linked open data: The essentials. A quick start guide for decision makers. 2012. Reference Source\n\nZuiderwijk A, Jeffery K, Janssen M: The potential of metadata for linked open data and its value for users and publishers. JeDEM. 2012; 4(2): 222–244. Reference Source\n\nZaveri A, Rula A, Maurino A: Quality assessment methodologies for linked open data: A systematic literature review and conceptual framework. 2012. Reference Source\n\nCaracciolo C, Stellato A, Morshed A, et al.: The AGROVOC linked dataset. Semantic Web. 2013; 4(3): 341–348. Reference Source\n\nAnibaldi S, Jaques Y, Celli F, et al.: Migrating bibliographic datasets to the Semantic Web: The AGRIS case. Semantic Web. 2015; 6(2): 113–120. Publisher Full Text\n\nRodríguez JM, Clement AJ, Farhan H, et al.: Publishing statistical data following the linked open data principles: The web index project. In Ordonez de Pablos, P. (ed). Cases on Open-Linked Data and Semantic Web Applications. Spain: IGI. 2013; 28. Publisher Full Text\n\nCelli F, Jaques Y, Anibaldi S, et al.: Pushing, Pulling, Harvesting, Linking: Rethinking bibliographic workflows for the semantic web. EFITA-WCCA-CIGR Conference, Turin, Italy, 24–27 June 2013. 2013. Reference Source\n\nTurpin A, Scholer F: User performance versus precision measures for simple search tasks. In Proceedings of the 29th Annual international ACM SIGIR Conference on Research and Development in information Retrieval. 2006; 11–18. Publisher Full Text\n\nJaques Y, Anibaldi S, Celli F, et al.: Proof and Trust in the OpenAGRIS Implementation. Proc. Int’l Conf. on Dublin Core and Metadata Applications. 2012. Reference Source" }	[ { "id": "8691", "date": "18 May 2015", "name": "Laura Privalle", "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\nThis is an informative article on the availability of a potentially very useful software tool of which I was not previously aware. The article presents clear examples of how to use the tool and the value it brings to the user. Others will be equally interested to learn of this website.", "responses": [] }, { "id": "9982", "date": "18 Aug 2015", "name": "Robert Paul Davey", "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, Robert Davey (TGAC, UK)will sign my name to my reviewwill review with integritywill treat the review as a discourse with you; in particular, I will provide constructive criticismwill be an ambassador for the practice of open scienceThe authors describe AGRIS, a metadata repository for an impressive cohort of bibliographic linked open data in the agricultural sciences.Managing large-scale data generative approaches (\"big\"/heterogeneous data) is only one side of the coin in the modern research era. With so much data available now (and this is only going to get worse), we need systems like AGRIS to make sense of the descriptions of data to ensure that researchers can find and reuse findings more easily, and more importantly integrate them into their own work.Whilst I too found the AGROVOC system to be a little slow and unresponsive, the AGRIS portal is relatively fast which shows the underlying power of the linked datasets, which the mashup interface represents nicely. I did attempt to use the LOD Live portion of the site, but all of the resources I tried to visualise came up with the \"no resource endpoint configured\". I'm not sure if this is a factor of the actual resource itself, or an issue with the query mechanism. Could the authors give some working examples?The paper mentions the use of Lucene, but are detailed technical documents about the software implementations that power the website available? Likewise, is there any relevant source code that would be suitable for release to the community? If so, links to this information might be useful.The paper reads well, and gives some insight into how such a platform is built, assessed and used. The ability to search for terms in multiple languages is something that is incredibly important, and should be commended.My only minor comments for revision would be that:the term \"mashup\" might not be well understood by many. A short description or refactoring of the term might add some clarity. I don't see the benefit of the 1.0, 2.0, etc section names. There are no subsections, so the article doesn't really need the ordered list style.", "responses": [] } ]	1	https://f1000research.com/articles/4-110
https://f1000research.com/articles/3-298/v1	08 Dec 14	{ "type": "Research Article", "title": "Effect of environmental and cultural conditions in medium pH and plant growth performance of Douglas-fir (Pseudotsuga menziesii) shoot culture", "authors": [ "Chien-Chih Chen", "Rick Bates", "John Carlson", "Chien-Chih Chen", "Rick Bates" ], "abstract": "The medium pH level of plant tissue culture has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies accordingly to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change and explant growth performance and 3) assess the effects of adding a pH stabilizer, 2-(N-morpholino)ethanesulfonic acid (MES) to Douglas-fir micropropagation medium. Spring buds, collected before breaking dormancy from juvenile and mature donor trees were utilized for these evaluations. Medium with or without MES, each at five medium pH levels was pre-adjusted before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. In general, medium with MES provided a more stable medium pH compared to pre-adjusted pH values under two storage conditions as well as with presence of explants over time. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. MES may help to maintain stable medium pH for bulk medium preparation. Our findings suggested a 21-day subculture practice may facilitate to sustain medium freshness, medium pH level and desirable explant growth.", "keywords": [ "medium pH", "Pseudotsuga menziesii", "Douglas-fir", "micropropagation", "2-(N-morpholino)ethanesulfonic acid", "MES" ], "content": "Introduction\n\nThe Christmas tree industry plays an important role within Pennsylvania agriculture as well as across the nation. The goal of this micropropagation project was to develop a true-to-type clonal propagation system to alleviate the cost of tree-to-tree variation by conventional seedling propagation. Understanding plant materials and their growing conditions may provide better assistance for later developmental stages in tissue culture.\n\nThe medium pH of plant tissue culture has been shown to be very important to many aspects of explant development and growth. Sensitivity or tolerance to medium pH change in vitro varies accordingly to specific requirements of individual species. Similar to soil pH, medium pH level may influence nutrient uptake (Ramage & Williams, 2002), cellular pH adjustment (Ballarin-Denti & Antoniotti, 1991), rooting and cellular growth (Leifert et al., 1992; de Klerk et al., 2008), plant gene expression and transcriptional pH responses in roots (Lager et al., 2010), and the efficiency of Agrobacterium-mediated transformation (Ogaki et al., 2008; Rai et al., 2012). Medium pH also can act to facilitate or inhibit nutrient availability in the medium such as ammonium uptake in vitro can be facilitated with a stable pH of 5.5 (Thorpe et al., 2008).\n\nMedium pH fluctuations may be attributed to medium components, autoclaving, ion exchange, and environmental conditions. Medium components may modify pH prior to and after autoclaving (Skirvin et al., 1986; Owen et al., 1991). Organic, inorganic salts, amino acids, vitamins, sucrose, gelling agents, and plant growth regulators are the common components added to tissue culture medium. Williams et al. (1990) reported adding agar significantly elevated medium pH prior to autoclaving in group of pre-adjusted pH from 3.5 to 5.5 of MS medium (Murashige & Skoog, 1962) but less increment was found in group of pre-adjusted medium pH from 5.5 to 7.0, or decrease in group of pre-adjusted medium pH from 7.0 to 8.0. In contrast, post-autoclaving medium pH increased in group of pre-adjusted pH of 3.5–4.5 but had more significant medium pH decrease in group of pre-adjusted pH of 5–8. Additions of synthetic or natural organic acids generally increase medium buffering ability (Thorpe et al., 2008). Organic compounds such as 2-(N-morpholino)ethanesulfonic acid (MES) could especially help to maintain suitable medium pH range for explant development (Parfitt et al., 1988; de Klerk et al., 2008; Yuan et al., 2012). MES and vitamin additions were also found to enhance embryo growth during the initiation stage of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) (Franco 1950) somatic embryogenesis (Pullman et al., 2005).\n\nAfter placing explants on the medium, medium pH fluctuations were also observed for various species. Medium pH of loblolly pine (Pinus taeda) grown in liquid suspension medium showed subsequent pH decrease from 5.5 to 4.6 within 5 days of incubation followed by pH increase to 6.0 in 19 days of incubation (Pullman et al., 2005). Significant medium pH decrease was also found after placing mulla mullas (Ptilotus exaltatus) shoots on MS medium for 4 weeks (Williams et al., 1990) and an U shaped curve of medium pH fluctuation was found in shoot tip culture of crabapple (Malus sp. cv. Almey) and pear (Pyrus communis L. cv. Seckel) on MS medium (Singha et al., 1987). According to Skirvin et al. (1986) and Thorpe et al. (2008), explant nutrient absorption in vitro is a function of ion exchange where deposition of free hydrogen ions (H+) and hydroxyl ion (OH-) in the medium may contribute to acidic or alkaline medium pH. In contrast, photooxidation induced chelating event bound free iron to reduce iron availability may also influence medium pH (Hangarter & Stasinopoulos, 1991). Secretion of plant secondary metabolites into culture medium is common in vitro (Dörnenburg & Knorr, 1995) but its role and function in altering medium pH and nutrient absorption are not clear.\n\nMedium pH fluctuations can involve many factors, and could eventually become problematic if lacking of care attentions. Douglas-fir shoot culture can be grown in a modified version (mDCR) of Douglas-fir cotyledon revised medium (DCR, Gupta & Durzan, 1985; Gupta & Durzan, 1987a). However, the interactions between the species and its growing medium are not well understood. To ensure an optimal shoot culture development and provide high quality shoots for later development of rooting protocol, medium pH can be a key indicator in determine subculture time. It may also be further utilized as a diagnostic tool for some abnormal growth symptoms such as necrosis, caused by low pH induced nutrient deficiency. The experimental system may be applied to studies of explant development and nutrient relationship in vitro (Singha et al., 1990). Hence, the objectives of this study are to 1) determine medium pH change over various times under storage conditions and in the presence of explants, 2) evaluate the effects of prolonged culturing on medium pH and explant growth performance, and 3) assess the effects of addition of a pH stabilizer, MES to Douglas-fir micropropagation medium.\n\n\nMaterials and methods\n\nSpring buds from juvenile (HF205 and HF210) and mature (PS-2) donor trees collected in 2006 were utilized for this study. These bud samples were collected prior to breaking dormancy. To classify juvenility, Douglas-fir trees of Lincoln seed source planted 10 years ago and not yet producing cones at the Penn State Horticulture Research Farm of Russell E. Larson Agricultural Research and Education Center at Rock Springs were the selected donors. The selected mature donor tree, as a result from a previous genetics study conducted by Gerhold (1984) was over 40 years old at the Penn State golf course. These bud samples were stored in a 4ºC cold room until further culture initiation. Preparation, sterilization, and dissection of collected bud sample were followed as per Traore et al. (2005).\n\nThis study consisted of a four-factor factorial design with three replications for each factor combination. Two juvenile genotypes, HF205 and HF210, and one mature genotype, PS-2 were entered for evaluations. Two types of media were used including mDCR only and mDCR with 2 g/L of MES (mDCR+MES) (M3671, Sigma-Aldrich, St. Louis, MO, USA). Levels of media pH were pre-adjusted to 3.6, 5.1, 5.7, 6.3, and 7.8 before adding 7 g of agar, MES, and autoclaving. Five dissected vegetative buds from each genotype were placed on each treatment combination. Controls consisted of medium without the presence of explants, at each pH level. They were placed in full dark versus light conditions in 25°C growth chambers. For treatments, explants were dissected and placed into mDCR versus mDCR+MES media for incubation for 1, 3, 5, 7, 14, 21, 28, and 35 days.\n\nDuring the dissection process, measurements of samples were taken for initial bud weight (mg) and petri dish (PD) weight (mg) with solidified media. After each treatment and incubation time, final media pH, final PD weight (mg), and explant final weight (mg) were recorded. Explant weight change (mg) was obtained by subtracting the initial bud weights from final explant weights. Medium pH was measured at five positions in the plates, between the explants, using a Thermo Orion PerpHecT pH meter (Thermo Fisher Scientific Inc., Waltham, MA, USA). Data analyses were performed using Minitab (Minitab Inc., State College, PA, USA), and graphs were generated by SigmaPlot (Systat Software Inc., Chicago, IL, USA), including ANOVA General Linear Model (GLM), Tukey Honestly Significant Difference test (HSD), and linear regression with significance set at p<0.05.\n\n\nResults\n\n\n\nAfter autoclaving, media pH shifts were found according to each pre-adjusted media pH. From 100 samples, mDCR medium showed a greater extend of pH fluctuations than mDCR+MES post-autoclaving. Media initially set at pH 3.6, 5.1, and 5.7 were increased for both media type (pH changes of 0.83, 0.58, 0.17 and 0.76, 0.22, 0.11 for mDCR and mDCR+MES, respectively) whereas medium initially at pH 7.8 were decreased (-0.66 and -0.59 for mDCR and mDCR+MES, respectively). Medium of pre-adjusted pH 6.3 showed decrease in mDCR (-0.11) but increase for mDCR+MES (0.05). In general, dark storage was better to maintain stable media pH than storage in light. Overall, media pH was 5.8 (n=400) when kept in the dark condition compared to pH 5.4 (n=400) in the light (P=0.000). Over the incubation times tested, media pH was maintained at fairly stable condition with a slight decreasing trend in the dark storage condition. In contrast, media pH had a stronger decreasing trend when incubated in the light. The mDCR+MES media maintained media with less pH change over the incubation times when compared to mDCR media in the light (Figure 1).\n\nMedium pH was pre-adjusted to 3.6, 5.1, 5.7, 6.3, and 7.8 prior to adding 7 g/L of agar, MES and autoclave. Medium was incubated in a growth chamber with full light or darkness at 25ºC until it reached incubation requirement. Post-autoclaving pH was recorded using a pH meter at each incubation time. Datapoints represent mean pH (n=5 for each datapoint), and were fitted with linear regression lines. Please see Dataset 1 for the raw data.\n\nAfter placing explants into the media, the pH of the medium was significantly influenced by all factors, genotype, media type, initial pH level, and incubation time (all P=0.000). Overall medium pH was 5.45 from medium incubated with PS-2 (n=1,355), which significantly greater than 5.41 and 5.19 from those incubated with HF210 (n=1,384) and HF205 (n=950), respectively. The medium pH from mDCR+MES (n=1,805) was significantly greater than the pH of mDCR only medium (n=1,884) (5.45 vs. 5.28, respectively) (P=0.000). Decreasing media pH over incubation time and variation among genotypes were both observed. Media with addition of MES was better able to maintain stable media pH up to 21 days of incubation from each of the 5 different initial pH levels (Figure 2). Regardless of initial pH level, incubation time had a strong significant effect on media pH (P=0.000). Combined two types of media, mean medium pH showed the lowest value of 5.04 (n=435) at 21-day of incubation. In contrast, the highest mean medium pH 5.88 was recorded at the 42-day of incubation (n=125). Overall medium pH at each initial pH (3.6, 5.1, 5.7, 6.3, and 7.8) showed significant differences between each other including 4.90 (n=744), 5.08 (n=740), 5.30 (n=745), 5.57 (n=740), and 5.99 (n=720), respectively. The media pH stabilizer MES demonstrated its ability to prevent media pH from dropping at the higher or lower ends of initial pH levels. Within individual genotype, both media type, and incubation time showed significant effects on media pH for all genotypes (P=0.000).\n\nGenotypes included one mature genotype, PS-2 and two juvenile genotypes, HF205 and HF210. After surface sterilization and dissection, inner vegetative buds from above three genotypes were placed on either mDCR or mDCR+MES medium for incubation up to 42 days. These buds were incubated in a growth chamber at 25ºC with light regime adjusted to 16-hour light followed by 8-hour darkness each day. Medium pH was recorded using a pH meter when sample reached each incubation requirement where five pH values were recorded within each petri dish. Datapoints represent mean pH. Please see Dataset 2 and 3 for the raw data.\n\nFor explants growth response, genotype (P=0.000), incubation time (P=0.000), and initial pH (P=0.012) showed significant effects on explant weight increment (mg). The addition of MES into the media did not show a significant effect on explant weight increment (P=0.281) (Figure 3). Overall, HF210 (30.86 mg, n=264) and PS-2 (22.50 mg, n=190) had a significant weight increment greater than HF205 (13.83 mg, n=205) (P=0.000). Explant weight increment of HF210 did not show a significant difference when compared with PS-2 (P=0.2903). However, a distinct trend in explant weight decrease was observed after 28 days of incubation in mDCR only medium for HF210. Initial medium pH of 3.6 had a significantly greater bud weight change (P=0.005) than medium pH 7.8 (25.89 vs. 19.23 mg, n=134 vs. 130, respectively). An increasing trend of bud weight change was observed but bud weight did not show significant increase during the first week of incubation.\n\nGenotypes included one mature genotype, PS-2 and two juvenile genotypes, HF205 and HF210. After surface sterilization and dissection, inner vegetative buds from above three genotypes were placed on either mDCR or mDCR+MES medium at each initial pH level (3.6, 5.1, 5.7, 6.3, and 7.8) for incubation up to 42 days. These buds were incubated in a growth chamber at 25ºC with light regime adjusted to 16-hour light followed by 8-hour darkness each day. Bud weight change was recorded using an electronic scale when sample reached each incubation requirement. Datapoints represent mean weight change (mg). Please see Dataset 4 for the raw data.\n\nComparing individual genotypes, medium type exhibited non-significant effect on explant weight increment of all three genotypes (P>0.05). For HF205, the weight differences were observed at the higher and lower ends of the given initial pH levels (Figure 4). Incubation time showed significant effect on explant weight increment for all three genotypes (P=0.000). For all three genotypes, explant weight did not show any significant differences for the first 7 days of incubation. However afterwards, explant weight growth dramatically increased for PS-2 and HF210. HF205 showed much less weight increment than the other two genotypes (Figure 5).\n\nAfter surface sterilization and dissection, inner vegetative buds from HF205 were placed on either mDCR or mDCR+MES medium at each initial pH level (3.6, 5.1, 5.7, 6.3, and 7.8) for incubation up to 42 days. These buds were incubated in a growth chamber at 25ºC with light regime adjusted to 16-hour light followed by 8-hour darkness each day. Bud weight change was recorded using an electronic scale when sample reached each incubation requirement. Vertical bars represent mean weight change (mg)±S.E. Means sharing the same letter indicate non-significant difference between means (P>0.05). Tukey’s (HSD) multiple comparison was used.\n\nAfter surface sterilization and dissection, inner vegetative buds from three genotypes were placed on either mDCR or mDCR+MES medium at each initial pH level (3.6, 5.1, 5.7, 6.3, and 7.8) for incubation up to 42 days. These buds were incubated in a growth chamber at 25ºC with light regime adjusted to 16-hour light followed by 8-hour darkness each day. Bud weight change was recorded using an electronic scale when sample reached each incubation requirement. Vertical bars represent mean weight change (mg)±S.E. Sample sizes according to the incubation time (1–42 days) were for HF205, n=45, 15, 15, 30, 30, 20, 20, and 30, for HF210, n=30, 30, 30, 30, 30, 31, 30, 38, and 15, and for PS-2, n=15, 30, 30, 30, 30, 16, 33, and 6. Means sharing the same letter indicate non-significant difference between means (P>0.05). Tukey’s (HSD) multiple comparison was used. Please see Dataset 4 for the raw data.\n\nAfter growing in medium for 28-days or more without being subcultured, explants showed various growth deformities such as chlorosis, delayed needle expansion, tip browning, browning of the bottom of explants and surrounding medium, vitrification, and even death. These symptoms occurred especially at the lower and higher ends of the initial pH levels, after prolonged culturing. Regardless of the given initial pH levels, explant growth did not show obvious delay at the early culture stages. The mentioned problems were only found at the later times of culturing. Moisture condensation was a common problem in the plate-based tissue culture system. Some of the deformities observed may have been associated with excessive amount of water droplets falling onto the medium surface or coming into contact with the explants.\n\n\nDiscussion\n\nIn general, mDCR medium with MES provided more stability of the pre-adjusted pH values after autoclaving in both the absence and presence of explants in the medium. We observed that after autoclaving, medium pH changed, but mDCR medium with MES showed less medium pH fluctuation than without MES. Storage of medium in the dark resulted in less medium pH fluctuation than storage under light. For mDCR medium incubated with explants, pH showed a gradual decrease that was followed by a sharp increase over the incubation time. However, mDCR+MES medium exhibited a slower decrease in pH or was followed by a convergent medium pH change for all pre-adjusted pH levels. Explant weight gain over time showed an inverse relationship with medium pH change, but also differed between juvenile and mature genotypes. The addition of MES did not show significant influence on explant weight growth. However, a distinct decrease in explant weight growth was observed after 28 days of incubation in the mDCR only medium.\n\nMedium storage is a common practice in tissue culture. The use of premade medium serves two main purposes. One is to hold the medium for a period of time to observe whether any contamination occurs in the medium. This ensures maximum explant growth performance achieved when antibiotics are not present in the medium. The other purpose is to facilitate timely arrangements of routine culture initiations and transfers. Owen et al. (1991) demonstrated that light affects post-autoclave medium storage, resulting in reduced medium pH over storage time. Our data confirmed their findings. Medium pH was more stable in the dark storage condition regardless of the presence of MES. The addition of MES could stabilize medium pH under the light condition, in both the presence and absence of explants.\n\nFluctuation of medium pH can be influenced by many factors. Hydrolysis, enzymatic break down, photooxidation and photolysis on light-sensitive medium components may all contribute to the fluctuation of medium pH. Sucrose hydrolysis often requires splitting of water molecules and breaking glycosidic bonds of the disaccharides. Once the breakage of glycosidic bonds has occurred, hydrogen ions from the splitting of water molecules bind with glucose, whereas hydroxyl groups bind with fructose. Since tissue culture medium often is adjusted to slight acidic conditions (pH 5.2–5.8), autoclaving provides a suitable temperature for catalyzing sucrose hydrolysis. Acid facilitated autocatalyzed sucrose hydrolysis was reported as being both pH and temperature dependant, where lower pH at a given temperature promotes more sucrose hydrolysis (Heidt et al., 1952; Wann et al., 1997). The availability of hydrogen ions in the acidic medium solution also depends on the buffering ability of the nutrient components (Thorpe et al., 2008). Furthermore, carbon sources, the amount of carbohydrates, and gelling agents act together to determine the amount of sucrose hydrolysis and the medium pH after autoclaving. As a result, medium with lower original pH may become higher while medium with higher original pH may become lower to reach equilibrium of the solution.\n\nAfter explants are introduced into the medium vessels, the sucrose is further converted into monosaccharides inter- and intra-cellularly by invertase or other plant enzymes (Thorpe et al., 2008). Egger & Hampp (1993) found the optimum activity of soluble acid invertase was at pH 4.1 in developing spruce (Picea abies (L.) Karst.) needles. They also reported that other sucrose synthesis enzymes, sucrose phosphate synthase, and sucrose synthase were pH dependant for their optimal activities (pH 7.7 and 6.7, respectively). Hence, as explants host numerous biological activities they must balance pH levels accordingly for each of the biochemical reactions. Ion uptake and release become the mechanism by which cells adjust for pH requirements. Dodds & Roberts (1995) attributed the fluctuation of medium pH after autoclaving may be a result of imbalance of anion and cation uptake.\n\nPhotochemistry may further induce degradation of photo-sensitive compounds in the medium, and trigger pH fluctuation. Photolysis is an event introduced by photons. For example, when a water molecule receives energy from photons during the photosynthesis process, photolysis occurs to generate electrons, hydrogen ions, and oxygen. If the free hydrogen ions are not bound by other substrates and excreted into the medium, they may cause medium pH fluctuation. Hangarter & Stasinopoulos (1991) reported a light induced Fe-catalyzed photooxidation of EDTA, which caused reduced root growth of the Arabidopsis thaliana ecotype Columbia. EDTA, an ion chelator, is considered to be a buffering agent in the tissue culture medium. After photooxidation occurred, formaldehyde and glyoxylic acid are produced, which can be toxic to explants, concomitant with increased chelated ferric oxide that explants can not readily use. This light induced change of buffering ability could definitely alter nutrient availability in the medium, and further affect the fluctuation of medium pH.\n\nMES can be utilized to stabilize medium pH for Douglas-fir micropropagation. MES has been employed in various tissue culture systems to maintain stable medium pH over extended cultural times. Park & Son (1992) reported the addition of MES alone in the medium, or together with Dithiothreitol, increased protoplast yield and viability from hybrid poplar protoplast culture system. Similarly, MES and arabinogalactan-protein, alone or combined, were found to maintain suitable medium pH and to enhance embryogenesis in white cabbage (Brassica oleracea var. capitata) microspore culture system (Yuan et al., 2012). For conifer species, MES was also utilized as pH stabilizer for silver fir (Abies alba L.) (Hartmann et al., 1992) and European larch (Larix decidua Mill.) (Korlach & Zoglauer, 1995) protoplast culture systems. Protoplasts, being a single cell without cell wall, are probably extremely sensitive to rapid pH changes. The effects of pH changes could be as important for shoot culture or other tissue culture prospects for explant productivity. Especially, MES may help to maintain stable medium pH for bulk medium preparation in large scale aspects of a propagation project.\n\nOur data suggested a 21-day subculture practice may be suitable for maintaining medium freshness, medium pH level, and desirable explant growth for Douglas-fir shoot culture. Although there might be specific pH requirements for individual species, explants of Douglas-fir genotypes showed various responses or adaptations to medium pH changes. Some genotypes may be able to tolerate or adapt better to fluctuations in medium pH, and to show continuous growth in a wide range of pH levels. The effects of MES and nutrient acquisition by explants in culture may require further investigations on specific aspects of nutrient dynamics regarding the effects of both medium and explants in vitro.\n\n\nData availability\n\nFigshare: http://dx.doi.org/10.6084/m9.figshare.1257689 (Chen et al., 2014).", "appendix": "Author contributions\n\n\n\nCCC performed the experiments, analyzed the data, and wrote the first draft of this manuscript. All authors contributed equally in data interpretations as well as writing and further refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Pennsylvania Department of Agriculture (grant number PDA ME 446711 to JEC) and the Schatz Center for Tree Molecular Genetics, the Pennsylvania State University.\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 would like to thank Eric Dice and all the members of tissue culture group from JEC lab for their assistance in tissue culture works. Thanks also go to Drs. Henry Gerhold, Larry Kuhns, Haiying Liang, James Sellmer, and Abdoulaye Traore for their valuable comments and suggestions, and for providing plant materials.\n\n\nReferences\n\nBallarin-Denti A, Antoniotti D: An experimental approach to pH measurement in the intercellular free space of higher plant tissues. Experientia. 1991; 47(5): 478–482. Publisher Full Text\n\nChen CC, Bates R, Carlson J: Media pH and explant weight changes over incubation times for Douglas-fir (Pseudotsuga menziesii) shoot cultures grown on different media. Figshare. 2014. Data Source\n\nDodds JH, Roberts LW: Experiments in plant tissue culture. (Cambridge University Press, New York). 1995. Reference Source\n\nDörnenburg H, Knorr D: Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzym Microb Technol. 1995; 17(8): 674–684. Publisher Full Text\n\nde Klerk G-J, Hanecakova J, Jasik J: Effect of medium-pH and MES on adventitious root formation from stem disks of apple. 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(ed), Plant propagation by tissue culture, 3rd edn volume 1, the background. (Springer, Dordrecht, The Netherlands). 2008; 115–173. Publisher Full Text\n\nTraore A, Xing Z, Bonser AL, et al.: Optimizing a protocol for sterilization and in vitro establishment of vegetative buds from mature Douglas-fir tree. Hortsci. 2005; 40(5): 1464–1468. Reference Source\n\nWann SR, Veazey RL, Kaphammer J: Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving. Plant Cell Tiss Organ Cult. 1997; 50(3): 221–224. Publisher Full Text\n\nWilliams RR, Taji AM, Winney KA: The effect of Ptilotus plant tissue on pH of in vitro media. Plant Cell Tiss Organ Cult. 1990; 22(22): 153–158. Publisher Full Text\n\nYuan S-X, Su Y-B, Liu Y-M, et al.: Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage. Plant Cell Tiss Organ Cult. 2012; 110(1): 69–76. Publisher Full Text" }	[ { "id": "7372", "date": "06 Feb 2015", "name": "Vibha Srivastava", "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 a descriptive paper analyzing pH fluctuations in the tissue culture media for the micropropagation of Douglas fir, and assessing the effect of MES, a commonly used compound in tissue culture media, for stabilizing media pH. Parameters such as the effect of incubation time (days) and dark/light in the presence or absence of MES were studied, in addition to the morphology of explant (buds) in MES + or - media. As expected, MES was found to be useful in stabilizing pH in different conditions and therefore recommended for the Douglas fir micropropagation media. This paper could be useful to the horticulturists, especially those working on micropropagation; though, the findings of this paper, in my opinion, are common knowledge in the field of tissue culture.I have one major criticism on the experimental design and data analysis, which compels me to approve this paper with reservations: only one concentration, 2 g/L, of MES was used throughout this study. No reasoning was presented towards selection of this particular concentration. MES is used in many tissue culture media at different concentrations. While the authors shy away from making any conclusions (regarding the role of MES), they suggest MES played a role in stabilizing pH. This conclusion cannot be substantiated without a dose-response curve and determination of optimum concentration.A minor criticism that I have is related to description in materials and methods. The tissue culture media used through out this study is simply referred by the acronym, mDCR. But no description of this media given is given, only reference provided. Whether it is MS/B5/N6 based media is not clear from this paper, and which other compounds are present in this media is not described.", "responses": [ { "c_id": "1209", "date": "10 Feb 2015", "name": "Chien-Chih Chen", "role": "Author Response", "response": "We would like to thank you for your comments on enhancing this manuscript. Once we received more reviews, we will include your suggestions and revise this manuscript." }, { "c_id": "1326", "date": "08 May 2015", "name": "Chien-Chih Chen", "role": "Author Response", "response": "We appreciate your comments. Please find our explanations regarding your concerns with the original version (and how they have been addressed in the new version of the article) below.We agree that MES is commonly used in tissue culture. It would have been better to establish a dose-response curve and determine the optimal concentration for our experimental system. But that would have involved testing the MES range with all of the starting pH levels and storage conditions and multiple explant genotypes. However, we were limited in the number of explant samples available from the Christmas tree seed orchard, and over the course of the research we did not have enough material to investigate all of the possible multi-factorial combinations. Instead, since we were mostly interested in learning how medium pH changes under storage and culture conditions, and how that effects the explant growth responses, we started with one MES level previously reported with conifer explant culture. As expected, that level of MES did maintain a better medium pH. This study was at the beginning of a long series of experiments to establish a protocol for an effective micropropagation method for a recalcitrant woody species, Douglas-fir, for large scale application. Because the one concentration of MES worked well, we then proceeded to a different set of experiments to test other factors involved in propagation and scale up. Based on our findings in this study, we could have even chosen to omit the addition of MES altogether, by subculturing explants before the medium pH falls too low. In that case, we could reduce cost and perhaps establish a more economic micropropagation protocol for the industry. The DCR medium itself is a modified version of the MS basal salts recipe developed for Douglas-fir by Gupta and Durzan (1985 and 1987). We further modified the DCR medium and classified it as mDCR, which is still based on MS. A comparison chart of the components between DCR vs. mDCR will be added in our revision." } ] }, { "id": "7619", "date": "10 Feb 2015", "name": "Isabel Arrillaga", "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 effect of the pH in the culture medium is a very important factor that often is not taken into account. This papers reports on the changes in the medium pH at several times during the tissue culture process and their effect on the explants growth. From this point of view the article is very interesting and deserves to be accepted for publication. Changes in the pH of the culture medium after and before autoclaving and during culture time have been studied in depth (see any of the editions of the book series Plant Propagation by Tissue Culture, George and Sherrington, Exegetics, London). The paper is well written and data well analyzed. I have found very interesting results as the effect of light on the medium pH during storage that can be of interest for plant tissue colleagues.Also, I have some questions to address to the authors.Why to use pH below 5?. Most of the culture media are adjusted to a pH from 5,7- to 6,0 before autoclaving. Did authors find any problems with low pH and agar gelification?. In our experience low pH affect media solidification. The effect of MES on medium pH stability is expected. From your data on Figures 3 and 5, I should not agree with your statement “21-day subculture practice may facilitate to sustain medium freshness, medium pH and desirable explant growth” (Abstract). It is true that pH of the medium increased after 21 of explant culture initiation, but it is also true that irrespective of the medium pH, explants sharply increased weight after 21 days in culture. These results should be emphasized and conclusions rewritten.", "responses": [ { "c_id": "1214", "date": "11 Feb 2015", "name": "Chien-Chih Chen", "role": "Author Response", "response": "Dr. Arrillaga, thank you so much for your comments. We are now working on the revision of this manuscript addressing reviewers' concerns." }, { "c_id": "1325", "date": "08 May 2015", "name": "Chien-Chih Chen", "role": "Author Response", "response": "We appreciate your comments. Please find our explanations regarding your concerns with the original version (and how they have been addressed in the new version of the article) below.The reason that we chose the larger range of pH levels was to expand upon observations from our routine practice in tissue culture medium preparation. Especially, this was to understand how Douglas-fir explants would respond to various levels of medium pH in vitro, while the 5.7 to 6.0 range derives more from in vivo conditions. The medium pH and explant weight increment curves provide detailed records relevant to both storage and tissue culture conditions, which we feel also expands upon the range of observations normally reported. You are correct. Low medium pH can affect agar gelling, and this is associated with types of gelling agents used and other additives (i.e. high concentrations of PEG) as well. We did have an issue with gelling, but not until the pre-adjusted medium pH was below pH 3.6 in our case. We agree that MES is a commonly used buffer in tissue culture.Because the pKa for MES is 6.15 at 25 °C, we felt it was necessary to observe what the buffering capacity of MES was with starting pH levels far from 6.15 that we wanted to investigate in medium under storage and with explants. If MES is added, what medium pH can be maintained, and for what duration, at such extreme pH levels under our test conditions?This is a very good point. As you can see from Fig. 3 and 5, explant weight increment according to various levels of pre-adjusted medium pH was genotype dependent. A broad evaluation including more genotypes would provide a more strong indication. Usually, explant weight increment was in a curvilinear relationship. We did observe a decreasing weight gain in HF210 after 28 days. Also based on Fig.2 and morphological observations, prolonged culture without subculturing could result in additional growth complications. Therefore, we would like to suggest a 21-day subculturing practice as optimal for Douglas-fir explants taking all the various factors and observations into account. We will provide a more precise conclusion in our revision." } ] } ]	1	https://f1000research.com/articles/3-298
https://f1000research.com/articles/4-41/v1	11 Feb 15	{ "type": "Research Note", "title": "F1000Prime: an analysis of discipline-specific reader data from Mendeley", "authors": [ "Robin Haunschild", "Lutz Bornmann", "Lutz Bornmann" ], "abstract": "We have used the F1000Prime recommended paper set (n= 114,582 biomedical papers) to inquire the number of Mendeley readers per (sub-) discipline via the Mendeley Application Programming Interface (API). Although the (sub-) discipline of Mendeley readers is self-assigned and not mandatory, we find that a large share (99.9%) of readers at Mendeley does share their (sub-) discipline. As expected, we find most readers of F1000Prime recommended papers work in the disciplines of biology and medicine. A network analysis reveals strong connections between the disciplines of engineering, chemistry, physics, biology, and medicine.", "keywords": [ "F1000Prime", "Altmetrics", "Mendeley", "paper", "evaluation" ], "content": "Introduction\n\nInterest in the broad impact of research (Bornmann, 2012, 2013) has resulted in new forms of impact measurements. Traditional forms of impact measurements using bibliometrics only allow the measurement of impact on research itself. These new forms which have been named as altmetrics (abbreviation of alternative metrics) pretend to measure the impact of research on other areas of society (than research) by counting the mentions of papers in social media: “Alternative metrics, sometimes shortened to just altmetrics, is an umbrella term covering new ways of approaching, measuring and providing evidence for impact” (Adie, 2014, p. 349). As altmetrics, the number of readers (on Mendeley), mirco-bloggers (on Twitter), and other consumers of research using social media are counted. Although scientometrics research on altmetrics is still in a very early phase (comparable to research on bibliometrics in the 1970s), the use of these data in research evaluation is already an issue. For example, altmetrics is considered in the Snowball Metrics project (Colledge, 2014). This project compiled a set of clearly defined indicators which will be used by participating universities (mostly Anglo-American universities) for research evaluation purposes. It seems that altmetrics will be used in practice before scientometrics research has produced standards on their reliable, fair and valid application (Weller, 2015).\n\nThis study uses one of the most important sources for altmetrics data, namely Mendeley. Mendeley “claims 3.1 million members. It was originally launched as software for managing and storing documents, but it encourages private and public social networking” (Van Noorden, 2014, p. 126). Since data from Mendeley can be received by an Application Programming Interface (API) without any problems and the coverage of the scientific literature has been pointed out as high (Priem, 2014), Mendeley is a very attractive data source for the reception of research. “Mendeley records the number of users that have listed it [i.e. an article], describing them as readers, whether or not they actually read it. Presumably, listing an article in Mendeley tends to reflect that an article has been read or will be read in the future, although there is no evidence that this assumption is true” (Thelwall & Maflahi, in press).\n\nIn this study, we match Mendeley data with data from F1000Prime. F1000Prime is a database with biomedical papers and their reviews by peers. It is intended as a support tool for researchers to receive hints for the most important literature. Since it is not clear who actually reads the F1000Prime recommended papers, we investigated the disciplines of researchers (and other people) who have read these papers. We are mainly interested in two questions: are F1000Prime papers only read by people from biomedicine or are people from other disciplines also interested? Which disciplines read F1000Prime papers frequently or seldom together? The latter question will be answered by using social network techniques.\n\n\nMethods\n\nF1000Prime is a post-publication peer review system of papers from medical and biological journals. This service is part of the Science Navigation Group, which publishes and develops information services for the professional biomedical community and the consumer market. Papers for F1000Prime are selected by a peer-nominated global \"Faculty\" of leading scientists and clinicians. The Faculty members rate the papers and explain their importance. This means that only a selected set of papers from the biomedical area covered is reviewed, and most of the papers are actually not (Kreiman & Maunsell, 2011; Wouters & Costas, 2012).\n\nThe Faculty nowadays numbers more than 5,000 members worldwide, assisted by further associates, which are organised into more than 40 subjects. Members can choose and evaluate any paper of interest; however, \"the great majority pick papers published within the past month, including advance online papers, meaning that users can be made aware of important papers rapidly\" (Wets et al., 2003, p. 254). Although many papers published in popular and high-profile journals (e.g. Nature, New England Journal of Medicine, Science) are evaluated, 85% of the papers selected come from specialised or less well-known journals (Wouters & Costas, 2012). The F1000Prime database is regarded as a useful aid for researchers (and other people working research-oriented) to obtain indications of the most relevant papers in the biomedical area: \"The aim of Faculty of 1000 is not to provide an evaluation for all papers, as this would simply exacerbate the ‘noise’, but to take advantage of electronic developments to create the optimal human filter for effectively reducing the noise\" (Wets et al., 2003, p. 253).\n\nWithin the first half of 2014 the reference manager Mendeley provided a new version of its API. Some restrictions of the previous API were lifted. For example, the usage statistics were previously provided in relative terms and only for the top three entries (Haustein & Larivière, 2014). The new API provides results in absolute numbers and not only for the top three but for all entries. Mendeley provides access to the readership status (e. g. professor, postdoc, or student) and the distribution of the Mendeley readership across scientific disciplines as well as countries via the API. Those sets of data can be correlated with other information available about papers (e. g. citations or Twitter counts).\n\nBefore one can start to use the Mendeley API, one has to register as a Mendeley user. Afterwards, registration of the desired application is necessary (http://dev.mendeley.com). Authentication with the API is done via OAuth 2.0. The credentials are set during registration of the application.\n\nWe used R (http://www.r-project.org/) to interface to the Mendeley API. It seems to us that using other interfaces does not change the functionality or responsiveness, but we did not try to use other interfaces. Mendeley provides sample codes for Javascript, Python, R, and Ruby (http://dev.mendeley.com/code/sample_code.html), whereby all requests to the API use HTTP GET and POST requests. Therefore, we suppose that any other scripting or programming language may be used. The reply is sent in Javascript Object Notation (JSON).\n\nWe requested user statistics for the F1000Prime publication set (n = 114,582 papers) using the PubMedID and DOI between the 4th and 6th of December 2014. We observed seemingly random connection problems. Sometimes those problems occurred after a few hundred or a few thousand requests. The largest chunk of requests we were able to get through the API without connection problems consisted of 47,629 papers. This large number of records is contrasted with smaller chunks of requests (between 1,049 and 9,307 records).\n\nMendeley provides a breakdown of the user count into sub-disciplines. The possible values for disciplines and sub-disciplines can be obtained directly from the API via the GET /disciplines endpoint. Each discipline has a certain number of sub-disciplines. The sub-discipline “miscellaneous” occurs in every discipline. Each Mendeley user can select a discipline and a sub-discipline from a drop-down menu. This piece of information is not mandatory, like the user’s location.\n\nPajek is used to create the F1000Prime readership network (http://pajek.imfm.si/doku.php; de Nooy, Mrvar & Batagelj, 2011) applying the spring embedder of Kamada & Kawai (1989). For detecting communities in the common readership of F1000Prime recommended papers, we used the VOS Clustering algorithm (Waltman, van Eck & Noyons, 2010), which is available in Pajek. The aim of this algorithm is to provide further insights into the structure of the network (Milojević, 2014).\n\n\nResults\n\nWe found 6,263,913 Mendeley readers for the F1000Prime publication set. 99.9% (n=6,257,603) of them share their discipline and sub-discipline. This is a much higher percentage than those who share their geographical location (Haunschild, Stefaner & Bornmann, in preparation). For the F1000Prime publication set, the vast majority (74.94%) of Mendeley users is found in the “miscellaneous” sub-discipline of all disciplines. Therefore, we added up all the readers of all sub-disciplines for each discipline. The results of our study are presented in Table 1. Nine disciplines have at least 1% of the readers of the F1000Prime publication set. The remaining 16 disciplines have less than 1%. As expected, most readers (81.78%) of the F1000Prime literature assign themselves to the biomedical (sub-) disciplines. All other disciplines comprise the remaining 15.19% of the F1000Prime readership at Mendeley. The third largest readership is found in the discipline psychology which is related to medicine. After chemistry, which is also related to biology, five other disciplines show readership values above 1% within the F1000Prime literature. Those disciplines seem rather unrelated to the field of biomedical research, especially environmental sciences (according to Figure 1, see also the description below). 3.05% of the F1000Prime readers at Mendeley come from other disciplines (not shown in Table 1). The disciplines with most readers below the threshold of 1% are: social sciences (0.67%), mathematics (0.42%), electrical and electronic engineering (0.27%), education (0.22%), and materials sciences (0.21%).\n\nOnly disciplines are shown with more than 1% readers (sorted in decreasing order).\n\nNetwork of F1000Prime recommended readers from arts and literature (AnL), astronomy and astrophysics (AsAs), biology (Bio), business administration (BuAd), chemistry (Chem), computer and information science (CIS), design (Des), earth sciences (ESci), economics (Eco), education (Edu), electrical and electronic engineering (EEE), engineering (Eng), environmental sciences (Env), humanities (Hum), law (Law), linguistics (Ling), management (Man), materials sciences (Mate), mathematics (Math), medicine (Med), philosophy (Phil), physics (Phys), psychology (Psy), social sciences (SoSc), sports and recreation (SpRe).\n\nWe also analyzed connections between the disciplines. These are shown in Figure 1. A paper which is read by Mendeley users of different disciplines (e.g. biology and physics) constitutes a connection between these disciplines. Therefore, a paper which is read by Mendeley users of the same discipline does not contribute to the network system, but a paper which is read by Mendeley users of many different disciplines contributes many connections to the network. The size of the vertices in Figure 1 reflects the numbers of readers for each discipline. The thicker and darker the edges between two disciplines, the more frequently they have read a F1000Prime paper jointly. The location of the discipline vertex also informs about the connectivity. The closer the vertex is located towards the center, the more connections to different disciplines are found. There are 25 disciplines and 300 links among those disciplines in the dataset. With a density of 1, the network is rather dense. The average node degree is 24. According to Figure 1, the strongest connection shows up between biology (Bio) and medicine (Med). The disciplines computers and information science (CIS), engineering (Eng), and chemistry (Chem) have rather strong connections to biology (Bio) and medicine (Med). The discipline arts and literature (AnL) shows a low amount of readers (0.15%, close to sports and recreation with 0.16%) as well as a good connection to other disciplines in the network.\n\nThe community detection algorithm detected two communities in the network with biology, medicine, engineering, chemistry, and physics as one community (yellow vertices) and all other disciplines as the other (green vertices).\n\n\nDiscussion\n\nThe (sub-) discipline of Mendeley readers is self-assigned and not mandatory. Still, we found that a large share (99.9%) of F1000Prime paper readers at Mendeley share their (sub-) discipline. Most readers (74.94%) assign the “miscellaneous” sub-discipline of their discipline to themselves. As the F1000Prime publication set is a collection of high-quality biomedical papers, it is expected that we find most readers in the disciplines of biology and medicine. We find strong connections between engineering, chemistry, physics, biology, and medicine as well as their rather high reader percentages. The connections of arts and literature to biology and medicine are much weaker. The disciplines arts and literature, engineering, materials sciences, and computer and information science have connections to many other disciplines, as the central location in the network indicates.\n\n\nData availability\n\nFigshare: Mendeley reader counts for F1000Prime papers. Doi: 10.6084/m9.figshare.1301463 (Haunschild & Bornmann 2014).", "appendix": "Author contributions\n\n\n\nWrote manuscript: RH and LB\n\nData acquisition: RH and LB\n\nData processing: RH\n\nData analysis: RH and LB\n\nProduced graphics: LB\n\nManuscript revision: RH and LB\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAdie E: Taking the Alternative Mainstream. EI Profesional de la Informacion. 2014; 23(4): 349–351. Publisher Full Text\n\nHaunschild R, Bornmann L: Mendeley reader counts for F1000Prime papers. Figshare. 2014. Data Source\n\nWeller K: Social Media and Altmetrics: An Overview of Current Alternative Approaches to Measuring Scholarly Impact. Incentives and Performance. In I. M. Welpe, J. Wollersheim, S. Ringelhan & M. Osterloh (Eds.), Springer International Publishing. 2015; 261–276. Publisher Full Text\n\nWets K, Weedon D, Velterop J: Post-publication filtering and evaluation: Faculty of 1000. Learned Publishing. 2003; 16(4): 249–258. Publisher Full Text\n\nWouters P, Costas R: Users, narcissism and control–tracking the impact of scholarly publications in the 21st century. Utrecht, The Netherlands: SURFfoundation. 2012. Reference Source" }	[ { "id": "7642", "date": "06 Mar 2015", "name": "Rodrigo Costas", "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 presents an analysis of F1000Prime recommended publications in combination with Mendeley users statistics. I think that in general terms the methodology is fine and the results are correct, having some descriptive interest. However, I have the following major remarks and also a number of other minor comments.Major remarks:The paper lacks in my view a solid justification of its research questions. The two research questions proposed in the paper (“are F1000Prime papers only read by people from biomedicine or are people from other disciplines also interested?”, and “Which disciplines read F1000Prime papers frequently or seldom together?”) are too general and basically the results reported suggest: “1. Yes, F1000 papers are mostly saved by biomedical Mendeley users” and “2. yes, there are reasonable connections between disciplines (e.g. Biology and Medicine) while some others are not easy to understand”. After all, the reader is left with the questions \"why is the analysis of the disciplines of the Mendeley users of F1000Prime recommended publications relevant? What have I learnt from this paper?\". For example, do Mendeley users link F1000 papers thematically in a special manner? Or, does the use of Mendeley readerships have a different characteristic in the thematic analysis of disciplines that wouldn't be possible with other methods (e.g. bibliographic coupling)? Is the Mendeley users ‘crowdsourced’ disciplinary classification valid/useful for the classification of F1000Prime papers? There are some methodological omissions. For example, what is the exact number of publications finally considered in the study? In the figshare dataset there are 147177 rows of data. If the article reports n=114582 papers (does this mean that Mendeley has a coverage of 78% of F1000Prime recommended papers?) Please, clarify this point. Also what are the publication years of the publications finally considered? In the paper there is only a brief comment to the fact that Mendeley may not necessary measure actual \"reads\" (at the end of the introductory section). In fact there seems to be some confusion in what are \"readers\" and readerships (or simply the act of adding papers by Mendeley users). For example, in the results section it is stated that \"we found 6,263,913 Mendeley readers\". This is a bit misleading. These 6 millions are events of the act of adding documents in their Mendeley libraries by an undetermined number of different Mendeley users. I recommend to revise the consistency of the vocabulary in this regard. This clarification is important for example to understand how the matrix of Mendeley readerships is constructed (see minor comment below). The results presented are not very surprising. Basically around 86% of F1000Prime publications are saved by Biomedical users, which is what would be expected considering the nature of F1000Prime. So what is the added value of this analysis? Are F1000 recommended publications more interdisciplinary than other biomedical publications as captured by Mendeley users? The network (Figure 1) and ‘community’ analysis are also not very informative. What does it mean that Bio, Med, Eng, etc. belong to the same community? I don't see the reason why Psy is not in the same community as Med. The authors say that biology is related to chemistry while not to environmental sciences. I don't see the logic of this result. Why chemistry is more linked to biology that Environmental sciences? (which intuitively I would expect to be related to biology). What does it mean the “central location” of engineering, material sciences and computer and information science by having connections to many other disciplines? Does it mean that users from these areas are more multidisciplinary than other Mendeley users? I think a much stronger case needs to be made to explain the value of these analyses and results.Other minor comments include:The Kreiman & Maunsell reference is missing. PubMedIDs and DOIs are used as the linking element. Although using PubMedIDs and DOIs is straightforward (and they have been used in other studies), problems with the metadata and ids recorded in Mendeley have been reported and need to be acknowledged (http://www.asis.org/SIG/SIGMET/data/uploads/sigmet2014/zahedi.pdf). It is stated that \"a paper which is read by Mendeley users of different disciplines ... constitutes a connection between these disciplines\". So, would this be a kind of \"Mendeley readerships coupling\"? For example Kraker et al. (2015) analyzed ‘co-readership’ networks and they briefly discussed the idea of bibliographic coupling and co-citation. In this paper a different approach as compared to Kraker and colleagues seems to be taken, i.e. here the focus seems to be more on readerships coupling. I think a discussion of the analytical approach would be necessary here. When explaining the construction of the matrix for the network analysis, how are the links exactly determined? In other words, from a matrix point of view, if the disciplines are the columns and the rows, what is exactly counted in the cells? To clarify this point would be very helpful to the reader and also for other potential scholars interested in the methodology.My main conclusion is that this paper is correct in technical terms and it has some descriptive merit, but it lacks a relevant focal point. Suggestions to improve the paper could include to turn it into a more methodological paper, so readers can learn in a step-wise mode how to produce and analyze Mendeley readerships coupling networks. Other more ambitious approaches would be to study for example if the disciplinary connections crowdsourced by Mendeley users match (or not) other classifications (e.g. Medline, WoS subject categories, etc.), thus the value of Mendeley and F1000Prime as tools to map disciplinary fields could be highlighted.I hope my comments are useful to the authors of the paper.", "responses": [ { "c_id": "1306", "date": "08 May 2015", "name": "Robin Haunschild", "role": "Author Response", "response": "“The paper lacks in my view a solid justification of its research questions. The two research questions proposed in the paper (“are F1000Prime papers only read by people from biomedicine or are people from other disciplines also interested?”, and “Which disciplines read F1000Prime papers frequently or seldom together?”) are too general and basically the results reported suggest: “1. Yes, F1000 papers are mostly saved by biomedical Mendeley users” and “2. yes, there are reasonable connections between disciplines (e.g. Biology and Medicine) while some others are not easy to understand”. After all, the reader is left with the questions \"why is the analysis of the disciplines of the Mendeley users of F1000Prime recommended publications relevant? What have I learnt from this paper?\". For example, do Mendeley users link F1000 papers thematically in a special manner? Or, does the use of Mendeley readerships have a different characteristic in the thematic analysis of disciplines that wouldn't be possible with other methods (e.g. bibliographic coupling)? Is the Mendeley users ‘crowdsourced’ disciplinary classification valid/useful for the classification of F1000Prime papers?”The discussion section has been extended. However, we abstained from having a very long discussion section, since the paper was intended as a shorter article.“There are some methodological omissions. For example, what is the exact number of publications finally considered in the study? In the figshare dataset there are 147177 rows of data. If the article reports n=114582 papers (does this mean that Mendeley has a coverage of 78% of F1000Prime recommended papers?) Please, clarify this point. Also what are the publication years of the publications finally considered?”We have revised the Methods section to clarify this point. The employed F1000Prime publication set consists of 114,582 unique papers. Each paper has at least one recommendation. The papers with multiple recommendations occur multiple times. Therefore, the F1000Prime publication set has 147,177 entries. Of course, we analyzed only the 114,582 unique papers.“In the paper there is only a brief comment to the fact that Mendeley may not necessary measure actual \"reads\" (at the end of the introductory section). In fact there seems to be some confusion in what are \"readers\" and readerships (or simply the act of adding papers by Mendeley users). For example, in the results section it is stated that \"we found 6,263,913 Mendeley readers\". This is a bit misleading. These 6 millions are events of the act of adding documents in their Mendeley libraries by an undetermined number of different Mendeley users. I recommend to revise the consistency of the vocabulary in this regard. This clarification is important for example to understand how the matrix of Mendeley readerships is constructed (see minor comment below).”We are aware of the fact that we measure reader counts (or bookmarks to papers) and not individual readers. We have revised the parts which might have given a different impression. Also, the results section acknowledges this fact in the revised version.“The results presented are not very surprising. Basically around 86% of F1000Prime publications are saved by Biomedical users, which is what would be expected considering the nature of F1000Prime. So what is the added value of this analysis? Are F1000 recommended publications more interdisciplinary than other biomedical publications as captured by Mendeley users? The network (Figure 1) and ‘community’ analysis are also not very informative. What does it mean that Bio, Med, Eng, etc. belong to the same community? I don't see the reason why Psy is not in the same community as Med. The authors say that biology is related to chemistry while not to environmental sciences. I don't see the logic of this result. Why chemistry is more linked to biology that Environmental sciences? (which intuitively I would expect to be related to biology). What does it mean the “central location” of engineering, material sciences and computer and information science by having connections to many other disciplines? Does it mean that users from these areas are more multidisciplinary than other Mendeley users? I think a much stronger case needs to be made to explain the value of these analyses and results.”First, a study is also valuable if an expected result is concluded. Second, this study also discovers unexpected readership connections. We added more explanations about the network analysis in the revised version of the paper.“The Kreiman & Maunsell reference is missing.”Unfortunately, many references got lost in a very late stage of the initial version of the manuscript. The Kreiman & Maunsell reference was cited in the text but did not appear in the reference list. We have recovered the lost references in the revised version.“PubMedIDs and DOIs are used as the linking element. Although using PubMedIDs and DOIs is straightforward (and they have been used in other studies), problems with the metadata and ids recorded in Mendeley have been reported and need to be acknowledged (http://www.asis.org/SIG/SIGMET/data/uploads/sigmet2014/zahedi.pdf).”As this manuscript was intended as a short article we have included a brief note regarding this in the new version of the paper.“It is stated that ‘a paper which is read by Mendeley users of different disciplines ... constitutes a connection between these disciplines’. So, would this be a kind of \"Mendeley readerships coupling\"? For example Kraker et al. (2015) analyzed ‘co-readership’ networks and they briefly discussed the idea of bibliographic coupling and co-citation. In this paper a different approach as compared to Kraker and colleagues seems to be taken, i.e. here the focus seems to be more on readerships coupling. I think a discussion of the analytical approach would be necessary here.”We have included the reference and a corresponding discussion in the introduction of the revised version of the paper.“When explaining the construction of the matrix for the network analysis, how are the links exactly determined? In other words, from a matrix point of view, if the disciplines are the columns and the rows, what is exactly counted in the cells? To clarify this point would be very helpful to the reader and also for other potential scholars interested in the methodology.”We have extended the description of the network analysis in the revised version of the paper." } ] }, { "id": "7640", "date": "31 Mar 2015", "name": "Stefanie Haustein", "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 study combines data from F1000 and Mendeley to analyze the (self-reported) disciplines of Mendeley users saving articles recommended in F1000. Research questions lack focus and clarity, analysis and discussions are weak, conclusions are absent. This is mainly due to the fact that the analysis focuses on a very small component of the data (sum of reader count per discipline), despite the fact that the two datasets contain many more interesting aspects that are worth analyzing. Many relevant previous studies are ignored.Due to the many shortcomings and weaknesses I do not approve the indexation of this article in its current state.Major revisions:The research questions lack clarity and the motivation of the study should not solely be based on the availability of datasets (Mendeley and F1000). It should be emphasized in how far this study is different from previous work, in particular Mohammadi & Thelwall, who analyzed very similar aspects on Mendeley and, in addition, compared the discipline of users to that of the citing papers. For the present study, it is not clear what the authors expect to find (how much biology readers are normal?) and what the data is able to show: Do papers recommended on F1000 have Mendeley users from more diverse disciplines than expected?It would be much more interesting and valuable to observe the effect of being recommended on F1000 by comparing Mendeley readership counts and disciplines of users of the dataset used in this study with a control set of papers that were not recommended. This could be achieved by analyzing and comparing the data for the population of PubMed articles for a certain set of recent years: Does the F1000 recommendation provide visibility to papers that increases the number of readers on Mendeley as well as the diversity of the audience in terms of disciplines and academic status? PubMed/Medline could also provide a meaningful subject classification for papers to measure interdisciplinary knowledge flows from authors to readers.The authors also need to clarify in how far the present study differs and distinguishes itself from their other publications on similar topics and the same datasets:1) Who reads F1000Prime publications?2) Who publishes, reads, and cites papers? An analysis of country information3) Usefulness of altmetrics for measuring the broader impact of research: A case study using data from PLOS and F1000Prime4) Validity of altmetrics data for measuring societal impact: A study using data from Altmetric and F1000Prime5) Overlay maps based on Mendeley data: The use of altmetrics for readership networks6) Usefulness of altmetrics for measuring the broader impact of research: A case study using data from PLOS (altmetrics) and F1000Prime (paper tags)7) The authors also mention Haunschild, Stefaner & Bornmann (in preparation), which seems to focus on the geographic location of Mendeley users of the same dataset. Could this aspect not be integrated in the present study? The reference list is particularly poor and not acceptable in its current form. There have been plenty of studies by Thelwall, Mohammadi, Costas, Zahedi, Kraker, Haustein and others that have evaluated Mendeley reader counts - these are completely ignored. The introduction should be additionally supported by core altmetrics publications by Priem (particularly his overview of altmetrics in Beyond Bibliometrics which includes a definition of altmetrics), Piwowar, regarding reference managers Taraborelli and Haustein & Siebenlist, as well as the above mentioned authors for research on altmetrics. Peter Kraker's work on readership networks based on Mendeley users needs to be considered as well.The parallels between early bibliometrics research and current altmetrics lack references either to the particular bibliometrics studies or detailed discussions of this parallel, for example in Haustein, Bowman & Costas.In addition, some of the references cited in the text are also not listed in the reference list. This needs proper revision. The dataset is not clearly defined. What is the F1000 Prime publication dataset? How and when was it retrieved? Are those all recommendations ever made in the database? What publications do the 114,582 papers refer to (journals, publications years, document types, discipline, research field, etc.)? What is the metadata quality of these entries; in particular, how many of these have a correct DOI, PMID or both? In how far does the availability of identifiers as well as the characteristics of papers (publication year, journals) influence and bias the matching with and availability in Mendeley? Regarding the matching of results: how many unique documents do the 6,263,913 reader counts refer to? What is the percentage of documents that could not be found in Mendeley? Readers/users should be distinguished from reader counts - to avoid the implication that there are 6.2 million readers. Mendeley has around 3 million users (2.8 million as of February 2014; Haustein & Larivière), who create reader(ship) counts by adding documents to their libraries. The description of the methods for the network analysis is too brief: How were co-occurrences calculated? How were they normalized? Due to the density of 1 (i.e., all nodes are connected), the network layout is not very meaningful and not easy to interpret, often counterintuitive. The informativeness of the network could be improved by removing weak links to obtain a more meaningful network structure, where central (i.e., well-connected) nodes are positioned in the center of the network and less important ones in the periphery. Moreover, similar nodes as detected by the clustering algorithm (yellow and green in Figure 1) should be placed close together. In addition, it would make sense to include self-loops for papers saved by users of the same discipline to highlight homogeneous user groups. As the authors use the VOSviewer clustering method, why was VOSviewer not chosen for the mapping? In my opinion, it provides much more meaningful robust networks and better visualizations than Pajek. Other alternatives are Gephi, GUESS, UCInet, etc.Regarding the interpretation of the network, the authors state that \"[t]he thicker and darker the edges between two disciplines, the more frequently [the users] have read a F1000Prime paper jointly\". Should it not rather be that \"[t]he thicker and darker the edges between two disciplines, the more frequently papers were saved by users of these two particular disciplines\"?The grouping into clusters seems to a certain extent counterintuitive: Why is environmental science grouped together with psychology instead of biology? Could this be introduced by (the lack of) normalization? These counterintuitive results need to be discussed! The discussion needs to be extended and a conclusion is missing. It is not clear what the study actually shows/proofs and in how far the few results (sum of readers per discipline) warrant a separate publication. The dataset of F1000 recommendations and Mendeley include many other pieces of interesting information such as the recommendation scores, F1000 tags (from F1000) and the geographic location and academic status of users (from Mendeley), which could be included to make the study much stronger and contribute to the understanding of readership counts and the effect of F1000 recommendations. In addition, subject classifications for the papers and locations of authors could be included to show if readers come from the same or different disciplines and countries as the paper and authors. I would also recommend the above mentioned extension of the study to include papers that were not recommended in F1000 to measure the effect of recommendations on readership counts. Combining these different aspects, one could investigate whether recommendations on F1000 lead to more diverse user groups on Mendeley in terms of discipline, country and academic status. For example, is a biology paper recommended and tagged as \"good for teaching\" on F1000 read by more Bachelor students from biology than a biology paper that was not recommended and tagged as such? Minor revisions:The first sentence \"Interest in the broad impact of research (Bornmann, 2012, 2013) has resulted in new forms of impact measurements.\" simplifies the situation too much: there is also the technological push and publishers' interest who resulted in the availability of new metrics, plus these metrics have not been validated as measuring impact yet. Also, the references to support interest in broad impact measures should refer to sources that show these interests such as REF etc. instead of papers by Bornmann, which claim that these interests exist. Regarding the use of altmetrics: apart from Snowball Metrics, they are also applied in the sense that various journals now show them to indicate the \"impact\" and use of articles (for example, PLOS journals, Nature, Wiley journals etc.). Funders have also declared interest in using these metrics (for example, see Dinsmore, Allen & Dolby). \"Since data from Mendeley can be received by an Application Programming Interface (API) without any problems\" - this is not completely true, there are a lot of issues with data quality and reliability for Mendeley, see for example: Bar-Ilan and Zahedi, Haustein & Bowman. These limitations need to be acknowledged in particular because the study is based on matching DOIs and PMIDs - Mendeley entries without these or incorrect IDs will be lost. What is the error rate introduced by using these identifiers only? In the methods, authors should specify what was done when problems with the API connection occurred. How was it insured that data was not lost due to these problems? It would be helpful to add the number of unique papers and mean (+ std. dev.) number of reader counts per paper per discipline to Table 1 and include also the other disciplines with less than 1% of reader counts.", "responses": [ { "c_id": "1307", "date": "08 May 2015", "name": "Robin Haunschild", "role": "Author Response", "response": "“The research questions lack clarity and the motivation of the study should not solely be based on the availability of datasets (Mendeley and F1000). It should be emphasized in how far this study is different from previous work, in particular Mohammadi & Thelwall, who analyzed very similar aspects on Mendeley and, in addition, compared the discipline of users to that of the citing papers. For the present study, it is not clear what the authors expect to find (how much biology readers are normal?) and what the data is able to show: Do papers recommended on F1000 have Mendeley users from more diverse disciplines than expected?”The motivation of the study is not solely based on availability of the datasets. The research questions do reflect this.We have added the reference (Mohammadi & Thelwall). Clearly, Mohammadi & Thelwall focus on a specific publication year and different disciplines than our study. Technical differences are highlighted in the Section Methods, Subsection Use of the Mendeley API. Mohammadi & Thelwall used the old API where only the top 3 categories in percentages. Our study used the new API where absolute reader numbers are provided and the top 3 restriction is no longer in place. All sub-disciplines with at least one reader are available in the API.We do not know how many readers from biology are normal for the F1000Prime publication set. This is one of the reasons why we pursued this research. As we have no real expectation value for F1000Prime readers from biology, it is not possible to judge if the observed reader counts are as expected, higher, or lower.“It would be much more interesting and valuable to observe the effect of being recommended on F1000 by comparing Mendeley readership counts and disciplines of users of the dataset used in this study with a control set of papers that were not recommended. This could be achieved by analyzing and comparing the data for the population of PubMed articles for a certain set of recent years: Does the F1000 recommendation provide visibility to papers that increases the number of readers on Mendeley as well as the diversity of the audience in terms of disciplines and academic status? PubMed/Medline could also provide a meaningful subject classification for papers to measure interdisciplinary knowledge flows from authors to readers.”While this is an interesting question, it is outside the scope of our current research question. Also, it is not easy (maybe even impossible) to answer it. Even if a paper was recommended into F1000Prime and has a very high Mendeley count, we do not know if this is due to the F1000Prime recommendation or not. Maybe, the paper is well written and interesting, attracted many Mendeley reader counts and was recommended into F1000Prime.“The authors also need to clarify in how far the present study differs and distinguishes itself from their other publications on similar topics and the same datasets:1) Who reads F1000Prime publications?”The paper 1 is actually the preprint version of the current paper. We uploaded the manuscript to Figshare after submitting it to F1000Research.“2) Who publishes, reads, and cites papers? An analysis of country information”The paper 2 is concerned about the academic status information of Mendeley readers of F1000Prime papers. Furthermore, the type of analysis is completely different.“3) Usefulness of altmetrics for measuring the broader impact of research: A case study using data from PLOS and F1000Prime”This is an old version of Paper 6.“4) Validity of altmetrics data for measuring societal impact: A study using data from Altmetric and F1000Prime”The paper 4 focusses on Twitter counts provided by Altmetric.“5) Overlay maps based on Mendeley data: The use of altmetrics for readership networks”The paper 5 uses a different data set (WoS publication year 2012) than our current paper. It focusses on the generation of overlay maps and is already in press in a different journal.“6) Usefulness of altmetrics for measuring the broader impact of research: A case study using data from PLOS (altmetrics) and F1000Prime (paper tags)”The paper 6 studied the intersection of altmetrics data from PLoS and F1000Prime publications. This intersection is rather small with 1082 papers. Our current paper studies Mendeley reader counts of 114,582 papers as noted in the Section Methods.“7) The authors also mention Haunschild, Stefaner & Bornmann (in preparation), which seems to focus on the geographic location of Mendeley users of the same dataset. Could this aspect not be integrated in the present study?”This paper is already in press and will be presented at the ISSI 2015 conference. Thus, it cannot be integrated into the present study. Furthermore, the topics of both papers are too different, so that it would not be possible to merge both into a concise article.Although the topics of the papers 2-7 might be similar (all deal with altmetrics), the focus of each paper is very different. In many cases, also the data set is very different.“The reference list is particularly poor and not acceptable in its current form. There have been plenty of studies by Thelwall, Mohammadi, Costas, Zahedi, Kraker, Haustein and others that have evaluated Mendeley reader counts - these are completely ignored. The introduction should be additionally supported by core altmetrics publications by Priem (particularly his overview of altmetrics in Beyond Bibliometrics which includes a definition of altmetrics), Piwowar, regarding reference managers Taraborelli and Haustein & Siebenlist, as well as the above mentioned authors for research on altmetrics. Peter Kraker's work on readership networks based on Mendeley users needs to be considered as well.The parallels between early bibliometrics research and current altmetrics lack references either to the particular bibliometrics studies or detailed discussions of this parallel, for example in Haustein, Bowman & Costas.”Priem’s overview of Altmetrics in the book “Beyond Bibliometrics” is already referenced in the text. Haustein, S., & Larivière, V. (2014) is also cited. We have extended the literature review in the new version of the manuscript somewhat. Considering that this was intended to be a shorter article, we think it is not appropriate to include an exhaustive literature review.“In addition, some of the references cited in the text are also not listed in the reference list. This needs proper revision.”We thank the referee for this comment. Unfortunately, a large part of the list of references got lost, due to a problem with our software in the final stages between submission and publication. We have included the lost references in the revised version.“The dataset is not clearly defined. What is the F1000 Prime publication dataset? How and when was it retrieved? Are those all recommendations ever made in the database? What publications do the 114,582 papers refer to (journals, publications years, document types, discipline, research field, etc.)? What is the metadata quality of these entries; in particular, how many of these have a correct DOI, PMID or both? In how far does the availability of identifiers as well as the characteristics of papers (publication year, journals) influence and bias the matching with and availability in Mendeley? Regarding the matching of results: how many unique documents do the 6,263,913 reader counts refer to? What is the percentage of documents that could not be found in Mendeley? Readers/users should be distinguished from reader counts - to avoid the implication that there are 6.2 million readers. Mendeley has around 3 million users (2.8 million as of February 2014; Haustein & Larivière), who create reader(ship) counts by adding documents to their libraries.”The employed F1000Prime publication set consists of 114,582 journal articles in journals such as Nature, PNAS, Science, Cell, PLoS ONE, etc. Additionally, there is at least one recommendation for each paper. We have added this information in the revised version of the manuscript. We checked the DOIs and PubMedIDs. There are only two wrong (duplicated) DOIs in the publication set. We found not a single PubMedID which is wrong. Considering that this was intended to be a shorter article, we have included a brief note regarding this in the new version of the paper.The data set is deposited at the Figshare link in the paper. A new Figshare link has been included which also includes a network file which can be loaded in Pajek to see detailed properties of the network.We have added descriptions of regarding the data set, problems of retrieval of reader data, and the relation between reader counts and unique documents.“The description of the methods for the network analysis is too brief: How were co-occurrences calculated? How were they normalized? Due to the density of 1 (i.e., all nodes are connected), the network layout is not very meaningful and not easy to interpret, often counterintuitive. The informativeness of the network could be improved by removing weak links to obtain a more meaningful network structure, where central (i.e., well-connected) nodes are positioned in the center of the network and less important ones in the periphery. Moreover, similar nodes as detected by the clustering algorithm (yellow and green in Figure 1) should be placed close together. In addition, it would make sense to include self-loops for papers saved by users of the same discipline to highlight homogeneous user groups. As the authors use the VOSviewer clustering method, why was VOSviewer not chosen for the mapping? In my opinion, it provides much more meaningful robust networks and better visualizations than Pajek. Other alternatives are Gephi, GUESS, UCInet, etc.”Based on these suggestions, we have replaced Figure 1 with a new version and extended the methodological description of the network analysis. VOSViewer has not chosen as visualization program because the co-occurences are shown as shorter distances but not as thicker connection lines. We prefer the thicker connection lines for this paper. Unfortunately, the self-loops could not be included due to system limits of Pajek.“Regarding the interpretation of the network, the authors state that ‘[t]he thicker and darker the edges between two disciplines, the more frequently [the users] have read a F1000Prime paper jointly’. Should it not rather be that ‘[t]he thicker and darker the edges between two disciplines, the more frequently papers were saved by users of these two particular disciplines’?The grouping into clusters seems to a certain extent counterintuitive: Why is environmental science grouped together with psychology instead of biology? Could this be introduced by (the lack of) normalization? These counterintuitive results need to be discussed!”We have revised the formulation accordingly and included more discussion on the counterintuitive results. Different normalization procedures lead to different results. Thus, we prefer to show the visualization without normalization.“The discussion needs to be extended and a conclusion is missing. It is not clear what the study actually shows/proofs and in how far the few results (sum of readers per discipline) warrant a separate publication. The dataset of F1000 recommendations and Mendeley include many other pieces of interesting information such as the recommendation scores, F1000 tags (from F1000) and the geographic location and academic status of users (from Mendeley), which could be included to make the study much stronger and contribute to the understanding of readership counts and the effect of F1000 recommendations. In addition, subject classifications for the papers and locations of authors could be included to show if readers come from the same or different disciplines and countries as the paper and authors. I would also recommend the above mentioned extension of the study to include papers that were not recommended in F1000 to measure the effect of recommendations on readership counts. Combining these different aspects, one could investigate whether recommendations on F1000 lead to more diverse user groups on Mendeley in terms of discipline, country and academic status. For example, is a biology paper recommended and tagged as \"good for teaching\" on F1000 read by more Bachelor students from biology than a biology paper that was not recommended and tagged as such?”Unfortunately, the other information from Mendeley (geographic location and academic status) are completely decoupled from the sub-discipline information. Therefore, it is not possible to define a “Bachelor student from biology” using current Mendeley data. We could create similar figures for academic status and geographic location, but they are not that interesting in this case. As a bio-medical publication set is studied, the vast majority of readers are expected from medicine and biology. To some extend this expectation is fulfilled, but some interesting readership connections between other disciplines and biology and/or medicine are found. We have no such expectation to test regarding location or academic status.“The first sentence 'Interest in the broad impact of research (Bornmann, 2012, 2013) has resulted in new forms of impact measurements.' simplifies the situation too much: there is also the technological push and publishers' interest who resulted in the availability of new metrics, plus these metrics have not been validated as measuring impact yet. Also, the references to support interest in broad impact measures should refer to sources that show these interests such as REF etc. instead of papers by Bornmann, which claim that these interests exist.”We revised this sentence.“Regarding the use of altmetrics: apart from Snowball Metrics, they are also applied in the sense that various journals now show them to indicate the \"impact\" and use of articles (for example, PLOS journals, Nature, Wiley journals etc.). Funders have also declared interest in using these metrics (for example, see Dinsmore, Allen & Dolby).”Thank you for the suggestion. We have included this into the introduction.\"’Since data from Mendeley can be received by an Application Programming Interface (API) without any problems’ - this is not completely true, there are a lot of issues with data quality and reliability for Mendeley, see for example: Bar-Ilan and Zahedi, Haustein & Bowman. These limitations need to be acknowledged in particular because the study is based on matching DOIs and PMIDs - Mendeley entries without these or incorrect IDs will be lost. What is the error rate introduced by using these identifiers only?”We have revised this sentence.“In the methods, authors should specify what was done when problems with the API connection occurred. How was it insured that data was not lost due to these problems?”We have added a more detailed description of the retrieval procedure.“It would be helpful to add the number of unique papers and mean (+ std. dev.) number of reader counts per paper per discipline to Table 1 and include also the other disciplines with less than 1% of reader counts.” We added also the other disciplines below 1% of the readers. Including also the number of unique papers, the mean number of readers, and the standard deviations would make the table much harder to understand. All raw data are deposited at a Figshare link so that people interested in other types of analysis can perform them on their own." } ] } ]	1	https://f1000research.com/articles/4-41
https://f1000research.com/articles/4-109/v1	08 May 15	{ "type": "Research Article", "title": "Short-term effect of acute and repeated urinary bladder inflammation on thigmotactic behaviour in the laboratory rat", "authors": [ "Rosemary H Morland", "Amparo Novejarque", "Wenlong Huang", "Rachel Wodarski", "Franziska Denk", "John D Dawes", "Tim Pheby", "Stephen B McMahon", "Andrew SC Rice", "Amparo Novejarque", "Wenlong Huang", "Rachel Wodarski", "Franziska Denk", "John D Dawes", "Tim Pheby", "Stephen B McMahon" ], "abstract": "Understanding the non-sensory components of the pain experience is crucial to developing effective treatments for pain conditions. Chronic pain is associated with increased incidence of anxio-depressive disorders, and patients often report feelings of vulnerability which can decrease quality of life. In animal models of pain, observation of behaviours such as thigmotaxis can be used to detect such affective disturbances by exploiting the influence of nociceptive stimuli on the innate behavioural conflict between exploration of a novel space and predator avoidance behaviour. This study investigates whether acute and repeated bladder inflammation in adult female Wistar rats increases thigmotactic behaviour in the open field paradigm, and aims to determine whether this correlates with activation in the central amygdala, as measured by c-Fos immunoreactivity. Additionally, up-regulation of inflammatory mediators in the urinary bladder was measured using RT-qPCR array featuring 92 transcripts to examine how local mediators change under experimental conditions. We found acute but not repeated turpentine inflammation of the bladder increased thigmotactic behaviour (decreased frequency of entry to the inner zone) in the open field paradigm, a result that was also observed in the catheter-only instrumentation group. Decreases in locomotor activity were also observed in both models in turpentine and instrumentation groups. No differences were observed in c-Fos activation, although a general increased in activation along the rostro-caudal axis was seen. Inflammatory mediator up-regulation was greatest following acute inflammation, with CCL12, CCL7, and IL-1β significantly up-regulated in both conditions when compared to naïve tissue. These results suggest that acute catheterisation, with or without turpentine inflammation, induces affective alterations detectable in the open field paradigm accompanied by up-regulation of multiple inflammatory mediators.", "keywords": [ "Pain", "Inflammation", "Open Field", "Cytokines", "Amygdala", "Behaviour", "c-Fos" ], "content": "1. Introduction\n\nPain is a complex experience, dependent on the interplay between sensory aspects and centrally-mediated affective, motivational, cognitive, and behavioural elements. The current translational difficulties in developing effective pain relief are associated in part with a pre-clinical focus on positive sensory signs, whereas the clinical burden of chronic pain comes largely from negative behavioural responses, such as avoiding behaviours perceived as exacerbating, and a general decrease in quality of life as a result of unpredictable spontaneous pain (Hummel et al., 2008; Suskind et al., 2013). This study uses an animal model to increase our understanding of the non-sensory components of pain by investigating whether acute and repeated visceral inflammation influence behaviour, if alterations are associated with activational changes in central brain areas implicated in generation of affective behavioural responses, and investigate the local changes in cytokine levels twenty-four hours after inflammation.\n\nVisceral pain affects 16–25% of the general population (Collett, 2013), and is experienced by virtually all at some point, if only transiently. Despite this, visceral pain is inherently difficult to study in animal models, due to sparse innervation and referred pain complicating precise location of origin. This is reflected in the literature. A PubMed search conducted on the 22nd June 2014 (search terms: (pain) AND (neuropathic) vs. (pain) AND (visceral)) revealed 12,938 results for pain with a neuropathic element, compared to 5035 for pain with a visceral component. Of these, 510 neuropathic studies were conducted in vivo, compared to only 40 for visceral (additional search term: AND (in vivo)). None of these in vivo visceral pain publications involved the open field paradigm (additional search paradigm: AND (open field)), which was one of the key reasons for this study. We chose the turpentine model of visceral inflammation due to its simplicity of induction and demonstrated phenotype of viscero-visceral hyper-reflexia and referred hyperalgesia (Jaggar et al., 1999), that is reversible with analgesics (Farquhar-Smith & Rice, 2001; Jaggar et al., 1998; Rice, 1995).\n\nTo detect negative behavioural responses in animals, complex behavioural outcomes are commonly used. Thigmotaxis is a behaviour characterised by a preference for movement along a surface (i.e. “wall-hugging”), which shows an inverse relationship with exploration of novel areas in rats. It is hypothesised as related to risk assessment and predator avoidance, with the presence of spontaneous or ongoing pain decreasing potentially risky behaviours, such as exploration. The open field paradigm is capable of detecting these subtle behavioural differences in experimental models of pain with varying aetiologies including antiretroviral therapy-induced neuropathy (Huang et al., 2013; Wallace et al., 2007; Wallace et al., 2008), chemotherapy-induced neuropathy (Barzegar-Fallah et al., 2014), spinal nerve transection (Blackbeard et al., 2012), spinal nerve ligation (Ewan & Martin 2014; Kontinen et al., 1999; Suzuki et al., 2007), chronic constriction injury (Grégoire et al., 2012), and post-traumatic peripheral nerve trauma (Medico et al., 2004).\n\nPain acts on multiple levels within the nervous system: sensory signals from the periphery are received centrally and interpreted in relation to previous experience and current circumstances to produce a behavioural response. The different subdivisions of the amygdala receive sensory input from areas such as the thalamus and spinal cord (capsulo-lateral portion of the central amygdala, via the lateral and basolateral amygdalae), and provides output via the medial nucleus of the central amygdala to the pre-frontal cortex and hypothalamus. This pattern of connectivity suggests involvement in the assessment and generation of emotional associations that form an integral part of the pain experience (Neugebauer et al., 2009). Studies have shown pain-associated increases in amygdala activity both clinically and pre-clinically in pancreatitis (Frøkjær et al., 2011), cluster headache (Seifert et al., 2011), back pain, arthritis (Baliki et al., 2008), fibromyalgia (Harris et al., 2009), and menstrual pain (Tu et al., 2010). In this study, we look at c-Fos immunoreactivity (a marker of persistent neural activation; Barth, 2007) in the amygdala with respect to open field activity, considering whether the presence of acute or persistent visceral inflammation influences c-Fos expression.\n\nCytokines and associated inflammatory mediators are up-regulated in pain conditions with an inflammatory component (Calvo et al., 2012; Cheppudira et al., 2009; Girard et al., 2011; Johansson et al., 2003; Nowak et al., 2012; Quan-Xin et al., 2012; Skurlova et al., 2010; Zhang et al., 2013). In particular, Dawes and co-workers demonstrated commonality between cytokines up-regulated following UVB-irradiation in clinical and pre-clinical models, showing the pro-inflammatory cytokine CXCL5 is up-regulated and capable of producing hypersensitivity in otherwise naïve rats (Dawes et al., 2011). In this study, we hope to show the profile of inflammatory mediators up-regulated following bladder inflammation and investigate whether this profile changes with repeated inflammation.\n\nThis study aims to investigate the acute and medium-term effects of visceral inflammation on thigmotactic behaviour, correlating differences in behaviour with both central activation (c-Fos immunoreactivity in the amygdala), and peripheral levels of inflammatory cytokines to further understand the behavioural implications of visceral inflammation.\n\n\n2. Materials and methods\n\nAll animal experiments conformed to British Home Office Regulations (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 (SI 2012/3039) and were performed under the authority of United Kingdom Home Office Project Licence 70/7162, adhering to the International Association for the Study of Pain (IASP) guidelines for in vivo research (Zimmermann, 1983; and http://www.iasp-pain.org). Experiments were designed according to Good Laboratory Practice standards (Macleod et al., 2009) and reported in accordance with the ARRIVE Guidelines (Kilkenny et al., 2010; Rice et al., 2013). Table 1 shows the major domains of good laboratory practice followed.\n\nFemale Wistar rats (Charles River, UK, RRID:RGD_737929), weighing 180–200 g on arrival, were housed in groups of 3–4 in individually ventilated cages with free access to food (RM1 (P), Special Diet Services, UK) and tap water. Animals were maintained under a 12 hour light cycle (07:00–19:00) in temperature and humidity-controlled conditions (25°C, 30%, both ± 5). Cages were cleaned weekly on a Tuesday (p.m.), and housed in a room containing mice and rats of both sexes. Animals were habituated to the holding room for a minimum 48 hours after delivery from the main campus facilities.\n\nSample size calculations used data from studies looking at the effect of neuropathic injury (spinal nerve transection) on frequency of inner zone entry in the open field (Morland et al., 2015 manuscript in preparation). Using a power (1-β) of 0.08 and alpha (α) value of 0.05, a sample size of 8 was calculated as sufficient to detect alterations in thigmotaxis.\n\nThere were three experimental groups: naïve, instrumentation (anaesthesia with catheterization and instillation of 0.5ml 100% olive oil), and turpentine (anaesthesia with catheterization, and inhalation anesthetic instillation of 0.5ml 50% turpentine). Full details of group sizes and exclusions are given in Figure 1. Female animals were selected for ease of catheterization.\n\nThe arrows on the right show the total number of animals used, with exclusions at each timepoint and final group sizes indicated in the central and right of the diagram respectively. Animals were lost during surgery due to complications associated with anaesthesia, and excluded from immunohistochemical analysis if hemispheric differentiation was not possible.\n\nAll behavioural experiments were conducted during the light phase (09:00–18:00) in a dedicated behavioural laboratory, with surgical procedures carried out in a separate but adjacent surgical room. In vivo studies were conducted in batches of 2–3 animals per group (n=6–9/batch) due to capacity and protocol constraints. Each animal was treated as a single experimental unit, with immunohistochemical analysis conducted using average values from a number of sequential sections from a single animal, as summarized in Table 2.\n\nUnder isoflurane anaesthesia, (1.5–3% in 2 L/min O2), bladder inflammation was induced as described by McMahon & Abel (1987). Briefly, a transurethral catheter (⌀ 1.02mm; Portex, UK) was introduced into the bladder, and position verified by applying gentle abdominal pressure to empty the bladder, before instillation of 0.5 ml olive oil or turpentine (50% in olive oil). The instillation was maintained under anaesthesia for 2 hours before removal of the catheter. The animal was allowed to recover in a separate area before returning to the home cage.\n\nBased on the above protocol, a model of persistent bladder inflammation was developed, involving three instillations, each one week apart. For both studies, surgery was conducted during the light phase and timed to ensure exactly 24 hours between instillation and exposure to the open field.\n\nTissue from whole bladders snap frozen during the saline phase of perfusion was used for RNA extraction. Briefly, samples were homogenized and total RNA obtained using a “hybrid” method of phenol extraction (Trizol, Invitrogen, USA) and column purification (RNeasy, Qiagen, USA). All samples were deoxyribonuclease (DNase, Qiagen, USA) treated to avoid genomic contamination. Purity and integrity confirmed with an RNA 6000 Nano Chip (Agilent, USA). Complementary DNA (cDNA) was synthesized from RNA using a SuperScript II reverse transcriptase kit (Invitrogen, USA).\n\nCustom-made Taqman array cards (as described by Dawes et al., 2011), featuring four sets of 92 different primer pairs, and four house-keeping genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal protein, β-actin, and β2-microtubulin. Inflammatory mediators were categorized into cytokines, chemokines, growth factors, enzymes, and ‘other’ (Table S1). Each cDNA sample was diluted with PCR-grade water and added in a 1:1 ratio to Taqman Universal master mix to produce a final concentration of 1 ng/µl cDNA. Samples were loaded into the appropriate ports (1 µl/well) according to manufacturer guidelines. Cards were placed into a 7900HT Fast Real-Time PCR system (Applied Biosystems, USA), and subjected to 40 cycles of amplification. Transcript expression was measured with the ΔΔCt (cycling time) method normalised to the geometric mean of the four housekeeping genes using the R package NormqPCR (https://r-forge.r-project.org/projects/qpcr/). Relative changes in transcript levels are presented as fold change (FC). When transcript numbers were undetermined for a given detector in <50% of samples, the average Ct value was calculated with the remaining data values. If transcript numbers were undetermined in >50% of transcripts for a given sample, a default Ct of 38 was assigned. If these conditions coincided, no FC value was calculated. Difference in gene expression were detected using the significance analysis of microarray (SAM) technique, involving calculation of false discovery rates (FDR), represented as a q statistic (Lin et al., 2008; Xiao et al., 2002).\n\nHumidity and temperature were maintained at 25°C and 30% humidity respectively. Light levels outside the isolation chamber during open field paradigms ranged from 70–300 lux. As rodents have a greater auditory range than humans, ultrasonic sound recordings were taken using a Mini-3 Bat Detector (Ultra Sound Advice, UK) to determine the background levels of high frequency sound generated by equipment. Fluorescent lighting and computer equipment emitted signals within the 20–50 kHz range - this equipment was switched on for the duration of each experiment and animals were allowed to acclimatise to the testing room for 30 min prior to testing.\n\nThigmotaxis. The open field paradigm was used to assess thigmotaxis. The open field arena (black, 100 cm2) was enclosed in an isolation chamber (115 × 115 × 255 cm) to minimise environmental interference. Light levels within the open field were set at 12 lux (measured in the centre of the arena). Animals were introduced into the near corner of the arena, facing the centre of the arena, and allowed to explore for 15 min. The arena was cleaned with 0.02% Distel (formerly Trigene, Tristel Solutions Ltd., UK) between trials. Behaviour was captured by high sensitivity camera (VCB 3372; Sanyo, Japan), and analysed using Ethovision XT 10.1 (Tracksys, UK (for Noldus, the Netherlands), RRID:rid_000100). The primary pre-determined outcome measure was frequency of entry into a virtual central zone (40 cm2). Secondary outcome measures were duration in the central zone, and rearing. Total distance travelled was used as a measure of general locomotor activity. Rearing was defined as both forelimbs elevated, either against a wall, or freestanding, and measured by a trained observer watching at 4 × playback speed.\n\nHistological studies. To capture peak c-Fos activation in response to the open field, fixation perfusion took place within 90–120min of open field exposure. Bladders were extracted and snap frozen in LN2 during the saline phase of perfusion fixation.\n\nAnimals were humanely killed with pentobarbital and transcardially perfused with 0.9% heparinized saline followed by 4% paraformaldehyde. The brain was removed and post-fixed in 4% paraformaldehyde for 6 hours and cryo-protected in 30% sucrose for at least 3 days prior to sectioning (50 µm) on a freezing microtome (model no. HM450, Thermo Scientific, USA). Sections were washed in phosphate buffered saline (PBS; 1(NaH2PO4.2H2O):9(Na2HPO4.12H2O):7(NaCl) in dH2O), quenched with 0.03% H2O2 (Sigma-Aldrich, UK), washed again in PBS, blocked for one hour in 5% normal goat serum (NGS, Millipore, UK; PBS with 0.3% TX (Triton X-100, BDH, UK)), before incubation overnight at 4°C with 1:20,000 polyclonal rabbit IgG anti-c-Fos (Santa Cruz Biotechnology Cat# sc-52 RRID:AB_2106783; 0.3% PBS-TX, 2% NGS). The following day, sections were washed in PBS and incubated for two hours at room temperature (20°C) with 1:250 Biotin-SP-conjugated Affinipure goat anti-rabbit IgG (F(ab’)2 fragment specific; Jackson ImmunoResearch, USA, Cat# 111-036-006 RRID:AB_2313586; 0.3% PBS-TX, 2% NGS). Staining was visualised using a Vectastain ABC kit (avidin-biotin-peroxidase complex, VectorLabs, UK) and DAB (3, 3′-Diaminobenzidine) with nickel salt intensification (VectorLabs, UK). Sections were mounted, counterstained with toluidine blue to enhance cyto-architecture, and cover-slipped using DePex mounting media (VWR, UK) prior to image capture.\n\nImage quantification. The bregma position of each mounted section was determined with reference to Paxinos & Watson (6th Edition, 2007), and only those between -1.44mm and -3.36mm (range of the central amygdala) were captured using a Leica DM R light microscope (Leica Microsystems, Germany). Image analysis was conducted using Photoshop CS5 (Adobe, USA), and the mean number of positively stained cells per mm2 calculated for each subdivision of the central amygdala (medial, lateral, and capsular), with lateralization and rostro-caudal axis position noted. A positively stained cell was defined as having a clearly defined dark rounded nucleus (blue/black) - sections without c-Fos positive cells in areas out-with the central amygdala were excluded. Image analysis was conducted blind, using individual animal identifiers rather than group codes until completion of analysis.\n\nThigmotaxis was analysed using 1-way ANOVA, with Kruskal-Wallis utilised for data that was not normally distributed. c-Fos data was analysed using 1-, 2-, and 3-way ANOVA taking account of hemispheric, sub-nuclear, and rostro-caudal designations of the central amygdala. Where overall ANOVA detected significant differences, multiple comparisons procedures were used - Holm-Sidak for normally distributed data, and Dunn’s multiple comparison for non-parametric tests. Correlations were assessed using Pearson correlation coefficients. Inflammatory mediator analysis was conducted using Significance Analysis of Microarrays (SAM), and the false discovery method (FDR), which take into account dependence between transcripts and uses non-parametric techniques. Significance was taken as q=0%.\n\nSummary statistics are expressed as mean (standard deviation; SD) when data was normally distributed, or median (interquartile range; IQR) when data failed normality testing. Significance was taken at p<0.05.\n\nAll statistical tests were performed, sample sizes calculated, and appropriate graphs generated using OriginPro v9.1 (OriginLab, USA) and SigmaPlot v10 (Systat Software Inc., USA; RRID:SciRes_000184).\n\n\n3. Results\n\n\n\nSee Figure 1 for a summary of exclusions and group sizes used. Bladder inflammation as a model of visceral inflammation was generally well tolerated; the main cause of mortality was surgical complications and related anaesthesia. In the repeated model, all deaths occurred during/following the final inflammation session. Following recovery from anaesthesia and return to the home cage, animals were alert and outwardly indistinguishable.\n\n3.1.1 Acute bladder inflammation. A total of 81/92 cytokine transcripts were analysed for magnitude and significance of transcript up-regulation following acute bladder inflammation. 11 markers (CCL1, CCL28, CTLA-8, CXCL17, IFNγ, IL-2, IL-3, IL-4, IL-9, IL-13, IL-27) were excluded from the acute inflammation model due to high cycle time (>38), indicative of low levels or issues in detection. The top ten up-regulated transcripts are shown in Table 3. Figure 1 shows the rank FC for all markers analysed. Significance analysis of microarrays (SAM) was used to identify 25 mRNAs that were significantly different in the turpentine group compared to naive (FC >1.5, Δ = 1.61, FDR=0%). Of these, 13 were classified as chemokines, 9 as cytokines, 2 as enzymes, 2 as growth factors, and 1 as ‘other’, as seen in Table 4.\n\nData presented as mean fold change (95% confidence interval), n=3–4 animals/group.\n\nSignificant fold difference in acute modela, repeated modelb, or bothc; Significance level, FDR q=0% denoted by bold text.\n\n3.1.2 Repeated bladder inflammation. A total of 75/92 cytokine transcripts were analysed for magnitude and significance of transcript up-regulation following repeated bladder inflammation. 16 markers (CCL1, CCL3, CCL25, CCL26, CCL28, CXCL3, IL-2, IL-3, IL-4, IL-9, IL-13, IL-19, IL-20, IL-27, C5, and IFNγ) were excluded from the repeated inflammation model due to high cycle time (>38 overall mean). One housekeeping gene (18s) was excluded as it was significantly up regulated. FC was therefore calculated normalized to the remaining housekeeping genes (HPRT, ACTB, and GAPDH). Figure 2 shows fold change rank for all cytokines analysed. The top 10 mRNA transcripts up-regulated following repeated bladder inflammation with turpentine are shown in Table 3. Using SAM, we identified 4 mRNAs that were significantly different in turpentine compared to naive (FC >1.5, Δ = 1.61, FDR=0%). Of these, 3 were classified as chemokines (CCL7, CCL12, and CXCL17, and 1 as cytokine (IL-1β), as seen in Table 4.\n\nFollowing significance of microarray analysis, only inflammatory mediators showing up-regulation were significant (q=0%).\n\n3.1.3 Comparison of Inflammatory Mediator Up-regulation Following Acute and Repeated Bladder Inflammation. From a possible list of 92 transcripts, 73 were detected in both models. Table 5 shows the mean rank, standard deviation, and super rank value (SRV) for each transcript examined. Of these IL-1β was significantly up regulated in both (FDR q=0%). Two other chemokines, CCL7 (acute: 272.74 FC; repeated: 7.15 FC), and CCL12 (acute: 17.96 FC; repeated: 7.15 FC), were also significantly up-regulated in both models (FDR q=0%).\n\n\n3.2 Open field behaviour\n\n3.2.1 Acute bladder inflammation. Thigmotactic behaviour was observed in animals from both instrumentation and turpentine groups. Animals in both these groups entered the inner zone less frequently compared to naïve (1-way ANOVA, p=0.009). Turpentine animals entered the inner zone 8.6 times (SD 5.84, p=0.025), whereas the instrumentation group averaged 6.3 entries (SD 3.68, p=0.0035), both significant compared to the naïve value of 13.9 entries (SD 6.86) as shown in Figure 3A.\n\nA/E - Frequency of entry to the inner zone, B/F - duration in the inner zone (s), C/G - rearing frequency, D/H - total distance travelled (cm).\n\nDuration in the inner zone was not significantly different between groups (Kruskal-Wallis 1-way ANOVA, p=0.192). The median time spent in the inner zone was 11.52s (6.68–29.20), 6.80s (2.88–12.96), and 8.64s (2.56–14.32) for naïve, instrumentation, and turpentine groups respectively (Figure 3C). Rearing behaviour was not significantly different between groups (1-way ANOVA, p=0.112), with mean rear counts of 51.31 (14.96), 43.20 (14.08), and 37.23 (19.70) for naïve, instrumentation, and turpentine groups respectively (Figure 3E).\n\nDistance travelled was reduced in both instrumentation (7411.62cm IQR 6584.73–7970.77) and turpentine (6744.29cm IQR 3843.16–8192.87) groups when compared to naïve (Dunn’s post-hoc test p<0.05, 8771.06cm IQR 7860.85–10,188.78; Figure 3G).\n\n3.2.2 Repeated Bladder Inflammation. No significant effect of group was observed on inner zone frequency (p=0.185) or duration (p=0.288), with naïve entering the inner zone an average of 11.5 (6.22) times with a duration of 13.76s (9.6–23.7), instrumentation averaging 8.7 (5.7) entries with a median duration of 14.48s (6.6–27.2), and turpentine entering the inner zone 7.25 (6.0) times, with a median duration of 6.96s (1.76–16.72) (Figure 3B and D). No difference was seen in rearing activity (p=0.638), with mean rear counts of 56.9 (13.9), 48.6 (7.7), and 52.22 (21.67) for naïve, instrumentation, and turpentine groups respectively (Figure 3F).\n\nA decrease in distance travelled was seen in both instrumentation (p=0.015, 6797.29cm SD 1637.10) and turpentine (p=0.00026, 5974.04 cm SD 2039.67) groups compared to naïve (8432.59cm SD 953.15; overall 1-way ANOVA p<0.001, Figure 3H).\n\n\n3.3 c-Fos immunoreactivity in the central amygdala in response to open field exposure\n\n3.3.1 Acute Bladder Inflammation. Higher levels of c-Fos immunoreactivity were seen in the left hemisphere (p=0.0031), localised to the CeM (p=0.032). No other differences in c-Fos immunoreactivity were observed in the central amygdala, and there was no effect of group, as shown in Figure 4, with full c-Fos density data in Table 6.\n\nA/D - Global CeA (central amygdala) c-Fos immunoreactivity, B/E - right CeA, C/F - left CeA.\n\n3.3.2 Repeated Bladder Inflammation. The CeC showed higher levels of f-cos immunoreactivity compared to the CeM (p=0.00485).\n\nA rostro-caudal gradient was seen with significantly higher levels of activation observed in the caudal regions (p=0.016). Significant differences were also observed within levels, with the rostral CeL (p=0.013) and caudal CeC (p=0.001) showing higher levels when compared to the CeM. c-Fos immunoreactivity in the CeM exhibited a rostro-caudal gradient, with the rostral region containing fewer positive cells compared to the intermediate (p<0.001) and caudal regions (p=0.002). There was no effect of group on c-Fos immunoreactivity (Figure 4 and Table 6).\n\nData presented as mean (95% confidence interval). Acute: n=8–13; Repeated: n=7–15. Data shown as mean (SD).\n\n\n3.4 Correlations\n\n3.4.1 Acute Bladder Inflammation. Overall, there was a positive correlation between rearing activity and c-Fos immunoreactivity in the rostral CeC (Pearson’s ρ=0.44, p=0.01).\n\nLooking at correlations within experimental groups, the naïve group showed the highest levels of correlation, with rearing activity positively correlated with CeC (Overall, right hemisphere, and rostral level; ρ=0.73–80, p<0.05) and CeM (caudal; ρ=0.72, p=0.03). Distance travelled was also positively correlated with caudal c-Fos immunoreactivity in the left central amygdala (ρ=0.8, p=0.02).\n\nIn the instrumentation group, there was a positive correlation between duration in the inner zone and c-Fos immunoreactivity in the intermediate CeL (ρ=0.76, p=0.05). No correlations were observed in the turpentine group.\n\nSee Table 7 and Table S2 for full details of all correlations.\n\nBold* denotes p>0.05. ABBREVIATIONS: N, Naïve; I, Instrumental; T, Turpentine; CeM, medial central amygdala; CeL, lateral central amygdala; CeC, capsular central amygdala.\n\n3.4.2 Repeated Bladder Inflammation. Overall, there were correlations between frequency, duration, and rearing and c-Fos immunoreactivity in the central amygdala. Frequency was positively correlated with CeL immunoreactivity in the left hemisphere (ρ=0.35, p=0.03), and CeM immunoreactivity in the right hemisphere (ρ=0.35, p=0.04) and caudal level (ρ=0.39, p=0.02). Duration in the inner zone was positively correlated with rostral CeL (ρ=0.33, p=0.05), caudal CeM (ρ=0.37, p=0.02), and c-Fos immunoreactivity in all intermediate divisions except the right hemisphere (ρ=0.34–0.41, p<0.05). Rearing was negatively correlated with CeM (overall, left, right, and rostral; ρ=-0.43- -0.67, p<0.05), right overall (ρ=-0.41, p=0.05), right CeL (ρ=-0.46, p=0.02), and caudal CeC (ρ=-0.52, p=0.01).\n\nWithin the naïve group, there were positive correlations between distance travelled and rostral c-Fos immunoreactivity (overall, left, and CeL; ρ=0.58–0.67, p<0.02). Frequency in the inner zone was positively correlated with caudal (overall, CeM; ρ=0.52–0.61, p<0.05) and rostral CeL c-Fos immunoreactivity (ρ=0.58, p=0.02). Duration in the inner zone was positively correlated with CeC (left, intermediate; ρ=0.59, p=0.02), CeL (left, rostral; 0.53–0.62, p<0.04), caudal CeM (ρ=0.66, p=0.01), left hemisphere (overall, intermediate, caudal; ρ=0.52–0.56, p<0.05), and intermediate (ρ=0.53, p=0.04). No correlations were seen with rearing behaviour.\n\nIn the instrumentation group, distance travelled was positively correlated with left CeL (ρ=0.71, p=0.02), and rearing was negatively correlated with left rostral c-Fos immunoreactivity (ρ=-0.93, p=0.02).\n\nIn the turpentine group, duration in the inner zone was correlated with intermediate CeM c-Fos immunoreactivity (ρ=0.64, p=0.02). Rearing behaviour was negatively correlated with c-Fos immunoreactivity in the CeM (overall, right, caudal; ρ=-0.71- -0.90, p<0.03), and in the right hemisphere (rostral, CeL; ρ=-0.76- -0.78, p<0.02).\n\nSee Table 7 and Table S2 for full details of all correlations.\n\n\n4. Discussion\n\nBoth acute and repeated bladder inflammation up-regulate inflammatory mediator expression, most notably IL-1β, CCL-7, and CCL-12. The fold change was higher in the acute model compared to the repeated, suggestive of a habituation effect when an animal is repeatedly exposed to an inflammatory stimulus. Thigmotactic alterations were observed in both instrumentation and turpentine groups following acute but not repeated bladder inflammation, although reductions in locomotor activity were observed in both models. Investigations into amygdalar c-Fos immunoreactivity following bladder inflammation were inconclusive.\n\nNumerous studies investigating cytokine expression profiles across experimental models of inflammatory pain show results similar to those reported here. IL-6, IL-10, and TNFα are highly expressed in normal bladder tissue (Pokrywczynska et al., 2013), and have pro-inflammatory actions (de Oliveira et al., 2011). Additionally, in a study examining cytokine expression following UVB irradiation, five markers also seen in our model of acute bladder inflammation were up-regulated: CCL-4, CCL-7, IL-1β, IL-24, and iNos/Nos2, of which IL-1β and CCL-7 were also seen in our repeated model (Dawes et al., 2011). In an acute model of pelvic pain (experimental prostatitis) there was increased expression of both CCL7 and CCL12, and seven other chemokines also seen in acute bladder inflammation (CCL2, CCL3, CCL4, CCL6, CXCL2, CXCL3, and XCL1), suggesting commonality in inflammatory mediators up-regulated in pelvic inflammatory models (Quick et al., 2012). Considering the effects of systemic cytokines on behaviour, peripheral IL-1β is associated with decreased activity in the open field, whether administered exogenously (Campbell et al., 2010; Song et al., 2005), or endogenously up-regulated in response to inflammatory stimuli such as lipopolysaccharide (LPS) (Swiergiel & Dunn, 2007) and intra-plantar carrageenan (Wen et al., 2014). In a study combining peripheral and systemic effects of cytokines with behaviour and c-Fos immunoreactivity in the amygdala, IL-1β, TNFα, and IL-6 reduced activity, with TNFα and IL-1β also associated with increased c-Fos immunoreactivity in the central amygdala (Skelly et al., 2013).\n\nOpen field behaviour was reduced in both turpentine and instrumentation groups following acute and repeated bladder inflammation. Distance travelled was decreased in both instrumentation and turpentine groups following acute and repeated bladder inflammation. Exaggerated thigmotactic behaviour (reduced frequency and duration of inner zone visits) was observed only in the acute model, but in both turpentine and instrumentation groups, suggesting catheterisation and instillation of vehicle (olive oil) could be capable of creating an aversive state. Notably, a similar trend was also seen in the repeated, although this did not reach significance due to high levels of variation. Previous studies examining cyclophosphamide (CYP)-induced cystitis revealed mixed effects on behaviour: male mice showed reduced rearing activity but increased time active in the open field four hours after acute injection (Olivar & Laird, 1999), however a recent study found no difference distance travelled in the open field following CYP treatment in female rats (Coelho et al., 2014). Other models of visceral inflammation show similarly mixed effects: trinitrobenzenesulfonic acid (TNBS)-induced acute pancreatitis increased immobility in male mice three weeks post-insult (Cattaruzza et al., 2013), but this effect was not seen in the cerulein-induced model of pancreatitis (Michalski et al., 2007). Chronic iodoacetamide (IAA)-induced gastritis reduced inner zone activity in female but not male rats (Luo et al., 2013), whereas acute intra-colonic instillation of mustard oil is reported to either reduce open field locomotor activity (Maia et al., 2006), or have no effect (Leite et al., 2012). A similar model of persistent colonic inflammation (deoxycholic acid/DCA) also failed to detect differences in distance travelled in the open field (Traub et al., 2008). However, intra-plantar CFA has been shown to induce thigmotactic behaviour without reducing distance travelled in the open field, up to 30 days after the original insult, suggesting the effects of acute inflammation can persist (Parent et al., 2012). Our data supports the idea that inflammation is capable of changing behaviour twenty-four hours after inflammation, but that this effect is not specific to introduction of an irritant into the bladder, and there is an element of habituation, as suggested by the reduced effects observed in the repeated model.\n\nThe presence of behavioural alteration in both surgical groups suggests the instillation procedure in itself is enough to alter behaviour. The volume instilled (0.5ml) is within the physiological range, accounting for diurnal variation (Herrera & Meredith, 2010), suggesting the pressure generated would be insufficient to initiate a noxious stretch response. Therefore, olive oil and/or catheterisation are capable of influencing open field behaviour. Anti-nociceptive effects of intra-peritoneal olive oil injection have been shown (Eidi et al., 2012), and there are numerous studies investigating its purported anti-inflammatory effects in relation to the Mediterranean diet (for review see Lucas et al., 2011), suggesting olive oil may have a protective rather than inflammatory effect. On the other hand, catheterisation is known as a risk factor for cystitis (Dayts, 2014; Tenke et al., 2014) and pelvic pain (Nazarko, 2014), suggesting further investigation is required into mechanisms involved in the behavioural alterations we saw in our instrumentation groups.\n\nIn the current study, we failed to detect an effect of instrumentation or turpentine on c-Fos immunoreactivity. Increased activity was seen in the lateral (CeL; rostral) and capsular (CeC; caudal) when compared with the medial central amygdala (CeM). The CeL and CeC often considered together as they both receive input from the spinal cord, and brainstem, as well as signals from higher regions such as the cortex, via the thalamus (Neugebauer et al., 2004). We noted significantly higher levels of activation in the caudal regions of the central amygdala, and although the biological significance of this is not known, it has been previously observed in a model of CYP-induced cystitis (Bon et al., 1998). No significant effects were seen in the acute model, suggesting the link between open field outcomes and central amygdala activation may be time-dependent, however the lack of robust correlations between behaviour and c-Fos immunoreactivity in this model could also be indicative of reduced involvement of the CeA in the inflammatory-mediated behavioural alterations we observed. Another factor to consider is the higher levels of immunoreactivity seen in the repeated model compared to the acute model. This is observed across all experimental groups, suggesting an environmental effect associated with differences in the housing of the animals.\n\nOther studies involving inflammatory models have shown associated increases in central amygdala activation: c-Fos immunoreactivity in the central amygdala was increased 60 minutes after acute intra-peritoneal injection of the gastric hormone CCK (cholecystokinin) (Rinaman, 2003), and intra-plantar formalin was associated with an increased c-Fos immunoreactivity detectable for up-to 2 (Rouwette et al., 2011). However, our data is not directly comparable with these studies as the acute timescale considered was considerably shorter than the twenty-four hours we studied. Furthermore, our experiment was designed to investigate whether recent visceral inflammation alters central amygdala activity in response to the open field, as opposed to c-Fos in response to noxious stimuli alone.\n\nA study looking at blood flow and proliferation (cell number and volume) found these measures increased in the central amygdala following spared nerve injury, without a concomitant alteration in elevated plus maze or open field behaviour (Gonçalves et al., 2008). Nonetheless, thigmotaxis has been observed in association with increased central amygdala activity in peripheral nerve trauma (Burke et al., 2013), and post-operative incisional models (Li et al., 2010), emphasising the complexity of central amygdala involvement the generation of thigmotactic behavioural alterations.\n\nWe found a significant negative correlations between CeM c-Fos immunoreactivity and rearing behaviour in the repeated turpentine group in particular, suggesting rearing behaviour may be suppressed by CeM activity, although we failed to detect significant alterations in rearing. c-Fos immunoreactivity in the CeM was generally lower than that seen in the CeL/C, as would be expected when considering the CeL and CeC are involved in modulation of incoming noxious signals, whereas the CeM is thought to be more involved in initiation of the behavioural response. Investigations into the direct effect of experimental pain states on amygdala activation have shown up-regulation in the central amygdala, (Dai et al., 1993; Hayashi et al., 2009; de Lange et al., 2005; Lehner et al., 2006; Rea et al., 2011; Rouwette et al., 2012; Yamashiro et al., 1998; Yang et al., 2010) but none have investigated differences between nuclear subdivisions.\n\nHigh levels of variability were observed across all data sets, suggesting innate behavioural variation. Numerous studies have shown evidence for a divergent response to stress, and typically note the presence of two behavioural phenotypes – those that have low activity and high neophobia (low activity/high “anxiety”), and those that show higher levels of activity, and low levels of neophobia (high activity/low “anxiety”) (Koolhaas, 2008; Koolhaas et al., 2010; Mällo et al., 2007; Kabbaj et al., 2000; Landgraf & Wigger, 2002). We noted the presence of distinctive behavioural phenotypes in all groups including naive, characterised by low or high thigmotaxis, and are developing techniques to investigate these differences further – ideally, animals would be behaviourally phenotypes prior to inflammation in order to determine the effects of trait characteristics on state responses. A previous study has shown differential c-Fos expression in animals with differing behavioural phenotypes, namely up-regulation of central c-Fos expression in low activity/high “anxiety” rats (Salomé et al., 2004). However, although the rats used in our study were the same strain (Wistar, Charles River, UK), those in the Salome study were bred for high and low anxiogenic phenotype in the elevated plus maze (F12), which would likely magnify underlying neurochemical differences present in the original strain. Additionally, the duration of open field exposure was 30 minutes, as compared to the 15 minutes in our study, and it is likely that this would affect the c-Fos activation profile as the animal habituates to its environment.\n\nTo fully elucidate the c-Fos response to open field exposure, and determine whether the increase in caudal activation we observed has biological significance, further experiments comparing animals with and without exposure to the open field are required. Investigating cytokine profiles in more individuals would allow further correlation between behavioural outcomes and physiological pathology, and could be particularly instructive in understanding the effect seen in the instrumentation groups. Studies have shown behavioural responses to systemic IL-1β are variable (Pertsov et al., 2009), and also that acute responses can vary with oestrus cycle in female rats (Avitsur et al., 1995), suggesting investigations taking this into account may increase the robustness of this study, although a literature survey conducted in 2014 found similar variability between male and female animals (Prendergast et al., 2014). Furthermore, there are potential refinements that could be applied to this model to facilitate testing behaviour at earlier time points, including reducing the duration of instillation and therefore anaesthesia required by using a more specific and/or potent irritant. Finally, testing of behavioural phenotype prior to treatment allocation would allow more detailed study of whether and how behavioural phenotype modulates responses to visceral inflammation.\n\nIn summary, bladder inflammation is associated with a robust up-regulation of peripheral cytokines implicated in pain, inflammation, and behavioural depression, and acutely with increased thigmotaxis, not specific to inflammation. The data on neural correlates are inconclusive, but as increased variation in these outcomes is observed, further studies are required to elucidate mechanisms responsible.\n\n\nData availability\n\nFigshare: Cytokine q-RT-PCR, c-Fos immunoreactivity and open field behaviour data in rats following bladder inflammation doi: 10.6084/m9.figshare.1394861 (Morland et al., 2015).", "appendix": "Author contributions\n\n\n\nRHM, AN, and ASCR conceived the study; RHM conducted the experiments, analysed the data and wrote the manuscript; AN assisted in experimental design; WH and RW contributed to preparation of the manuscript; FD, JDD, and SBM provided support for cytokine analysis; TP provided assistance during immunohistochemical preparations; ASCR supervised the project. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed\n\n\nGrant information\n\nThis work was primarily funded by an MRC CASE award in conjunction with Pfizer to the London Pain Consortium (Wellcome Trust Strategic Award 083259). The manuscript was written under joint-funding from the Europain Collaboration, which has received support from the Innovative Medicines Initiative Joint Undertaking, under grant agreement AG2013/3347, and NC3Rs (P41508).\n\n\nSupplementary materials\n\nARRIVE Checklist. Click here to access the file.\n\nBold denotes p<0.05.\n\n\nReferences\n\nAvitsur R, Donchin O, Barak O, et al.: Behavioral effects of interleukin-1 beta: modulation by gender, estrus cycle, and progesterone. Brain Behav Immun. 1995; 9(3): 234–41. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "8596", "date": "19 May 2015", "name": "Gordon Munro", "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\nThe manuscript submitted by Morland et al. has assessed whether rats with acute or more prolonged bladder inflammation induced by turpentine oil exhibit thigmotactic behaviour as assessed in the open field paradigm. In addition, regulation of inflammatory mediators within the bladder and c-Fos immunoreactivity within the central amygdala was assessed. The authors provide a clearly defined hypothesis which they address appropriately with a number of the methodologies at hand. In the absence of a direct method to assess pain-like behaviour in the various groups I like that the authors assessed inflammatory mediator profiles using a well established methodology previously utilized in other experimental pain models (e.g. UV burn) where pain-like behaviours were measured directly. The manuscript is well written. The data have been analysed using appropriate statistical tests and the results are extensively described. Although the data ultimately reveal that instrumentation rather than inflammation per se can sufficiently induce thigmotactic behaviour this should not detract from the value of this work. Rather, the extensive description of study design, powering, randomization, blinding, exclusions and availability of their data for the scrutiny of their peers makes this work in many ways a first, and an extremely valuable addition to the field in my opinion. This said I have a number of issues with some of the included work as described in more detail below. Major pointsThe value of the current work would have been considerably enhanced if the experimental design had included a separate arm in which vehicle treated rats were treated with an anxiolytic dose of e.g. diazepam, or a motor impairing dose of an opiate like morphine with corresponding plasma samples obtained for purposes of drug exposure. Despite the extensive detail provided within the Methods section I find it extremely difficult to judge the sensitivity of their open field paradigm without the availability of this kind of information. The inclusion of the c-Fos data in the context of the current manuscript is to be blunt, both confusing and distracting. This is not because the data are inconclusive per se, in that no differences in c-Fos immunoreactivity were noted between instrumentation or turpentine treated groups. I would have expected that the authors might have seen a more robust pattern/magnitude of c-Fos immunoreactivity if rats in these 2 groups had been sacrificed at 90 minutes after induction of the procedure rather than the following day following open field performance. Whilst careful consideration and extensive reporting of experimental design was provided in relation to performing behavioural analysis, I just can’t see that the same is the case for the purposes of assessing ‘Amygdala activation’. Incidentally, without knowing precisely the function and phenotype of the c-Fos positive cells included in their analysis, I would suggest that this term is misleading – I think the authors might be aware of this as indicated at the bottom of page 14. The authors specify that sequential sections were obtained from each rat. I would presume that in a number of cases depending upon the exact plane of the sections that the same positive neurones were measured more than once. Accordingly, with section numbers ranging from 1-7 per rat as indicated in Table 2 this must introduce some form of bias between treatments. This issue goes against the grain of the rigorous experimental designs employed for purposes of behavioural analyses As such, I would strongly recommend that the authors remove all c-Fos related method and results from the manuscript. Alternatively, they could provide videocapture of all sections used in their analyses and provide this for open review to see if others can replicate their findingMinor pointsWhen performing c-Fos immuno were all sections processed within the same experimental run? If not, were assays blocked appropriately to contain representative samples from each of the treatment groups? Following on from the above, a generalized positive control group (with a background in neuroendocrinology I would have included a group of rats treated with eg hypertonic saline and measured c-Fos expression within specific hypothalamic nuclei such as the SON or PVN) would have provided a general measure of assay sensitivity. Can the authors comment on whether all rats were tested during similar stages of the oestrus cycle? Does this matter?", "responses": [] }, { "id": "8597", "date": "19 May 2015", "name": "Nicholas Andrews", "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\nThis is very detailed description of an interesting study attempting to bring together behaviour, anatomy and neurochemical responses. The methods are very clearly described. I have only a few minor points for the authors to consider.IntroductionThe case is made for the amygdala in relation to pathways involved in pain relay but not made for combining with open field. The open field arguably elicits thygmotaxis through a neophobic and spatial effects which might relate more closely to the hippocampus. A little more in the introduction discussing the known relationship between the amygdala and open field behaviour would be helpful to the reader and place the choice of test in greater context.ResultsCan the reduction in frequency of entries into the centre be explained by a general reduction in motor activity? If this is accounted for in an ANCOVA does the reduced time in the centre become more or less pronounced?Was velocity altered between the groups? Might also reflect wariness?DiscussionIs it possible that the variation of responses in the open field is due to the lack of clear salient stimuli for causing the thygmotaxis? A more discrete training stimulus as evident in conditioned fear-based paradigms may give less variability. Have the authors considered using other such tests, especially in light of the strong body of evidence relating conditioned fear to amygdala function?", "responses": [] }, { "id": "8593", "date": "22 May 2015", "name": "Hugo Leite-Almeida", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript entitled “Short-term effect of acute and repeated urinary bladder inflammation on thigmotactic behaviour in the laboratory rat” authors report their observations on the effects of acute and repeated bladder inflammation in open-field associated behavioral parameters like locomotion, thigmotaxis and rearings. In addition, the expression of inflammatory mediators in the bladder was analyzed by RT-qPCR and c-fos expression was quantified in the amygdala. Authors observed that turpentine driven inflammation in the bladder resulted in the up-regulation of inflammatory mediators particularly following acute challenge; these observations were not controlled for the effect of catheterization. Turpentine inflammation was associated with a decreased number of entries in the central area of the open-field area; in this case authors controlled for the effect of catheterization alone and observed that this group displayed a similar phenotype. Again, acutely challenged subjects were more affected. C-fos expression in the amygdala following open-field exposure was inconclusive.The manuscript is well-written and is in many aspects exemplar, adhering to the best practices in data reporting (sample size calculation, inclusion/exclusion criteria for experimental subjects, randomization, among others). Also, authors made available an interesting set of raw data permitting reanalysis to anyone interested. Finally, authors stress the importance of having alternative measures in pain models that go beyond sensory testing. There are however some aspects that need clarification:Authors preformed an exhaustive characterization of inflammation-associated transcripts expressed in the bladder; it is not clear what was the gain of this approach in the context presented by the authors in the introduction to the study; after all the model involved trauma (associated with the catheterization) and the injection of turpentine, therefore inflammation in the bladder was not an unexpected outcome. On the contrary, I would see some advantages if authors would have treated animals with anti-inflammatory drugs or, more importantly, if the strategy was applied in CNS tissues (namely in the amygdala); indeed a number of studies have associated the up-regulation of inflammatory mediators with anxiety-like behaviors.\n\nInstrumentation and turpentine animals present a robust decrease in the distance travelled in the open-field most probably as a result of inflammation-driven sickness behavior. Because open-field readouts depend on animal’s ability to move, other behavioral parameters can be indirectly affected. Have authors controlled for this? Have other measures be taken like animals’ weight, grooming, body temperature, etc? These would be particularly important in the repeated model. c-fos density values presented for naïve animals present an increase over 5 fold between acute and repeated models (in the global analysis; similar increases can be found in the segmented analysis); apart from open-field exposure naïve animals should by definition be equal. My concern is that such effect can mask biologically relevant variations of c-fos expression (e.g. ceiling effect).", "responses": [] } ]	1	https://f1000research.com/articles/4-109
https://f1000research.com/articles/3-317/v1	30 Dec 14	{ "type": "Study Protocol", "title": "Promoting self-management through adherence among heart failure patients discharged from rural hospitals: a study protocol", "authors": [ "Lufei Young", "Sue Barnason", "Van Do", "Sue Barnason", "Van Do" ], "abstract": "Background Heart failure is one of the most prevalent chronic conditions in adults, leading to prolonged morbidity, repeated hospitalizations, and placing tremendous economic burden on the healthcare system. Heart failure patients discharged from rural hospitals, or primarily critical access hospitals, have higher 30-day readmission and mortality rates compared to patients discharged from urban hospitals. Self-management improves heart failure patients’ health outcomes and reduces re-hospitalizations, but adherence to self-management guidelines is low. We propose a home based post-acute care service managed by advanced practice nurses to enhance patient activation and lead to the improvement of self-management adherence in heart failure patients discharged from rural hospitals.Objective This article describes the study design and research methods used to implement and evaluate the intervention.Method Our intervention is a 12-week patient activation (Patient AcTivated Care at Home [PATCH]) to improve self-management adherence. Patients were randomized into two parallel groups (12-week PATCH intervention + usual care vs. usual care only) to evaluate the effectiveness of this intervention. Outcomes were measured at baseline, 3 and 6 months.DiscussionThis study aimed to examine the effectiveness of a rural theory based, advance practice nurse led, activation enhancing intervention on the self-management adherence in heart failure patients residing in rural areas. Our expectation is to facilitate adherence to self-management behaviors in heart failure patients following discharge from rural hospitals and decrease complications and hospital readmissions, leading to the reduction of economic burden. Clinical Trial Registration Information: ClinicalTrials.gov; https://register.clinicaltrials.gov/ NCT01964053", "keywords": [ "heart failure", "self-management", "patient activation", "adherence" ], "content": "Study rationale\n\nHeart failure is one of the most prevalent chronic diseases among the adult population1 and hospitalizations account for the majority of costs related to heart failure treatment2. Rural hospitals had higher 30-day readmission rates for heart failure patients than urban hospitals (28% vs. 25%)3,4 (http://www.uppermidwestrhrc.org/pdf/Readmissions_finalreport_110310.pdf). Self-management is key to improving heart failure patients’ health outcomes5 and reducing re-hospitalizations6,7. Non-adherence to self-management guidelines accounted for 50% of hospital readmissions in heart failure patients8,9.\n\nCompared to urban residents, patients in rural communities face greater challenges in managing their heart failure10. Difficulties include lack of local cardiac services and heart failure specialists3,10, lack of heart failure specific self-management guidance from providers11,12, less hospital discharge education at critical access hospitals, lack of follow-up by providers13,14, poor communication between the patient and providers, difficulty in traveling long distances for follow-up appointments and associated problems (time, fatigue, and cost)11, and feeling isolated and unsupported15,16. Despite these identified needs, effective programs to support heart failure patients in managing this complex, chronic condition in rural communities have not been reported10. In addition, there is lack of reimbursement for programs that promote heart failure patients engaging in self-management behaviors over time. Innovative programs, such as the proposed PATCH program, are needed to assist heart failure patients’ self-management adherence.\n\nThe effective interventions to improve adherence to heart failure self-management behaviors are primarily disease management programs17 which require intensive resources and are mainly delivered in urban areas with comprehensive medical care centers. The limitations of existing interventions to promote self-management adherence in rural heart failure patients include: lack of theoretical guidance for the development of a rural-based intervention18,19, unclear mechanism of intervention8,17,20, and reliance on self-report measures of self-management adherence21–23.\n\nOur study will fill the gap of knowledge and evidence existing in the current literature about self-management interventions by: 1) identifying and appraising new intervention mechanisms to improve self-management behaviors; 2) testing the feasibility and efficacy of a rural theory-based intervention designed to assist rural heart failure patients in managing their chronic condition; and 3) evaluating the use of biomarkers (i.e., brain natriuretic peptide [BNP] and sodium concentration collected from a spot urine sample) to assess the adherence of self-management behaviors.\n\n\nConceptual framework\n\nSelf-management adherence is defined as the ability to follow and engage in self-management behaviors recommended in heart failure treatment guidelines (e.g., monitor daily weight, follow a restricted sodium diet, take medication as prescribed, exercise regularly, and keep follow-up appointments)24. We have proposed the patient activation intervention PATCH (Patient AcTivated Care at Home Model) for this study based on components of Lorig’s chronic disease self-management model25, Hibbard’s patient activation theory26,27, Bandura’s conceptualization of self-efficacy28, and Long and Weinert’s rural nursing theory19 (Figure 1). According to Long and Weinert’s rural nursing theory, rural patients are more likely to accept help and care during times of crisis19. Therefore, the intervention is triggered by the patient’s hospitalization and initiated during their hospital stay when they feel most vulnerable and receptive to the idea of making behavioral change to avoid readmission. Rural patients’ belief about self-reliance (responsibility for one’s own care) supports the use of Hibbard’s patient activation theory26.\n\nIn summary, the goal of the PATCH intervention is to increase adherence to self-management behaviors, leading to improved clinical biomarkers (BNP and urine sodium concentration) and fewer hospital readmissions that are considered to be threats to their health beliefs (health is to work, be productive and function in one’s own role)19. Our central hypothesis, based upon our preliminary data, is that patients with higher activation levels, as assessed by the Hibbard patient activation measure, will have significantly better self-management adherence. Given the significant challenges of managing heart failure patients in rural settings, it is essential to examine the feasibility, acceptability, and size of the effects of PATCH on adherence to self-management behaviors and readmissions.\n\nWe test our intervention with the following aims:\n\nAim 1. To evaluate the immediate and extended effects of the patient activation intervention on self-management adherence, we measure adherence using clinical biomarkers and self-report of self–management behaviors. Our working hypothesis (H1) is that subjects in the intervention group have better self-management adherence than the usual care group over time (3 and 6 months).\n\nAim 2. To evaluate the immediate and extended effects of the patient activation intervention on the specific health outcome, we measure hospital readmission rates. Our working hypothesis (H2) is that subjects in the intervention group have lower readmission rate than the usual care group over time (30 days, 3 and 6 months).\n\nAim 3. To evaluate the mechanism of the patient activation intervention. Our working hypothesis (H3) is that the scores on self-management knowledge, self-efficacy for self-management, patient activation, and self-management strategies in the intervention group are higher than the usual care group at the end of the intervention (3 months) when the maximum difference for each variable is expected.\n\nAim 4. To evaluate the feasibility of the PATCH intervention for a future larger clinical trial, which includes evaluation of enrollment (recruitment efficiency, attrition, problems and solutions), intervention fidelity (delivery, receipt, enactment), data collection, subject acceptability of the intervention, and estimation of effect sizes for sample size determination.\n\n\nMethods/design\n\nStudy participants were recruited and enrolled between October 2013 and December 2014 from two rural critical access hospitals. The principal investigator and research assistants who have ethical access at each study site were responsible to identify the potential participants, screen for eligibility and recruitment (Figure 2).\n\nThis study is a prospective, two-group, randomized experimental design with three data collection points (baseline, 3 months and 6 months). Heart failure patients discharged from the rural hospitals were randomized into two groups: the intervention or control groups.\n\n1. Control group received only usual care. Usual care refers to the standardized care received after hospital discharge, including the written discharge information and the scheduled follow-up doctor appointments. Standardized discharge instructions, as recommended by CMS and the Joint Commission, include: activity level, diet, discharge medications, weight monitoring, and what to do if symptoms worsen.\n\n2. Intervention group received usual care and the 12 weeks of PATCH intervention. The intervention comprised of two phases in which the in-hospital discharge education session was followed by 12 weeks of post-discharge education sessions delivered by telephone.\n\nBecause this is a preliminary study, sample size was estimated for two-sided statistical tests using a liberal α level of .10. For Aim 1, a repeated-measures ANOVA with an average between-group difference of Cohen’s f=.25 (a medium effect) and a within-subject correlation of ≤.6 would require 41 patients per group to have power=.80. With this sample size, a z-test of independent proportions would have power=.79 if the group proportions meeting guidelines differed by approximately .25, a value reached or exceeded by medication, diet, and weighing adherence in most of the intervention trials reviewed29. The sample of 82 also would be large enough to estimate proportions ± .07–.13 with 90% confidence (the precision depends on the value of the proportion and whether it was calculated within-group or for the entire sample). Allowing for 15% attrition, 48 patients per group (total N=96) are recruited.\n\nInclusion criteria. Patients were eligible for the study if they: 1) were age 21 or older; 2) had heart failure as one of their discharge diagnoses; 3) had New York Heart Association (NYHA) class II to IV (http://www.abouthf.org/questions_stages.htm) or had NYHA class I symptoms and at least one other heart failure-related hospitalization or emergency department visit in the year prior to the study; 4) were discharged to home; 5) passed a mini-cognitive screen30; 6) understood English; and 7) had access to a phone.\n\nExclusion criteria. Patients were not eligible for the study if they: 1) had depressive symptoms (received a score of 3 or above on the Patient Health Questionnaire-2 (PHQ-2))31 (http://www.cqaimh.org/pdf/tool_phq2.pdf); 2) had documented medical diagnosis or diagnostic evidence of liver cirrhosis; 3) had documented medical diagnosis or diagnostic evidence of renal failure defined as serum creatinine greater than 2.0mg/dl; and 4) had documented medical diagnosis or diagnostic evidence of end stage and/or terminal illness (e.g. cancer) affecting their abilities to perform self-management behaviors.\n\n\nPATCH intervention\n\nThe intervention group received usual care and the PATCH intervention. The intervention was comprised of two phases in which the in-hospital discharge education session was followed by 12 weeks of post-discharge education sessions delivered by telephone. The telephone delivery mode was a reliable method to reach patients living in rural counties where internet service was often unreliable and costly. In addition, telephone contact was preferred by many elderly patients because of the complexity of navigating and manipulating other communication platforms32.\n\nDuring Phase I (in-hospital discharge education session), the intervention was delivered in the hospital to capture a “teachable moment” when patients had recently experienced deteriorated health and recognized the need to better manage their heart failure. The intervention was focused on assessing the patient’s intent and readiness to assume a self-management role or encouraging the patient to assume this role (patient activation level 1) and building knowledge, skills and confidence specific to areas of knowledge deficit identified by the patient (patient activation level 2). The teaching materials included: 1) an educational workbook developed by Dr. Darren DeWalt at the Cecil G. Sheps Health Services Research Center for heart failure patients (http://www.nchealthliteracy.org/comm_aids/Heart_Failure_Intervention_eng_v1.pdf), 2) the Agency for Healthcare Research and Quality (AHRQ) guide book for patients discharged from hospitals (http://www.ahrq.gov/qual/goinghomeguide.pdf) and 3) the personal stories about living with heart failure posted on the American Heart Association webpage (http://www.heart.org/HEARTORG/Conditions/HeartFailure/HeartFailureToolsResources/Heart-Failure-Personal-Stories_UCM_306386_Article.jsp). The overall goal was to establish the initial patient-provider relationship and encourage patients to take an active role in self-management. At discharge, each participant from the intervention group received an intervention toolkit containing the heart failure self-management workbooks, an electronic talking pillbox and a digital scale.\n\nDuring Phase II (post-discharge phone education sessions), a total of 11 phone contacts were made with the patient (twice a week for the first 2 weeks, once a week for weeks 3–6, and every other week for weeks 7–12). Each session focused on 1–2 topics and confirmed the patient’s understanding of the knowledge and skills delivered during their hospital stay. The goals for the Phase II intervention were to establish a therapeutic patient-provider relationship and to monitor and reinforce self-management behaviors. Each session started with an informal assessment of the patient’s activation level and the intervention strategies were modified based on the results. The length of the intervention and number of sessions were similar to Wolever’s study that showed effects of a telephone delivered patient activation intervention on the improvement of self-management behaviors in type 2 diabetic patients33.\n\nTable 1 describes the outcome variables specified in the study aims, the study instruments used, their psychometric characteristics, and data collection points.\n\nA two-sided, alpha level of 0.10 was used to identify trends in the group differences on outcomes because this is an exploratory study. Descriptive statistics are reported at baseline, 3 months and 6 months. Chi –square tests were used to evaluate the difference between proportions. We used t-tests to compare the averages of continuous variable between groups.\n\nFor the continuous outcomes (days of self-weighing and taking prescribed medication, physical activity outcomes, and level of BNP and urine Na/Cr), linear mixed model methods are used to compare the groups across the 6-month period, adjusting for baseline levels on the respective outcome. We used ANOVA analysis for repeated measures. Kaplan-Meier method is used for survival analysis to estimate the difference of hospital readmission occurrence between groups.\n\n\nDiscussion\n\nThis study will examine the effectiveness of a rural theory based, advance practice nurse led, activation enhancing intervention on the self-management adherence in heart failure patients residing in rural areas. The findings of this study could fill the gap of knowledge in self-management research in rural heart failure populations.\n\nThe long-term goals of this research are to: 1) test this patient activation intervention in other rural patient populations with multiple chronic conditions; 2) develop a rural based patient activation conceptual framework to guide the design and implementation of interventions to promote life-long self-management adherence in rural and underserved communities; and 3) develop a point of care tool kit for heart failure patients to provide timely feedback about their performance in managing their chronic conditions.\n\nOur expectation is to facilitate adherence to self-management behaviors in heart failure patients following discharge from rural hospitals and decrease complications and hospital readmissions, leading to the reduction of economic burden.", "appendix": "Author contributions\n\n\n\nAs the principle investigator (PI) of the study, Dr. Young contributed the essence part of this manuscript from study’s conception and design; acquisition of data and preparation of the manuscript.\n\nOther authors contributed extensively to the work presented in this paper. Dr Barnason, as co-PI, contributed to the development of conceptual framework, intervention protocol, outcomes and their instrument selection, manuscript revisions, as well as other essential part of manuscript preparation. Dr. Do contributed in analysis of data, literature search, citation and reference editing, table and figure editing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch reported in this publication was supported by the National Institutes Nursing Research of the National Institutes of Health under award number 1R15NR 13769-01A1. The sponsor had no role in conducting the study, preparing and disseminating the study results. The authors are the recipients of the funding provided by the National Institutes Nursing Research of the National Institutes of Health. She has full access to the study data and takes responsibility for their integrity and the accuracy of the data analysis.\n\n\nAcknowledgements\n\nResearch reported in this publication was supported by the National Institutes Nursing Research of the National Institutes of Health under award number 1R15NR 13769-01A1. The sponsor had no role in conducting the study, preparing and disseminating the study results. The authors are the recipients of the funding provided by the National Institutes Nursing Research of the National Institutes of Health. She has full access to the study data and takes responsibility for their integrity and the accuracy of the data analysis.\n\n\nEthical approvals\n\nThe study protocol was approved by the Institutional Review Board at the University of Nebraska Medical Center. The written informed consent was obtained from all study participants prior to enrollment. In analyzing data and reporting, all participants will be anonymized.\n\n\nRegistration number and name of trial registry\n\nClinical Trial Registration Information: ClinicalTrials.gov; https://register.clinicaltrials.gov/ NCT01964053\n\n\nReferences\n\nGiamouzis G, Kalogeropoulos A, Georgiopoulou V, et al.: Hospitalization epidemic in patients with heart failure: Risk factors, risk prediction, knowledge gaps, and future directions. J Card Fail. 2011; 17(1): 54–75. PubMed Abstract \| Publisher Full Text\n\nManning S: Bridging the gap between hospital and home: a new model of care for reducing readmission rates in chronic heart failure. J Cardiovasc Nurs. 2011; 26(5): 368–76. PubMed Abstract \| Publisher Full Text\n\nJoynt KE, Jha AK: Who has higher readmission rates for heart failure, and why? Implications for efforts to improve care using financial incentives. Circ Cardiovasc Qual Outcomes. 2011; 4(1): 53–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGamble JM, Eurich DT, Ezekowitz JA, et al.: Patterns of care and outcomes differ for urban versus rural patients with newly diagnosed heart failure, even in a universal healthcare system. Circ Heart Fail. 2011; 4(3): 317–323. PubMed Abstract \| Publisher Full Text\n\nLee CS, Tkacs NC, Riegel B: The influence of heart failure self-care on health outcomes: hypothetical cardioprotective mechanisms. J Cardiovasc Nurs. 2009; 24(3): 179–87, quiz 188–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiordano A, Scalvini S, Zanelli E, et al.: Multicenter randomised trial on home-based telemanagement to prevent hospital readmission of patients with chronic heart failure. Int J Cardiol. 2009; 131(2): 192–9. 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PubMed Abstract \| Publisher Full Text\n\nHart LG, Larson EH, Lishner DM, et al.: Rural definitions for health policy and research. Am J Public Health. 2005; 95(7): 1149–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiegel B, Moser DK, Anker SD, et al.: State of the science: promoting self-care in persons with heart failure: a scientific statement from the American Heart Association. Circulation. 2009; 120(12): 1141–63. PubMed Abstract \| Publisher Full Text\n\nLee HJ, Winters CA: Testing rural nursing theory: Perceptions and needs of service providers. Online J Rural Nursing and Health Care. 2004; 4(1): 51–63. Reference Source\n\nLong KA, Weinert C: Rural nursing: developing the theory base. Sch Inq Nurs Pract. 1989; 3(2): 113–127. PubMed Abstract\n\nClark AM, Freydberg CN, McAlister FA, et al.: Patient and informal caregivers’ knowledge of heart failure: necessary but insufficient for effective self-care. Eur J Heart Fail. 2009; 11(6): 617–621. 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PubMed Abstract \| Publisher Full Text\n\nJessup M, Abraham WT, Casey DE, et al.: 2009 focused update: ACCF/AHA Guidelines for the Diagnosis and Management of Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the International Society for Heart and Lung Transplantation. Circulation. 2009; 119(14): 1977–2016. PubMed Abstract \| Publisher Full Text\n\nLorig K: Self-management of chronic illness: A model for the future. Generations. 1993; XVII(3): 11–14. Reference Source\n\nHibbard JH, Stockard J, Mahoney ER, et al.: Development of the patient activation measure (PAM): Conceptualizing and measuring activation in patients and consumers. Health Serv Res. 2004; 39(4 Pt 1): 1005–1026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHibbard JH, Mahoney ER, Stock R, et al.: Do increases in patient activation result in improved self-management behaviors? Health Serv Res. 2007; 42(4): 1443–1463. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandura A: Self-efficacy: The exercise of control. New York: WH: Freeman and Company; 1997. Reference Source\n\nvan der Wal MH, Jaarsma T, van Veldhuisen DJ: Non-compliance in patients with heart failure; how can we manage it? Eur J Heart Fail. 2005; 7(1): 5–17. PubMed Abstract \| Publisher Full Text\n\nBorson S, Scanlan J, Brush M, et al.: The mini-cog: a cognitive 'vital signs' measure for dementia screening in multi-lingual elderly. Int J Geriatr Psychiatry. 2000; 15(11): 1021–7. PubMed Abstract \| Publisher Full Text\n\nLi C, Friedman B, Conwell Y, et al.: Validity of the patient health questionnaire 2 (PHQ-2) in identifying major depression in older people. J Am Geriatr Soc. 2007; 55(4): 596–602. PubMed Abstract \| Publisher Full Text\n\nCudney SA, Weinert C, Phillips LD: Telephone technical support: an essential adjunct to a computer intervention for rural chronically ill women. Comput Inform Nurs. 2007; 25(4): 221–227. PubMed Abstract \| Publisher Full Text\n\nWolever RQ, Dreusicke M, Fikkan J, et al.: Integrative health coaching for patients with type 2 diabetes: a randomized clinical trial. Diabetes Educ. 2010; 36(4): 629–639. PubMed Abstract \| Publisher Full Text\n\nWu JR, Chung M, Lennie TA, et al.: Testing the psychometric properties of the medication adherence scale in patients with heart failure. Heart Lung. 2008; 37(5): 334–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTryon WW: The reliability and validity of two ambulatory monitoring actigraphs. Behav Res Methods. 2005; 37(3): 492–7. PubMed Abstract \| Publisher Full Text\n\nArtinian NT, Magnan M, Sloan M, et al.: Self-care behaviors among patients with heart failure. Heart Lung. 2002; 31(3): 161–72. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7376", "date": "19 Jan 2015", "name": "Pascale Salameh", "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\nThis is an excellent work, well justified and adequately addressed. However, some points should be met:Study design: References are needed for interventions. More details are particularly needed for the major PATCH intervention.Figure 2: Why not add the number of patients for each step, since the study is already over?Statistical analysis: Why adjust over baseline value of the measure in question only? Adjustment over other baseline variables may also be needed in case randomization did not succeed to equilibrate them. For readmission, we think that Cox regression model would be more adequate than KM analysis.One more point: In several locations in the manuscript, the authors say it is an exploratory work. Why? What is lacking?", "responses": [ { "c_id": "1309", "date": "07 May 2015", "name": "Lufei Young", "role": "Author Response", "response": "\"Study design: References are needed for interventions. More details are particularly needed for the major PATCH intervention.\"Author Response: Thank you for your suggestion. We added the reference and more detail for our intervention.\"Figure 2: Why not add the number of patients for each step, since the study is already over?\"Author Response: Our study is still collecting data for 3 months and 6 months so we haven’t gotten the data for these steps yet. Moreover, given that our manuscript is a research protocol, we think the data result is not mandatory\"Statistical analysis: Why adjust over baseline value of the measure in question only? Adjustment over other baseline variables may also be needed in case randomization did not succeed to equilibrate them. For readmission, we think that Cox regression model would be more adequate than KM analysis.\" Author Response: Thank you for valuable comment. We will adjust all relevant baseline variables in case randomization did not succeed to attenuate the differences between groups. The aim of the study is to examine the effects of 12-week patient activation enhancing intervention on self-management adherence. Identifying the potential predictors of readmission and assessing how intervention affecting the identified predictors of readmission are not the main aim of the study, which was not designed to examine predictors of readmission. Therefore, Kaplan-Meier method is preferred due to its simplicity. In the future manuscript, we may use Cox regression model with multiple predictors for readmission if our survival analysis need to adjust for the other risk factors.\"One more point: In several locations in the manuscript, the authors say it is an exploratory work. Why? What is lacking?\"Author Response: The purpose of this study is to evaluate the feasibility of the PATCH intervention and gather initial data to support a larger investigation. But in order to avoid misunderstanding, the author will remove the word, “exploratory\" as suggested." } ] }, { "id": "7154", "date": "28 Jan 2015", "name": "Huo Yong", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Young et al. does an excellent job aiming to propose an intervention and evaluation of it for self-management among heart failure patients discharged from rural hospitals. With the prevalence of heart failure, self-management of patients plays a critical role in their improvement of quality of life, relief of exacerbation of symptoms and readmission to hospitals. Patients discharged from rural hospitals especially need education and guidance to improve adherence. The authors tried to solve this needs and described in detail the innovative intervention of a 12-week patient activation (Patient AcTivated Care at Home [PATCH]). And this paper is well-organized in clarifying the rational and design of the study to evaluate feasibility and efficacy of PATCH. Moreover, the authors showed us a vision of implementation of this intervention. The genesis of this study based on solid nursing and patients’ activation theories. And a prospective, two-group, randomized experimental design with three data collection points (baseline, 3 months and 6 months) were chosen to assess the feasibility and efficacy of the intervention. Sample size was carefully estimated. Also, different measures on biomarkers, outcomes and self-management were designed. In order to prevent the possible cognitive or psychotic confounding factors, simple screening tests were required to conduct prior to enrollment. The article was well constructed, the study was well designed, and interpretation was prudent.However, there are some comments and suggestions for authors to consider. First, is it practical for a rural heart failure patient to complete so many questions on measurements of self-management knowledge, self-efficacy, patients’ activity and strategies at one visit? Suggest simplified measurable scales. Second, for measurements of adherence of self-management, only data at 3 months were collected. Will it help to analyze extended effects of the intervention by record data at 6 months?Finally, this work is a good reminder for all the cardiologists to pay more attention to the management and education of heart failure patients. The novel intervention of PATCH might acts as an example to help more and more heart failure patients.", "responses": [ { "c_id": "1310", "date": "07 May 2015", "name": "Lufei Young", "role": "Author Response", "response": "\"First, is it practical for a rural heart failure patient to complete so many questions on measurements of self-management knowledge, self-efficacy, patients’ activity and strategies at one visit? Suggest simplified measurable scales.\"Author Response: We greatly appreciate your suggestion and will use objective measures for activity level, adherence and knowledge in the larger scale study.\"Second, for measurements of adherence of self-management, only data at 3 months were collected. Will it help to analyze extended effects of the intervention by record data at 6 months?\"Author Response: This is a feasibility study aimed to examine the magnitude of intervention on variable of interest. To determine the mechanism of intervention, the group comparison was made at 3 months when the 12-week intervention was just completed so the maximal differences would be expected between intervention and control groups in self-management knowledge, self-efficacy for self-management, patient activation, and self-management strategies. Another reason to assess 3-month only is to reduce subject burden. The population studied is characterized as elderly living with heart failure and other debilitating chronic conditions. Fatigue and functioning declination are primary symptoms in this population.\"Finally, this work is a good reminder for all the cardiologists to pay more attention to the management and education of heart failure patients. The novel intervention of PATCH might acts as an example to help more and more heart failure patients.\"Author Response: We really appreciate your recognition of the practice implication of this study. Thank you so much." } ] }, { "id": "7608", "date": "18 Mar 2015", "name": "Kevin T. Fuji", "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\nThis is a well-written and well thought-out protocol for an exploratory (or pilot) approach to the use of the PATCH intervention in critical access hospitals. There are just a few additional points of clarification that would further strengthen the paper. During Phase II (post-discharge phone education sessions), what happens if patients cannot be reached? How many attempts are made to reach each patient? At what point of non-contact is the patient excluded from the study?The authors might consider adding a third column to Table 1 – “Data Analysis” which could describe the data analysis procedures used to analyze each variable. In Figure 1, where is the description for “Level 2 Building Knowledge, Skills & Confidence” that matches the other Levels in the figure?", "responses": [ { "c_id": "1311", "date": "07 May 2015", "name": "Lufei Young", "role": "Author Response", "response": "\"During Phase II (post-discharge phone education sessions), what happens if patients cannot be reached? How many attempts are made to reach each patient? At what point of non-contact is the patient excluded from the study?\"Author Response: During Phase II, we tried to reach patients 3 times. If we fail to reach patients after 3 attempts, the patient will be excluded from the study. Fortunately, we haven’t had to exclude any patient during Phase II \"The authors might consider adding a third column to Table 1 – “Data Analysis” which could describe the data analysis procedures used to analyze each variable.\"Author Response: Author added analysis procedures will be used for each variable as suggested. \"In Figure 1, where is the description for “Level 2 Building Knowledge, Skills & Confidence” that matches the other Levels in the figure?\"Author Response: We added the description for level 2 of patient activity level in the figure." } ] } ]	1	https://f1000research.com/articles/3-317
https://f1000research.com/articles/4-108/v1	06 May 15	{ "type": "Clinical Practice Article", "title": "Early vascular unclamping reduces warm ischaemia time in robot-assisted laparoscopic partial nephrectomy", "authors": [ "Kevin Lah", "Devang Desai", "Charles Chabert", "Christian Gericke", "Troy Gianduzzo", "Kevin Lah", "Devang Desai", "Charles Chabert", "Christian Gericke" ], "abstract": "Introduction: The aim of this study was to assess the outcomes of early vascular release in robot-assisted laparoscopic partial nephrectomy (RAPN) to reduce warm ischaemia time (WIT) and minimise renal dysfunction. RAPN is increasingly utilised in the management of small renal masses. To this end it is imperative that WIT is kept to a minimum to maintain renal function.Methods: RAPN was performed via a four-arm robotic transperitoneal approach. The renal artery and vein were individually clamped with robotic vascular bulldog clamps to allow cold scissor excision of the tumour. The cut surface was then sutured with one or two running 3-0 V-LocTM sutures, following which the vascular clamps were released. Specific bleeding vessels were then selectively oversewn and the collecting system repaired. Renorrhaphy was then completed using a running horizontal mattress 0-0 V-LocTM suture.Results: A total of 16 patients underwent RAPN with a median WIT of 15 minutes (range: 8-25), operative time 230 minutes (range: 180-280) and blood loss of 100 mL (range: 50-1000). There were no transfusions, secondary haemorrhages or urine leaks. There was one focal positive margin in a central 5.5 cm pT3a renal cell carcinomas (RCC). Long-term estimated glomerular filtration rate (eGFR) was not significantly different to pre-operative values.Conclusion: In this patient series, early vascular release effectively minimised WIT and maintained renal function without compromising perioperative safety.", "keywords": [ "Robot-assisted laparoscopy", "Partial nephrectomy", "Renal function", "Renal Mass" ], "content": "Introduction\n\nThe incidence of diagnoses of renal masses is on the rise with increased use of abdominal imaging1. Nephron-sparing surgery (NSS), such as robotic-assisted laparoscopic partial nephrectomy (RAPN) is increasingly recommended to preserve long-term renal function2,3. This is particularly important given the increasing prevalence of diabetes mellitus and hypertension in the community4,5. The American Urological Association recommends NSS as the treatment of choice for most T1 renal masses6. NSS has been shown to reduce the long-term risks of renal dysfunction, and cardiovascular morbidity, as well as overall mortality when compared to radical nephrectomy7,8. The duration of warm ischaemia time (WIT) during partial nephrectomy (PN) is critically important in achieving these outcomes. Thompson and colleagues9 found that a WIT of 25 minutes or longer was significantly associated with new onset of stage IV chronic kidney disease and concluded that “every minute of ischaemia counts” in the preservation of renal function10. Ongoing refinement of the technique over the last decade to minimise ischaemia time has been the challenge in mastering PN.\n\nLaparoscopic PN (LPN) is a not a procedure for any novice laparoscopic urologist11. The difficulties in tumour excision and reconstruction of the collecting system and renal cortex can be improved by the use of robot-assisted laparoscopic instruments. This allows a greater degree of motion and dexterity which may allow the surgeon to reduce WIT. Other techniques to decrease WIT have included the use of intra-arterial hypothermic perfusion12, intra-corporeal placement of ice slush13 and specific refinements in clamping renal artery and vein, including the early vascular release technique14. This study presents the initial experience of early vascular release as a means to minimise WIT during RAPN.\n\n\nMethods\n\nWith UnitingCare Health Ethics Committee approval, nr. 2013.25.96, the outcomes of RAPN of two oncology fellowship-trained surgeons (TG and CC) were analysed. Data from 16 consecutive patients was prospectively collected between July 2011 and September 2013.\n\nPre-operative data included: age, gender, body mass index, American Society of Anesthesiologists Physical status classification (ASA), side of the mass, pre-operative renal function (estimated glomerular filtration rate), tumour size on imaging and relevant medical history (i.e. previous abdominal surgery). Intraoperative data included: operative duration, console time, WIT, estimated blood loss and intraoperative complications. Operative duration was defined as skin-to-skin time, and console time defined as the time during which the robotic interface was used during the procedure. Perioperative data included: complications, day 1 post-operative renal function and length of hospital stay. Post-operative data included renal function at 6 months, and tumour histology, stage, margin and size. Pathological analysis was performed by a single uropathologist experienced in partial nephrectomy assessment.\n\nRAPN was performed via a four-arm transperitoneal approach in a modified lateral position with a 30° tilt and the table at 20–30° contralateral tilt. An ipsilateral ureteric catheter is routinely used to allow retrograde instillation of methylene blue to check collecting system integrity. The kidney was mobilized in the standard fashion whereby the colon was reflected medially, the ureter elevated with the fourth arm and the duodenum kocherised for right-sided tumours. The renal vessels were then isolated and looped with vessel loops. Gerota’s fascia was then incised and the kidney defatted in order to localise the mass. Care was taken to leave perinephric fat directly on the mass. The renal mass was then assessed with intraoperative ultrasound to ascertain its depth and to plan the margin of incision. Both renal artery and vein were clamped using robotic bulldog clamps and the tumour excised with cold scissor dissection. Renorrhaphy was performed using one or two 2-0 V-LocTM absorbable polyglyconate knotless sutures (Covidien Inc.), following which the bulldog clamps were released. This early release allowed specific bleeding vessels to be immediately positively identified and specifically suture ligated with figure-of-8, 3-0 vicryl sutures. Collecting system defects were specifically repaired and the integrity assessed with retrograde instillation of methylene blue. Cortical reconstruction was performed using a single, running horizontal mattress 12 inch 0-0 V-LocTM suture. Floseal® (Baxter Corp.) was applied to the closed defect.\n\nNumerical data was summarised using median and range (Microsoft Excel), and analysed using the Wilcoxon signed-rank test (www.socscistatistics.com) where appropriate. A P < 0.05 was considered to indicate statistical significance. Demographics and categorical data were summarised in table format.\n\n\nResults\n\nA total of 16 patients underwent RAPN. There were ten males and six females with a median age of 66.5 (range 48 to 80 years). There were nine left sided lesions and seven right sided lesions. The majority of the masses were exophytic with a median size of 2.65 cm. Table 1 shows the demographics.\n\nASA: American Society of Anesthesiologists Physical status classification\n\nPerioperative data are recorded in Table 2. Day 1 eGFR was marginally reduced compared to pre-operative levels (p < 0.01). However this was not clinically significant and by 6 months eGFR had returned to baseline (p = 0.11). The median operative time was 230 minutes with 192.5 minutes of console time. Median WIT was 15 minutes and median blood loss was 100 mL. The median hospital length of stay was 2 days. There were no transfusions, urine leaks, or post-operative haemorrhage. Histopathology demonstrated nine clear cell renal cell carcinomas (RCC), three papillary RCC, three angiomyolipoma and three eosinophilic variant clear cell RCC (Table 3). Tumour abutted the resection margin in one case of a central 5.5 cm mass which demonstrated renal sinus and vascular invasion on frozen section. A completion nephrectomy was then immediately performed given these high-risk features and also given that the contra-lateral kidney and pre-operative renal function were normal. There was no residual tumour in the remaining kidney. One patient had a grade 1 Clavien-Dindo classification who had self-resolved neuralgic pain. No cases were converted to an open operation. Our study is compared with international data in Table 4.\n\neGFR: estimated glomerular filtration rate. [p value] compared with pre-op.\n\nRCC: Renal cell carcinoma; AML: Angiomyolipoma\n\nWIT: Warm ischaemia time\n\n\nDiscussion\n\nRAPN is an effective surgical alternative in NSS in which the ultimate goal is to achieve the “trifecta” of a negative cancer margin, minimal decrease in renal function and an absence of complications14. The use of robotic technology can assist in achieving these outcomes and in particular, minimise renal dysfunction by reducing WIT. Laparoscopic partial nephrectomy not only has a steep learning curve to achieve acceptable WIT but also requires skills that are challenged by its technical difficulties, including the use of instruments that have limited degrees-of-freedom. The robot application in PN allows a three-dimensional vision with magnification and instrument arms that are versatile with its EndoWrist® technology, providing increased angle and maneuverability for tumour excision and repair of the renal defect15.\n\nVarious methods have been employed in the past to lessen the ischaemic injury. Intra-arterial hypothermic perfusion was used in early series of LPN12 as was intra-corporeal placement of ice slush13. However endovascular hypothermia came with the added risks of placing an arterial catheter and administration of extra fluid for patients with poor cardiopulmonary performance status. Intra-corporeal cooling reduced the working space and exposure of the hilum.\n\nThere has been an ongoing refinement of clamping techniques to reduce WIT, progressing from conventional clamping of both renal artery and vein10 to early unclamping14,16,17. With the introduction of early unclamping, where the clamp is released once a central medullary running suture is placed and the rest of the kidney repaired with the revascularised perfused kidney, Nguyen and Gill16 were able to decrease WIT in LPN from 31.3 minutes to 13.9 minutes. The overall complications including estimated blood loss were not significantly different compared with the standard clamping technique. Furthermore, surgeons have pushed the boundaries of minimal ischaemia by selectively micro-clamping arteries supplying the tumour18,19 and in one study, without clamping and aiming for “zero ischaemia”20. This clamp-less group of eight patients had significantly reduced operative time but an increased blood loss. The transfusion and renal dysfunction were similar to the clamped group.\n\nThe technique of early vascular release has been translated from LPN to RAPN17. San Francisco et al. described 12 patients who underwent RAPN with early vascular release (Table 4)17. In that series, the median WIT was 16 minutes, median operative time 227 minutes, median estimated blood loss 150 mL and length of stay 2 days. These results are comparable to the present study (Table 4).\n\nKaouk and colleagues21 reported their single-institution’s 252 RAPNs with early unclamping technique when deemed appropriate. Their study confirmed that the longer the duration of WIT, the greater the decrease in renal dysfunction at 1 month. Kucharczyk and colleagues reported an Australian series of 50 consecutive patients undergoing RAPN by a single surgeon22. The mean WIT was 17.8 minutes, operative time was 151 minutes and estimated blood loss was 171.1 ml. They achieved no positive malignant surgical margins and a clinically stable renal function post-operatively.\n\nIn this series, the outcomes of early vascular release were comparable to the literature with short WIT and no morbidity from intraoperative or postoperative haemorrhage or urine leak. The limitations of this study include a small sample size, and the lack of a randomised comparison to other techniques.\n\nIn conclusion, early vascular release following tumour excision during RAPN resulted in short WIT with minimal morbidity and preserved renal function.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data, 10.5256/f1000research.6276.d4676923\n\n\nConsent\n\nAll patients gave consent for collection of data for research purposes.", "appendix": "Author contributions\n\n\n\nKL and DD prepared the first draft of the manuscript and analysed the collected data from TG and CC. TG, CC and CG contributed to the design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGill IS, Aron M, Gervais DA, et al.: Clinical practice. Small renal mass. N Eng J Med. 2010; 362(7): 624–34. PubMed Abstract \| Publisher Full Text\n\nLjungberg B, Cowan NC, Hanbury DC, et al.: EAU guidelines on renal cell carcinoma: the 2010 update. Eur Urol. 2010; 58(3): 398–406. PubMed Abstract \| Publisher Full Text\n\nUzzo RG, Novick AC: Nephron sparing surgery for renal tumors: indications, techniques and outcomes. J Urol. 2001; 166(1): 6–18. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization. World Health Statistics 2012. Reference Source\n\nWild S, Roglic G, Green A, et al.: Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diab Care. 2004; 27(5): 1047–1053. PubMed Abstract \| Publisher Full Text\n\nNovick AC, Campbell SC, Belldegrun A, et al.: Guideline for management of the clinical stage 1 renal mass. Am Urological Assoc. 2009. Reference Source\n\nMiyamoto K, Inoue S, Kajiwara M, et al.: Comparison of renal function after partial nephrectomy and radical nephrectomy for renal cell carcinoma. Urol Int. 2012; 89(2): 227–232. PubMed Abstract \| Publisher Full Text\n\nWeight CJ, Larson BT, Fergany AF, et al.: Nephrectomy induced chronic renal insufficiency is associated with increased risk of cardiovascular death and death from any cause in patients with localized cT1b renal masses. J Urol. 2010; 183(4): 1317–1323. PubMed Abstract \| Publisher Full Text\n\nThompson RH, Lane BR, Lohse CM, et al.: Renal function after partial nephrectomy: effect of warm ischemia relative to quantity and quality of preserved kidney. Urology. 2012; 79(2): 356–360. PubMed Abstract \| Publisher Full Text\n\nThompson RH, Lane BR, Lohse CM, et al.: Every minute counts when the renal hilum is clamped during partial nephrectomy. Eur Urol. 2010; 58(3): 340–345. PubMed Abstract \| Publisher Full Text\n\nRashid P, Goad J, Aron M, et al.: Laparoscopic partial nephrectomy: integration of an advanced laparoscopic technique. ANZ J Surg. 2008; 78(6): 471–475. PubMed Abstract \| Publisher Full Text\n\nJanetschek G, Abdelmaksoud A, Bagheri F, et al.: Laparoscopic partial nephrectomy in cold ischemia: renal artery perfusion. J Urol. 2004; 171(1): 68–71. PubMed Abstract \| Publisher Full Text\n\nGill IS, Abreu SC, Desai MM, et al.: Laparoscopic ice slush renal hypothermia for partial nephrectomy: the initial experience. J Urol. 2003; 170(1): 52–56. PubMed Abstract \| Publisher Full Text\n\nHung AJ, Cai J, Simmons MN, et al.: “Trifecta” in partial nephrectomy. J Urol. 2013; 189(1): 36–42. PubMed Abstract \| Publisher Full Text\n\nFicarra V, Novara G, Volpe A, et al.: Robot-assisted vs traditional laparoscopic partial nephrectomy: the time for meta-analysis has not yet arrived. BJU Int. 2013; 112(4): E334–E336. PubMed Abstract \| Publisher Full Text\n\nNguyen MM, Gill IS: Halving ischemia time during laparoscopic partial nephrectomy. J Urol. 2008; 179(2): 627–632; discussion 632. PubMed Abstract \| Publisher Full Text\n\nSan Francisco IF, Sweeney MC, Wagner AA, et al.: Robot-assisted partial nephrectomy: early unclamping technique. J Endourol. 2011; 25(2): 305–308. PubMed Abstract \| Publisher Full Text\n\nGill IS, Eisenberg MS, Aron M, et al.: “Zero ischemia” partial nephrectomy: novel laparoscopic and robotic technique. Eur Urol. 2011; 59(1): 128–134. PubMed Abstract \| Publisher Full Text\n\nAbreu ALC, Gill IS, Desai MM: Zero-ischaemia robotic partial nephrectomy (RPN) for hilar tumours. BJU Int. 2011; 108(6 Pt 2): 948–954. PubMed Abstract \| Publisher Full Text\n\nWhite WM, Goel RK, Haber G, et al.: Robotic partial nephrectomy without renal hilar occlusion. BJU Int. 2010; 105(11): 1580–1584. PubMed Abstract \| Publisher Full Text\n\nKaouk JH, Hillyer SP, Autorino R, et al.: 252 Robotic partial nephrectomies: Evolving renorrhaphy technique and surgical outcomes at a single institution. Urology. 2011; 78(6): 1338–1344. PubMed Abstract \| Publisher Full Text\n\nKucharczyk JR, Basto M, Landau A, et al.: Early experience and operative technique of robotic-assisted partial nephrectomy. ANZ J Surg. 2014. PubMed Abstract \| Publisher Full Text\n\nLah K, Desai D, Chabert C, et al.: Dataset 1 in: Early vascular unclamping reduces warm ischaemia time in robot-assisted laparoscopic partial nephrectomy. F1000Research. 2015. Data Source" }	[ { "id": "8579", "date": "12 May 2015", "name": "Prem Rashid", "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\nThis paper on the early experience of robotic LPN highlights the complexity of the procedure and the possible outcomes with good background preparation. The results are helpful to guide those embarking on LPN or robotic assisted LPN. It is well written. The methodology and conclusions are sound.", "responses": [] }, { "id": "8577", "date": "13 May 2015", "name": "Mark William Louie-Johnsun", "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\nThis article highlights the early solid results of two oncology fellowship-trained surgeons performing robot assisted laparoscopic partial nephrectomy. The short WIT with minimal perioperative complications highlights this complex operation can be performed expertly in well trained surgeons even in their early experience.", "responses": [] } ]	1	https://f1000research.com/articles/4-108
https://f1000research.com/articles/4-107/v1	06 May 15	{ "type": "Review", "title": "Photoperiodic and circadian bifurcation theories of depression and mania", "authors": [ "Daniel F. Kripke", "Jeffrey A. Elliott", "David K. Welsh", "Shawn D. Youngstedt", "Jeffrey A. Elliott", "David K. Welsh", "Shawn D. Youngstedt" ], "abstract": "Seasonal effects on mood have been observed throughout much of human history. Seasonal changes in animals and plants are largely mediated through the changing photoperiod (i.e., the photophase or duration of daylight). We review that in mammals, daylight specifically regulates SCN (suprachiasmatic nucleus) circadian organization and its control of melatonin secretion. The timing of melatonin secretion interacts with gene transcription in the pituitary pars tuberalis to modulate production of TSH (thyrotropin), hypothalamic T3 (triiodothyronine), and tuberalin peptides which modulate pituitary production of regulatory gonadotropins and other hormones. Pituitary hormones largely mediate seasonal physiologic and behavioral variations. As a result of long winter nights or inadequate illumination, we propose that delayed morning offset of nocturnal melatonin secretion, suppressing pars tuberalis function, could be the main cause for winter depression and even cause depressions at other times of year. Irregularities of circadian sleep timing and thyroid homeostasis contribute to depression. Bright light and sleep restriction are antidepressant and conversely, sometimes trigger mania. We propose that internal desynchronization or bifurcation of SCN circadian rhythms may underlie rapid-cycling manic-depressive disorders and perhaps most mania. Much further research will be needed to add substance to these theories.", "keywords": [ "depression", "mania", "bipolar disorder", "circadian rhythm", "suprachiasmatic nucleus (SCN)", "photoperiod", "triiodothyronine (T3)", "thyrotropin (TSH)" ], "content": "Review and theoretical interpretation\n\nIn this presentation, we review the seasonality of mood disorders and the photoperiodic control of seasonality among mammals, in order to present new theories of the causes of depression and mania. The seasonal timing of daily light exposures reorganizes cellular circadian clocks in the bilateral suprachiasmatic nuclei (SCN) of the hypothalamus above the optic chiasm to regulate the evening rise and duration of melatonin secretion1–4. In mammals, an exquisite mechanism in the pars tuberalis (PT) interprets daylength (photoperiod) from the duration of nocturnal melatonin secretion and particularly from the early morning termination time of secretion. Melatonin offset influences thyrotropin (TSH) and tuberalin hormone synthesis by the PT5,6. These PT products then influence pituitary hormones and induce deiodinase 2 (DIO2), increasing hypothalamic triiodothyronine (T3), which maintains seasonal gonadal fertility. We propose that this same mechanism may be a key controller of mood. Inadequate hypothalamic T3 may cause depression, whereas excessive hypothalamic T3 may mediate mania. Peculiar aspects of the induction of mania by bright light or sleep restriction suggest a theory that mania may be promoted by bifurcation of the circadian phasing of neuronal firing in two distinct populations of SCN neurons. Finally, we will propose particular inquiries where more research is needed to explore and validate these theories.\n\n\nMood and seasonality\n\nTo understand mood disorders, we must hope to understand seasonality. Associations of mood changes with the seasons have been observed since antiquity7. Likewise, seasonality in suicide was recognized by the ancients and has been studied with modern scientific methods for over a century8–10. In the short dark days of winter, many people, especially bipolars (people who have experienced mania or hypomania)11, experience a tendency towards low mood, usually mild. Winter, however, may not be the season for the most serious manifestations of depression9. The seasonal peaks for suicide and for hospital admissions for depression are in April or May in many Northern Hemisphere data sets. Additionally, peaks in mania are often observed in May or June, and both depression and mania sometimes express secondary peaks in the fall7,11–13.\n\nIn small mammals in temperate climates, a quiescent interval or even hibernation in winter may be followed by a spring mating season sometimes highlighted by increased venturesome wandering or migrations, increased aggressiveness, rutting behaviors among males, and ovulation and mating receptivity among females. Perhaps these winter and spring behaviors resemble some aspects of depression and mania, respectively. According to Wehr et al., seasonality in primates is quite variable and seems to be influenced by complexities of food availability, latitude, and body size: since in tropical and equatorial environments, the rainy season may be more influential than temperature or day length14. Many human groups have a peak in conception close to the spring equinox, sometimes with a secondary peak in the fall or at Christmas15,16. Perhaps human populations have become variable partly because human groups have moved to new latitudes and climates without much time for evolutionary adaptation17. Moreover, seasonal reproductive trends have tended to flatten as modern lighting and heating became available15. Because of the wide range of environmental adaptations and recent migrations, human populations may have diversities in seasonal behaviors and mood more complex than common mammalian models.\n\n\nPhotoperiodic and molecular control of seasonal responses through melatonin\n\nIn mammalian species as diverse as hamsters and sheep, seasonal behaviors are largely regulated by the photoperiod: the interval of daylight within each 24 hours, also called the photophase2,18. Gross locomotor activity varies with photoperiod: for example, among nocturnal rodents, activity is compressed into the short nights of summer, but expands in duration as nights grow longer in winter. The photoperiod regulates seasonal responses specifically through SCN control of nocturnal pineal melatonin secretion. Melatonin generally increases after dusk and terminates by dawn, under control of the SCN circadian timing system as regulated by day length4. It has been shown that in mammals, pineal melatonin secretion is under control of a multisynaptic pathway arising primarily from the dorsomedial SCN AVP cells. The interval of melatonin secretion is short during the short nights of summer and longer during the long nights of winter in most animals, whether nocturnal or diurnal. Melatonin has been considered a neuroendocrine signal of the night or scotophase (the dark inverse of the photophase). There is evidence that nocturnal melatonin secretion feeds back on SCN neurons to modulate certain components of the circadian molecular clockwork19. The interval of nightly locomotor activity among nocturnal rodents and the interval of sleep propensity among diurnal adult humans both correspond roughly, but not exactly, to the interval of melatonin secretion by the pineal. A wealth of studies suggest that it is the duration of the photoperiod-regulated nocturnal melatonin secretion that controls seasonal increases in gonadal size and the adaptive timing of mammalian breeding activities20.\n\nWhether mammals are nocturnal or diurnal in activity, most SCN neuronal firing occurs during the day. High-level multiunit firing expands in duration in long days. Thus, in diurnal animals, multiunit activity and waking physical activity tend to occur together, whereas among nocturnal animals they are inverse.\n\nA useful hypothesis has been that two coupled circadian oscillators interact to regulate nocturnal activity, e.g., in rodents: an evening oscillator (E) has been linked to the burst of locomotor activity beginning about dusk and a morning oscillator (M) may be primarily responsible for timing the cessation of locomotor activity before dawn20. In theory, the E and M oscillators spread apart during long nights, allowing an increased span of nocturnal locomotor activity in winter, whereas in summer, the long hours of daylight and short nights squeeze the interval between E and M, resulting in a shorter duration of nocturnal locomotor activity. Likewise, the evening increase and morning decline of melatonin secretion seem to be influenced by the separate timing of evening and morning oscillators, i.e. their phase-timing relationships20. The expansion of the nocturnal interval between E and M varies inversely with the compression of the diurnal interval of rapid SCN neuronal firing, and vice versa. Perhaps nocturnal locomotor activity and melatonin secretion are inhibited by daytime SCN neuronal firing.\n\nRecent work in nocturnal rodents has revealed that these E and M oscillators are embodied in groups of coupled neurons located in the SCN3,21,22. The daylight photoperiodic input, sensed mainly by intrinsically blue-light-sensitive retinal ganglion cells23, is transmitted by their axons to a ventrolateral and largely rostral “core” region of each SCN, where a key neurotransmitter is vasoactive intestinal polypeptide (VIP)24. The core neurons send VIP axons to surrounding dorsomedial “shell” regions (mainly caudal), entraining the shell neurons, which then transmit arginine vasopressin (AVP) signals to other regions such as the hypothalamic paraventricular nucleus, as well as feeding back on the core25. It might seem plausible that the core would encompass the evening oscillator and the shell the morning oscillator26, yet recent studies suggest a more complex tri-dimensional distribution of cell groups27. From another perspective, the most caudal SCN cells seemingly correspond to the morning oscillator28, and some of the rostral cells correspond to the evening oscillator21,29, but there is at least one additional cell group in the rostral SCN region which may be more closely linked to shell than core21,30,31. Under the influence of long photoperiods (short nights), the caudal morning oscillator tends to phase advance several hours, drawing closer to the rostral evening oscillator32, as the interval of behavioral activity of nocturnal rodents is compressed, but inversely, daytime intense SCN neuronal firing expands in duration. There seems to be greater spatial and neuropharmacologic complexity than the rostral-caudal or core-shell dichotomies suggest, and there is insufficient evidence to firmly link particular SCN neuronal populations to E and M or particular features of motor activity and melatonin secretion. Also, there may be differences among species. Unfortunately, many of the studies of SCN responses to photoperiod have been conducted in laboratory-bred mice that do not synthesize melatonin (and therefore, lack melatonin feedback upon SCN neuronal phases).\n\nDuring the night, melatonin can be suppressed acutely by light. Rather dim light will suppress melatonin among nocturnal rodents, and brighter (but still dim) light will likewise shift circadian phases in nocturnal rodents33,34. Much brighter light, brighter than most contemporary indoor illumination, is usually required to suppress melatonin in humans35, and even brighter light resembling sunlight or bright cloud cover is required for maximally strong resetting of the human clock through circadian phase shifting36. However, there are exceptions to the rule that bright light is required for melatonin suppression and phase shifting in humans, perhaps related to nocturnal dark adaptation of the eyes37–39. Effects of dawn simulation during sleep (with closed eyes) may imply that the circadian system is especially sensitive to light towards the latter half of nocturnal sleep when the greatest retinal dark adaptation might have occured37. Moreover, the phase-shifting sensitivity of the hamster phase-response curve is modified by long and short photoperiods40, and the same might be true in humans.\n\n\nMelatonin regulation of molecular biology in the pars tuberalis region\n\nThe duration of nocturnal melatonin secretion regulates seasonal gonadal growth and breeding through hypothalamic regulation of the most active thyroid hormone, T3 (triiodothyronine); as will be discussed, T3 is likewise crucial to mood. The importance of T3 in photoperiodic control was recognized in Japanese quail41 and then confirmed in mammalian species. In mammals, melatonin binds to a dense supply of melatonin receptors in the pars tuberalis (PT) in the rostral anterior pituitary just below the hypothalamic median eminence42. A primary effect of melatonin in PT is control of the transcription factor EYA3 (Figure 1). In the summer when the interval of melatonin secretion ends early, EYA3 is strongly transcribed in PT in the early morning about 12 hours after dark, a time when circulating melatonin is low43. While TEF binds to a D-Box motif on the TSHB promoter in PT, SIX1 binds to an adjacent So1 site on the promoter, and EYA3 binds either to SIX1 or to a nearby site on the TSHB promoter5,6. Together, EYA3, SIX1, and TEF combine to promote pars tuberalis transcription of the TSHB gene. TSHB transcription leads to translation of the thyroid stimulating hormone beta chain, which hybridizes with the TSHA polypeptide to form the active dimer, thyroid stimulating hormone (TSH). PT TSH then passes retrograde into the 3rd cerebral ventricle CSF5,6,18,44. Very high local concentrations of TSH in the 3rd ventricle bind to TSH receptors on ependymal tanycytes lining the ventricular surface, which in turn promotes transcription of a deiodinase (DIO2) that converts T4 to T3, especially in the tanycytes. This produces high concentrations of T3 in the third ventricle and adjacent hypothalamic region, close to TRH (thyrotropin releasing hormone) cells which homeostatically respond to T3 feedback41,43,45. Since T3 passes into the brain poorly, most brain T3 is produced within the brain and substantial portions by these 3rd ventricle tanycytes46. PT production of TSH is not influenced by homeostatic feedback from TRH and T3, and unique PT glycosylation of TSH prevents the small amounts of TSH produced by PT from directly influencing the thyroid47.\n\nA, Depicted is some of the circadian gene network that times transcription through pathways leading to E-box activation (green) or which deactivates transcription and E-box promoter action (red) in a night owl or depressed person. B, The yellow line illustrates normal melatonin secretion commencing shortly before the preferred nocturnal sleep time and terminating about the time of awakening near dawn, so that preferred sleep times and sleepiness normally correspond. The yellow dotted line illustrates how in DSP, melatonin secretion offset may become delayed, with correspondingly delayed sleep propensity. C, The gene EYA3 reaches a sharp peak in pars tuberalis transcription about 12 hours after darkness onset (solid orange line), but if melatonin is still elevated (due to long nights of winter, long time in bed, or DSP), the EYA3 peak is largely suppressed (dashed orange line). Bright lights (light bulb and sun symbols) conversely suppress and advance melatonin offset (red arrows), disinhibiting EYA3. D, After short nights in summer, EYA3, SIX1 and TEF coactivate near a D-box on the TSHB promoter. TSHB hybridizes with TSHA, releasing active TSH into 3rd ventricle CSF6,44. E, TSH circulates retrograde to promote DIO2 which converts T4 to T3. F, T3 promotes synthesis and release of gonadotropin hormones, implementing summer reproduction and good mood. Revised with permission from Kripke et al., Psychiat. Invest., 201499.\n\nT3 promotes the secretion of GnRH into portal blood, leading to increased pituitary release of LH and FSH, which augment testosterone, estrogens, and progesterone, thus promoting seasonal reproduction43,48. Melatonin is thought to suppress prolactin secretion, either through direct effects on the PT transmitted to the anterior pituitary through PT tuberalin peptides or through actions of CSF T3 upon hypothalamic TRH and dopamine secretion49, which then influence pituitary prolactin secretion42,50–52. Interestingly, the PT tuberalins derived from the gene TAC1 may promote pituitary ACTH, GH, TSH, LH and FSH secretion, as well as influencing prolactin52.\n\nDuring the long nights of winter, melatonin may remain elevated during those early morning hours when maximal EYA3 transcription is scheduled. Because in winter, elevated morning melatonin inhibits PT EYA3 and TAC1 transcription, PT TSH production is inhibited, thus reducing expression of DIO2, production of T3 by tanycytes, and ultimately inhibiting gonadal maintenance in winter. Note that in humans, reduced hypothalamic T3 would lead to loss of libido, a major component of depression. Conversely, increased libido is a typical component of mania.\n\nAlthough sheep are autumn short-day breeders, much of the melatonin control of T3 among rodents and sheep is similar5,6,18,44,53. Almost all mammals, both long-day and short-day breeders, produce more prolactin in the summer. Humans may be an unexplained exception with greater blood prolactin in winter54, but results from different genders and populations seem inconsistent. Wehr found that long scotophases were associated with longer nocturnal elevations of prolactin among both men and women, suggesting that human prolactin may be higher when melatonin is higher55,56. One study of afternoon prolactin found slightly higher prolactin during winter in premenopausal females, but patients with winter depression (either unipolar or bipolar) had much lower prolactin than controls in both summer and winter57. Any causal role for prolactin in mood swings seems uncertain.\n\n\nMore studies relating thyroid homeostasis to mood\n\nDuring the middle of the 20th century, Richter demonstrated that lesions of the rat pituitary-thyroid axis produced periodic cycles of activity resembling rapid mood cycles. Richter pointed out the relationship of thyroid impairments to the manic-depressive mood cycles that had been described in early clinical studies58,59. Despite this hint, generations of psychiatrists studying thyroid effects on mood may have been frustrated or misled by the poor correlations between peripheral blood indices of thyroid function and mood, which may result from poor correlations between the T3 concentrations in the blood versus the T3 concentrations in the basal hypothalamus that might be the crucial determinant of mood symptoms. There is a variety of evidence for subclinical hypothyroidism in unipolar and bipolar depression60, and the antidepressant response to sleep deprivation is related to the TSH response and to variations in the activity of circulating TSH that are attributable to the degree of sialylation61.\n\nNevertheless, much evidence has accumulated that functional brain hypothyroidism is associated with depression and with bipolar and rapid-cycling manic-depressive symptoms46,59,62–66. Because functional brain hypothyroidism may not be indicated by standard blood thyroid indices reflecting thyroid regulation outside the brain, seemingly supraphysiologic oral doses of thyroxine may be required to benefit mood67. It may be necessary to add T3 to T4 supplementation67. Besides mood affects, elevated hypothalamic T3 increases appetite, which might help counter the loss of appetite associated with depression68. Moreover, there are now several genetic polymorphisms in thyroid-regulation genes reported to influence depression and mania, perhaps through influences on hypothalamic T3 regulation69. A DIO2 polymorphism is associated with the lifetime incidence of major depression70. Two other DIO2 polymorphisms have been related to poor mental health71. There are several other polymorphisms known to influence thyroid metabolism, though their possible role in hypothalamic T3 regulation seems inadequately explored72,73. A TEF promoter SNP has been reported to be associated with depression74. Also, humans have a very common single nucleotide polymorphism labeled rs1321108 in the So1 binding site of the TSHB promoter, altering it and possibly influencing the promoter functions of EYA3 and SIX1. Contemporary genome-wide association studies (GWAS) have not confirmed that these polymorphisms (SNPs) are associated with major depression or bipolar disorder. However, in bipolar disorder and separately in major depressions, whole genome expression studies have observed reduced DIO2 RNA expression in a frontal basal brain area (P=0.008), not confirmed by overall meta-analysis; DIO3 was increased in the same study and in the same area (P=0.005) in bipolars but not in meta-analysis whereas DIO3 was decreased in major depression (P<0.02); the major T3 receptor in the hypothalamus, THRA, was reduced in that area (P=2.32E-06) and in an adjacent site in major depression and increased among bipolars in two assays but not in meta-analysis; TEF is increased in frontal cortex of bipolars by meta-analysis (P<0.05), especially in fronto-basal cortex, but not in MDD75,76. In summary, there are now scattered clinical and genetic findings indicating that photoperiodic PT control of hypothalamic T3 levels might interact with other aspects of thyroid regulation to contribute to causal pathways both for bipolar mania and for development of depression.\n\nBright light is known at times to trigger mania77, whereas darkness therapy is an effective acute anti-manic treatment78,79. Because morning bright light immediately suppresses melatonin, early morning light should lead to increased EYA3 production, with consequent elevations of hypothalamic T3, whereas darkness would lower hypothalamic T3. Peripheral hyperthyroidism may produce mental disturbances that sometimes resemble mania, so perhaps hypothalamic excess T3 is largely responsible for the manic phenotype62. If a bipolar person had a genetic tendency towards circadian phase delay, in the Spring as the days grow longer and dusk occurs later in the evening, the EYA3 peak might rise later in the day, while an early dawn might mask (suppress) melatonin and thus disinhibit the EYA3 peak. Hypothalamic TSH and T3 might then become excessively elevated, leading to an April-June peak in mania. Another factor is short sleep or a night without sleep, that tends to predict mania80,81, and may often involve increased light exposure at night. A consistent finding is that airplane passengers travelling from west to east (so that they would be exposed to daylight before their normal melatonin offset) tend to become manic, whereas travelers from east to west (so that darkness might retard melatonin offset) are more likely to become depressed82,83. The east-going air travel effect is consistent with the antidepressant effect of advancing sleep84. Nevertheless, since bright light or air travel do not make most people manic at any time of year, a more complicated interaction of factors is likely involved.\n\n\nSleep and photoperiodic mechanisms\n\nSleep restriction (wake therapy) may have dramatic antidepressant effects and may sometimes trigger mania85. Indeed, a single night of sleep loss often seems to trigger the onset of mania81. The antidepressant effect of sleep restriction seems partly (but not entirely) mediated by light at night86–88. Whether sleep loss influences PT production of TSH apart from light effects on melatonin appears to be unknown. We have located no data regarding effects of sleep deprivation upon TSH and T3 in the 3rd ventricle CSF. Remaining awake past a normal nocturnal bedtime produces a sudden increase in blood TSH89. With normal human sleep, blood TSH falls abruptly at sleep onset, though the extent to which this is due to darkness or to some aspect of sleep itself is uncertain.\n\nA combination of partial sleep restriction (“wake therapy”), phase-advancing the timing of sleep (and awakening), and morning bright light have an enhanced and almost immediate antidepressant action, but there are insufficient comparative controlled trials to prove that this innovative triple combination is more antidepressant than bright light treatment alone84,87. One may speculate that the triple combination treatment could further limit melatonin inhibition of EYA3 transcription and therefore lead to more enhanced hypothalamic TSH and T3 synthesis. In certain models, morning light exposure by itself produces circadian rhythm phase advances accompanied by temporary abbreviation of the duration of melatonin secretion20,90,91, though this abbreviation was not documented in our own studies demonstrating light-induced advances of melatonin92.\n\n\nWinter depression, other depression, and photoperiodic control of mood\n\nThe mechanism of winter depression (Seasonal Affective Disorder or SAD) can be understood theoretically from the photoperiodic mechanisms that we have reviewed. The long nights of winter prolong nocturnal melatonin secretion and delay the morning melatonin secretion offset, particularly among SAD patients (with possible gender inconsistencies)93,94. Winter depressives tend to have circadian-phase-delayed melatonin as well as perhaps an expanded duration of secretion94,95, either of which can cause delayed melatonin offset. A delayed offset of melatonin would inhibit pars tuberalis EYA3 and TAC1 production among winter depressives just as in laboratory rodents, thus inhibiting hypothalamic T3 production. This theory is supported by the distinct antidepressant effectiveness of early morning bright light which suppresses late night melatonin91,96 as well as by the antidepressant effectiveness of morning propranolol and atenolol, beta blockers that can also suppress melatonin96–98. This theory is likewise supported by the high prevalence of depression among people with delayed sleep phase disorder, as explained in Figure 199.\n\nIt has been shown that depressed people tend to display circadian rhythm phase delays at all times of year, most notably in the melatonin offset100–104. A tendency towards eveningness (mild symptoms of circadian delay) is associated with lack of remission of depression105. For bipolar manic-depressives, eveningness (e.g., sleep phase delay) is a characteristic trait partly independent of mood state106. Bipolar patients in remission display an actigraphic sleep interval that is longer (though with more midsleep awaking and poorer sleep efficiency107, perhaps suggesting longer time-in-bed rather than increased actual total sleep time), and this predicts depression relapse108. Further, when depressed, bipolars are more likely to experience long sleep than unipolar depressives109, which might indicate a particular tendency of the morning oscillator and melatonin offset to delay among bipolars. In one study, bipolars had later peaks of nocturnal melatonin than controls, but melatonin offsets were not recorded104. Further, there is evidence from small samples of patients that bipolars display a long cellular free-running circadian cycle in their fibroblasts in tissue culture110,111, presumably of genetic origin. If a similar trend towards a longer cellular circadian period were found in SCN cells, it would contribute to a delayed melatonin circadian rhythm and delayed offset. Depression is most often associated with insomnia, but bipolar depression is also commonly associated with long or excessive sleep. Long sleep and delayed melatonin offset are associated112. It is possible that simply because they spend a longer time in bed, both people with insomnia and long sleepers may delay their first substantial morning light exposures113, thus allowing a delayed melatonin offset to mask their EYA3 peak transcription.\n\nA specific genetic contribution to phase delay may arise from polymorphisms in CACNA1C, a calcium channel component which mediates light-induced shifts of circadian phase114, perhaps through effects on both GSK3B and also on CREB (CREB mediates light stimulation of the SCN)115. CACNA1C is one of the loci most strongly associated with bipolar disorder in GWAS studies (as well as less strongly associated with schizophrenia and major depressive disorder)116. A polymorphism in ASMT, the last gene in the melatonin synthesis pathway, is associated with circadian phase delay and perhaps with inadequate melatonin synthesis (factors that combined might augment or inhibit EYA3), and with depression and bipolar disorder117,118. In addition, there have been quite a few reports of genetic variants associated with affective disorders in genes participating in circadian oscillator regulation, but we feel there has as not been adequate replication of these findings, including our own99,119–124.\n\nThere is evidence that depressed people experience below-average daytime illumination overall compared to the population as a whole125,126. Depression may result at least in part from light deficiency, especially morning light deficiency, whether from the winter season, circadian phase delays, long sleep, or various social, behavioral, or occupational factors. Moreover, depressive symptoms are treated successfully by morning bright light treatment as well as by manipulations of sleep and circadian phase at any time of year84,127. Data from a population survey suggested that adults who were more depressed spent longer times in bed, possibly because they experienced more light at night113. Even though light at night when found in ordinary households is associated with depression, the reported light intensities do not seem bright enough to substantially reduce total nocturnal melatonin production113,128,129.\n\nSome theoretical difficulties should be acknowledged. There is evidence that a minority of patients with winter depression may have advanced melatonin in reference to their sleep times130, at least as measured by the dim-light melatonin onset. There might be phase-advance as well as phase-delay variants of nonseasonal depression and bipolar illness as well131 that possibly might result if the EYA3 peak becomes more phase-advanced than does melatonin offset. Seasonal summer depression is more difficult to explain, but might arise from people staying indoors in hot weather, thus prolonging nocturnal melatonin secretion, although direct hypothalamic suppression of thyroid function by summer heat might also be involved.\n\n\nComplexities in control of mood\n\nGiven our proposed theory of winter depression, it is difficult to explain Spring seasonal peaks in hospitalizations for depression, suicide, and mania e.g., April and May in the northern hemisphere. Some authorities have hypothesized that these Spring peaks are due to prolongation or exacerbation of depressions that begin as winter depression, or rebounds therefrom, but there are few specific data to support this view. A genetic trend towards phase delay in melatonin offset may explain how depressions due to inadequate pars tuberalis EYA3 and low hypothalamic T3 might occur at any time of year, but a genetic predisposition to delay does not explain why symptoms should peak near or just after the Spring equinox. We may speculate that a genetic tendency to delay could exacerbate effects of the spring transition to longer days. Spring lengthening of days results in a delay shift of the evening oscillator caused by later sunsets, which we combine with the “Daylight Savings” advance in the time standard that influences time in bed. The combined effect makes sunset suddenly much later by our adopted time standard. As days grow longer in Spring, a balancing advance shift of the morning oscillator might be anticipated due to earlier dawns, but melatonin offset was not found earlier in summer among normal urban subjects, perhaps partly because of the shift in time standard132. In contrast, melatonin offset was found to be earlier in summer than winter among winter depressives, with some differences between men and women132. An endogenous tendency to delay among depressives could make advance of the morning oscillator in Spring especially indolent, especially if combined with a relative unresponsiveness of the morning oscillator to phase-advancing light exposures. Numerous studies indicate some asymmetry of responses to light stimuli causing swifter light-stimulated delay phase shifts versus advances as photoperiods vary90,114,133. We may speculate that perhaps the spring transition to a shorter scotophase combined with the Daylight-Savings time reference could produce a delay in melatonin offset despite an earlier EYA3 peak, accentuating the morning melatonin masking of EYA3 with consequent depression. On the other hand, in a rat model, light-induced phase advance may temporarily suppress pineal n-acetyltransferase (NAT), thus suppressing melatonin production90. These competing processes promoting possible increased or decreased melatonin masking of EYA3 in the Spring might produce the paradoxical peaks of both depression and mania at about the same season, depending on various factors influencing susceptibility in a diverse population. Whether Spring tends to promote depression or mania may depend on the extent to which the later sunset delays melatonin offset more than the EYA3 peak, despite an earlier dawn. An unexplained mystery is how the peak of EYA3—seemingly about 12 hours after dark--is controlled and synchronized.\n\n\nRapid cycling and circadian oscillator desynchronization\n\nA feature of some bipolar syndromes that is particularly hard to understand is the appearance of rapid cycling, that is, episodes of depression and/or mania which come and go at least four times a year. In extreme cases, mania and depression may alternate every few days or even every other day81,134. Halberg hypothesized that such mood swings could be caused by a free-running desynchronized circadian rhythm with a cycle longer than 24 hours, so that its peak drifted later each day relative to the 24-hour light-dark cycle135. One might consider this an “external desynchronization” model, that is, where all of the body’s internal rhythms might remain synchronized to each other, but they might free-run progressively later and later, beating in and out of phase with the external environment, particularly, its light-dark cycle. Both Kripke, Wehr and their colleagues tried to document such non-24-hour rhythms among rapid-cycling patients, but apart from a very few intriguing examples that did not seem fully persuasive, they had little success78,131,136,137.\n\nOn the other hand, Wehr reported clear demonstrations that repeated 48-hour sleep wake cycles are at times observed among bipolar patients, in association with 48-hour cyclic manic-depressive symptoms81. Wehr attributed these 48-hour cycles to an “internal desynchronization” model wherein the temperature rhythm and many other circadian rhythms remained synchronized to the 24-hr environment, but the period of sleep-wake (and some associated rhythms) decelerated so much as to double cycle length and produce 48-hour rhythms. Overt symptoms depended on when critical intervals of these two sets of rhythms were in or out of phase. In temporal isolation and cave experiments, circadian rhythms of core temperature and related functions have at times been observed to free-run with cycle periods of about 25.0 hr., while the sleep-wake rhythm might internally desynchronize to cycles as long as 36 hr. or even 48–50 hr138,139. Because in these internal desynchronization models, sleep-wake cycles tend to be much more unstable and generally slower, debate has emerged about whether sleep-wake should be considered a “weak” non-linear circadian oscillator or alternatively a homeostatic relaxation oscillator that should not be classified as circadian. In any case, the internal desynchronization observed in isolation studies has not generally been recognized to cause mood disturbances in cave and isolation experiments, despite a few severe psychoses reported in such experiments. The absence of daylight and the dark surrounding of cave environments might have been a protective antimanic factor. The isolation and cave studies did prove that cycles of alternating long and short sleep or even 48-hr sleep-wake cycles could arise as a consequence of circadian internal desynchronization140.\n\nIn rats, overall SCN firing is higher in the day (when the animals mainly sleep) than at night, and the duration of multiunit neuronal firing is longer in long photoperiods. However, within either the photophase or scotophase, firing is somewhat higher in wake and in REM than in SWS141. SCN metabolic activity is likewise higher in the day and further increased by light exposure142. In rats, when core and shell were internally desynchronized by 22-hour light-dark cycles, both the SCN core and shell remained associated with slow-wave sleep, but REM sleep and body temperature were more exclusively associated with activity of the shell neurons143. It would appear from the responses to phase-shifting light-dark cycles, considering the two-process model of sleep-wake control, that the more-directly-light-responsive core is associated better with the homeostatic aspect of sleep-wake regulation, whereas the shell is better associated with the circadian modulation of sleep wake. In diurnal mammals, also, SCN firing tends to be higher during the day times when these animals tend to be awake, but whether firing of SCN core or shell augments or suppresses sleep among diurnal animals is unknown to us, and therefore, it would be uncertain how to relate the SCN core and shell division to internal desynchronization in humans.\n\n\nCircadian oscillator bifurcation\n\nPerhaps we may gain further insight into mechanisms that could trigger mania by considering circadian rhythm bifurcation, which is the division of the circadian rhythm into two components, with the two peaks being separately entrainable. Circadian research has developed certain laboratory models that “bifurcate” nocturnal rodent activity into two circadian components (bouts of activity) about 12 hours apart from each other. These two activity bouts can be entrained in a stable manner by a special light-dark cycle consisting of two photophases (light intervals) and two scotophases (dark phases) within each 24 hours (for example, light-dark-light-dark hours abbreviated as LDLD7:5:7:5)144. A large set of studies utilizing Syrian hamsters, Siberian hamsters, and mice have demonstrated stable entrainment of bifurcated scotophase activity bouts, body temperature peaks, and melatonin peaks145. Taken together, these studies lead to the hypothesis that LDLD entrainment bifurcates the neural oscillators in the SCN into two or more components, each driving activity, body temperature, and melatonin secretion. Bifurcated-rhythm hamsters develop and maintain summer gonadal size and presumed reproductive fertility146, perhaps because the duration of each bout of melatonin secretion is brief (as in the short nights of summer). The bifurcated activity components and bifurcated melatonin secretion in the scotophases represent the control of two independently-entrainable circadian pacemakers which are yet mutually coupled, and which will fuse into a single component if the bifurcated photophase is withdrawn71,147. In this model, the circadian bifurcation seems to result from two different populations of neurons in the SCN that assume almost opposite phases, though the two bifurcated SCN populations appear to be bilaterally symmetrical148,149. It should be emphasized that the two scotophases and two photophases per 24 hr day do not need to be absolutely symmetrical, especially once the bifurcation has occurred. When the bifurcated photoperiod is replaced by constant dark and the bifurcated activity bouts rejoin each other, the melatonin secretion components presumably also fuse. The two activity bouts can be recoupled either by the day-scotophase activity component delaying or by the day component advancing in reference to the night-scotophase activity component (Figure 2)71.\n\nIn this diagram, each line of the ordinate represents a 24-hour day and the abscissa represents the 24 hours within that day. The grey shading depicts very dim light or darkness, whereas the white background represents daylight and artificial light. The light-dark cycle is modelled as commencing with LD16:8 and transitioning in the middle days to LDLD8:4:8:4, with return to LD16:8 in the final days. The orange shading represents SCN multiunit neuronal firing that gradually splits apart and bifurcates into two antiphase patterns of firing during LDLD8:4:8:4, representing two distinct populations of coupled SCN neurons. During LD16:8, firing might be spread out over a longer interval in the light than is shown, but there may be insufficient data to model the pattern of neuronal timing more exactly. After return to LD16:8 or to continuous darkness (DD), the two components of neuronal firing gradually fuse together again. The blue regions represent melatonin secretion during the dark intervals. Suppressed by neuronal firing and light suppression, it is plausible that melatonin secretion would be partly or completely inhibited during the transitions from LD16:8 to LDLD8:4:8:4 and back again, during which melatonin secretion would bifurcate and then fuse again. These patterns are theoretical, because the transitions of neuronal firing and melatonin secretion from an LD pattern to a bifurcating LDLD pattern and back again have never been observed simultaneously in detail, certainly not in a diurnal mammal.\n\nConceivably, LDLD bifurcation into two circadian oscillator components produces two peaks in EYA3 transcription, neither of which is well-suppressed by melatonin, thus promoting increased PT TSH production and increased tanycyte production of T3.\n\nThe attainment of bifurcated circadian activity cycles by LDLD lighting cycles and the phase-shifting effects of light in general can be enhanced in nocturnal rodents by very dim illumination during the dark scotophases, for example, 0.005 lux147. This would be less than 1% of bright moonlight and too dim to suppress melatonin. Since a dim-light scotophase causes the duration of the rodent nocturnal activity phase (alpha) to expand, it has been inferred that dim light weakens the coupling between separate circadian neuronal populations71,147,150,151. Coupling refers to the mutual influence of one oscillator on another. Conceivably, even dim light might suppress LHX1, a transcription factor (known to be suppressed by bright light) that mediates expression of VIP and the AVP receptor AVPR1A, thus impairing coupling of SCN neurons152.\n\nIn humans, Worthman and Melby have found that in the tropical and subtropical environments in which, for the most part, our species developed, daytime napping is quite pervasive, especially near the middle of the day153. One wonders if the bifurcated activity patterns which are observed in hot climates in equatorial regions, where the heat of the day leads to mid-day sun avoidance, might also mimic a bifurcated LDLD laboratory photophase and the resultant bifurcated circadian organization. However, the common human daytime sleep episodes usually described do not constitute half of 24-hour sleep. In primitive surroundings, illumination intensities are substantial during daytime sleep, unlike the dim scotophase in the rodent circadian bifurcation model. When people in equatorial climates or in summer at high latitudes remain awake in the cooler night, often using artificial light, or among night shift workers, a somewhat-bifurcated bright-light photophase might be combined with dim light exposure during the scotophases.\n\nWe do not know much about human circadian responses to bifurcated sleep conditions such as those suggested by napping. We are unfamiliar with any evidence that bifurcated melatonin secretion may be produced or that the two sleep episodes come to represent two independently-entrainable circadian oscillators. Unfortunately, we have virtually no information concerning what levels of light at night might produce phenomena in humans similar to the dim night light effects promoting circadian bifurcation in hamsters and mice, if indeed this scotophase effect occurs in humans at all, and we have no definite data concerning what conditions might produce true bifurcated circadian oscillators among humans. Our own pilot studies attempting to induce bifurcated melatonin rhythms with LDLD cycles produced only a few bimodal melatonin peaks of unequal amplitude, and we are unsure if longer exposure to LDLD or addition of dim light to the dark scotophases might have led to more convincing bifurcation. Although it has been asserted that irregular sleep cycles may induce bipolar relapses and mania154, there is no evidence that specific conditions producing bifurcated sleep patterns in humans predispose to depression or mania. Moreover, it is likely that interactions of genetic variations with environmental factors are required to trigger major mood disorders.\n\n\nPhase jumping caused by light and singularity\n\nRelated to circadian rhythm bifurcation, another peculiar phenomenon called “phase jumping” should be considered. When bright light compresses the effective primary scotophase excessively (e.g., to around 4 hours in some nocturnal rodents, LD20:4), and an alternative secondary scotophase is made available, the originally nocturnal activity bout may “jump” to a relative antiphase orientation in the newly opened scotophase147. This phase jumping is likewise augmented by dim light during the scotophase, and might result partly from light-suppression of LHX1, VIP, and AVPR1A. Possibly during the very long summer days that occur at higher latitudes and with certain patterns of artificial light, phase jumping might be triggered. Similarly, it is conceivable that severe sleep restriction (e.g., no more than 4 or 5 hours in bed) would trigger human phase jumping if coupled with other permissive conditions (e.g., a daytime retreat with darkness or very dim lighting).\n\nUsing mice bred with a PER1 or PER2-bound luciferase, SCN slices can be monitored in vitro over time. Luciferase luminescence is then a marker of the molecular circadian clock phase of individual SCN neurons. In slices from mice housed in the dark or in LD12:12 (that is a photoperiod of 12 hours light and 12 hours dark), the peak times of PER2-luciferase activity do not differ more than a few hours in various SCN regions, nor is the neuronal timing determined simply by core-shell VIP-AVP or rostral-caudal parameters21,27. As the duration of the photophase is incrementally increased, the phase distribution of SCN neurons broadens substantially, until in LD20:4, a population of mostly-core neurons and a population of mostly-shell neurons are 6–12 hr out-of-phase with each other21,155, somewhat resembling rodent LDLD bifurcation experiments. Released into DD (continuous darkness), the mutual coupling of these two neuronal pacemaker populations pulls them back into alignment, either through relative advances or delays of the core-like population in reference to the shell-like population. After exposure to such atypical photoperiods, the coupling of the two groups of pacemaker neurons might undergo a full 360° circadian phase rotation in reference to each other155. Thus, over many days, one pacemaker component might steadily delay (or advance) relative to another, reminiscent of the non-24-hour components and internal desynchronization previously hypothesized to trigger rapid mood cycling in humans.\n\nAnother phenomenon which might possibly be involved in mania is a complex of light pulses that may drive a circadian system to its singularity point, apparently stopping the clock156. It is possible that an anti-phase orientation of the core and shell could at times produce an appearance of SCN singularity while both core and shell remain inversely oscillatory157. Stopping or severely attenuating SCN rhythmicity could have profound consequences for brain function, e.g., memory158. Bright constant light may also suppress circadian activity rhythms in rodents159. Human circadian rhythms are occasionally driven through an apparent singularity by phase-shifting stimuli160, but in the presence of a synchronizing environment, the circadian rhythms appear to recover after a few days. It is conceivable that an interval of singularity in at least one portion of the SCN, e.g., the core, is an element in sudden switches into mania.\n\n\nCore and shell circadian bifurcation, melatonin bifurcation, and mania\n\nTo recapitulate, both a bifurcated photophase, e.g., LD7:5:7:5 or a very long photophase, e.g., LD20:4 can evidently phase shift two SCN neuronal populations towards a near-antiphase SCN pacemaker organization from which a 360° phase rotation between the two pacemakers might evolve. In rodents, dim light during the scotophases enhances this bifurcation. The anti-phase orientation of two SCN neuronal populations could result in internal circadian desynchronization somewhat resembling the observations in temporal isolation, cave experiments and LD phase shifts, but appearing much less overt than the full external circadian desynchronization hypothesized by Halberg for rapid mood cycles of several days135. Indeed, we do not know exactly how internal circadian desynchronization or resynchronization (reorganized phase relationships among SCN components) might best be documented among humans. The best clue comes from experiments in which a bifurcated LDLD cycle produced bifurcated locomotor activity in Siberian hamsters that was associated with two temporally dissociated episodes per day of melatonin production, one melatonin secretion interval seemingly coupled to the SCN core oscillator and the other to the shell28,145. Note, behavioral and reproductive data from related studies indicated that these short intervals of melatonin production would be associated with gonadal fertility146, permitting an inference that hypothalamic TSH and T3 were produced at long-day concentrations or greater.\n\nCollecting blood, saliva, or urine samples for melatonin every few hours from severe manics has been so challenging that until recently we could locate no substantial body of round-the-clock observations of melatonin from manics which might reveal if a bifurcated rhythm or a shifted phase relationship among distinct SCN oscillator components are likely to be associated with mania. Remarkably, an outstanding group of investigators has now overcome the challenges of collecting 24-hour saliva samples from manics for assaying melatonin. Their exciting new evidence shows that during acute mania, bipolars indeed produce two antiphase separated peaks of melatonin secretion, one at night and one in the day, much like the melatonin secretion of bifurcated Siberian hamsters145,161. The investigators suggested that the bifurcated peaks in melatonin secretion might be due to disruption of coupling between SCN oscillators. There had been previous observations of two largely-merged peaks of melatonin as well as possibly some rare unmerged double-peaks even among normal subjects162, or a possible small daytime peak among occasional winter depression patients96, but we do not know of situations apart from mania in which fully-separated and relatively equal and symmetrical antiphase melatonin peaks have been observed in humans. We can speculate that bifurcated melatonin excretion could prove a valuable marker of a bifurcated antiphase orientation of human SCN neuronal populations, and perhaps this antiphase SCN organization is the specific circadian disorder of mania.\n\nSince in normal humans, waking activity is highest in the day, but melatonin is highest at night encompassing the hours of sleep, we might expect that among manics with bifurcated melatonin secretion, bifurcated locomotor activity or bifurcated sleep-wake patterns might also be observed. The trail-blazing study of human melatonin in manics displaying two peaks as described above did not record sleep-wake or activity161, but some informative wrist activity plots in mania were published by Wehr’s group81,163. In the plots from Wehr’s observations, we could not discern any persuasively bifurcated activity rhythms—on the other hand, brief daytime cessations of activity in these plots prevent us from being certain that a bifurcated activity rhythm did not occur among the manics recorded.\n\nAnother finding related to this theory of bifurcated SCN pacemaker components in mania comes from the following clinical observations. Rapid-cycling bipolars seem less likely to suffer relapse if bright light treatment is given near midday164, a time when bright light might tend to reverse a bifurcation between SCN pacemaker components.\n\nWe have an interesting model of potential circadian sleep bifurcation among human shift workers. On the one hand, it has been asserted that circadian behavioral irregularities such as those produced by shift work schedules promote mania154. On the other hand, mania is not generally noted among shift workers, although it is often convenient for night shift workers to divide their sleep between several hours in the morning just after the night shift and an additional 1–3 hours in late afternoon or evening before going to work. So far as we know, bifurcated melatonin rhythms have not been described among such night shift workers, perhaps because many shift their melatonin rhythms little from their day-work pattern. Also, the dim lighting during most night shifts might be protective. To the extent that attempts to use bright light to promote alertness during night shift work are effective in shifting melatonin secretion rhythms, such lighting might increase the risks of triggering mania or depression. Evidence for possible bifurcation in sleep-wake has been reported from certain circadian isolation studies, but there was no evidence for mania in these studies, in which melatonin was not assessed165.\n\n\nComplex light stimuli: possibilities triggering circadian bifurcation and mania\n\nNow we may synthesize hypotheses of how mania could result from a disorder of photoperiodic regulation. From the poor sleep and often early awakening of manics, it appears that some aspect of circadian regulation becomes disordered during mania. The triggering of mania by a single night’s sleep loss or midsleep awakening or perhaps more chronic sleep compression or phase shifts81,83,166 raises the question of whether a sudden internal phase shift between two SCN component oscillators may produce a switch into mania. If a melatonin-related SCN oscillator component became delayed well past dawn, perhaps because of sleep loss and use of artificial lighting late at night, then morning light might further delay that component past noon, triggering internal circadian desynchronization or bifurcation. Dim light at night might facilitate the sudden phase shift, since a person suffering severe sleep disturbance for any reason is likely to turn on artificial lighting irregularly at night, and this may weaken coupling of SCN component circadian oscillators. We currently have no evidence base from which to judge what intensities and timings of light might be most likely to produce internal desynchronization or altered phase of circadian oscillator components in humans, but it does appear that bright morning light perceived at or before the usual time of awakening might contribute77,91. Likewise, since sleep restriction in the second half of the night seems almost as effective as whole-night sleep deprivation in its antidepressant effects167, it seems likely that sleep in this second half of the night is most critical to preventing mania. The often-discussed shortened sleep and elevated mood experienced by Scandinavians near the summer solstice might be a modest human replica of the LD20:4 response, or the impressive peak in violent suicides in Greenland at about the same season might be an even more dramatic model10,168. At present, we have only one strong study showing that the circadian system is actually bifurcated during mania (as indicated by melatonin). The difficulties of collecting such data must be acknowledged, but perhaps we know better now what measurements are needed.\n\nA seeming paradox arises from our hypothesis that mania may arise from excessive phase delays of an oscillator component within the SCN, in part due to genetic tendencies to delay, since mania is more often described as a condition of early awakening and phase advance161. Perhaps the explanation is that should bright daytime light cause an SCN component to delay more than 180°, it becomes advanced from the perspective of the other component. The mutual coupling of SCN oscillator components might be expected to resolve any transient internal desynchronization within a few days, but perhaps bright light exposures both soon after awakening and again past mid-wake would stabilize persistent mania, just as LDLD lighting can prevent bifurcated circadian oscillations from resolving among hamsters. Likewise, it becomes logical that either bright light at some critical time of day or round-the-clock darkness would tend to resolve antiphase malsynchronization of the two SCN oscillator components. The empirical observations that mania may resolve when a patient is treated with the delaying drug lithium or with a dark environment79 may be consistent with these speculations. Perhaps the process of resolution of mania can be monitored by studying the evolution of the two peaks of melatonin in mania.\n\n\nConclusion and needs for future research\n\nTo conclude, we have proposed that photoperiodic mechanisms, interacting with inadequate or untimely illumination and a genetic tendency for phase delay, produce depression. Among bipolars, we propose that combined with genetic susceptibilities, abnormal bright light illumination patterns trigger mania by producing internal desynchronization and perhaps bifurcation of SCN circadian oscillator components, thus leading to photoperiodic malregulation and excess hypothalamic T3 production. Data supporting a photoperiodic mechanism triggering depression are already quite strong: both evidence for delayed melatonin offsets and delayed awakenings among depressed patients and evidence that forcing the melatonin offset earlier (with bright light treatment or propranolol) is antidepressant. Data supporting circadian bifurcation as the cause of mania do not extend beyond the seminal observation of a bifurcated melatonin excretion pattern in one study of manics161 and some support among other scattered and uncertain clinical observations. The hypotheses presented have many limitations including missing elements of the proposed neurobiologic mechanisms, an insufficient evidence base, some apparent inconsistencies with available data, and insufficient testing of predictions. These hypotheses are presented as a call for much further study and testing of predicted effects, both among laboratory animals and among consenting human volunteers.\n\nHere are some key areas for future research:\n\nFurther observations of bifurcated 24-hour melatonin secretion among manics are needed, extended by descriptive longitudinal data during the development and remission of mania. Likewise, data on sleep-wake, activity, and core temperature are needed to correlate with melatonin changes during the evolution of mania.\n\nBlood measurement of PT TSH47 in humans could confirm impaired secretion of PT TSH in depression, a positive response to light, and excessive PT TSH in mania.\n\nLong-term longitudinal descriptive monitoring of bipolar patients (becoming increasingly practical with the growing mass-market for health-monitoring actigraphic wrist bands) should be initiated to try to identify what lighting patterns trigger depression and mania, and how the consequent activity patterns evolve. Considering the ethical obligation to “do no harm,” we do not recommend attempts to trigger depression or mania experimentally, but in the long run, observational research may lead to testable preventive interventions.\n\nMore clinical trials are needed to optimize bright light treatment timing, sleep-wake phase-advance, and sleep restriction combinations in relieving depression. Likewise, more clinical trials are needed to clarify what manipulations of light or darkness or melatonin agonists might cause mania to remit.\n\nSystematic dose-response studies should define what levels (and color-spectrum) of dim light might facilitate loosening of SCN component oscillator coupling in humans, with possible resultant increased melatonin secretion durations, facilitated phase-shifting, and perhaps facilitated circadian bifurcation of sleep and locomotor activity behaviors in humans.\n\nSystematic experiments should search for photoperiod manipulations which can produce circadian bifurcation in humans, and measure the related endocrine and mood responses.\n\nShift workers with bifurcated sleep patterns should be re-examined to see if bifurcated melatonin secretion results, and if this correlates with mood disorders. Studies of various transmeridional air travel effects may also clarify the roles of varying light exposure patterns.\n\nSystematic experiments should examine if very long photoperiods, e.g., 20:4, when coupled with an inserted phase of darkness or dim illumination, can produce phase-jumping in humans, with consequences in activity, sleep wake, melatonin secretion, and mood. Both among humans and laboratory animals, data are needed as to whether phase jumping produces bifurcated melatonin secretion patterns.\n\nWe would like to see testing of the hypothesis that the circadian bifurcation produced by LDLD skeleton photoperiods produces bifurcated large EYA3 peaks in PT and consequent increased third ventricle TSH and T3. Perhaps this could be tested in a diurnal mammal, possibly using microdialysis of TSH or T3 in the third ventricle CSF near PT. Possibly in-vivo MR spectral imaging could be an alternative to microdialysis.\n\nBlood measurement of PT TSH47 could be useful to assess effects of circadian bifurcation in rodents.\n\nWe would like to understand the molecular mechanism controlling the timing of the morning peak in PT EYA3, absent melatonin inhibition.\n\nWe would like clarification of how PT regulation of hypothalamic T3, which does impact TRH secretion, interacts with the peripheral homeostatic regulation of thyroid metabolism.\n\nWe would like to see further study of the SCN neurophysiology related to LDLD-induced circadian bifurcation, including clarification of core-shell and anterior-posterior SCN functional differentiation, clarification of the SCN neuroanatomical structures, and exploration of which SCN-efferent neurotransmitters such as AVP and VIP mediate bifurcated secretion of melatonin when it occurs. Likewise, clarification of the contrasts and overlap between the evening-morning oscillator models and core-shell oscillator models is needed.\n\nSimilarly, we would like to see clarification of the neuronal firing patterns in the core and shell regions of the SCN after LDLD bifurcation as they relate to the component circadian oscillators in both nocturnal and diurnal rodents.\n\nWe would like more data on the effects of LDLD circadian bifurcation in rodents on reproductive endocrine functions.\n\nWe would like to see how LDLD-induced circadian bifurcation influences rodent behavioral models of depression and mania.", "appendix": "Author contributions\n\n\n\nDFK conceived this review and prepared the first draft. All authors took part in previous experiments related to the physiology reviewed, contributed ideas, text, and critique, helped revise the manuscript, and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDFK was supported by NHLBI HL71560 and HL61280. JAE was supported by HL61280 and ONR N000141310285. DKW was supported by a Veterans Affairs Merit Award (1I01BX001146) and a NARSAD Young Investigator Award. SDY was supported by R01 HL095799.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis manuscript was inspired by “Clockwatchers” seminars of the UCSD Center for Circadian Biology, especially by presentations from the laboratory of Professor Michael R. Gorman.\n\n\nReferences\n\nMoore RY: Neural control of the pineal gland. Behav Brain Res. 1996; 73(1–2): 125–30. 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Publisher Full Text\n\nFrank E, Hlastala S, Ritenour A, et al.: Inducing lifestyle regularity in recovering bipolar disorder patients: results from the maintenance therapies in bipolar disorder protocol. Biol Psychiatry. 1997; 41(12): 1165–73. PubMed Abstract \| Publisher Full Text\n\nEvans JA, Leise TL, Castanon-Cervantes O, et al.: Dynamic interactions mediated by nonredundant signaling mechanisms couple circadian clock neurons. Neuron. 2013; 80(4): 973–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrone BP, Chang D, Bourgin P, et al.: Acute light exposure suppresses circadian rhythms in clock gene expression. J Biol Rhythms. 2011; 26(1): 78–81. PubMed Abstract \| Publisher Full Text\n\nSumova A, Illnerova H: Effect of photic stimuli disturbing overt circadian rhythms on the dorsomedial and ventrolateral SCN rhythmicity. Brain Res. 2005; 1048(1–2): 161–9. PubMed Abstract \| Publisher Full Text\n\nFernandez F, Lu D, Ha P, et al.: Circadian rhythm. Dysrhythmia in the suprachiasmatic nucleus inhibits memory processing. Science. 2014; 346(6211): 854–7. PubMed Abstract \| Publisher Full Text\n\nOhta H, Yamazaki S, McMahon DG: Constant light desynchronizes mammalian clock neurons. Nat Neurosci. 2005; 8(3): 267–9. PubMed Abstract \| Publisher Full Text\n\nWeitzman ED, Kripke DF: Experimental 12-hour shift of the sleep-wake cycle in man: Effects on sleep and physiologic rhythms. In: LC, Tepas DI, Colquhoun WP, Colligan MJ, editors. Advances in Sleep Research: Biological Rhythms, Sleep and Shift Work. New York: Spectrum; 1981; 93–110.\n\nNovakova M, Prasko J, Latalova K, et al.: The circadian system of patients with bipolar disorder differs in episodes of mania and depression. Bipolar Disord. 2015; 17(3): 303–14. PubMed Abstract \| Publisher Full Text\n\nWehr TA, Schwartz PJ, Turner EH, et al.: Bimodal patterns of human melatonin secretion consistent with a two-oscillator model of regulation. Neurosci Lett. 1995; 194(1–2): 105–8. PubMed Abstract \| Publisher Full Text\n\nWehr TA, Turner EH, Shimada JM, et al.: Treatment of rapidly cycling bipolar patient by using extended bed rest and darkness to stabilize the timing and duration of sleep. Biol Psychiatry. 1998; 43(11): 822–8. PubMed Abstract \| Publisher Full Text\n\nLeibenluft E, Turner EH, Feldman-Naim S, et al.: Light therapy in patients with rapid cycling bipolar disorder: preliminary results. Psychopharmacol Bull. 1995; 31(4): 705–10. PubMed Abstract\n\nCampbell SS, Zulley J: Evidence for circadian influence on human slow wave sleep during daytime sleep episodes. Psychophysiology. 1989; 26(5): 580–5. PubMed Abstract \| Publisher Full Text\n\nWehr TA, Lewy AJ, Wirz-Justice A, et al.: Antidepressants and a circadian rhythm phase-advance hypothesis of depression. In: Collu R, et al., editors. Brain Peptides and Hormones. New York: Raven Press; 1982; 263–76.\n\nParry BL, Meliska CJ, Martinez LF, et al.: Late, but not early, wake therapy reduces morning plasma melatonin: Relationship to mood in Premenstrual Dysphoric Disorder. Psychiatry Res. 2008; 161(1): 76–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBjorksten KS, Kripke DF, Bjerregaard P: Accentuation of suicides but not homicides with rising latitudes of Greenland in the sunny months. BMC Psychiatry. 2009; 9: 20. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8564", "date": "01 Jun 2015", "name": "Klaus Martiny", "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\nThis comprehensive review of seasonality, mood, circadian regulation, and their inter-relatedness is of great value for the creation of new hypotheses in the field and gives impetus for a number of new studies. The title is appropriate even though the paper touches on a number of additional subjects such as separate morningness and eveningness oscillators and the regulation of thyroid function in relation to mood. The abstract covers the content of the paper. Many theories are presented and perhaps the red thread is sometimes a little difficult to follow in the narrative. Of the many ideas presented the hypothesis regarding separate oscillators for morningness and eveningness is of immediate interest. It should be investigated how this phenomenon is reflected in the rating scales used to assess chronotype and time-dependent preferences. Do we need separate scales for morningness and eveningness? In the quest for understanding the higher prevalence of suicide in springtime authors do not include the widely accepted psychological concept that for a person with persistent depression, the contrast of seeing other persons elation and increased energy in springtime, could contribute to the phenomenon. This raises a more general point: why is the interaction or cascading effect between cognition (thought and emotion) and circadian physiology not included in our understanding of the regulation of mood? A few critical points: Authors uses the word sleep restriction for sleep deprivation (wake therapy). Sleep restriction in the sense of a continuous reduction in sleep time does not induce an antidepressant effect. The effect of sleep deprivation depends on sleep abstinence for a substantial part or the whole of a single night AND the timing of recovery sleep days. Also the effect of sleep deprivation probably relates to the relation between sleep and the activity of the raphe nuclei on the brain-stem and this should be mentioned. Figure 1 is rather busy and could be simplified to enhance readability. On page 5 the effect of darkness as an antimanic treatment is defined as “effective”. At this point we should probably regard the evidence of dark therapy, however interesting, as preliminary (and difficult to carry out). The statement, also on page 5, that the effect of sleep deprivation is partly caused by light, is not quite substantiated by the one reference dealing with this issue. Finally, references should be checked for content in relation to their relation to issues discussed in the paper with a more critical attention to the quality of the studies (case reports and pilot studies).", "responses": [ { "c_id": "1414", "date": "05 Jun 2015", "name": "Daniel F. Kripke", "role": "Author Response", "response": "We appreciate Dr. Martiny's thoughtful review as well as his important contributions to light treatment of depression. Some of the interesting issues he raises deserve further discussion. As Dr. Martiny points out, the concept of chronotype and the usual methods of measuring chronotype do not recognize the partially-independent phases of morning-oscillator and evening-oscillator components. However, the relative timing of evening and morning components seems crucial to the photoperiodic responses in animals and apparently in humans. Although our presentation emphasizes the melatonin offset, the dim light melatonin onset or DLMO has become popular as a more-easily-measured phase marker. It is likely that the DLMO is correlated with the timing of the EYA3 peak in pars tuberalis. As demonstrated in much animal work and to some extent at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1988787/ Figure 6, melatonin onsets and offsets may have slightly different phase-response curves. Factor analysis of morningness-eveningness scales has likewise suggested the presence of partly independent morning-related and evening-related factors, so we agree that there could indeed be value in generating separate morning and evening subscales from existing chronotype questionnaires. As Dr. Martiny pointed out, the dramatic antidepressant response that he has successfully employed clinically depends on sleep abstinence for a substantial part of the night and also upon the timing of recovery sleep. The term sleep restriction is commonly used for milder reductions of sleep duration continuing over weeks or longer. Cognitive-behavioral treatments of insomnia that include an element of sleep restriction seem to be antidepressant, e.g., see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353033/ , though it is unclear whether time-in-bed or EEG sleep curtailment or earlier morning light exposure is most determinative. The mood effects of long-term mild sleep restriction need more clarification, especially for those with long sleep. Although there is a large literature decrying the effects of sleeping too little, more mortality and as much morbidity (including depression) is associated with unusually long reported sleep durations. As Dr. Martiny mentioned, many of the ideas assembled in our review are based on meager experimental evidence, so more study is clearly needed." } ] }, { "id": "9288", "date": "02 Jul 2015", "name": "Robert D. Levitan", "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\nThis is a thoughtful and timely review of an important topic in mood disorders i.e. the circadian regulation of mood and its relevance to the understanding and treatment of major mood disorders. Despite significant efforts over many decades, and incremental improvements in the treatment of depression and bipolar disorder, many patients do not respond to standard treatments. At a clinical level it is apparent that the vast majority of patients with major mood disorders have a significant disruption in one or more aspects of circadian regulation whether it be sleep, activity rhythms, social behavior, energy regulation, hormonal function etc. The current paper adds significantly to the literature in both proposing new ways of thinking about circadian dysregulation and mood and proposing a series of studies that follow from the models proposed. The proposed studies also exemplify well the new Research Domain Criterion strategy to understanding complex pathology.Overall, the paper has a strong emphasis on basic mechanisms, often at a molecular level, which can be difficult to follow at times. It might be of interest for the authors to balance this approach with a consideration of the possible evolutionary/adaptive significance of the mechanisms under consideration as this might provide further context for their arguments. For example, what might be the advantage to a particular species of having the ability to bifurcate circadian rhythms? Or do the authors propose that this bifurcation only occurs in pathological situations or when the system is stressed beyond its normal physiological control?In terms of specific mechanisms, while the current paper emphasizes melatonin studies at many points, it can be argued that the neurotransmitter dopamine is as important to daytime activity rhythms and arousal as melatonin is to sleep rhythms and physiology at night. Accumulating evidence suggests that there are several ways by which melatonin and dopamine interact and modulate one another, including basic circadian rhythmicity (reviewed by Zisapel, 2001). Furthermore, there is a large body of work demonstrating dopaminergic dysfunction in mood disorders and bipolar disorder in particular. It may be that the best way forward for future work on circadian physiology and mood is to consider these two systems as one integrated system and to understand their interplay in the regulation of mood. How this might relate to the proposed hypotheses and research program might be of great interest going forward.", "responses": [] } ]	1	https://f1000research.com/articles/4-107
https://f1000research.com/articles/4-52/v1	24 Feb 15	{ "type": "Research Article", "title": "Theoretical modelling of epigenetically modified DNA sequences", "authors": [ "Alexandra Teresa Pires Carvalho", "Maria Leonor Gouveia", "Charan Raju Kanna", "Sebastian K. T. S. Wärmländer", "Jamie Platts", "Shina Caroline Lynn Kamerlin", "Alexandra Teresa Pires Carvalho", "Maria Leonor Gouveia", "Charan Raju Kanna", "Sebastian K. T. S. Wärmländer" ], "abstract": "We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT) methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.", "keywords": [ "Epigenetics", "DNA modifications", "DNA methylation", "Density functional theory", "hybrid QM/MM calculations", "DNA model systems" ], "content": "Introduction\n\nThe standard four-letter alphabet used to encode genetic information in DNA is a central tenet of molecular biology. However, in vivo chemical modification of bases can expand this alphabet markedly, giving rise to a host of important biological phenomena1. Epigenetic modifications, most importantly DNA methylation and histone variation, have the potential to affect gene expression, and are believed to play a major role in the complex pattern of development and differentiation of multi-cellular organisms. Fascinatingly, such modifications may be heritable despite not affecting DNA sequence, although the mechanism(s) by which this could be achieved are currently unknown.\n\nThe most common and biologically important such modification involves methylation of the 5 position of cytosine (C) to form 5-methylcytosine (5-mC), illustrated in Figure 1. This does not strongly affect the ability of the base to pair with guanine (G), and in mammals is generally found in CpG sequences, though bacteria and plants display less sequence specificity2. Oxidation of 5-mC can form 5-hydroxymethylcytosine (5-hmC), which is believed to be involved in regeneration of C via ten-eleven translocation (TET) proteins. Moreover, recent work has shown that 5-formylcytosine (5-fC), and 5-carboxycytosine (5-caC) are present in stem cells and organs of mice3.\n\nThe structural consequences of cytosine methylation and related modifications were the focus of a recent study4 that used X-ray crystallography to show that incorporation of 5-mC or 5-hmC at different points in the d(CGCGAATTCGCG) dodecamer has a negligible effect on both local (base pair) and global (helical) geometry, although specific preference for the orientation of the hydroxyl group in the latter was clearly evident. However, while elegant, the resolution of these studies (between 1.42 and 1.99 Å) may mean that subtle structural changes could go unnoticed. Therefore, molecular modelling, whether based on quantum or classical mechanics, has the potential to contribute significantly in this field. Quantum mechanical models, typically using density functional theory (DFT), have been used to examine the base pairing and stacking of both unmodified (wild-type) and 5-mC DNA. Many groups, including those of Fonseca-Guerra5–7, Šponer8–13, Leszczynski14–16 and others have used DFT to great effect in understanding the structure and properties of unmodified DNA. Regarding epigenetic modifications in particular, Acosta-Silva et al.17 showed in this manner that methylation enhances stacking interactions, and can produce local distortions in base-pair step parameters, most notably slide. Yusufaly et al. used similar calculations to show that methylation can induce over-twisting as well as softer modes for distortion from the global energy minimum18. We recently employed classical mechanics to examine not only the structure but also the flexibility of different DNA sequences with methyl and hydroxymethyl substituents19. Through use of extended molecular dynamics (MD) simulations, we showed that structural effects are subtle, but that epigenetic modifications can give rise to changes in twist, roll and tilt angles that are markedly sequence-dependent. Moreover, introduction of 5-mC within a sequence that already contains hydrophobic groups in the major groove strongly affects hydration patterns, whereas an isolated 5-mC has a lesser effect on solvation and structure.\n\nIn this work, we use DFT and QM/MM methods to examine model systems containing modified cytosines. These range from individual base pairs, through double-stranded trimers, to heptamers. By including the sugar-phosphate backbone, sodium counterions and solvent we suggest that these are more realistic models than previous work using similar methods. However, a trimer of DNA brings us close to the size limit for application of DFT with the computing resources available to us. We therefore test and employ hybrid QM/MM methods for larger systems, in which the central bases are treated with dispersion-corrected DFT, while outer bases, sugar-phosphate backbone and solvent (where appropriate) with a molecular mechanics approach, thus allowing accurate and efficient description of systems consisting of hundreds of atoms.\n\n\nComputational methodology\n\nThe initial structures of model systems were built in the canonical B-DNA geometry, using the w3DNA server20. Hydrogen atoms were added to the system according to expected protonation states at physiological pH using the Molecular Operating Environment (MOE) software package, and Na+ were added manually in the vicinity of each phosphate group to produce an overall neutral structure. Where relevant, the central cytosine was also manually modified, and the results of all simulations were analysed using the X3DNA software package21,22. Atomic coordinates of wild-type, methylated and hydroxymethylated DNA dodecamers were obtained from X-ray structures deposited in the Protein Data Bank (PDB IDs: 1BNA, 4GJU, 4GLG, 4GLH and 4GLC)23, and truncated to 5´-ATTCGCG-3´ heptamers containing a single modification on the central C. All DNA termini were capped with methyl groups for simplicity.\n\nAll DFT calculations were performed with the Gaussian09 simulation package20, using Grimme’s B97-D functional24, that includes an explicit correction for the missing dispersion term in conventional DFT functionals, with either def2-TZVP or 6-31+G(d,p) basis set. This was previously recommended after thorough benchmarking for thermochemistry, kinetics, and non-covalent interactions25. All such calculations took advantage of the density fitting approximation, and where appropriate included the effect of aqueous solvation via the use of the polarized continuum model (PCM)26. Binding energies are corrected for the effects of basis set superposition error using the counterpoise method27.\n\nHybrid QM/MM calculations were performed using the ONIOM approach with electrostatic embedding28, as implemented in Gaussian09. The boundary between the quantum and classical regions was chosen as the N-C1’ glycosidic bond in the relevant nucleotide. The QM regions were saturated by the use of a “link” hydrogen atom placed along the N-C1’ vector at an idealized distance, and were modelled at the B97-D/6-31+G(d,p) level of theory, again within PCM water. The MM part of these calculations employed the AMBER force field parm9629, as defined within Gaussian09. The subtractive nature of the ONIOM method means that undefined terms in the MM expression do not contribute to the overall energy if the relevant atoms are entirely within the QM region, making it ideally suited for the purposes of the current study. We note that this approach has been widely adopted for QM/MM studies of DNA and related structures30–32. Pure molecular mechanics (MM) geometry optimisation was also performed using the GROMACS simulation package33 and the AMBERParmbsc0 force field34, including RESP charges derived for modified bases in our previous work19, in explicit aqueous phase, specifically TIP3P water35 with Na+ and Cl- counter ions to create a neutral system.\n\n\nResults and discussion\n\nTo examine the effect of modifications on base pairing we examined the structure and energy of gas-phase CG pairs in both hydrogen bonded and stacked orientations, with results reported in Table 1 and Table 2 respectively. These data show that methylation has little effect on the geometry or stability of the Watson-Crick base pair. The presence of a hydroxymethyl slightly weakens the N4-H4…O6 H-bond, perhaps due to the proximity of CH2OH and NH2 groups, reported as X…H4 in Table 1. Formyl has a larger effect overall, lengthening N3…H1-N1 and O2…H2-N2 H-bonds and hence reducing binding by over 3 kcal/mol. The pattern of changes induced by carboxylate is different from all other modifications, lengthening the peripheral H-bonds N4-H4…O6 and O2…H2-N2 markedly, but shortening N3…H1-N1. Despite this weakening, the carboxylate-substituted cytosine binds most strongly to guanine, presumably due to ion-dipole interactions within the anionic system. Both formyl and carboxylate contain close O…H4 contacts, but overall the proximity of these groups does not appear to be related to strength or geometry of binding.\n\na X refers to the atom of the substituent on position 5 closest to H4.\n\na Cent…Cent refers to the distance between centroids of 6-membered rings; Dihedral refers to the angle between mean planes of rings.\n\nAs well as the effect on H-bonding, epigenetic modifications can alter the stacking behaviour of DNA bases. Table 2 reports geometrical details, as well as binding energies, of the five modified cytosines considered here stacked with guanine. All such calculations started from the idealised B-DNA orientation (Cent…Cent = 4.390 Å, Dihedral = 4.9°), and overall this is retained in our gas-phase DFT optimisation. Table 2 shows that methylation leads to closer contact and greater stabilisation between bases, as might be expected due to the increased polarizability of this modified base. Hydroxymethylation leads to the most stable pair considered here, largely due to a strong H-bond between the H—O of hydroxymethyl and O6 of guanine (H…O = 1.770 Å), whereas formylation leads to longer, weaker interaction between bases. Carboxylate-substituted cytosine is the only case considered here that loses the approximately parallel orientation of bases. This appears to be driven as much by repulsion between the carboxylate group and C=O6 of guanine as by H-bonding.\n\nWhile these gas-phase dimers give useful information on the intrinsic effect of modifications on cytosine’s ability to interact with guanine, environmental effects including the DNA sequence, sugar-phosphate backbone and solvent will play a major role in determining their effect in real systems. In order to better simulate the behaviour of modified cytosines in real systems, structures of double-stranded d(GCG) and d(ACA), as well as epigenetic modifications to the central cytosine were optimized using DFT in continuum solvent (PCM), and the resulting geometries of the local base pairs were analysed in the coordinate frame recommended by Olson et al.36. Unlike the free dimers considered above, modifications have only subtle effects on this larger structure, which retains the overall canonical B-DNA shape of the unmodified WT structure.\n\nFollowing Zubatiuk et al.37, we summarise key aspects of trimer structure, which are displayed graphically in Figure 2 and Figure 3. The corresponding values are tabulated in Table S1 of the Supporting Information, with the base step and local helical parameters tabulated in Table S2. As with Zubatiuk et al.14, base pair step parameters are averaged over 3´ and 5´ directions. In the GCG oligomer, methylation has only a small effect on base pair distances, but does alter the propeller angle by over 4°. Hydroxymethylation has a larger effect on the GCG oligomer, especially on the stagger, buckle and propeller, whereas the stretch and opening parameters are much less affected. Formyl does not strongly affect base pair distances but does change angles substantially, especially buckle and propeller, which change by as much as 10°. In contrast, carboxylate induces a large change in stagger but only small changes in angular geometry. Base pair step parameters for d(GCG) in general are less affected than those for the base pair noted above, with the exception of formyl which exhibits smaller slide and less negative roll values than unmodified DNA.\n\nThe corresponding data are provided in Table S1.\n\nThe corresponding data are provided in Table S1.\n\nRather larger changes are evident on modification of d(ACA), as shown in Figure 3. In this case, even methylation induces significant changes in distances, especially stagger which increases by 0.1 Å, and angles (buckle and propeller change by 8 and 13°, respectively). At the base pair step level, methylation gives rise to substantial increase (0.9 and 1.5 Å) in slide and more negative roll in both 3´ and 5´ directions. Less apparent in Figure 3, but still notable, are changes in rise that are 0.1 and 0.3 Å smaller in the methylated structure, reflecting the greater stacking that results from addition of a methyl group. Other modifications induce different patterns of structural change: for the central base pair these changes are typically smaller than for methylation, but for base pair steps much larger changes are found in some parameters. Most notable of these are slide, which changes by over 3 Å and roll (up to 17°) in the 3´ direction, in a similar way to that reported previously for smaller systems17,18. Other parameters such as the width of the DNA strand, measured as the distance between C1´ nuclei, and virtual angles λY and λR, which describe the pivoting of complementary bases in the base-pair plane, vary only slightly from the idealised values for B-DNA.\n\nThe oligomers considered so far are close to the limit of our computational capabilities of current DFT methods (the largest structure, carboxylated d(ACA), has 962 electrons in 2743 basis functions), such that longer sequences cannot currently be routinely studied in this manner. However, they are too small to correctly represent how DNA behaves in a real system, where the conformations adopted by each base pair step depend on the neighbouring step. Moreover, simulations of nucleic acids are known to suffer problems due to greater elasticity of the terminal part of the structure (the so called “end-effect”38). For these reasons, these small oligomers are inadequate models to probe the effects of epigenetic modifications on the structure of DNA. We therefore turn to hybrid QM/MM methods, in which a subset of the atoms in the system is treated with DFT, and the remainder of the system with much faster molecular mechanics methods. In order to test the validity of this approach, methylated GCG was optimized using either only two or six bases in the QM region (Figure 4). These tests show that including only two bases in the QM region leads to significant differences in geometry to that obtained from DFT, particularly in the stagger and buckle coordinates. In contrast, including six bases in the QM region reproduces the DFT structure reasonably well. Similar observations were made from analogous treatment of methylated ACA (data not shown).\n\nThe corresponding values are shown in Tables S3 and S4.\n\nAs a further test, we also compared DFT and QM/MM derived structures with those optimised using the force field parameters developed in our previous work. Figure 5 shows the base-pair parameter values of the methylated structure d(GC´G) for the different methods. The MM structures provide very close values to those obtained by both QM/MM and DFT approaches, showing slight difference only in the stagger and propeller angle. We can therefore conclude that for small DNA oligomers, DFT, QM/MM and MM methods can all produce almost equally adequate DNA structures, but that QM/MM and MM approaches are more similar to one another than those obtained from DFT alone.\n\nThe corresponding values are shown in Tables S3 to S6.\n\nQM/MM geometry optimization with six bases in the QM region was then applied to a set of larger DNA sequences. The experimental structure of Renčiuk et al.4 obtained using X-ray diffraction (PDB Entry 4GLG) was truncated to a sequence of 7 base pairs, i.e. 5´-ATT CGCG-3´, and the central 6 bases (TCG//CGA) assigned as QM atoms. The remaining atoms, including crystallographic water molecules and counterions, were assigned to the MM layer, and the entire system was geometry optimized. The resulting optimized structure of the system with methylated C in the central position is shown in Figure6. Base pair and base pair step geometries of wild type, methylated, hydroxymethylated structures optimised with QM/MM, along with experimental values for methylated C, are shown in Figure 7.\n\nA purple sphere highlights the methylation position, and water molecules and counterions have been omitted for clarity.\n\nThe corresponding values are shown in Table S2.\n\nWe find that the structural effect of methylation is larger in this longer sequence than in the trimers considered above. Particularly, the optimized values of shear, stagger and buckle of the central base pair differ markedly between the methylated and WT forms of DNA. In contrast, the base pair step parameters exhibit rather smaller changes. For the hydroxymethylated structures, we observe similar profiles to the methylated structures. Furthermore, our simulations also allow us to probe the preferred orientation of the hydroxymethyl group: our DFT calculations predict a slight preference for the OH group to point in 3´ over 5´ and an optimized of C6-C5-C5A-O5 torsion angle of 118.4°, while previous MD simulations show this torsion to vary between 85 and 120° over 100 ns of simulation19. This is in good agreement with the experimental and theoretical results of Renčiuk et al.39, who reported values between 72 and 133° using X-ray diffraction methods.\n\n\nConclusions\n\nThrough use of modern, dispersion-corrected DFT and hybrid QM/MM methods, we have examined the structural consequences of epigenetic modifications of DNA. Concentrating on methylation and related modifications of cytosine, we show that the overall Watson-Crick base-pairing is retained, with rather small changes to hydrogen bond and stacking geometries. Despite this, some modifications have a substantial effect on the strength of intermolecular interactions: hydroxymethyl and formyl groups reduce H-bonding strength, while carboxylate increases this markedly.\n\nSituating these modifications within the double-stranded DNA trimers GCG and ACA allows us to examine the effects on the central CG base pair and base pair steps. Base pair geometries undergo rather larger changes within ACA than in GCG, with changes in buckle and propeller angles particularly apparent. Changes to base pair steps are smaller, although some changes in shift and slide values due to modifications are evident. Optimised geometries also act as a useful test of hybrid QM/MM methods. These can reproduce DFT structures if all six bases are included in the QM region, but if only the central base pair is treated with QM significant differences result. This approach is then applied to heptamers derived from a recent X-ray crystallography; here again, the central base pair is found to be significantly disrupted, whereas base pair step parameters are largely retained.\n\nThe studies reported here deal solely with static structures, but it is well-known that DNA is a flexible system that is in constant motion at biologically relevant temperatures. In previous work, we showed that long timescale molecular dynamics was able to highlight subtle differences in structure, flexibility and solvation resulting from incorporation of 5-mC and 5-hC in several different DNA sequences. The work reported here gives new insight into the intrinsic effects of epigenetic modification of cytosine, complementing our previous molecular dynamics study19 as well as providing support for the molecular mechanics force field chosen for that work.\n\n\nData availability\n\nFigshare: Data of theoretical modelling of epigenetically modified DNA sequences. Doi: 10.6084/m9.figshare.131044840", "appendix": "Author contributions\n\n\n\nSKTSW, JP and SCLK conceived the study. AC, JP and SCLK designed the research. AC, LG and CRK contributed to design of the research. JP carried out the research. JP and SCLK prepared the first draft of the manuscript. All authors analysed data and contributed to preparation of the manuscript. All authors were involved in the revision of the draft manuscript, and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Swedish Research Council (Vetenskapsrådet, 2010-5026) and funding from the Sven and Ebba Christina Hagbergs Foundation.\n\nWe confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nJAP is grateful to Advanced Research Computing @ Cardiff (ARCCA) for use of computing facilities.\n\n\nReferences\n\nKorlach J, Turner SW: Going beyond five bases in DNA sequencing. Curr Opin Struc Biol. 2012; 22(3): 251–61. PubMed Abstract \| Publisher Full Text\n\nColot V, Rossignol JL: Eukaryotic DNA methylation as an evolutionary device. Bioessays. 1999; 21(5): 402–11. 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Data Source" }	[ { "id": "7803", "date": "27 Mar 2015", "name": "Célia Fonseca Guerra", "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\nThis paper presents an interesting theoretical studty on epigenetically modified DNA. Legend of Figure 1: Please include the numbering, so that non-experts can follow the rest of the text. Page 4: “The presence of a hydroxymethyl slightly weakens the N4-H4...O6“ Is there an internal hydrogen bond that is competing with the N4-H4•••O6 hydrogen bond? Please explain.Page 4 “Formyl has a larger effect overall, lengthening N3...H1-N1 and O2...H2-N2 H-bonds and hence reducing binding by over 3 kcal/mol. ” This can easily be understood because N3 and O2 become less negative due to the electron withdrawing effect. See Chem. Eur. J. 2006, 12: 3032-3042, Chem. Eur. J. 1999, 5: 3581-3594 and Chem. Eur. J. 2011, 17: 8816-8818 and use these publications to explain these effects on the hydrogen bonds. The epigenetic modifications can be considered to be substituent effects and therefore the changes in the hydrogen bonds can be easily explained.Table 1: What are the hydrogen bonds lengths meant here? N4•••O6 or H4•••O6. The preference would be N4•••O6.", "responses": [ { "c_id": "1304", "date": "06 May 2015", "name": "Lynn Kamerlin", "role": "Author Response F1000Research Advisory Board Member", "response": "We again thank the reviewer for the time taken to referee the manuscript. Please see our point-by-point response below.Legend of Figure 1: Please include the numbering, so that non-experts can follow the rest of the text.Numbering has been added to Figure 1. Page 4: “The presence of a hydroxymethyl slightly weakens the N4-H4...O6“ Is there an internal hydrogen bond that is competing with the N4-H4•••O6 hydrogen bond? Please explain.The OH group of hydroxymethyl is found to lie close to H4, but the lengths reported in Table 1 put this “contact” outside typical ranges of N-H…O hydrogen bonds, such that we prefer not to refer to a hydrogen bond, but rather the proximity of groups. Page 4 “Formyl has a larger effect overall, lengthening N3...H1-N1 and O2...H2-N2 H-bonds and hence reducing binding by over 3 kcal/mol. ” This can easily be understood because N3 and O2 become less negative due to the electron withdrawing effect. See Chem. Eur. J. 2006, 12: 3032-3042, Chem. Eur. J. 1999, 5: 3581-3594 and Chem. Eur. J. 2011, 17: 8816-8818 and use these publications to explain these effects on the hydrogen bonds. The epigenetic modifications can be considered to be substituent effects and therefore the changes in the hydrogen bonds can be easily explained.We completely agree that these trends can be understood as substituent effects, and have therefore added both text to reflect this and the suggested references to the relevant section of the Results and Discussion. Table 1: What are the hydrogen bonds lengths meant here? N4•••O6 or H4•••O6. The preference would be N4•••O6.H-bond lengths are reported as H…Y, since the alternative depends on angular geometry of the X-H…Y system. In any case, full coordinates have been deposited as Supporting Information in case interested parties wish to extract X…Y distances." } ] }, { "id": "8191", "date": "17 Apr 2015", "name": "Katja Petzold", "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 presents a study of different simulation methods to investigate different modification of dCMp in base pairs or dsDNA and their influence of the surrounding structure. Disparate statement in the abstract – please modify/clarify: “The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects” versus “but these leave the overall helical shape of DNA essentially intact.” larger changes but DNA shape of helix intact? Please clarify statement in introduction: “Fascinatingly, such modifications may be heritable despite not affecting DNA sequence, although the mechanism(s) by which this could be achieved are currently unknown.” In respect to enzymes (e.g. DNA methyltransferase) known to transfer methylation from parent to daughter strands? Please enhance figures for clarity.A: Fig 1: numbering of atoms, full name of modifications, example GC WC base pair geometry and info on parameters “role, rise twist etc…”.B: Fig. 2-5 & 7: please keep coherent direction of sequences e.g. GC/GC (5’-3’/5’-3’) in Fig. 2 vs Fig. 4 GC/CG, or coherent naming of modification: Fig. 2 – no indication which nucleotide is modified, Fig. 4: C’, Fig. 5: (5-mC), if mis-understood – please clarify.C: Please give more detail in each of the Figure caption (e.g. construct, reference structure – Fig. 7).D: Fig. 7: what is the reference “along with experimental values for methylated C” – are the values shown here the X-ray structure values or the X-ray Structure values optimized with QM/MM for the WT? – if it is the optimized data, than I suggest to add the experimental data uncorrected as well. It is difficult to estimate the significance of the changes in structural parameters between different cytosine modifications or different simulation methods, as there are no errors/standard deviations are presented. I would suggest using a set of X-ray/NMR structures with the same sequence and/or modifications to create a standard deviation for the different parameters to give the analysis more significance (than I can estimate if a difference of 0.03Å is of importance or not: “Formyl has a larger effect overall, lengthening N3…H1-N1 and O2…H2-N2 H-bonds and hence reducing binding by over 3 kcal/mol.” Difference in h-bond length from wtC is 0.025Å and 0.049Å, respectively – seems very small, but if all GC wc bp are within of 0.01Å distance, this would be significant).Important for: Table 1&2 – as well important for Fig. 2-5 & 7, please adjust. Describe structural/distortion findings in structure/sketches. E.g.: “largely due to a strong H-bond between the H—O of hydroxymethyl and O6 of guanine (H…O = 1.770 Å)” for a better understanding of how the structures are supposed to look like. Formality: p5 first sentence “Following Zubatiuk et al.37,” should be “Following Olson et al.37,” OR “Following Zubatiuk et al.14,” More information on the Methods and Materials would be appreciated: E.g. How extensive where the simulations/optimizations? What were the energy cutoffs?...", "responses": [ { "c_id": "1303", "date": "06 May 2015", "name": "Lynn Kamerlin", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank the referee for the time taken to review our manuscript. Please find a point-by-point response below, with our responses italicised. Disparate statement in the abstract – please modify/clarify: “The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects” versus “but these leave the overall helical shape of DNA essentially intact.” larger changes but DNA shape of helix intact? We do not see these statements as contradictory: we show that there are indeed substantial changes in H-bonding and stacking interactions, but that these are not sufficient to disrupt the overall helical structure. We have, however, now explicitly included this in the abstract to prevent reader confusion. Please clarify statement in introduction: “Fascinatingly, such modifications may be heritable despite not affecting DNA sequence, although the mechanism(s) by which this could be achieved are currently unknown.” In respect to enzymes (e.g. DNA methyltransferase) known to transfer methylation from parent to daughter strands? This is certainly one key mechanism, but this is not the place to discuss in detail the biology of epigenetics, which is covered at length in references cited. We have made this point more explicit in the introduction and refer the reader to reference 1 for further information about currently proposed mechanisms. Please enhance figures for clarity. The figures have been modified as outlined below and we hope the improved version is now clearer to the reader.A: Fig 1: numbering of atoms, full name of modifications, example GC WC base pair geometry and info on parameters “role, rise twist etc…”.Numbering has been added to Figure 1, as has a representation of CG base pair. Roll, rise, twist etc. are widely used in DNA studies and should not need re-definition here.B: Fig. 2-5 & 7: please keep coherent direction of sequences e.g. GC/GC (5’-3’/5’-3’) in Fig. 2 vs Fig. 4 GC/CG, or coherent naming of modification: Fig. 2 – no indication which nucleotide is modified, Fig. 4: C’, Fig. 5: (5-mC), if mis-understood – please clarify.The legend for figures 2 to 4 has been altered to explain that central C has been modified.C: Please give more detail in each of the Figure caption (e.g. construct, reference structure – Fig. 7).Legend for Figure 7 has been expanded to clarify source of data.D: Fig. 7: what is the reference “along with experimental values for methylated C” – are the values shown here the X-ray structure values or the X-ray Structure values optimized with QM/MM for the WT? – if it is the optimized data, than I suggest to add the experimental data uncorrected as well.This was an oversight from a previous draft: Figure 7 does not contain experimental data, and this has been removed from the manuscript. Inclusion of further data from experiment would make this figure too cluttered and difficult to read. It is difficult to estimate the significance of the changes in structural parameters between different cytosine modifications or different simulation methods, as there are no errors/standard deviations are presented. I would suggest using a set of X-ray/NMR structures with the same sequence and/or modifications to create a standard deviation for the different parameters to give the analysis more significance (than I can estimate if a difference of 0.03Å is of importance or not: “Formyl has a larger effect overall, lengthening N3…H1-N1 and O2…H2-N2 H-bonds and hence reducing binding by over 3 kcal/mol.” Difference in h-bond length from wtC is 0.025Å and 0.049Å, respectively – seems very small, but if all GC wc bp are within of 0.01Å distance, this would be significant). It is indeed difficult to estimate the significance of changes in geometry: these static DFT and QM/MM calculations do not yield standard deviations. It would indeed be interesting to extract experimental information to estimate variability across structures, but this would be a whole new project, and is therefore out of the scope of the present work.Important for: Table 1&2 – as well important for Fig. 2-5 & 7, please adjust.As outlined above, we do not have suitable data with which to adjust these tables and figures. Describe structural/distortion findings in structure/sketches. E.g.: “largely due to a strong H-bond between the H—O of hydroxymethyl and O6 of guanine (H…O = 1.770 Å)” for a better understanding of how the structures are supposed to look like. We have added a figure for this structure to supporting information, and stress that all optimised coordinates have been deposited should readers wish to assess further detail. Formality: p5 first sentence “Following Zubatiuk et al.37,” should be “Following Olson et al.37,” OR “Following Zubatiuk et al.14,”We thank the referee for spotting this error, and have corrected it to Following Zubatiuk et al.14,” More information on the Methods and Materials would be appreciated: E.g. How extensive where the simulations/optimizations? What were the energy cutoffs?... All DFT and QM/MM calculations used Gaussian09 default convergence criteria for SCF calculation and geometry optimisation: a statement to this effect has been added to the methods section. Details of MM calculations are identical to those from our previous work (ref 19): again, a statement has been added to this effect." } ] } ]	1	https://f1000research.com/articles/4-52
https://f1000research.com/articles/4-106/v1	06 May 15	{ "type": "Data Note", "title": "Genomic resources of two landsnail, Aegista diversifamilia and Dolicheulota formosensis, generated by Illumina paired-end sequencing", "authors": [ "Chih-Wei Huang", "Wen-Lung Wu", "Wen-Lung Wu" ], "abstract": "Despite the land snail harboring high biodiversity and dominance on land, just a few genetic markers are available for phylogeographic and phylogenetic research. We sequenced the partial genome of two land snail species that belong to the speciose family Bradybaenidae in East Asia: Aegista diversifamilia and Dolicheulota formosensis. The raw sequences were generated by Illumina paired-end sequencing and can be accessed in the Sequence Read Archive under the accession numbers SRR1918809 (A. diversifamilia) and SRR1920140 (D. formosensis).", "keywords": [ "Helicoidea", "Next-generation sequencing", "Pulmonata", "Stylommatophora" ], "content": "Introduction\n\nStylommatophora is the dominant gastropod clade on land. The family Bradybaenidae is mainly distributed in Asia and diversified in East Asia. However, only a handful of genetic markers are available for phylogenetic research. We selected two land snail species as representatives of Bradybaenidae: Aegista diversifamilia Huang et al., 2014 and Dolicheulota formosensis (H. Adams, 1866). We subjected these species to Illumina paired-end sequencing in the hope that this will expand and provide valuable genetic resources for phylogeographic and phylogenetic research of gastropods.\n\n\nMaterials and methods\n\nGenomic sequences were generated from one fresh specimen preserved at -20°C (A. diversifamilia) and one two-year-old ethanol-preserved specimen (D. formosensis), respectively. The living individual of A. diversifamilia was collected from leaf litter under lowland broadleaf forest in Xiulin Township, Hualien County, Taiwan in October 2013. D. formosensis was collected from the tree trunk of a lowland broadleaf forest in Mudan Township, Pingtung County, Taiwan in June 2012. Snails were brought back to laboratory and rinsed with ddH2O to eliminate soil and other particles attached to the shell. The living individual of A. diversifamilia was preserved in a -20°C refrigerator. The individual of D. formosensis was relaxed under water for 12 hours and fixed by blanching with boiling water for several seconds. The specimen of D. formosensis was transferred to 95% ethanol solution and the ethanol was changed every 24 hours for 48 hours. The specimen of D. formosensis was finally preserved in 95% ethanol.\n\nFor A. diversifamilia, the shell was removed after the snail was crushed. Soft tissue was rinsed with TEK buffer (50 ml 1M Tris-HCl pH 7.5, 50 ml 0.2M EDTA pH 7.5, 15g KCl, pH 7.5) and then used for genomic DNA extraction with AxyPrep™ Multisource Genomic DNA Miniprep Kit (Axygen Bioscience) following the manufacturer’s protocol. For D. formosensis, partial foot tissue was rinsed with ddH2O several times to remove ethanol and then used for DNA extraction through a modified phenol-chloroform method (Jiang et al., 1997).\n\nThe quantity of extracted DNA solution was evaluated by Qubit® dsDNA HS Assay Kit (Life Technologies). The DNA concentration was 131 ng/μL (total mass 20.96 μg) and 52.9 ng/μL (5.92 μg) for A. diversifamilia and D. formosensis, respectively. The DNA solution was send to BGI (Shenzhen, China) for paired-end library construction with insert size of approximately 500 base pairs using a Paired-End DNA Sample Prep Kit (Illumina). Sequencing was performed using Illumina HiSeq2000 in BGI. Raw sequences data can be accessed in Sequence Read Archive under accession number SRR1918809 for A. diversifamilia and SRR1920140 for D. formosensis.\n\n\nEthics policies\n\nEthical approval for the animals used was not required. Both species used in this study were common and not under list of protected species in Taiwan.", "appendix": "Author contributions\n\n\n\nAll authors were involved in experimental design, manuscript preparation and approval of the final version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was partially supported by the grant to Wen-Lung Wu from the Center for Information Technology Innovation and Biodiversity Research Center, Academia Sinica.\n\n\nAcknowledgments\n\nWe would like to thank Genomics BioSci & Tech (Taiwan) for the assistance of experimental design. Thanks for BGI (Shenzhen, China) for the service of sequencing.\n\n\nReferences\n\nAdams H: Descriptions of fifteen new species of land and freshwater shells from Formosa, collected by Robert Swinhoe, Esq., consul at Taiwan in that island. Proceedings of Zoological Society of London. 1866; 1866: 316–319. Reference Source\n\nHuang CW, Lee YC, Lin SM, et al.: Taxonomic revision of Aegista subchinensis (Möllendorff, 1884) (Stylommatophora, Bradybaenidae) and a description of a new species of Aegista from eastern Taiwan based on multilocus phylogeny and comparative morphology. ZooKeys. 2014; 445: 31–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang L, Wu WL, Lin YS: Efficient methods for isolating mitochondrial DNA from fresh or fixed molluscan specimens. Zoological Studies. 1997; 36(1): 74–78. Reference Source" }	[ { "id": "8910", "date": "22 Jun 2015", "name": "Juan Opazo", "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\nThis data note is reporting massive amount of genetic data for two landsnail species, with the aim that this resource will serve as a platform to develop phylogenetic and phylogeographic makers. The goal of this note is well justified given that for one of the genera, Dolicheulota, there are no NCBI entries, whereas for the other the diversity of them is low. Samples were sequenced using standard protocols in a facility that has experience in sequencing protocols. I am convinced that this data will be useful to the scientific community, especially to those interested in the evolutionary history of gastropods.", "responses": [] } ]	1	https://f1000research.com/articles/4-106
https://f1000research.com/articles/4-104/v1	01 May 15	{ "type": "Research Note", "title": "In silico analysis suggests repurposing of ibuprofen for prevention and treatment of EBOLA virus disease", "authors": [ "Veljko Veljkovic", "Marco Goeijenbier", "Sanja Glisic", "Nevena Veljkovic", "Vladimir R. Perovic", "Milan Sencanski", "Donald R. Branch", "Slobodan Paessler", "Marco Goeijenbier", "Sanja Glisic", "Nevena Veljkovic", "Vladimir R. Perovic", "Milan Sencanski", "Donald R. Branch", "Slobodan Paessler" ], "abstract": "The large 2014/2015 Ebola virus outbreak in West Africa points out the urgent need to develop new preventive and therapeutic approaches that are effective against Ebola viruses and can be rapidly utilized. Recently, a simple theoretical criterion for the virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection was proposed. Using this method the ‘drug space’ was screened and 267 approved and 382 experimental drugs as candidates for treatment of the Ebola virus disease (EVD) have been selected. Detailed analysis of these drugs revealed the non-steroidal anti-inflammatory drug ibuprofen as an inexpensive, widely accessible and minimally toxic candidate for prevention and treatment of EVD. Furthermore, the molecular mechanism underlying this possible protective effect of ibuprofen against EVD is suggested in this article.", "keywords": [ "drug space", "NSAID", "molecular libraries" ], "content": "Introduction\n\nThe recent Ebola virus outbreak in West Africa caused (as of April 8, 2015) a total of 25,556 confirmed cases, including 10,587 deaths (World Health Organization, Ebola data and statistics). Although reports of new, confirmed cases of Ebola seemed to decrease in April of 2015 to approximately 30 cases per week, medical and aid organizations are warning that the current crisis is not over. Furthermore, there remains a risk for future epidemics. Therefore, there is an urgent need to develop new preventive and therapeutic approaches that can be rapidly utilized. New drugs for the treatment of EVD should be safe, efficacious, easy to manufacture and inexpensive in order to be successfully deployed in African countries. In contrast to the significant progress recently achieved in development of an effective Ebola virus vaccine1, therapeutic options are still limited2. The main obstacle represents identification of an appropriate therapeutic target, which has been largely hampered by the time and money-consuming development of new drugs, especially for neglected diseases. Furthermore, the registration of newly identified drugs, like favipiravir, for potential usage in EVD patients takes a relatively long period of time, even though it has been shown to be effective in non-human primate models. In order to avoid these obstacles, several approaches for repurposing of approved drugs for the treatment of EVD patients have been proposed in literature3–7.\n\nRecently, we proposed a relatively simple theoretical criterion for the fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection8. Using this criterion, which is based on calculation of the average quasi-valence number (AQVN) and the electron-ion interaction potential (EIIP) - parameters determining long-range interaction between biological molecules9–13 - we selected 267 approved and 382 experimental drugs as candidates for treatment of EVD. Further detailed analysis of these drugs, including molecular docking, revealed ibuprofen as an inexpensive, widely accessible and minimally toxic candidate for potential prevention and treatment of EVD. The molecular mechanism underlying the possible protective effect of ibuprofen against EVD is suggested.\n\n\nMaterial and methods\n\nRecently, we proposed a theoretical criterion for selection of candidate inhibitors of Ebola virus infection8. This criterion is based on the calculation of EIIP and AQVN which are determined by following equations:\n\n\n\nwhere:\n\ni - Type of the chemical element\n\nZi - Valence of the i-th chemical element\n\nni - Number of the i-th chemical element atoms in the compound\n\nm - Number of types of chemical elements in the compond\n\nN - Total number of atoms\n\n\n\nThe EIIP values calculated according to the equation (2) are in Rydbergs (Ry = 13.6 eV).\n\nThe AQVN and EIIP molecular descriptors determine the long-distance (>5Å) intermolecular interactions in biological systems14. This approach showed that molecules which potentially block Ebola virus infection are placed within AQVN range (2.3–2.7) and EIIP range (0.829–0.954 Ry), respectively. Using this theoretical criterion the drug library encompassing 267 approved drugs selected as candidate inhibitors of the Ebola virus infection (Candidate Ebola Drugs Database, CEDD) was established15. This drug library was used for selection of an approved drug, which represents the optimal candidate for prevention and treatment of EVD.\n\nThe modelling of glycoprotein GP1 from Ebola virus was previously described in 16.\n\nLigands were built in VEGA ZZ17, protonated according to physiological conditions and optimized on semi empirical PM6 level of theory using MOPAC 2009.\n\nLigand and receptor were prepared in VEGA ZZ17. The docking was carried out with Autodock Vina (version 1.1.2.)18. In both cases the whole receptor conformational space was searched, using grid boxes with dimensions 60×60×60 and 30×30×30Å3. The docking was carried out with weighting of hydrophilic interactions, and the corresponding parameter value in the docking configuration was set to -1.20 (compared to default weight: hydrogen = -0.587439). The exhaustiveness was set to 250. The conformations with lowest binding energy values were chosen. The calculations were carried out on the PARADOX Cluster computer.\n\n\nResults and discussion\n\nWe screened the CEDD library15 for optimal candidates for prevention and treatment of EVD. To achieve this end, we used the following criteria: a) high efficacy of binding of drug to the Ebola virus glycoprotein GP1; b) low toxicity and accessibility (nonprescription drugs); and c) low cost. By mining of CEDD using these criteria, we selected ibuprofen as the best candidate. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID). Its mode of action, as for other NSAIDs, is not completely understood, but may be related to prostaglandin synthetase inhibition. It achieves this effect on prostaglandin synthesis by inhibiting cyclooxygenase (COX I and II isotypes), an enzyme that is present in at least one isoform most of the tissues of the body and which is responsible for production of not only prostaglandins but also prostacyclins and tromboxane.\n\nGenerally, ibuprofen is minimally toxic drug. A meta-analysis of eight placebo-controlled studies showed that application of non-prescription doses of ibuprofen (800–1200 mg/day) over 10 days caused no significant adverse events19. Similar results were obtained in a prospective study of ibuprofen use in healthy volunteers taking the maximum permitted, non-prescription dose of ibuprofen (1200 mg/day) for 10 days20. It was demonstrated that administration of ibuprofen in children is safe. A randomized, community-based study on the safety of ibuprofen in febrile children aged 2 years old (5 or 10 mg/kg) showed that the risk of hospitalization for gastrointestinal bleeding associated with this drug was 17 per 100,00021. It was also shown that fecal blood loss associated with application of prescription doses of ibuprofen (2400 mg/day) did not exceed the normal range22. Even in the case of post-surgical usage for pain control in children undergoing tonsillectomy, ibuprofen administration is considered to be safe and does not increase risk of hemorrhages23. Accordingly, we believe that it would be crucial to better understand the potential risk and benefits of ibuprofen usage in experimental models of EVD. Taken together, these data indicate that ibuprofen could be safely used in nonprescription doses in EVD patients, with potential antiviral effects as well as to alleviate the symptoms.\n\nTo assess specificity of ibuprofen as a potential inhibitor of the Ebola virus infection, distribution of all approved NSAIDs in AQVN/EIIP space was analyzed. Results presented in (Table 1 and Figure 1) show that only ibuprofen and its isomer dexibiprofen are located within the domain of the AQVN/EIIP space. Interestingly, most of the experimentally verified inhibitors of the Ebola virus infection are located in the same space (e.g. chloroquine, amodiaquine, brincidofovir, etc)8. This suggests that ibuprofen and dexibuprofen are the only drugs from the NSAID group that potentially have anti-Ebolavirus effects, which should be tested both in vitro and in vivo.\n\nDomain of inhibitors of Ebola virus infection8 is marked in red.\n\nA previous in silico study suggested that Elastin Microfibril Interface Located Proteins (EMILINs) are involved in interaction between GP1 and endothelial extracellular matrix (ECM)16. Docking of ibuprofen to the GP1 model gave conformations which bound to EMILINs binding domain on GP116. The binding site of ibuprofen on GP1 spans the edge of this region and consists of Thr 338, Ser 340, Gln 344 and Ala 41516. The intermolecular interactions between ibuprofen and binding site amino-acids include hydrogen bonds of carboxyl group with Thr 338, Ser 340 and Gln 334. Additional stabilization is provided through aromatic and hydrophobic interaction with Ala 415. This binding site is placed between two loops, which provide the possibility of stabilizing a particular conformation, and therefore possibly blocking receptors. The appropriately high binding energy of -9.0 kcal/mol favors this assumption. The binding conformation is presented in Figure 2. At this stage it can be hypothesized that ibuprofen prevents interaction between Ebola virus and ECM by blocking the interaction between GP1 and EMILIN. There are some literature data that support our current hypothesis. EMILIN-1 is a glycoprotein expressed in the vascular tree that binds to the TGF-β1 precursor and prevents its processing by cellular protease furin24. It was shown that Emilin-1 knockout mice display increased TGF-β1 signaling in the walls of their blood vessels, leading to peripheral vasoconstriction and arterial hypertension25. These matrix-dependent changes in the vascular hemodynamics caused by TGF-β1 and EMILIN-1 are important because they ultimately affect the cardiovascular morbidity and mortality rates. Recently, it was shown that activation of the TGF-β1 signaling pathway by Ebola virus plays an important role in pathogenesis of EVD26. These findings suggest the possibility that binding of GP1 to EMILIN-1 prevents its interaction with TGF-β1, which results in activation of TGF-β1 signaling pathway. Binding of ibuprofen to GP1 could prevent GP1/EMILIN-1 interaction allowing EMILIN-1 to keep control of TGF-β1 signaling pathway.\n\nGreen dotted lines: hydrogen bonds; grey: hydrophobic interactions.\n\nIn conclusion, presented results should encourage further investigation of ibuprofen and ibuprofen-inspired drugs as inexpensive, low-toxic and wide-accessible candidates for prevention and its usage in the treatment of EVD.\n\n\nData availability\n\nF1000Research: Dataset 2. Approved and experimental drugs selected as candidate for treatment of EVD, 10.5256/f1000research.6110.d4287715", "appendix": "Author contributions\n\n\n\nVV, SP and MG conceived and designed the study. VP developed the analysis tools. VV, SG, NV, MS and DB analyzed the data. VV, SP and MG wrote the paper. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant no. 173001).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAgnandji ST, Huttner A, Zinser ME, et al.: Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe - Preliminary Report. N Engl J Med. 2015. PubMed Abstract \| Publisher Full Text\n\nOlszanecki R, Gawlik G: Pharmacotherapy of Ebola hemorrhagic fever: a brief review of current status and future perspectives. Folia Med Cracov. 2014; 54(3): 67–77. PubMed Abstract\n\nMadrid PB, Chopra S, Manger ID, et al.: A systematic screen of FDA-approved drugs for inhibitors of biological threat agents. PLoS One. 2013; 8(4): e60579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKouznetsova J, Sun W, Martínez-Romero C, et al.: Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerg Microb Infect. 2014; 3: e84. Publisher Full Text\n\nEkins S, Freundlich JS, Coffee M: A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus [v2; ref status: indexed, http://f1000r.es/4wt]. F1000Res. 2014; 3: 277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLitterman N, Lipinski C, Ekins S: Small molecules with antiviral activity against the Ebola virus [v1; ref status: indexed, http://f1000r.es/523]. F1000Res. 2015. 4: 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkins S, Coffee M: FDA approved drugs as potential Ebola treatments [v2; ref status: indexed, http://f1000r.es/554]. F1000Res. 2015. 4: 48. PubMed Abstract \| Publisher Full Text\n\nVeljkovic V, Loiseau PM, Figadere B, et al.: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection [v1; ref status: indexed, http://f1000r.es/51s]. F1000Res. 2015; 4: 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeljkovic V, Veljkovic N, Este J, et al.: Application of the EIIP/ISM bioinformatics concept in development of new drugs. Curr Medic Chem. 2007; 14(4): 441–53. PubMed Abstract \| Publisher Full Text\n\nVeljkovic V, Mouscadet JF, Veljkovic N, et al.: Simple criterion for selection of flavonoid compounds with anti-HIV activity. Bioorg Medic Chem Lett. 2007; 17(5): 1226–32. PubMed Abstract \| Publisher Full Text\n\nVeljkovic N, Glisic S, Perovic V, et al.: The role of long-range intermolecular interactions in discovery of new drugs. Exp Opin Drug Disc. 2011; 6(12): 1263–70. PubMed Abstract \| Publisher Full Text\n\nMaga G, Veljkovic N, Crespan E, et al.: New in silico and conventional in vitro approaches to advance HIV drug discovery and design. Exp Opin Drug Discov. 2013; 8(1): 83–92. PubMed Abstract \| Publisher Full Text\n\nVeljkovic N, Glisic S, Prljic J, et al.: Simple and general criterion for “in silico” screening of candidate HIV drugs. Curr Pharm Biotechnol. 2013; 14(5): 561–9. PubMed Abstract \| Publisher Full Text\n\nVeljkovic V: A theoretical approach to preselection of carcinogens and chemical carcinogenesis. Gordon & Breach New York. 1980. Reference Source\n\nVeljkovic V, Loiseau PM, Figadère B, et al.: Dataset 2 in: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Res. 2015. Data Source\n\nVeljkovic V, Glisic S, Muller CP, et al.: In silico analysis suggests interaction between Ebola virus and the extracellular matrix. Front Microbiol. 2015; 6: 135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedretti A, Villa L, Vistoli G: VEGA--an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming. J Comput Aided Mol Des. 2004; 18(3): 167–173. PubMed Abstract \| Publisher Full Text\n\nTrott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010; 31(2): 455–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKellstein DE, Waksman JA, Furey SA, et al.: The safety profile of nonprescription ibuprofen in multiple-dose use: a meta-analysis. J Clin Pharmacol. 1999; 39(5): 520–32. PubMed Abstract\n\nDoyle G, Furey S, Berlin R, et al.: Gastrointestinal safety and tolerance of ibuprofen at maximum over-the-counter dose. Aliment Pharmacol Ther. 1999; 13(7): 897–906. PubMed Abstract \| Publisher Full Text\n\nLesko SM, Mitchell AA: The safety of acetaminophen and ibuprofen among children younger than two years old. Pediatrics. 1999; 104(4): e39. PubMed Abstract\n\nPorro GB, Corvi G, Fuccella LM, et al.: Gastro-intestinal blood loss during administration of indoprofen, aspirin and ibuprofen. J Int Med Res. 1977; 5(3): 155–60. PubMed Abstract\n\nYaman H, Belada A, Yilmez S: The effect of ibuprofen on postoperative hemorrhage following tonsillectomy in children. Eur Arch Otorhinolaryngol. 2011; 268(4): 615–7. PubMed Abstract \| Publisher Full Text\n\nRaman M, Cobb MH: TGF-beta regulation by Emilin1: new links in the etiology of hypertension. Cell. 2006; 124(5): 893–5. PubMed Abstract \| Publisher Full Text\n\nZacchigna L, Vecchione C, Notte A, et al.: Emilin1 links TGF-β maturation to blood pressure homeostasis. Cell. 2006; 124(5): 929–42. PubMed Abstract \| Publisher Full Text\n\nKindrachuk J, Wahl-Jensen V, Safronetz D, et al.: Ebola virus modulates transforming growth factor β signaling and cellular markers of mesenchyme-like transition in hepatocytes. J Virol. 2014; 88(17): 9877–92. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8913", "date": "05 Jun 2015", "name": "Mattia Mori", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the work of Velijkovic et al., in silico methods were used with the aim of identifying inhibitors of Ebola virus infection. Particularly, authors focused their investigation on approved and experimental drugs and proposed ibuprofen as a candidate for prevention and treatment of Ebola virus. It is well known that Ebola virus represents an international health emergency and that there is an urgent need for therapeutics and prophylactic drugs.In the present manuscript, authors applied a previously described and well-established criterion, based on EIIP and AQVN calculation, to select ibuprofen as the best candidate for Ebola virus disease treatment. Moreover, a mechanism of action consisting on GP1 inhibition has been proposed.It is the opinion of the present referee that some minor issues should be fixed before the manuscript may be considered as acceptable for indexation:Since molecular docking of ibuprofen was performed against the putative target GP1, it would be important to report the predicted binding energy (in kcal/mol) or score, and to compare this value with estimated or experimental affinity of reference GP1 ligands. Moreover, in a repurposing perspective, it would be interesting if authors could comment on the approximated dosage of ibuprofen they expect for providing anti-Ebola activity. Ibuprofen has a serum half-life of 1.8 to 2.0 hours. Did authors consider this aspect when claiming that ibuprofen could be also administered to patients to prevent Ebola virus disease. Liver and gastrointestinal tissues are among the most injured by Ebola virus infection. Moreover, it is well known that ibuprofen can cause serious gastrointestinal toxicity and should be used with caution in person with coagulation defects. Did authors considered ibuprofen side effects in the context of Ebola infection treatment? Following the question 1., a comment on the expected dosage would help to clarify most of these issues. It is not clear how ibuprofen can alleviate the symptoms of Ebola virus disease, also considering the proposed mechanism of action.", "responses": [ { "c_id": "1430", "date": "23 Jun 2015", "name": "Thomas Hoenen", "role": "Reader Comment", "response": "With respect to reviewer comment #3 it is worth pointing out that Medicines Sans Frontieres, the WHO, the CDC, and other health organizations specifically advise AGAINST the use of Ibuprofen and other NSAIDs due to the risk of adverse effects. This is an important point that should, in my opinion, be addressed by the authors.See also:http://www.ammi.ca/media/69846/Ebola%20Clinical%20Care%20Guidelines%202%20Sep%202014.pdfhttp://www.slamviweb.org/es/ebola/FHFfinal.pdfwww.cdc.gov/vhf/ebola/ppt/ebola-101-cdc-slides-for-us-healthcare-workers.pptxThe short version:http://www.who.int/csr/disease/ebola/photos/ebolablock-poster15.jpg" }, { "c_id": "1532", "date": "24 Aug 2015", "name": "Veljko Veljkovic", "role": "Author Response", "response": "Response to Mattia Mori It was demonstrated that several host proteins interact with GP1. This indicates (i) that more than one receptor/co-receptor is involved in the Ebola virus infection and (ii) that entry inhibitors have different mode of actions because they could bind different targets. All experimentally proven entry inhibitors of Ebola virus are selected in vitro and their exact binding site on GP or receptor/co-receptor is not known. For these reasons, there are no “reference GP ligands” whose binding properties could be compared with ibuprofen.Without initial experimental data any suggestion of dosage of ibuprofen in treatment of EVD would be highly speculative because the low toxicity of this drug allows its application in a very broad dosing range (100 – 2400 mg per day). Although the plasma half-life of ibuprofen is short (1.9 to 2.2 hours), without experimental data it is not possible to predict antiviral effect of the residual concentration of this drug. Fourteen to twenty-four hours after administration of single dose of 800 mg of ibuprofen, the plasma level of this drug and its metabolites is < 250 ng/mL. For comparison, after administration of the therapeutic dose of 300 mg of Maraviroc, the plasma concentration of this HIV entry inhibitor after 10 hours is <100 ng/mL (Abel et al., 2009). Application of ibuprofen in the early stage of disease should be safe because hemorrhagic effect of EVD appears in the late stage of the illness. This is confirmed by the safe use of ibuprofen in the treatment of Ebola patients in Sierra Leone during the 2014/2015 outbreak without side effects (Ansumana et al., 2015). We proposed that ibuprofen acts as an entry inhibitor preventing spread of virus in the body of Ebola patients (like HIV entry inhibitor Maraviroc which prevents spread of virus in the body of HIV patients). Response to reader’s comment (Thomas Hoenen) Despite the recommendation of WHO, CDC and some other organizations against ibuprofen in therapy of EVD, this drug was safely used in treatment of EVD patients in Sierra Leone during 2014/2015 Ebola outbreak (Ansumana et al. 2015)." } ] }, { "id": "8796", "date": "23 Jun 2015", "name": "Ayub Darji", "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\nCurrent manuscript by Veljkovic et al., deals with the identification of inhibitors against Ebola virus infection. Authors took advantage of existing approved and experimental drugs and, based on in silico analysis, proposed ibuprofen as a potential candidate for prevention and treartment of Ebola virus infection. EIIP and AQVN calculation, previously described by authors, was employed to define ibuprofen as the best candidate against Ebola virus infection.The manuscript is adequately presented and can be considered as acceptable for indexation.Minor points:Ebola virus infection is initiated by interaction between the virus glycoprotein GP1 and its cognete receptor(s). Putative receptor binding domain of GP1, key residues involved in GP1 protein folding or structure and a putative receptor binding pocket has been proposed and mapped to the N-terminal 150 amino acids of GP1 (33-185 residues).Authors should include in the discussion as how interaction of ibuprofen with GP1 (residues, Thr 338, Ser 340, Gln 344 and Ala 415) will influence the receptor binding domain.", "responses": [ { "c_id": "1531", "date": "24 Aug 2015", "name": "Veljko Veljkovic", "role": "Author Response", "response": "It was previously suggested that Elastin Microfibril Interface Located Proteins (EMILINs) are involved in interaction between GP and endothelial extracellular matrix (ECM) (Veljkovic et al. 2015) The binding site of ibuprofen on GP overlaps with the proposed domain of GP involved in GP/EMILINs interaction. This suggests that ibuprofen could modulate the virus/ECM interaction." } ] } ]	1	https://f1000research.com/articles/4-104
https://f1000research.com/articles/3-187/v1	11 Aug 14	{ "type": "Method Article", "title": "The ACCE method: an approach for obtaining quantitative or qualitative estimates of residual confounding", "authors": [ "Eric G. Smith" ], "abstract": "Background: Nonrandomized studies typically cannot account for confounding from unmeasured factors. Method: A method is presented that exploits the recently-identified phenomenon of “confounding amplification” to produce, in principle, a quantitative estimate of total residual confounding resulting from both measured and unmeasured factors. Two nested propensity score models are constructed that differ only in the deliberate introduction of an additional variable(s) that substantially predicts treatment exposure. Residual confounding is then estimated by dividing the change in treatment effect estimate between models by the degree of confounding amplification estimated to occur, adjusting for any association between the additional variable(s) and outcome. Results: A hypothetical example is provided to illustrate how the method produces a quantitative estimate of residual confounding if the method’s requirements and assumptions are met. Previously published data is used to illustrate that, whether or not the method routinely provides precise quantitative estimates of residual confounding, the method appears to produce a valuable qualitative estimate of the likely direction and general size of residual confounding. Limitations: Uncertainties exist, including identifying the best approaches for: 1) predicting the amount of confounding amplification, 2) minimizing changes between the nested models unrelated to confounding amplification, 3) assessing the association of the introduced variable(s) with outcome, and 4) deriving confidence intervals for the method’s estimates (although bootstrapping is one plausible approach). Conclusions: To this author’s knowledge, it has not been previously suggested that the phenomenon of confounding amplification, if such amplification is as predictable as suggested by a recent simulation, provides a logical basis for estimating total residual confounding. The method's basic approach is straightforward. The method's routine usefulness, however, has not yet been established, nor has the method been fully validated. Rapid further investigation of this novel method is clearly indicated, given the potential value of its quantitative or qualitative output.", "keywords": [ "Confounding is a central challenge for virtually all nonrandomized studies. Recent research1–4 has revealed that propensity score methods may actually increase", "or “amplify”", "the residual confounding remaining after their application. In general", "this recently recognized property of propensity score methods has been viewed as a limitation or complication to the use of propensity scores", "for understandable reasons. More recently", "however", "a study has indicated that the degree of confounding amplification (also termed “bias amplification”4) occurring between propensity score models appears to be quantitatively predictable (at least in simulation)5. Not yet recognized", "to my knowledge", "is the extremely valuable corollary that results: the predictability of confounding amplification should", "in principle", "permit extrapolation back to an unamplified value of the total residual confounding originally present prior to amplification. (Throughout this manuscript “confounding” refers to baseline confounding. Confounding occurring after treatment initiation from differential discontinuation of the intervention in the treatment group and comparison group is not addressed", "but some consideration is given to post-initiation confounding and a possible related approach to addressing to its estimation is briefly discussed in Appendix 1.3.b). In this manuscript and the associated appendices", "I describe the general framework and detailed specifics of a new method designed to use amplified confounding to estimate total residual confounding and an unconfounded treatment effect estimate." ], "content": "Introduction\n\nConfounding is a central challenge for virtually all nonrandomized studies. Recent research1–4 has revealed that propensity score methods may actually increase, or “amplify”, the residual confounding remaining after their application. In general, this recently recognized property of propensity score methods has been viewed as a limitation or complication to the use of propensity scores, for understandable reasons. More recently, however, a study has indicated that the degree of confounding amplification (also termed “bias amplification”4) occurring between propensity score models appears to be quantitatively predictable (at least in simulation)5. Not yet recognized, to my knowledge, is the extremely valuable corollary that results: the predictability of confounding amplification should, in principle, permit extrapolation back to an unamplified value of the total residual confounding originally present prior to amplification. (Throughout this manuscript “confounding” refers to baseline confounding. Confounding occurring after treatment initiation from differential discontinuation of the intervention in the treatment group and comparison group is not addressed, but some consideration is given to post-initiation confounding and a possible related approach to addressing to its estimation is briefly discussed in Appendix 1.3.b). In this manuscript and the associated appendices, I describe the general framework and detailed specifics of a new method designed to use amplified confounding to estimate total residual confounding and an unconfounded treatment effect estimate.\n\nThe basic logic of this method is straightforward, but its performance in practice has yet to be confirmed. Testing of this method on both simulated and real-world data is clearly needed. Under specific circumstances, this method may theoretically provide a quantitative estimate of total residual confounding, including from unmeasured factors. Whether and how often this is attainable in practice remains to be determined. This manuscript also illustrates, however, that even when this method is not able to provide a precise quantitative estimate of residual confounding, it may provide a very helpful qualitative estimate of the likely direction and general size of residual confounding. This manuscript is intended to provide detailed information to the research community to facilitate the rapid evaluation of the practical feasibility of this proposed approach.\n\n\nMethod\n\nThe “Amplified Confounding-based Confounding Estimation (ACCE) Method” depends on the use of two propensity score models, one (“Model 1”) nested in the other (“Model 2”) so that Model 2 contains all the Model 1 covariates plus an additional variable or variables. Importantly, these added variable(s) should be sufficiently associated with treatment exposure to produce discernible confounding amplification. That is, the variables introduced to the model should further predict treatment exposure sufficiently to substantively increase differences between the treatment groups in the prevalences of those confounding factors that are not present in the either model.\n\nIn principle, the original confounding existing prior to amplification can be estimated by extrapolation backwards if the proportional amount of confounding amplification and quantitative change in the treatment effect occurring between two propensity score models can be estimated with precision. For example, 2-fold confounding amplification that changed the observed treatment effect odds ratio (OR) from 1.10 in Model 1 to 1.21 in Model 2 (a difference in coefficients of 0.09531) would imply that residual confounding initially existed in Model 1 at such a magnitude as to entirely explain the initial, Model 1 treatment effect (β = 0.09531, or approximately OR = 1.10). (Please see Endnote A, provided at the end of the manuscript, for more detail). That is, doubling the residual confounding doubled the observed treatment effect estimate, implying all the original treatment effect estimate was due to residual confounding. Attention is needed during the method’s implementation, however, to ensure that changes between the two models distinct from confounding amplification are minimized to the extent feasible (Appendix 1).\n\nThe method requires an ability to estimate the proportional amount of confounding amplification occurring between two propensity score models. Two very different approaches suggest themselves. One approach would be to estimate amplification from existing or future simulation research based on particular metrics of exposure prediction. An example of this approach is research published5 using the linear measure of exposure prediction, R2. This work demonstrated that, for propensity score stratification or matching approaches, a linear relationship exists between unexplained variance in exposure and confounding amplification across the range of R2 = 0.04 to 0.56. This simulation study5, using a propensity score based on a linear probability model, also made the important demonstration that different unmeasured confounders appear to be amplified to a highly similar degree. Whether this is true in real-world datasets, or is simply a byproduct of this simulation, clearly merits further investigation. (Further discussion is provided in Appendix 2.2). Additional research is clearly needed to determine if a similarly predictable relationship exists for other metrics of exposure prediction (such as those proposed for logistic regression6,7), and whether apparent nonlinearities between the prediction of exposure and confounding amplification at more extreme ranges of prediction5 can be addressed quantitatively.\n\nA second approach would be to adopt an “internal marker” strategy: deliberately withholding a measured covariate from both models to allow the increase in its imbalance between treatment groups in Model 2 to serve as an approximate indicator of the proportional confounding amplification that has occurred. It is possible, however, that the “internal marker” strategy might consistently yield at least a slight degree of underestimate of the amount of confounding amplification (Appendix 2.1).\n\nA key assumption of this ACCE method is that residual confounding attributable to different confounders is uniformly or relatively uniformly amplified in Model 2 compared to Model 1. This important characteristic has been observed in the initial simulation that this method draws upon5, but some possibility still exists that the quantitative predictability of amplification that was observed may be merely a consequence of the particular conditions of this simulation (Appendix 2.2).\n\nThe addition of a variable(s) to Model 2 will almost always alter the amount of residual confounding present compared to Model 1, independent of its effect producing confounding amplification (i.e., true instrumental variables are rare). A challenge arises in that it is not the total residual confounding in Model 1 (the quantity being sought) that is amplified in Model 2, only the fraction of that residual confounding that remains after introduction of the introduced variable. Because of this, adjustments are needed that reflect the confounding attributable to the introduced variable. However, to estimate total residual confounding through this method, such adjustments must occur to two quantities: 1) the change in the treatment effect estimate between Model 1 and Model 2, and 2) the Model 1 treatment effect estimate.\n\nTo make these adjustments, I propose obtaining coefficients for the introduced variable from regression models of the outcome that include all other propensity score covariates. (Please see Endnote B for more detail). This regression coefficient for the introduced variable may be biased by partially reflecting the associations with outcome of those unmeasured confounders that are correlated with the introduced variable. However, the adjustment that is needed at this step of the Method needs to reflect both the change resulting from both the improved balance in the introduced variable and from the less extensive changes in the balance of correlated variables that result. To the extent that correlations between the introduced variable and unmeasured confounders produce biases in the introduced variable-outcome association that are similar in size to the amount of increased balance occurring in these covariates, an adjustment that partially reflects the unmeasured covariates could actually be advantageous. The degree of similarity in how correlation affects the introduced variable-outcome association compared to how such correlation affects the balance between treatment groups for the correlated variables is currently uncertain. This is an area worthy of further research.\n\nOnce this introduced variable regression coefficient(s) is estimated, the Bross equation8 is used to estimate the confounding attributable to the introduced variable(s) and its correlates in both Model 1 and Model 2. (The Bross equation8, which recently has been used by Schneeweiss and colleagues in their high-dimensional propensity score algorithm9, quantifies the amount of confounding attributable to a confounder by combining the strength of the association between the covariate and outcome and the imbalance in the covariate between the treatment groups. Please see the demonstration of its use in Appendix Table 1). The amount of such confounding in Model 1 is then subtracted from the amount in Model 2 to produce an estimate of the amount of the change in the treatment effect estimate between Model 1 and Model 2 that is attributable to increased balance in the introduced variable(s) and its correlates. This estimate then is subtracted from the overall treatment effect estimate change from Model 1 to Model 2 to produce the quantity being amplified (the residual confounding in Model 1 separate from the introduced variable). (Please see Endnote C for more detail).\n\nThe degree to which this step functions successfully to separate the effect of confounding amplification from any change in the treatment effect estimate attributable directly to the improved balance in the introduced variable has yet to be determined, especially if the introduced variable is correlated with other uncontrolled confounders. However, the theoretical potential to perform the proposed adjustment suggests that this method possibly might provide a quantitatively or qualitatively accurate estimate of an unconfounded treatment effect in circumstances in which instrumental variable analysis may not be possible. At a minimum, the method may prove to provide a relatively accurate estimate of an unconfounded treatment effect in the special case in which the introduced variable is suspected to be largely uncorrelated with important unmeasured confounders. Stated in other words, unlike instrumental variable analysis, it is possible that associations between the exposure-predicting introduced variables and outcome simply complicate, but do not preclude, the use of the method. Further research, however, is clearly needed to determine whether this is the case.\n\nThe final step involves two substeps. First, divide the result from Step 3 (the change in the treatment effect estimate from Model 1 to Model 2, adjusted to remove the change produced by increased balance in the introduced variable(s) and its correlates) by the amount of confounding amplification. This calculation derives by extrapolation an estimate of the total residual confounding in Model 1 except for the confounding attributable to the yet-to-be-introduced variable(s). Finally, subtract both that extrapolated estimate of residual confounding and the confounding attributable to the yet-to-be-introduced variable from the Model 1 treatment effect estimate. (Please see Endnote C for more detail). The result is, in general principle, an estimate of the unconfounded treatment effect.\n\nThe accuracy of this estimate, however, is not yet established. The largest uncertainty in this estimate, as discussed above, likely involves the accuracy of the adjustments proposed in Steps 3 and 4 in the context of unmeasured confounders correlated with the introduced variable. In addition, the consistent predictability of confounding amplification needs to be further established. The degree to which other differences between the models can be sufficiently minimized to prevent them from biasing the quantitative estimate of confounding amplification also deserves investigation.\n\nOther research needs include: 1) determining whether random variability particularly reduces the method’s usefulness in smaller samples; 2) developing a methodology, such as bootstrapping, to estimate the variance for the final effect estimates; and 3) investigating whether multiple variables can be introduced together if needed to produce sufficient amplification. Nevertheless, the potential significance of a method that may produce estimates of total residual confounding and unconfounded treatment effects from nonrandomized studies should spur research into the method’s feasibility.\n\n\nResults\n\nConsider an example in which the (confounded) Model 1 treatment effect estimate equals OR = 1.265 (with an R2 of 0.25), the (confounded) Model 2 treatment effect estimate equals OR = 1.2985 (with an R2 of 0.50), the introduced variable has an association of approximately OR = 1.05 with outcome, an 80% prevalence in the treated group and 20% prevalence in the comparison group in Model 1, and a 52% prevalence the treated group and 48% prevalence in the comparison group in Model 2. (This example assumes a linear propensity score model but a logistic regression outcome model. Please see Endnote E for more detail). What is observed is an increase in the treatment effect estimate away from the null in Model 2. This change away from the null occurs despite tight control in Model 2 (but not Model 1) of a variable (the “introduced variable”) that is not only highly predictive of exposure but is also, to some degree, a confounder that would have been expected to have biased the treatment-outcome association at least modestly away from the null in Model 1. This suggests, in the absence of confounding amplification, that the tight control of this covariate in Model 2 would ordinarily result in a less biased treatment effect estimate moving towards, not away from, the null. Furthermore, given the mere 1.5-fold amplification of confounding that would be expected to result (0.75 remaining variance unexplained in Model 1 versus 0.50 variance remaining unexplained in Model 2, or 0.75/0.50 = 1.5), the fact that this modest confounding amplification is sufficient to move the treatment effect estimate away from the null despite tight control of a confounder with an OR = 1.05 implies that a substantial proportion of the Model 1 effect estimate is attributable to confounding (biasing away from the null). Specifically, these findings would imply that more than half of the original, sizeable “treatment effect” estimate (OR = 1.265) was attributable to residual confounding, and would suggest a genuine unconfounded treatment effect estimate of only OR = 1.10. (Please see Supplementary Table 1 for complete calculations).\n\nThus, despite the fact that the treatment effect estimates for Model 1 and Model 2 are both confounded, knowledge of the amount of expected confounding amplification allows the comparison of the effect estimates of models (with appropriate adjustments) to yield an estimate of an unconfounded treatment effect.\n\nThe study of Patrick et al.10 provides sufficient detail to fortuitously provide a partial opportunity to test some aspects of the ACCE methodology on real-world data. Obviously, this study was not constructed to illustrate the ACCE Method; therefore it is being used post hoc to explore the potential of the method. As a result, the data provided include several additional uncertainties beyond those that would accompany a deliberate implementation of the ACCE Method. However, by permitting an examination of the performance of even a partial version of the ACCE Method, this study illustrates the potential value this method may have as a probe indicating whether substantial residual confounding is likely (and its likely direction), even in circumstances in which a firm quantitative estimate of residual confounding is not able to be derived.\n\nPatrick et al.10 derived a substantial number of propensity scores during their analyses of the association between statins and both all-cause mortality and hip fracture outcomes. Of note, two of the propensity scores used (for both outcomes) included an important pair in which one propensity score was nested within a slightly larger propensity score identical to the original propensity score except for the addition of a single covariate (glaucoma diagnosis). Glaucoma diagnosis was considered to be a potential instrumental variable in these analyses. First, glaucoma diagnosis was associated extremely strongly with treatment exposure (since the treatment group compared to statins for both analyses consisted of users of medications for glaucoma). Patients with a glaucoma diagnosis had an odds ratio for statin exposure of 0.07 (that is, patients with glaucoma diagnosis had approximately a 14× greater odds of being in the comparison treatment group than the statin treatment group). Second, it is plausible (although not provable) that glaucoma diagnosis lacks a substantial association with the outcomes of all-cause mortality and hip fracture, and thus may be functioning as an instrumental variable or near-instrumental variable. (Although not termed an “instrumental variable” originally10, such a term was used for glaucoma diagnosis in these analyses in a subsequent manuscript describing these findings11).\n\nPatrick et al.10 reported both effect estimates and a measure of prediction (the c statistic) for the original (“Model 1”) model and after adding the “introduced variable” (i.e., glaucoma diagnosis) (“Model 2”). This permits an examination of the valuable qualitative findings that might result even when the ACCE Method is unable to produce a precise quantitative estimate of residual confounding. In this somewhat artificial case, the partial version of the ACCE Method that can be implemented is unlikely to produce precise quantitative estimates of residual confounding for several reasons, including the fact that the relationship of the c statistic to confounding amplification has yet to be explored, unlike the relationship between R2 and confounding amplification. In addition, the partial version of the method that can be implemented does not include the possible checks of model similarity in confounding control, patient sample, and intervention delivered (e.g., dose) described in Appendix 1. Of particular importance, this partial, illustrative version of the method does not include any adjustment to account for the association of the introduced variable (glaucoma diagnosis) with outcome (using information estimated from a full multivariate regression containing the other propensity score covariates). This lack of adjustment somewhat limits this example, since even a small association with outcome of a covariate with such an imbalance in prevalence between the treatment groups may contribute substantively to overall confounding. In fact, the manuscript notes that the minimally-adjusted hazard ratio (HR) for glaucoma diagnosis (adjusted for age, age2, and sex) is >1.175 or <1/1.175 for both outcomes. (The actual age and-sex-adjusted HR observed is HR≈0.85 for both outcomes [Amanda Patrick, Personal Communication]). What is lacking, however, is the glaucoma diagnosis HR adjusted for all the covariates in the propensity score model, rather than just age and sex. (This adjustment would involve including a total of 143 covariates for the mortality analysis and 120 covariates for the hip fracture analysis10). This fully-adjusted HR would provide information about whether or not the age and sex-adjusted glaucoma diagnosis HR might be related to aspects of care-seeking, care access, health attitudes, or other factors that might be also represented by other covariates (leaving a much lesser or close-to-null association for glaucoma diagnosis in the actual analysis). Most importantly, this fully-adjusted association would provide the quantity needed to help calculated the estimate of the unconfounded treatment effect estimate for Model 1 (Steps 3 and 4 of the method).\n\nDespite the limitation of not having a fully-adjusted regression coefficient for the glaucoma diagnosis-outcome association, as well as the other substantial limitations mentioned above, application of even this highly partial version of the ACCE Method appears to provide useful qualitative estimates of residual confounding for these two analyses (all-cause mortality and hip fracture).\n\nTable 1A shows that in the all-cause mortality analyses, addition of the introduced variable (glaucoma diagnosis) moves the treatment effect estimate away from the null by a modest amount. This implies that the total residual confounding (including residual confounding from unmeasured factors) likely biases, but only very modestly, towards observing a larger effect size for statins than is genuinely present. This result is consistent with the effect estimate derived from available randomized data. In contrast, Table 1B shows that in the hip fracture analyses, addition of the same introduced variable changes the observed treatment effect HR from 0.76 to 0.69. This is a much more sizeable change in the treatment effect estimate, implying a larger quantity of underlying residual confounding biasing the estimate away from the null. If glaucoma diagnosis is in fact a near-instrumental variable, the results would imply that the unconfounded hip fracture treatment effect estimate is considerably closer to null, the approximate value that the authors expect to be the genuine treatment effect based on randomized data12.\n\nA. Nested Models differing by single variable with observed strong association with exposure and expected minimal association with outcome.\n\na Reference 10, Table 2 and Discussion.\n\nb Reference 10, Results section text (4th paragraph).\n\nc For residual confounding not to be modest (relative to treatment effect estimate) either 1) the introduced variable would have to have a substantial association with increased mortality risk. (This seems rather unlikely, since the age and sex-adjusted HR is in the protective direction [HR ≈ 0.85; M. Patrick, personal communication], but cannot be rigorously excluded), or 2) the amplification would have to be distinctly minor (e.g., approximately 1.25×). It is assumed here that amplification from the c statistic occurs in similar fashion as with R2 in the simulation of Reference 5; that is, that the change in the remaining unexplained variance of exposure predicts amplification. This has not been established for the c statistic (and it is generally appreciated that the c statistic is not a very desirable metric for comparisons between models). Nevertheless, while we do not know the amplification precisely, amplification would appear to have to be much less than that observed by Reference 5 in similar ranges of exposure prediction using R2 for the partial ACCE method applied here to predict a large amount of residual confounding in this analysis. Furthermore, whatever the amplification is, it is likely to be highly similar between Table 1A and Table 1B. Thus, the conclusion concerning the relative amount of unmeasured confounding in the all-cause mortality compared to the hip fracture analyses given in Table 1B is likely to be valid (as long as the fully-adjusted glaucoma diagnosis association does not differ markedly for the two outcomes).\n\nB. Nested Models differing by single variable with observed strong association with exposure and expected minimal association with outcome.\n\na Reference 10, Table 2 and Discussion.\n\nb Reference 10, Results section text (4th paragraph).\n\nc Given that the all-cause mortality and hip fracture analyses have propensity score c statistics suggesting highly similar predictions of exposure, seemingly the only likely plausible scenario by which the all-cause mortality analysis could be more confounded than the hip fracture analysis is if the introduced variable of glaucoma diagnosis has a substantially stronger protective association with outcome after control for the other propensity score covariates than the association between glaucoma diagnosis and all-cause mortality. Since these results are not available (i.e., results from an extensive multivariate regression), such a possibility cannot be rigorously excluded. Some difference might even be plausible given that considerably less is known about the predictors of hip fracture (and what is known may be less represented in healthcare databases) than for all-cause mortality. It can be inferred, however, that the magnitude of this difference would need to be substantial for the ACCE Method to suggest that the hip fracture analysis is less confounded than the all-cause mortality analysis. In an actual implementation of the ACCE method, highly-adjusted multivariate regression of the introduced-variable-outcome association would be conducted involving all or many (if the number of outcomes did not permit all the covariates to be simultaneously included) of the propensity score covariates.\n\nEven if glaucoma diagnosis is not functioning as a near-instrumental variable, as long as the full multivariate regression coefficients for glaucoma diagnosis are even somewhat similar between the models, these two analyses considered together suggest the presence of considerably more residual confounding in the hip fracture analysis than the all-cause mortality analysis. (Please see Endnote F for more details). This is a conclusion independently suggested by the randomized trial meta-analyses12,13 cited by the authors. That is, based on the differences between the propensity score findings compared to the randomized trial meta-analyses (i.e., the hip fracture HR differed much more from previous randomized findings than the all-cause mortality HR), the general supposition would be that the hip fracture analysis is likely to be considerably more confounded than the all-cause mortality analysis. The ACCE Method, even when applied in a very partial and qualitative form, suggests the same conclusion. In this fashion, the ACCE Method may prove useful for estimating at least the likely general size and direction of residual confounding in the many circumstances where substantial randomized trial data is not available to guide one’s interpretation. This capacity of the method to provide even a qualitative estimate of residual confounding may constitute an important analytic advance.\n\n\nDiscussion\n\nThis paper presents a relatively straightforward four-step method exploiting the phenomenon of confounding amplification to potentially provide quantitative estimates of total confounding and unconfounded treatment effects. To my knowledge, it has not been previously recognized that the phenomenon of confounding amplification, if predictable (as suggested by recent simulation5), provides a potential mechanism to estimate total residual confounding. The fundamental approach of deliberately introducing amplified confounding into an analysis to evaluate the total residual confounding existing prior to amplification appears to possess both clear logic and considerable promise. The method hinges on part on whether the recently observed predictability of confounding amplification is found to be a generally observed phenomenon; in addition, at this stage it is unclear whether the method will need particularly large sample sizes to be routinely useful in providing quantitative estimates. Nevertheless, although aspects of the method’s implementation and precise accuracy are not yet fully resolved, further research is clearly indicated given the potential value of a new approach that may advance efforts to remove confounding from nonrandomized treatment effect estimates.\n\nFurthermore, even if subsequent research determines that the estimates from this approach typically are sufficiently imprecise as to limit the quantitative usefulness of the method, this general approach may have considerable value as a semi-quantitative or qualitative “probe” of whether a substantial amount of residual confounding likely exists. It is hoped that the description of the method provided here is sufficient to permit the larger research community to immediately begin participating in the validation and refinement of this novel approach.\n\nThe ACCE method is fundamentally a conceptually simple approach, but one that may require some care in its implementation (e.g., in the need to structure the two models so as to minimize other changes that might influence the treatment effect estimate while obtaining sufficient confounding amplification). The value of this method will depend on how often in practice it provides a useful quantitative or qualitative estimate of residual confounding. Answering this question will involve more detailed and precise examination of both simulated and real-world data, and almost certainly will involve the contributions of multiple research teams.\n\nUseful avenues for validation research likely include: 1) the predictability of the relationship between a particular metric measuring prediction of exposure and confounding amplification and/or the potential substitutability of an “internal marker” as an alternative approach; 2) approaches to, or circumstances that would, ensure other changes between the models (in patient sample, intervention received, and the degree of control achieved for measured, included confounders) are minimized; 3) confirming that multivariate regressions provide an accurate measure of the change in confounding resulting from balancing of the introduced variable in Model 2 (and thus permits adjustment for the direct and indirect contributions of the introduced variable(s) to confounding in Model 1); 4) determining how easily multiple introduced variables can be used if a single introduced variable does not produce sufficient confounding amplification; and 5) determining whether sufficiently precise results can be routinely obtained from the ACCE Method despite the effects of random variability in treatment effect estimates, since this method requires the accurate detection of what may be fairly small changes in treatment effect estimates. It may prove that, for this reason, this method may be most useful when applied to particularly large databases; however, some recent studies using propensity score-based stratification do suggest that quite subtle changes in relative risk or hazard ratio from application of slightly different propensity score models can be detected9,10. Finally, an obvious need exists for methodology to develop confidence limits around the effect estimates emerging from the ACCE method. The procedure of bootstrapping would be one obvious candidate approach.\n\nSimulation studies, given that the genuine treatment-outcome association is able to be specified by the investigator, may be the most immediate approach to addressing these research needs and evaluating the performance of this method in general. (Such simulations would be similar to the recent simulation study initially observing that confounding amplification may be predictable5, and others that have considered the impacts of unmeasured confounding13,14). Real-world studies might investigate whether the method appears to accomplish the task of making results from nonrandomized studies better parallel results from randomized trials15.\n\nRegardless of its ultimate precision, this method may prove beneficial for nonrandomized comparative effectiveness research in general, as well as especially beneficial for studies in which substantial residual or unmeasured confounding is expected. For example, many studies of mental health and/or behavioral interventions might be expected to have substantial unmeasured confounding, since the important elements of the conversation between provider and patient that contributes to judgments of the severity of the patient’s condition and helps influence treatment decisions often may go unrecorded even in the patient’s chart, and thus becomes unmeasurable.\n\nAnother notable use would be to enhance medication surveillance efforts. By providing even a highly approximate estimate of unmeasured confounding in a few simple steps, the ACCE Method could help more accurately indicate which prominent “signals” (either in effectiveness or safety) observed during the screening of large datasets appear to be less confounded (and thus are a particular priority for further investigation).\n\n\nConclusions\n\nThis paper has outlined a relatively straightforward yet novel method to potentially obtain a quantitative estimate of total residual confounding. This total residual confounding estimate (which would include confounding from unmeasured as well as measured factors) then allows, in principle, for an estimate of unconfounded treatment effects to be calculated. This paper has described the steps involved in applying this method, offered a very preliminary examination of the performance of a simple, partial version of this method using published data, and outlined research needs for refinement and validation of this method. Given the importance of a method that may potentially help remove confounding from nonrandomized treatment effect estimates, further investigation of this method by multiple research groups is clearly warranted. Even if the ACCE method is eventually shown to have limitations or evolves from the form proposed here, the method’s general approach of deliberately amplifying confounding to reveal existing residual confounding may have enduring analytic value. The ACCE Method and its underlying logic therefore have the potential to constitute a substantial advance for nonrandomized intervention research, and follow-up research should be rapidly conducted.\n\n\nEndnotes\n\nA. Not addressed in this simple example is the fact that, in almost all implementation of this Method (i.e., all implementations other than introducing a true instrumental variable), these calculations would need to adjust for the association with outcome of the variable(s) introduced into Model 2 to produce the amplification. This is discussed subsequently in Steps 3 and 4.\n\nB. These regressions could be performed either within treatment arms or across both treatment arms while including an indicator for treatment arm, as well as a covariate(s) for treatment arm-introduced variable interaction(s). Comparing the results of all these approaches may be useful.\n\nC. A key area for additional investigation is whether the effects upon the treatment effect estimate of the increasing balance in Model 2 in variables correlated with the introduced variable is adequately reflected by the adjustment proposed in Steps 3 and 4. This proposed adjustment does separate the residual confounding associated, directly or indirectly, with the introduced variable (which is being controlled in Model 2 and therefore cannot amplify) from the residual confounding being amplified. However, whether this separation and calculation fully captures the change in confounding attributable to the resulting increase in control, even if modest, of unmeasured confounders correlated with the introduced variable is unclear. Even if this adjustment should prove only incompletely effective in capturing the change in confounding attributable to correlated covariates, it may be determined that sometimes this is a relatively small source of error. The method would be also expected to exhibit its strongest performance when introduced variable(s) can be chosen that are suspected to be largely uncorrelated with potential unmeasured confounders. Please see Appendix 2.2 for further discussion.\n\nD. Subtraction of both these quantities is necessary because, as pointed out in Step 3, the process of adding the introduced variable to Model 2 means that the amplification that occurs in Model 2 is not amplification of all the residual Model 1 confounding, but only the remaining Model 1 residual confounding (i.e., minus the contribution of the introduced variable and its correlates). Therefore, the value for the original residual confounding in Model 1 that is extrapolated from the amplified value does not include the contribution of the yet-to-be-introduced variable(s) and its correlates. The contribution to Model 1’s original residual confounding that is attributable to the yet-to-be-introduced variable(s) and its correlates must be subtracted, along with the extrapolated remaining residual confounding, from the Model 1 treatment effect estimate to estimate an unconfounded treatment effect.\n\nE. This example assumes a linear propensity score model but a logistic regression outcome model because the existing simulation demonstrating proportional confounding amplification is for a linear propensity score model5. Still to be determined is whether linear, rather than logistic, outcome models will need to be used for the ACCE method’s estimates to be the most accurate, due to the need to compare risks of outcome between Model 1 and Model 2. A requirement for linear outcome models, if it exists, would add complications; however, it may prove that these complications are relatively minor drawbacks in the context of permitting the ACCE’s Method’s estimation of residual confounding and an unconfounded treatment effect estimate. This is another worthwhile area for additional research. Meanwhile, the published data examples suggests the ACCE Method may contribute useful information to guide inferences from nonrandomized studies even when only outcomes from nonlinear analyses (i.e., hazard ratios from Cox regression) are available. However, these examples are premised on the assumption that the c statistic can serve at least as an approximate index of confounding amplification.\n\nF. This comparison can be made in this straightforward fashion since for the two analyses, the change in the prediction of exposure (in this case the c statistic) was highly similar (all-cause mortality: Model 1 c = 0.82, Model 2 c = 0.90; hip fracture: Model 1 c = 0.81, Model 2 c = 0.89). Thus, the resulting confounding amplification would be expected to be generally similar.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Health Services Research and Development (HSR&D). Specifically, this work was supported by a VA HSRD&D Career Development Award (09-216) and by support from the Center for Healthcare Organization and Implementation Research.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip.t\n\n\nAcknowledgements\n\nThe author would like to thank the numerous individuals who provided important encouragement or support of this manuscript throughout its development. Specific thanks should go to the friends and colleagues who provided very helpful reviews of manuscript drafts, including Brian Sauer, James Burgess, Lawrence Herz, David Hoaglin, Susan Eisen, Katherine Hoggatt, Guneet Jasuja, Keith McInnes, Donald Miller, C. Arden Pope, Karen Quigley, Kevin Rader, David Smith, Marcia Valenstein, and Amy Borg. The author also wants to thank John Brooks for providing a timely and thoughtful email response clarifying aspects of his simulation, and Amanda Patrick for generously discussing her analyses and providing the quantitative value for the age-and-sex adjusted glaucoma diagnosis hazard ratio and thanks to Jeroan Allison for the suggestion to consider bootstrapping as an approach to generating confidence intervals. However, the author alone is responsible for the ideas advanced in this manuscript, as well as the final form of the manuscript and associated documentation and whatever errors or oversights they may contain. In addition, the author would like to specifically thank the Health Services Research and Development Office of the Veterans Health Administration for their generous funding of his Career Development Award that helped provide valuable protected time to dedicate to the development of the ideas in this manuscript.\n\n\nSupplemental appendices\n\nThe essence of the proposed method described in the manuscript can be summarized in a very simple explanatory example. If one knew that turning up the volume on a television or radio doubled the volume (if, for instance, if the volume settings were genuinely proportional, so that turning up the volume from a setting of “20” to “40” doubled the sound produced), and one knows the actual volume obtained after this doubling occurred, it should be both possible and simple to extrapolate back to determine what the original volume was. That is, it would not be necessary to know the original volume if you knew these other two quantities (the final volume, and the proportion by which the volume changed). In nonrandomized intervention studies, one never knows exactly the “volume”, or amount, of total confounding, so an additional wrinkle is employed of measuring the change in the overall effect estimate that occurs between two models when only the amount of confounding is deliberately changed (through amplification). To make this example even more closely comparable, consider the scenario in which one knew that a certain sound system apparatus would unfortunately double (i.e., “amplify”) the static, or white noise, in whatever sound is being broadcast. If one knew the original total, or overall, volume (on a linear scale) was “90 units”, and this changed to 93 units when the particular apparatus was used, then we would know that the static made up 3 units of the original sound (since doubling it added 3 units). This would mean that the volume of sound devoid of any static was 87 units. (I deliberately refer to an imaginary linear unit of sound, rather than using the highly-familiar units of “decibels”. Decibels use a logarithmic scale, which would make the example less easily appreciated). It would not be necessary to know beforehand the value of the sound volume without static; rather, knowing the static had doubled and how much the sound volume had changed would permit extrapolation backwards to determine the unknown, devoid-of-static value. “Static” in this example can be seen as analogous to residual baseline confounding, and the sound volume without static as analogous to the unconfounded treatment effect.\n\nWhen applying this approach to comparative effectiveness research, however, the details of the approach are very important. While only the amplification of confounding is being deliberately changed, it is very easy to unintentionally introduce changes to additional aspects of the models besides simply the amplification of confounding. These appendices therefore highlight the major points that I have been able to identify to date which appear to warrant some consideration in the application of the method.\n\nSome readers may view this level of detail concerning important details of the method to be premature, since the method has not yet been extensively validated. I hope instead that these Appendices will both facilitate the method’s rigorous validation and promote sophisticated use of the method going forward. The details discussed below may not prove as important if the method is ultimately determined to provide only general, highly-approximate qualitative estimates of residual confounding. If, however, this method indeed appears to provide quantitative estimates of residual confounding in some circumstances, the details discussed below and even further details yet to be identified may prove important to consider or address.\n\n\nReferences\n\nBhattacharya J, Vogt W: Do instrumental variables belong in propensity scores? In: NBER Technical Working Paper no 343. Cambridge, MA: National Bureau of Economic Research. 2007. Reference Source\n\nWooldridge J: Should instrumental variables be used as matching variables? East Lansing, MI: Michigan State University; Unpublished manuscript. Accessed July 21, 2014. 2009. Reference Source\n\nPearl J: On a class of bias-amplifying variables that endanger effect estimates. In: Proceedings of the Twenty-Sixth Conference on Uncertainty in Artificial Intelligence (UAI 2010); Corvallis, OR: Association for Uncertainty in Artificial Intelligence: Accessed November 8, 2013. 2010; 2425–2432. Reference Source\n\nPearl J: Invited commentary: understanding bias amplification. Am J Epidemiol. 2011; 174(11): 1223–1227; discussion pg 1228–1229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooks JM, Ohsfeldt RL: Squeezing the balloon: propensity scores and unmeasured covariate balance. Health Serv Res. 2013; 48(4): 1487–1507. PubMed Abstract \| Publisher Full Text\n\nDeMaris A: Explained variance in logistic regression: A Monte Carlo study of proposed measures. Sociol Methods Res. 2002; 31(1): 27–74. Publisher Full Text\n\nSteyerberg EW, Vickers AJ, Cook NR, et al.: Assessing the performance of prediction models: a framework for traditional and novel measures. Epidemiology. 2010; 21(1): 128–138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBross ID: Spurious effects from an extraneous variable. J Chronic Dis. 1966; 19(6): 637–647. PubMed Abstract \| Publisher Full Text\n\nSchneeweiss S, Rassen JA, Glynn RJ, et al.: High-dimensional propensity score adjustment in studies of treatment effects using health care claims data. Epidemiology. 2009; 20(4): 512–522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatrick AR, Schneeweiss S, Brookhart MA, et al.: The implications of propensity score variable selection strategies in pharmacoepidemiology: an empirical illustration. Pharmacoepidemiol Drug Saf. 2011; 20(6): 551–559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMyers JA, Rassen JA, Gagne JJ, et al.: Effects of adjusting for instrumental variables on bias and precision of effect estimates. Am J Epidemiol. 2011; 174(11): 1213–1222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToh S, Hernandez-Diaz S: Statins and fracture risk. A systematic review. Pharmacoepidemiol Drug Saf. 2007; 16(6): 627–640. PubMed Abstract \| Publisher Full Text\n\nSturmer T, Schneeweiss S, Rothman KJ, et al.: Performance of propensity score calibration--a simulation study. Am J Epidemiol. 2007; 165(10): 1110–1118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrookhart MA, Schneeweiss S, Rothman KJ, et al.: Variable selection for propensity score models. Am J Epidemiol. 2006; 163(12): 1149–1156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneeweiss S, Patrick AR, Sturmer T, et al.: Increasing levels of restriction in pharmacoepidemiologic database studies of elderly and comparison with randomized trial results. Med Care. 2007; 45(10 Supl 2): S131–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSturmer T, Rothman KJ, Avorn J, et al.: Treatment effects in the presence of unmeasured confounding: dealing with observations in the tails of the propensity score distribution--a simulation study. Am J Epidemiol. 2010; 172(7): 843–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernan MA, Robins JM: Authors’ response, part I: observational studies analyzed like randomized experiments: best of both worlds. Epidemiology. 2008; 19(6): 789–792. Publisher Full Text\n\nToh S, Garcia Rodriguez LA, Hernan MA: Confounding adjustment via a semi-automated high-dimensional propensity score algorithm: an application to electronic medical records. Pharmacoepidemiol Drug Saf. 2011; 20(8): 849–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlkin I, Tate RF: Multivariate correlation models with mixed discrete and continuous variables. Ann Math Statist. 1961; 32(2): 448–465. Publisher Full Text\n\nVanderWeele TJ, Shpitser I: A new criterion for confounder selection. Biometrics. 2011; 67(4): 1406–13. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6843", "date": "27 Nov 2014", "name": "Mark Lunt", "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\nThis article outlines a very interesting approach to using propensity score methods to correct for unmeasured confounding. That was not the aim of the propensity score, and current methods are not able to do this, so it potentially represents a considerable advance. The idea is conceptually a simple one, related to the well-established use of instrumental variables to control for unmeasured confounding. However, I have not come across this idea before, and the author is to be congratulated on his originality. Having said that, I was a little disappointed in the presentation of the method. I do not feel that I am in a position to apply this method to any of my own data. One reason that I took so long over the review was that I wanted to be certain I fully understood the method by applying it myself, but it has become obvious that I will not be able to in a reasonable timescale. Greater precision in the presentation would have been welcome, whether that was explicit mathematical formulae, or simply causal diagrams showing how the various biases arose and which causal paths contributed to which estimates. This has been done very well in some of the references. For example, the relation between bias amplification and 1-R2 is given a clear mathematical basis in reference 4, and I would regard this as more convincing than simulation evidence. I’m sure that the author would agree with me that there is a lot of work to be done on this method before it can be applied routinely. I hope that this paper does spark that research, and that the author gets the credit he deserves for coming up with this potentially very useful idea.", "responses": [ { "c_id": "1244", "date": "29 Apr 2015", "name": "Eric Smith", "role": "Author Response", "response": "I would like to thank both reviewers for their thoughtful, insightful, and encouraging reviews. I particular appreciate their openness to a new methodology to attempt to estimate residual/unmeasured confounding. I am very glad to see that they recognized the value in disseminating and exploring a methodology that takes a very different approach (and possibly an approach that is more broadly applicable) than some of the limited number of alternatives currently available to tackle the problem of unmeasured confounding. Their specific comments were also extremely valuable. Both reviewers suggested that the manuscript would benefit from greater clarity; therefore I have revised and enhanced the presentation of the method quite substantially. The major ways I have done this is to: 1) expand the description of the method in the text and adding cross-references to the exact steps in the Appendix Table (which has also been expanded); 2) adding 3 additional hypothetical examples to communicate more incrementally the rationale for the method; 3) reorganized the manuscript Table so it reads more vertically than horizontally; 4) attempted to be more precise and detailed in my language; and, perhaps most importantly, 5) expressed the entire method mathematically in a single Summary Equation to help facilitate its understanding. The main manuscript text is substantially longer as a result of this increased explanation, but hopefully less ambiguous at key points. Some of the increase in length results from the more detailed description of the method, but much of the increase relates to the more detailed hypothetical examples, which some readers may not even feel a need to review. Similarly, the Appendices are considerably longer, but the reader is encouraged to pick and choose whether they want to review some, none, or all of these based entirely on their interest. Another important comment was Dr. Lunt’s comment that considerably further work needed to be done on the method. I couldn’t agree more, and it is my hope that the dividend that results from laying out the method in such detail is that multiple research groups can quickly advance this research. As I try to anticipate and highlight as fully as possible, there are a number of important uncertainties. These uncertainties range from such fundamental points as how consistently predictable the phenomenon of confounding amplification actually is, how accurately the difference between effect estimates can be determined, and how accurate are the proposed Bross equation-based corrections for the contribution of the Introduced Variable and, to a partial degree, its correlates, on the estimates of the change in treatment effect estimate as well as the starting Model 1 treatment effect estimate. Indeed, it is not even certain whether the method can be applied to some common logistic model effect estimates (e.g., odds ratio). I have even identified two more potential sources of uncertainty that are now included and discussed in the text and appendices: whether the introduced variable-outcome regression coefficient would potentially also suffer from at least some confounding amplification, and whether possible “constraints” might exist to achievable confounding amplification in real-world settings. So I am in complete agreement with Dr. Lunt that this manuscript represents only the very start of what hopefully will be steady advance of knowledge about this method and its value relative to other proposed approaches addressing unmeasured confounding. To my point of view, this is all the more reason to seek to enlist the greater research community in this effort. Nevertheless, it is important to note that approaches suggest themselves to address or minimize many of these uncertainties, although much investigation is needed. In addition, I want to emphasize a key point: while a number of uncertainties exist relevant to the actual performance of the method, it is my intention that, with this version of the manuscript, that there be no substantial uncertainty concerning the specific approach that is actually being proposed. I paid close attention to the fact that Dr. Matthews and Dr. Lunt (who has published on bias amplification) appeared uncertain about how to apply the method as described in Version 1. I hope in this version that I have communicated the method clearly enough that the vital next step can take place: testing the method in simulated and real-world datasets. It is for this reason – to facilitate the ability of as many interested research teams as possible to contribute to the method’s evaluation and evolution – that I have taken particular pains to expand communication concerning the overall logic, and underlying rationale, of the method and each of its steps. There are certainly places in which my proposed solutions to potential challenges for the method may prove imperfect or suboptimal (some possibilities might include the use of a regression coefficient and the Bross equation to take account confounding from the Introduced Variable-outcome relationship, the suggested approach to addressing possible confounding amplification in the Introduced Variable-outcome coefficient, and/or the favoring of stratification over matching to increase comparability of Model 1 and Model 2 mentioned in Appendix 2). It is my firm hope that other research groups can contribute by suggesting other approaches to accomplishing that particular objective within in the method, or even other angles concerning how to exploit confounding amplification to help estimate residual confounding. Therefore I wanted to be particularly clear in explaining the method so that the objective to be accomplished in each step was clear. This communication has been done through expanded text, calculations, examples, metaphors, technical Appendices, and the Summary Equation. I also outline the clear initial and subsequent steps for research as I see them (most centered on simulation) in the Discussion. Hopefully the manuscript is now sufficiently clearer so that collaborative investigation and elaboration of this method can take place. I thank the reviewers for encouraging me to much more carefully clarify the logic and approach of the method, and I hope they think that I have succeeded in that task. In closing, I would like to address the remaining specific points brought up by the reviewers:Dr. Lunt (Reviewer 1): As mentioned above, I am extremely grateful for Dr. Lunt’s observation for noting that the denominator of equations 3-6 in Reference 4 (Pearl, 2011) does indeed appear to support the 1-R2 relationship predicting the proportional amount of confounding amplification separate from the Brooks and Ohsfeldt (2013) simulation. This is potentially quite important, for it suggests that application of the technique might not need to be limited to an R2 of ≤ 0.56 (one of the concerns of the 2nd reviewer, Dr. Matthews). It does, however, increase the need to understand why the Brooks and Ohsfeldt simulation begins to exhibit nonlinear confounding amplification above R2 of 0.56. Dr. Matthews (Reviewer 2): Dr. Matthews asked a number of helpful questions concerning important details involved in implementing the method that I see now were not addressed as directly and thoroughly as they might have been. So that many readers can easily benefit from his helpful inquiry concerning recommendations on how to choose an instrumental variable without having to access my response to this comment, I have added an entire Appendix (Appendix 7) devoted in large part to this topic. In addition to offering practical suggestions on implementing the method, based on current knowledge, this Appendices also attempts to anticipate the likely trade-offs involved in optimizing one characteristic of the method potentially at the cost of another characteristic (e.g., wanting to maximize confounding amplification while minimizing differences between the two models that are separate from confounding amplification). Regarding Dr. Matthew’s 2nd major point, the simulation research that I hope follows this manuscript will likely provide the best guidance on what approaches should be taken if the R2 is < 0.04 or > 0.56. It should be noted, however, that, until that research is available, it is to be hoped that almost all propensity score models will succeed in achieving an R2 of at least 0.04. Furthermore, one remedy for circumstances in which Model 2 exceeds an R2 of 0.56 seemingly would be simply to remove measured covariates from the propensity score model until Model 2’s R2 is ≤ 0.56. This is a pragmatic, but not a perfect solution, since as pointed out in Appendix 3.2, such a step places extra weight on the method achieving an accurate estimate of residual/unmeasured confounding, since more of that type of confounding now exists. Also, as discussed in Appendix 4, if variables have to be removed from the propensity score, priority should be given to removing variables with little or no correlation with the Introduced Variable(s) and retaining in the propensity scores, to the extent possible, variables that correlate with the Introduced Variable(s) I also thank Dr. Matthews for pointing out the mislabeling of the outcome in Table 1. As mentioned, in addition to correcting this error, I have entirely restructured this Table to make it read more vertically than horizontally, at least in regard to the information pertaining to Model 1 versus Model 2. Regarding the “IntV” terminology in Supplementary Table 1, I have retained this abbreviation. “IntV” is my attempt to propose a nomenclature (abbreviation) for the introduced variable that will separate it from instrumental variables (which, unfortunately, share the same initials). “InV” might also be useable, but I felt the extra letter of “IntV” as an abbreviation for the term “Introduced Variable” made sense because the abbreviation was less likely to appear to be simply an erroneous typing of “IV.” I have also made the following minor changes: Capitalized “Introduced Variable(s)” to make each of its mentions more noticeable, since this variable or variables plays a key role in the method. Expanded the discussion of the potential impacts of correlations between various types of variables on the method’s estimates, and added Appendices that explore potential threats to the accuracy of the Introduced Variable-outcome regression coefficient, that provide explanation of the method’s components (and key uncertainties) in reference to the terms of the ACCE Method Summary Equation, and that begin to explore the use of sets of Introduced Variables and the practical trade-offs to be considered when implementing the method. Tried to be consistent with my language concerning “confounding amplification”: “proportional confounding amplification” refers to the percentage increase in residual confounding predicted by 1-R2, some other measure of exposure prediction, or an internal marker, while “quantitative confounding amplification” refers to the numerical change in the treatment effect estimate (technically, the change in the treatment effect estimate adjusted for the impact of increased balance in the Introduced Variable(s)). Replaced the term “multiple” Introduced Variable(s) with the term “set of Introduced Variables” to make it clearer I am referring to simultaneously insertion of several to many Introduced Variables, rather than the sequential use of different single Introduced Variables. Clearly labeled the Hypothetical Examples as Hypothetical Examples, moving them out of “Results.” Changed the examples from “odds ratio” to “risk ratio” due to concerns that noncollapsibility of the odds ratio might interfere with the subtraction of the Model 1 and Model 2 treatment effect estimates necessary to estimate the quantitative effect of confounding amplification. Invented the term “amplifiable fraction of residual confounding” to hopefully better communicate that (if the Introduced Variable(s) has any association with outcome) it is only the residual confounding separate from that which is attributable to the Introduced Variable(s) (which is not amplified) that is able to be amplified. Hopefully this has made this clearer. Removed the somewhat redundant word “Supplementary” from “Supplementary Appendix Table.” Corrected a minor subtraction error in the Appendix Table, Equation 3b (and subsequent steps), that had no substantive impact on the estimates of total residual confounding and the unconfounded treatment effect estimate. Also corrected a notation error in Step 4a where “M2” had been written “M3” by mistake." } ] }, { "id": "7091", "date": "05 Jan 2015", "name": "Gregory Matthews", "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\nThe authors present a manuscript describing a procedure that allows for the quantification of the total amount of residual confounding prior to bias amplification caused by propensity score models. I believe the procedure described in reasonable, and my biggest concerns with this manuscript are the presentation of the approach, which I had a hard time following initially. I think this paper is deserving of indexing as it is, but could be substantially improved with clearer presentation. Specific Comments:The authors talk about creating two models (Model 1 and Model 2) that are nest within each other in such a way that Model 2 contains all the variables in Model 1 plus one/several extra variable/s. It seems like there are money choices for this extra variable/s from among the possible variables. Do the authors have any specific advice on how this or these should be chosen? They do mention that this variable should be chosen to have ``discernible confounding amplification\", but isn't it possible that there are many acceptable choices that will satisfy this criteria? In that case is there any advice on how to choose between the good candidate variables?\n\nIn Step 2 of the description of the method,the authors mention that the when $R^2$ is between 0.04 and 0.56 there is a linear relationship between unexplained variance and confounding amplification. I believe that this threshold is then used in Supplementary table 1 when they state that the step should be taken only if R^2 is less than 0.56. Should this step not be taken if R^2 is less than 0.04? Do the authors have any advice on what to do when R^2 is greater than 0.56? Minor Comments:Should the outcome in Table 1B be hip fracture rather than all cause mortality? Supplementary Table1, 3a I think this is a typo: ``IntV:\"", "responses": [ { "c_id": "1243", "date": "29 Apr 2015", "name": "Eric Smith", "role": "Author Response", "response": "I would like to thank both reviewers for their thoughtful, insightful, and encouraging reviews. I particular appreciate their openness to a new methodology to attempt to estimate residual/unmeasured confounding. I am very glad to see that they recognized the value in disseminating and exploring a methodology that takes a very different approach (and possibly an approach that is more broadly applicable) than some of the limited number of alternatives currently available to tackle the problem of unmeasured confounding. Their specific comments were also extremely valuable. Both reviewers suggested that the manuscript would benefit from greater clarity; therefore I have revised and enhanced the presentation of the method quite substantially. The major ways I have done this is to: 1) expand the description of the method in the text and adding cross-references to the exact steps in the Appendix Table (which has also been expanded); 2) adding 3 additional hypothetical examples to communicate more incrementally the rationale for the method; 3) reorganized the manuscript Table so it reads more vertically than horizontally; 4) attempted to be more precise and detailed in my language; and, perhaps most importantly, 5) expressed the entire method mathematically in a single Summary Equation to help facilitate its understanding. The main manuscript text is substantially longer as a result of this increased explanation, but hopefully less ambiguous at key points. Some of the increase in length results from the more detailed description of the method, but much of the increase relates to the more detailed hypothetical examples, which some readers may not even feel a need to review. Similarly, the Appendices are considerably longer, but the reader is encouraged to pick and choose whether they want to review some, none, or all of these based entirely on their interest. Another important comment was Dr. Lunt’s comment that considerably further work needed to be done on the method. I couldn’t agree more, and it is my hope that the dividend that results from laying out the method in such detail is that multiple research groups can quickly advance this research. As I try to anticipate and highlight as fully as possible, there are a number of important uncertainties. These uncertainties range from such fundamental points as how consistently predictable the phenomenon of confounding amplification actually is, how accurately the difference between effect estimates can be determined, and how accurate are the proposed Bross equation-based corrections for the contribution of the Introduced Variable and, to a partial degree, its correlates, on the estimates of the change in treatment effect estimate as well as the starting Model 1 treatment effect estimate. Indeed, it is not even certain whether the method can be applied to some common logistic model effect estimates (e.g., odds ratio). I have even identified two more potential sources of uncertainty that are now included and discussed in the text and appendices: whether the introduced variable-outcome regression coefficient would potentially also suffer from at least some confounding amplification, and whether possible “constraints” might exist to achievable confounding amplification in real-world settings. So I am in complete agreement with Dr. Lunt that this manuscript represents only the very start of what hopefully will be steady advance of knowledge about this method and its value relative to other proposed approaches addressing unmeasured confounding. To my point of view, this is all the more reason to seek to enlist the greater research community in this effort. Nevertheless, it is important to note that approaches suggest themselves to address or minimize many of these uncertainties, although much investigation is needed. In addition, I want to emphasize a key point: while a number of uncertainties exist relevant to the actual performance of the method, it is my intention that, with this version of the manuscript, that there be no substantial uncertainty concerning the specific approach that is actually being proposed. I paid close attention to the fact that Dr. Matthews and Dr. Lunt (who has published on bias amplification) appeared uncertain about how to apply the method as described in Version 1. I hope in this version that I have communicated the method clearly enough that the vital next step can take place: testing the method in simulated and real-world datasets. It is for this reason – to facilitate the ability of as many interested research teams as possible to contribute to the method’s evaluation and evolution – that I have taken particular pains to expand communication concerning the overall logic, and underlying rationale, of the method and each of its steps. There are certainly places in which my proposed solutions to potential challenges for the method may prove imperfect or suboptimal (some possibilities might include the use of a regression coefficient and the Bross equation to take account confounding from the Introduced Variable-outcome relationship, the suggested approach to addressing possible confounding amplification in the Introduced Variable-outcome coefficient, and/or the favoring of stratification over matching to increase comparability of Model 1 and Model 2 mentioned in Appendix 2). It is my firm hope that other research groups can contribute by suggesting other approaches to accomplishing that particular objective within in the method, or even other angles concerning how to exploit confounding amplification to help estimate residual confounding. Therefore I wanted to be particularly clear in explaining the method so that the objective to be accomplished in each step was clear. This communication has been done through expanded text, calculations, examples, metaphors, technical Appendices, and the Summary Equation. I also outline the clear initial and subsequent steps for research as I see them (most centered on simulation) in the Discussion. Hopefully the manuscript is now sufficiently clearer so that collaborative investigation and elaboration of this method can take place. I thank the reviewers for encouraging me to much more carefully clarify the logic and approach of the method, and I hope they think that I have succeeded in that task. In closing, I would like to address the remaining specific points brought up by the reviewers:Dr. Lunt (Reviewer 1): As mentioned above, I am extremely grateful for Dr. Lunt’s observation for noting that the denominator of equations 3-6 in Reference 4 (Pearl, 2011) does indeed appear to support the 1-R2 relationship predicting the proportional amount of confounding amplification separate from the Brooks and Ohsfeldt (2013) simulation. This is potentially quite important, for it suggests that application of the technique might not need to be limited to an R2 of ≤ 0.56 (one of the concerns of the 2nd reviewer, Dr. Matthews). It does, however, increase the need to understand why the Brooks and Ohsfeldt simulation begins to exhibit nonlinear confounding amplification above R2 of 0.56. Dr. Matthews (Reviewer 2): Dr. Matthews asked a number of helpful questions concerning important details involved in implementing the method that I see now were not addressed as directly and thoroughly as they might have been. So that many readers can easily benefit from his helpful inquiry concerning recommendations on how to choose an instrumental variable without having to access my response to this comment, I have added an entire Appendix (Appendix 7) devoted in large part to this topic. In addition to offering practical suggestions on implementing the method, based on current knowledge, this Appendices also attempts to anticipate the likely trade-offs involved in optimizing one characteristic of the method potentially at the cost of another characteristic (e.g., wanting to maximize confounding amplification while minimizing differences between the two models that are separate from confounding amplification). Regarding Dr. Matthew’s 2nd major point, the simulation research that I hope follows this manuscript will likely provide the best guidance on what approaches should be taken if the R2 is < 0.04 or > 0.56. It should be noted, however, that, until that research is available, it is to be hoped that almost all propensity score models will succeed in achieving an R2 of at least 0.04. Furthermore, one remedy for circumstances in which Model 2 exceeds an R2 of 0.56 seemingly would be simply to remove measured covariates from the propensity score model until Model 2’s R2 is ≤ 0.56. This is a pragmatic, but not a perfect solution, since as pointed out in Appendix 3.2, such a step places extra weight on the method achieving an accurate estimate of residual/unmeasured confounding, since more of that type of confounding now exists. Also, as discussed in Appendix 4, if variables have to be removed from the propensity score, priority should be given to removing variables with little or no correlation with the Introduced Variable(s) and retaining in the propensity scores, to the extent possible, variables that correlate with the Introduced Variable(s) I also thank Dr. Matthews for pointing out the mislabeling of the outcome in Table 1. As mentioned, in addition to correcting this error, I have entirely restructured this Table to make it read more vertically than horizontally, at least in regard to the information pertaining to Model 1 versus Model 2. Regarding the “IntV” terminology in Supplementary Table 1, I have retained this abbreviation. “IntV” is my attempt to propose a nomenclature (abbreviation) for the introduced variable that will separate it from instrumental variables (which, unfortunately, share the same initials). “InV” might also be useable, but I felt the extra letter of “IntV” as an abbreviation for the term “Introduced Variable” made sense because the abbreviation was less likely to appear to be simply an erroneous typing of “IV.” I have also made the following minor changes: Capitalized “Introduced Variable(s)” to make each of its mentions more noticeable, since this variable or variables plays a key role in the method. Expanded the discussion of the potential impacts of correlations between various types of variables on the method’s estimates, and added Appendices that explore potential threats to the accuracy of the Introduced Variable-outcome regression coefficient, that provide explanation of the method’s components (and key uncertainties) in reference to the terms of the ACCE Method Summary Equation, and that begin to explore the use of sets of Introduced Variables and the practical trade-offs to be considered when implementing the method. Tried to be consistent with my language concerning “confounding amplification”: “proportional confounding amplification” refers to the percentage increase in residual confounding predicted by 1-R2, some other measure of exposure prediction, or an internal marker, while “quantitative confounding amplification” refers to the numerical change in the treatment effect estimate (technically, the change in the treatment effect estimate adjusted for the impact of increased balance in the Introduced Variable(s)). Replaced the term “multiple” Introduced Variable(s) with the term “set of Introduced Variables” to make it clearer I am referring to simultaneously insertion of several to many Introduced Variables, rather than the sequential use of different single Introduced Variables. Clearly labeled the Hypothetical Examples as Hypothetical Examples, moving them out of “Results.” Changed the examples from “odds ratio” to “risk ratio” due to concerns that noncollapsibility of the odds ratio might interfere with the subtraction of the Model 1 and Model 2 treatment effect estimates necessary to estimate the quantitative effect of confounding amplification. Invented the term “amplifiable fraction of residual confounding” to hopefully better communicate that (if the Introduced Variable(s) has any association with outcome) it is only the residual confounding separate from that which is attributable to the Introduced Variable(s) (which is not amplified) that is able to be amplified. Hopefully this has made this clearer. Removed the somewhat redundant word “Supplementary” from “Supplementary Appendix Table.” Corrected a minor subtraction error in the Appendix Table, Equation 3b (and subsequent steps), that had no substantive impact on the estimates of total residual confounding and the unconfounded treatment effect estimate. Also corrected a notation error in Step 4a where “M2” had been written “M3” by mistake." } ] } ]	1	https://f1000research.com/articles/3-187
https://f1000research.com/articles/4-76/v1	24 Mar 15	{ "type": "Opinion Article", "title": "Challenges and opportunities for early-career Teaching-Focussed academics in the biosciences", "authors": [ "Katharine Hubbard", "Sarah Gretton", "Katherine Jones", "Lucy Tallents", "Sarah Gretton", "Katherine Jones", "Lucy Tallents" ], "abstract": "Twenty-seven percent of academics in UK Higher Education (HE) are in Teaching-Focussed positions, making major contributions to undergraduate programmes in an era of high student expectations when it comes to teaching quality. However, institutional support for Teaching-Focussed academics is often limited, both in terms of peer networking and opportunities for career development. As four early-career stage Teaching-Focussed academics working in a variety of institutions, we explore what motivated our choices to make teaching our primary academic activity, and the challenges that we have faced in doing so. In addition to highlighting the need for universities to fully recognise the achievements of teaching staff, we discuss the role that the various biosciences learned societies have in supporting Teaching-Focussed academics. We identify that there is a need for the learned societies to come together and pool their expertise in this area. The fragmented nature of the Teaching-Focussed academic community means that clear sources of national support are needed in order to best enable the next generation of bioscience educators to reach their full potential.", "keywords": [ "Higher", "education", "teaching" ], "content": "Introduction\n\nThere are 1.8 million undergraduates studying in UK universities, with Biological Sciences students accounting for 10.3% of the undergraduate population (The Higher Education Statistics Agency, HESA, 2013). Students place a high value on teaching quality; scores for overall satisfaction in the National Student Survey are most strongly correlated with the scores for quality of learning and teaching (Buckley, 2012). Undergraduates expect that their experience of higher education represents value for money, and want to be taught in small-scale classes by experienced and qualified teaching staff (Kandiko & Mawer, 2013). Providing high quality teaching is a therefore a significant component of Biology departments, many of whom rely on Teaching-Focussed academics to deliver aspects of their undergraduate programmes. At the Society for Experimental Biology (SEB) Education Meeting in December 2014, a major theme that emerged was the challenges facing those at the early stages of their teaching careers. Many of the difficulties faced by Teaching-Focussed academics have been well documented (Cashmore & Ramsden, 2009; Cashmore et al., 2013; Locke, 2014; Young, 2006). Here we present our experiences as early-career teaching orientated academics in a range of UK Higher Education (HE) institutions to explore both the challenges and opportunities of working in teaching roles.\n\nTeaching-Focussed academics are employed on a range of different bases; some are on Teaching-Focussed contracts and are responsible just for covering a given number of hours of contact time, but an increasing number are on Teaching and Scholarship contracts, with an explicit part of their role being to advance understanding of teaching and learning. The HESA includes Teaching-Focussed academics in their surveys of academic staff (note that the HESA uses the term Teaching-Only), revealing that those on Teaching-Focussed contracts represent around 25% of all academic staff, rising to 50% of staff in pre-1992 universities that are not members of the Russell Group (HESA, 2013; Locke, 2014). The employment profiles of Teaching-Focussed academics are significantly different to those on Teaching and Research or research-only contracts. There are roughly equal proportions of male (48%) and female (51%) Teaching-Focussed academics, contrasting with Teaching and Research where there is a gender bias (60% male). Teaching-Focussed roles are primarily filled by academics on part time, fixed term contracts; only 18% have a full time open ended contract, compared with 74.5% of those on Teaching and Research contracts (see Figure 1). While 12% of academic staff are on zero-hours contracts, this number rises to 47% of Teaching-Focussed staff (University and College Union, 2013). A large proportion of those with responsibility for delivering undergraduate programmes therefore have limited job security. This is perhaps symptomatic of a wider conflict within HE teaching; universities are relying on Teaching-Focussed contracts to fulfil their teaching requirements, yet at an institutional level there is often little support for those in Teaching-Focussed roles to become established members of the academic community.\n\nArea of boxes represents number of individuals. Data from HESA, 2012–3.\n\n\nWhy have Teaching-Focussed academics?\n\nWhen preparing this article all of us described experiencing a bias in careers advice we had received, with a heavy focus on the traditional research dominated model of academia. One of us commented “When I finished my PhD I went to a careers event in 2005 [where] I was told there was no such thing a job for those who only wanted to teach at University and yet here I am with a permanent Teaching-Focussed contract 8 years later”. Teaching-Focussed academics take a diversity of routes towards their roles; some move into teaching at later career stages, but we represent a group of academics who have actively chosen to focus on teaching at an early-career stage having recognised we have the skills and passion for it. As such, we have much to offer our respective institutions, independent of the type of university we work in.\n\nIn having teaching and learning as a primary focus we have opportunity to consider which teaching methods are most likely to cultivate curiosity, motivation and independence in learners. In our experience, staff employed on Teaching-Focussed contracts are more likely to explore the pedagogical literature and adopt evidence-based best practice compared to staff for whom teaching is only a small part of their role. Teaching-Focussed staff therefore represent a powerful force to embed student-centred learning across the whole range of academic institutions. Our enthusiasm for understanding learning and teaching makes us an intellectual and practical asset for colleagues whose research dominates their time. We can help them to develop their understanding of pedagogical philosophies and theories, suggest different learning activities, observe their work and act as critical friends or mentors, providing a springboard for their own transformation into self-reflective educators.\n\n\nChallenges in establishing a Teaching-Focussed academic career\n\nWhile all early-career academics face significant challenges, there are unique challenges to early-career Teaching-Focussed academics that are easily overlooked. Teaching is influenced by differing drivers from research, which may result in early-career teachers having reduced choice in setting the scope of their activities compared to their research equivalents. The teaching agenda is often set by more senior staff, leaving less flexibility for early-career individuals to teach the subjects that they find the most intellectually stimulating. Funding for teaching is more restricted and less transparently advertised than research fellowships and grants, leaving those on Teaching-Focussed contracts at a disadvantage when external funding success is a promotion criterion. Teaching outputs may also not be transferrable between institutions due to copyright ownership issues, therefore making sharing good practice and demonstrating impact more difficult than for equivalent research outputs.\n\nThe barriers faced by early-career teachers are often in acquiring and maintaining a job in the first place (as indicated by the significant number of teaching staff on part-time and/or fixed-term contracts; Figure 1). Unlike research which has a well-defined (though not necessarily flawless) path to lectureship, currently there appears to be no clear route to a teaching dominant academic role in the sciences within most UK academic institutions, although some have created Teaching-Focussed career paths (e.g. University of Bristol). Most advertised teaching fellow positions require a PhD and teaching experience, but the opportunities to gain teaching experience and/or training during a PhD or post-doctoral position are mixed and often limited, with supervisors prioritising the publication of papers over the development of non-research skills. For those combining teaching and research at early career stages, teaching commitments mean that it may not be possible to achieve the same quantity or impact of research publications, creating a barrier to progressing to the more prevalent research-teaching posts or returning to research-only positions. In contrast, research staff retain the opportunity to move sideways to teaching at any stage in their career because the importance of demonstrating high-impact teaching, and recruiting on that basis, is not yet universally recognised (High Level Group on the Modernisation of Higher Education, 2013).\n\nTeaching is typically organised according to research departments, leaving Teaching-Focussed positions a relative rarity within a research-dominated environment. While cross-disciplinary teaching support networks do exist within many institutions, their form and membership can be nebulous and lack prominence. This means that we have to look further afield to find a group of peers with whom we can discuss pedagogical concepts and their applications, compared to our research-focussed colleagues. The vocabulary and style of pedagogical literature can be quite different than that of bioscience, and the lack of immediate peers can make engaging with the literature more challenging. Not only does this make current research harder to interpret and implement, but it acts as a hurdle to preparing manuscripts when authors are less clear about the expectations of journal editors and their audience; something which we experienced ourselves when writing this paper. These factors can lead to a feeling of isolation amongst Teaching-Focussed academics, which several of us have experienced during our careers (see case studies) and which can be frustrating and demoralising.\n\nFor those that are successful in acquiring a permanent contract the challenges continue. It has been long documented that teaching in HE is deemed a low status activity (High Level Group on the Modernisation of Higher Education, 2013; Young, 2006). Recently though, and particularly with the changes to student finance in England and Wales, reward and recognition for teaching and related activities has been put under the spotlight across the HE sector (Cashmore & Ramsden, 2009; Cashmore et al., 2013). The Academy of Medical Sciences, The Physiological Society, Heads of University Biosciences and the Society of Biology recently published a survey of over 250 bioscience academics from a range of institutions, career stages and contract types (Academy of Medical Sciences et al., 2014). Only 57% of respondents reported that their institution has a clear strategy for evaluating staff teaching contributions. Furthermore, 55% of respondents indicated scepticism that teaching is considered equally to research in professorial promotions, with an additional 24% stating that professorial promotions based on teaching achievement were not possible at their institution.\n\n\nSupport structures for those in teaching positions\n\nWhat support structures exist for bioscientists who embark on Teaching-Focussed careers in Higher Education? An increasing number of Universities now offer accredited Postgraduate Certificates in Teaching in Higher Education courses or equivalents. However, these courses can be difficult to access for the large numbers of teaching dominant staff on part-time and/or fixed term contracts, those who do have time allocated for continuing professional development (CPD) in their positions, or are in institutions who do not offer this support. There is therefore a need for professional development at a national level; in the past this has been provided by the Higher Education Academy who made available a wealth of resources, primarily through the Centre for Bioscience. Withdrawal of government funding in recent years has unfortunately resulted in the loss of the Subject Centre in 2011 and the Biosciences discipline lead in 2014. The HEA does maintain however a role in providing professional recognition for teaching through the UK Professional Standards Framework (Turner et al., 2013), and also provides accreditation for courses and training schemes.\n\nAll academics can attest to the value of professional networks, which are often established at the early-career stage at conferences. However, what happens to these networks if you take a different academic path and you find yourself just as interested in how to educate others in your science as the science itself? It is here that the Teaching-Focussed academic can risk becoming an island, with restricted funding, losing those opportunities for renewed inspiration and sharing of good practice. This is especially true of those early in their career that have not yet established links across institutions. With the HEA no longer offering subject-specific support, an obvious alternative are the learned societies, who have always played an important role in providing networking opportunities for academics. Education meetings run by organisations such as SEB are hugely important in providing Teaching-Focussed academics to meet and exchange ideas; none of the authors of this piece knew each other before attending SEB meetings. Learned societies also have a valuable role to play in filling the gap in terms of external mechanisms of validation; for example, the Society of Biology has taken over the HEA funded HE Bioscience Teacher of the Year award.\n\nDiscussions on supporting bioscience teaching at both the SEB education meeting (2014) and at a recent HEA Biosciences meeting (University of Newcastle, 2014) have included the fact that the learned societies for biology in the UK are very fragmented. The Society of Biology, The Society for Experimental Biology, The Physiological Society, The Biochemical Society, The British Ecological Society and others all have Education sections. While having separate societies makes sense for research activities, the challenges facing educators in the different areas of bioscience are quite similar. For the early-career bioscience teacher it can be unclear which society is the natural ‘home’ for them, and belonging to all societies would be prohibitively expensive. This contrasts with the support provided to Teaching-Focussed staff in the physical sciences, where the Institute of Physics or the Royal Society of Chemistry have central roles in coordinating educational activities.\n\n\nOpportunities and future prospects - a supportive network with learned societies?\n\nIn the absence of the HEA as a national body to support biosciences teaching, we feel there is a need for learned societies to come together and organise interdisciplinary events on the theme of education and outreach. Learned societies combining their efforts in terms of education would allow expertise of different organisations to be shared more easily, and provide a clearer sense of identity for Teaching-Focussed academics. At the SEB meeting (December 2014), we discussed whether educational meetings should be integrated within society-wide meetings or whether meetings should be specialised on education, perhaps across several learned societies. The advantage for removing the segregation between “research” and “teaching” academics, may be to improve the status of teaching and spread good practice further than simply “preaching to the choir”. The advantage of more specialist education meetings is that common themes often arise in learning and teaching across a wide variety of disciplines, as seen by the attendance of physicists at the SEB meeting. Subject-specific meetings may therefore lose the chance to learn from pedagogical advances in other disciplines.\n\nSocieties also have a potential role in supporting the development of junior academics through mentoring, which is one intervention that has been shown to improve career prospects (Eby et al., 2008). Currently there is no formal mentoring process in the wider HE bioscience community; an informal community of practise exists via the HEA’s Bioscience PedR JISC email list and conferences. The education committees of the learned societies could potentially take an active role in supporting the next generation of bioscience educators by coordinating mentoring relationships, which would be invaluable to those in institutions without an existing community of Teaching-Focussed academics. The British Ecological Society already has a successful mentoring scheme for female ecologists; an equivalent scheme hosted by the combined learned societies could result in real gains in supporting Teaching-Focussed academics.\n\nThe role of education committees on learned societies are also expanding. As one of us noted, “In my 5 years on the British Ecological Society Education, Training and Careers Committee, the emphasis of the committee changed from one that predominantly focussed on increasing the impact of teaching ecology in schools by working with teachers and policy makers to an ever expanding portfolio that now includes internships for undergraduates, outreach work at music festivals and supporting lecturers in delivering innovative field teaching”. If learned societies can continue to expand their reach beyond the traditional research domain, they will play an important part in fostering a collaborative approach to teaching in higher education, and concurrently support the teaching careers of early career academics.\n\nIn conclusion, early-career Teaching-Focussed academics make valuable contributions to bioscience departments, and institutions should be enabling these academics to achieve their full potential, both in terms of their immediate teaching responsibilities to their students and their long term career progression. There is wide variation in the support universities provide, with some institutions nurturing a high quality environment for teaching staff, while others lag behind. However, even with the best possible local institutional support, the fragmented nature of the Teaching-Focussed community means that national organisations are essential to bring otherwise isolated individuals together. As the HEA is unlikely to fund subject-specific initiatives again without a major increase in funding, it falls to the learned societies to provide cross-institutional support for Teaching-Focussed academics, particularly through providing bioscience education conferences. Presenting at education-focussed sessions enables sharing of good practice, external validation of teaching activities, and ultimately, as this article demonstrates, opportunity for new collaborations. As one of us noted, “I never planned my route through academia or imagined myself as an educator; it is through my involvement in learned societies that I have met the key people that allowed me to see that there is no “one-size-fits-all” concept of an academic”.\n\n\nCase study 1: Sarah Gretton, University of Leicester\n\n‘I started my career in academia initially in a technical post, followed by a PhD and then a post-doctoral research position, all at Russell group institutions. I was fortunate during my PhD to have the opportunity to demonstrate in laboratory sessions and take tutorials. Unfortunately, during my post-doctoral post the opportunities to teach didn’t arise and I started considering carefully what I enjoyed and realised I found communicating biology far more fulfilling than spending my time in a fairly solitary capacity focused on a narrow, detailed research area. Despite publishing a handful of papers I decided not to embark on another research position and applied and acquired an hourly- paid lecturing position at a Post-92 institution and a fixed-term, part-time Teaching Fellow contract at my current institution.\n\nMy experience at these two intuitions differed quite significantly. The hourly paid post did not provide any training/mentoring and I really felt thrown in at the deep end. In contrast at my current institution I was observed early on in my practise and embarked on a PG Cert in Academic Practice in Higher Education and was encouraged to develop new resources and initiatives. For these reasons at the start of the following academic year I took on more teaching at my current institution rather than the Post-92 institution, despite it being a much longer commute. After 3 and half years juggling temporary and part-time positions, I was given an open-ended Teaching Fellow contract.\n\nI have been very fortunate at my current institution, as there is a small but thriving community of Natural Scientists interested in the scholarship of teaching and learning who meet regularly. I have been mentored by incredibly supportive line managers experienced in HE education, who have found funding for me to attend educational conferences. This has been very formative in my practice, and has given me the support and confidence to successfully bid for teaching grants, present and publish my educational research. Last year I received a university teaching award and now I lead our college research theme into teaching and learning.’\n\n\nCase study 2: Lucy Tallents, University of Oxford\n\n‘I have a standard fixed-term postdoctoral (research) contract, although my role is 100% teaching and technical assistance. I’m passionate about wildlife conservation, and believe that my conservation impact will be greater through developing the capacity of in-country nationals rather than doing my own research. During my Masters and PhD I trained wildlife conservation professionals in field and analytical skills, tutored fellow PhD students and post-docs, and demonstrated in undergraduate statistics labs. For my first post-doc I was tasked to develop a post-graduate diploma in conservation research skills. I rapidly realised that I didn’t have sufficient understanding of curriculum design, how to structure engaging learning activities, and how to effectively assess student learning, let alone the process of getting a new course approved by my institution. I joined a CPD course in learning and teaching run by the University of Oxford’s Learning Institute and was lucky to be mentored by an inspirational educator, Dr Chris Trevitt, who has since left Oxford. He supported me to submit a portfolio to the HEA to gain recognition as a Fellow. It was challenging to do this alongside creating my course and then teaching it almost single-handedly, but the exposure to pedagogical theory and the opportunity to discuss ideas and applications with my peers was invaluable.\n\nAfter relying on this local support at a crucial time, I lost contact with my peers as our career paths diverged, and I now feel quite isolated both within my research group and the wider institution. This is especially true now that my focus has switched to online learning, which is less familiar to colleagues both within my research discipline and those who teach in other fields. While I have seen increasing support and training put in place for those who are new to teaching, I think that a gap still exists in support for early/mid-career teaching professionals. I’m keen to explore ways to connect people who are interested in student-centred teaching, and are figuring out how to navigate a Teaching-Focussed career path.’\n\n\nCase study 3: Katharine Hubbard, University of Cambridge\n\n‘I have been passionate about teaching from the earliest days of my academic career. During my PhD I did large amounts of small group teaching and laboratory class demonstration, and created a number of resources to support teaching within my department. I then did a post-doc at the University of California, San Diego, however I really missed the contact with undergraduates. Despite having a strong publication record, I realised my strengths were in teaching and this is what I wanted to spend my career doing. I came back to the UK and spent 2 years teaching for the University on an informal freelance basis, where the lack of job security was very stressful. I was then appointed to a Teaching By-Fellowship at Churchill College, and was appointed to my current Teaching-Focussed role within the Department of Plant Sciences on a full time, fixed term basis 2 years ago.\n\nIn my current job I find that my contributions to my college, department and inter-departmental teaching are highly valued at a personal level, including by the current Head of Department and other senior academics, but that in terms of the institution I am very much a ‘square peg in a round hole’. I am the only early-career Teaching-Focussed academic in biological sciences, which often feels very isolated as I lack immediate peer support. Getting good career advice is difficult as there are few who understand the Teaching-Focussed route. The University is currently debating whether there should be a formal career structure for those on Teaching-Focussed contracts, but at present career development opportunities are unclear. I love teaching in HE and I achieve some of the highest student feedback in my department, but the isolation is challenging. Meeting other Teaching-Focussed academics at conferences has been invaluable to me, as I have made connections with others who care about teaching and learning in a way that I don’t have within my local institution.’\n\n\nCase study 4: Katherine Jones, Bangor University\n\n‘My journey into lecturing was not something I planned but the result of a rather slow identification of my own strengths and interests, combined with the rather more dull need to remain employed. This led to me working in eight universities in three countries in just one decade. After 6 years in Cambridge and Oxford, I started my first post-doctoral position in Canada, thinking only of research; an extension of my PhD when I had viewed teaching as an enjoyable side-activity that I only engaged in when I needed money or had time for a bit of “CV building”. At the end of this post-doc, unemployment looming, a colleague in my research area encouraged me to teach research skills at a research institute in Nigeria for 6 months. This proved a pivotal turning point in my career, and I realised then that teaching could have as much impact as research. The freedom my Nigerian students gave me to experiment with my own teaching style, has also proved invaluable in my lectureship, where I have had confidence to take risks in teaching and innovate.\n\nAfter my time in Nigeria, I returned to post-doctoral life, punctuating contracts with some Open University teaching that again cemented my desire to teach. Keeping research active enabled me (to the surprise of most people, including myself) to obtain a short-term lectureship at my current institution. It was here, that I took a risk. I decided to go against my research-led job contract, ignore advice of most senior staff, except the open-minded head of school at the time, and decided to love my job and ignore my h-index. I loved teaching, so that’s what I mostly did. This risk paid off, since my institution has begun, like many UK universities to value teaching more, and my department has been supportive of my move to a more Teaching-Focussed contract, whilst keeping a job contract that is now permanent rather than fixed-term. I am not sure my career route would have been for everyone (the constant moving was stressful), but I would say that you can’t simply copy the career routes of older staff - higher education is in constant flux, so the strength to calculate your own trajectory is important, although this is not without risk.’", "appendix": "Author contributions\n\n\n\nKH proposed the writing of the article and coordinated the preparation of the manuscript. All authors contributed to the writing of the draft manuscript, and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAcademy of Medical Sciences. Improving the status and valuation of teaching in the careers of UK academics. Academy of Medical Sciences, The Physiological Society, Heads of University Biosciences and the Society of Biology, 2014. Reference Source\n\nBuckley A: National Student Survey: Analysis of national results for 2011. York, The Higher Education Academy, 2012. Reference Source\n\nCashmore A, Cane C, Cane R: Rebalancing promotion in the HE sector: is teaching excellence being rewarded? Genetics Education Networking for Innovation and Excellence: the UK’s Centre for Excellence in Teaching and Learning in Genetics (GENIE CETL), University of Leicester, The Higher Education Academy, 2013. Reference Source\n\nCashmore A, Ramsden P: Reward and recognition in higher education: Institutional policies and their implementation. The Higher Education Academy and the Genetics Education Networking for Innovation and Excellence (GENIE) CETL, University of Leicester, 2009. Reference Source\n\nEby LT, Allen TD, Evans SC, et al.: Does Mentoring Matter? A Multidisciplinary Meta-Analysis Comparing Mentored and Non-Mentored Individuals. J Vocat Behav. 2008; 72(2): 254–267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHigh Level Group on the Modernisation of Higher Education. Report to the European Commission on improving the quality of teaching and learning in Europe’s higher education institutions. 2013. Reference Source\n\nKandiko CB, Mawer M: Student Expectations and Perceptions of Higher Education. London: King’s Learning Institute, 2013. Reference Source\n\nLocke W: Shifting academic careers: implications for enhancing professionalism in teaching and supporting learning. The Higher Education Academy, 2014. Reference Source\n\nThe Higher Education Statistics Agency. Data for 2012/13, 2013. Reference Source\n\nTurner N, Oliver M, McKenna C, et al.: Measuring the impact of the UK Professional Standards Framework for Teaching and Supporting Learning (UKPSF). The Higher Education Academy, 2013. Reference Source\n\nUniversity and College Union. The Use of Zero Hours Contracts in Further and Higher Education. 2013. Reference Source\n\nYoung P: Out of balance: Lecturers’ perceptions of differential status and rewards in relation to teaching and research. Teaching in Higher Education. 2006; 11(2): 191–202. Publisher Full Text" }	[ { "id": "8085", "date": "26 Mar 2015", "name": "Anne Tierney", "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 subject is one that is close to my heart, and on reading it I recognised all of the issues and challenges that the authors have described. The paper is an honest account of the realities faced by \"teaching-focused\" academics in Life Sciences, and is well written and presented. I have selected \"approved with reservations\" for the following reason: the authors rightly state,\"The vocabulary and style of pedagogical literature can be quite different than that of bioscience, and the lack of immediate peers can make engaging with the literature more challenging. Not only does this make current research harder to interpret and implement, but it acts as a hurdle to preparing manuscripts when authors are less clear about the expectations of journal editors and their audience; something which we experienced ourselves when writing this paper.\"In addition, identifying relevant literature is also a challenge. I believe that the paper would be strengthened by the addition of relevant pedagogical literature, and ask the authors to consider inclusion of the following papers to strengthen their argument:Roxå, T., Olsson, T. & Mårtensson, K. (2007) Scholarship of Teaching and Learning as a strategy for institutional change, in Enhancing Higher Education, Theory and Scholarship, Proceedings of the 30th HERDSA Annual Conference, Adelaide, 8-11 July 2007: pp 487 available online at: http://www.herdsa.org.au/wp-content/uploads/conference/2007/papers/p233.pdf [accessed 26/03/2015]This paper explores the role of the scholarship of teaching and learning for institutional change, and the importance of local and external networks, both of which were touched upon in the paper. Kreber, C. (2005) Reflection on teaching and the scholarship of teaching: Focus on science instructors, Higher Education, 50, 323-359 available at http://link.springer.com/article/10.1007%2Fs10734-004-6360-2 [accessed 26/03/2015]Carolin Kreber's work surrounds the importance of reflective practice. This paper concentrates on science instructors, and so is particularly relevant for this paper, although Carolin's other work is also relevant. Again, reflective practice was discussed by the authors, and the inclusion of this paper strengthens the argument.The final suggestion I have is the case studies. I like them very much and feel that they bring the paper to life, but at the moment they feel like an appendix. I would advise that you move them forward into the body of the paper, as they highlight some of the challenges and issues that you have talked about in general, but as you experience them as individuals. Moving them forward in the paper also allows you to then discuss them in the conclusion section.I enjoyed reading this paper very much, and your experiences as early-career academics resonated with my own, and colleagues' experiences. Thank you very much for writing this paper, and I hope my suggestions will strengthen your argument.", "responses": [] }, { "id": "8082", "date": "26 Mar 2015", "name": "Graham Scott", "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\nThe Opinion Article presents the views of four individuals who have reflected upon their own experiences in the context of a shift in the make up of the UK Higher Education Biosciences teaching community. The article makes a contribution to an on going discussion in the UK (and further afield) but brings to that discussion a fresh perspective (in my opinion observations on the experience of new teachers are too often made by those not new to teaching themselves). The authors suggest that the UK Higher Education Academy (HEA) no longer provides targeted support to teachers in the biosciences. Although it is the case that the HEA no longer support a Biosciences Subject Centre and their current emphasis seems to be focussed more on interdisciplinary/generic support the authors also refer (p4) to a “recent HEA Biosciences meeting” taking place in 2014 – this is an apparent contradiction. Although I like the inclusion of the useful case studies (they provide a clear picture of the context of the authors and the lens through which they have constructed their argument) the structure of this paper is unusual. I feel that the reader progresses naturally through the main text and into the case studies and then reaches the end of the paper rather than coming to its conclusion. The authors might consider a subtitle to more clearly signpost the main concluding paragraph. I am happy to recommend the indexation of this paper. I think that it makes a useful contribution to a current discussion in bioscience education and I feel that it should give all members of the bioscience teaching faculty cause for reflection.", "responses": [] } ]	1	https://f1000research.com/articles/4-76
https://f1000research.com/articles/4-102/v1	29 Apr 15	{ "type": "Research Article", "title": "Laryngeal mask placement in a teaching institution: analysis of difficult placements", "authors": [ "Anastasia D Katsiampoura", "Peter V Killoran", "Ruggero M Corso", "Chunyan Cai", "Carin A Hagberg", "Davide Cattano", "Anastasia D Katsiampoura", "Peter V Killoran", "Ruggero M Corso", "Chunyan Cai", "Carin A Hagberg" ], "abstract": "Background: Laryngeal mask airway (LMA) placement is now considered a common airway management practice. Although there are many studies which focus on various airway techniques, research regarding difficult LMA placement is limited, particularly for anesthesiologist trainees. In our retrospective analysis we tried to identify predictive factors of difficult LMA placement in an academic training program.Methods: This retrospective analysis was derived from a research airway database, where data were collected prospectively at the Memorial Hermann Hospital, Texas Medical Center, Houston, TX, USA, from 2008 to 2010. All non-obstetric adult patients presenting for elective surgery requiring general anesthesia, were enrolled in this study: anesthesiology residents primarily managed the airways. The level of difficulty, number of attempts, and type of the extraglottic device placement were retrieved.Results: Sixty-nine unique Laryngeal Mask Airways (uLMAs) were utilized as a primary airway device. Two independent predictors for difficult LMA placement were identified: gender and neck circumference. The sensitivity for one factor is 87.5% with a specificity of 50%. However with two risk factors, the specificity increases to the level of 93% and the sensitivity is 63%.Conclusion: In a large academic training program, besides uLMA not been used routinely, two risk factors for LMA difficulty were identified, female gender and large neck circumference. Neck circumference is increasingly being recognized as a significant predictor across the spectrum of airway management difficulties while female gender has not been previously reported as a risk factor for difficult LMA placement.", "keywords": [ "airway management", "LMA", "anesthesia", "neck circumfrence" ], "content": "Introduction\n\nSince its introduction into clinical practice in 19831, the laryngeal mask airway (LMA) has found a place in everyday anesthesia practice2–4, including its use as a primary airway device in the elective or pre-hospital emergency settings, as well as a rescue airway device in either settings5,6. Additionally, the LMA placement has become a common airway management technique, particularly in ambulatory surgery2,3, and is associated with shorter recovery time, earlier patient discharge and lower associated costs7,8. Even if the LMA is considered a very safe airway device9 with a low incidence of complications, there may be situations where it either does not function properly or is difficult to place10. Importantly, the association between difficult LMA placement and increased incidence of Difficult Mask Ventilation (DMV) has been recognized11.\n\nAppropriate sizing is critical for correct LMA application12, while the selection of the device type seems to play a less significant role, yet the prediction of the correct size is not easy. This can be attributed to the absence of a coherent and universal standard sizing system13. Most of the manufacturers suggest a weight-based size selection, however there is no consistency between weight and oropharyngeal anatomy14.\n\nAlternative recommendations for the selection of the appropriate size of a LMA, regarding age, height and gender, as well as anatomical landmarks, are still under investigation15–17.\n\nAs a result, the concepts of difficult LMA placement and effective usage have prompted new research, focusing on the prediction of difficult LMA placement18.\n\nA simple, objective, predictive score to identify patients at risk of difficult LMA placement at the bedside does not currently exist, however to achieve such score a comprehensive airway assessment based analysis of risk identification needs to be accomplished first. Based on recorded outcomes at a major teaching hospital that utilized a comprehensive airway assessment19 we aimed to identify predictive factors for difficult LMA placement.\n\n\nMethods\n\nData for this retrospective analysis were derived from a database of airway assessments, management plans, and outcomes collected prospectively from August, 2008 to May, 2010 at a Level 1 academic trauma center (Memorial Hermann Hospital, Texas Medical Center, Houston, TX, USA)11. The study was sponsored by an educational grant from the Foundation for Anesthesia, Education and Research (FAER), and other educational funds from the Department of Anesthesiology at University of Texas Medical School at Houston. After obtaining IRB approval, (HSC-MS-07-0144) all non-obstetric adult patients presenting for elective surgery requiring general anesthesia were enrolled in this study (n=8364). All uLMA placements were carried out by anesthesiology residents. In the ‘mother study’, residents were randomized into two groups—an experimental group, which used a comprehensive airway assessment form11,20 in addition to the existing anesthesia record, and a control group, which used only the existing anesthesia record. For the purpose of the present analysis, only the experiment (n=2348) group data was utilized, since the comprehensive airway assessment needed to be linked to the airway device that was utilized. We identified 110 cases-used of LMA, disposable laryngeal mask (uLMA, North America, San Diego, CA), and 69 of those as primary airway device, which we utilized for our analysis. Difficult LMA placement was defined as either inability to physically place a LMA device or inadequacy of ventilation, oxygenation, or airway protection after placement that required conversion to an alternative technique. The level of difficulty and the number of attempts of the uLMA placement were documented by the anesthesiology residents.\n\n\nStatistical analysis\n\nSixty nine uLMA placements were completed and an analysis was performed (based on “per protocol” and not intention to treat). The mean and standard deviation were used to summarize continuous variables, and frequency (percentage) was summarized for categorical variables. A two-tailed sample t-test was applied to compare continuous variables and Chi-square or Fisher exact tests as appropriate were performed for categorical variables between patients with or without uLMA placement difficulty. Using multivariate logistic regression models, the variables associated with uLMA placement difficulty were identified. All variables with a p-value ≤0.25 in univariate analysis and variables of known biological importance (e.g., age and BMI) were entered into a full model. A backward selection method was used to identify significant independent predictors. A receiver-operating-characteristic (ROC) area under the curve was also calculated to evaluate the resulting model’s predictive value, (Figure 1) as well as adjusted odds ratios and their 95% confidence intervals. Continuous variables were included after the dichotomization and the best cut-off was determined by maximizing the sum of sensitivity and specificity using the ROC curve. Age distribution for our population was assessed by using descriptive statistics including mean, standard deviation, and median values. All statistical analyses were conducted using SAS 9.3 (SAS Institute, Cary, NC, USA). A p-value <0.05 was considered significant.\n\nTwo independent predictors for LMA difficulty were identified using logistic regression: Female and NeckCirc of 44 or greater. The area under the curve was 0.69. The area under the curve was calculated to evaluate the resulting model’s predictive value. The adjusted odds ratios and their 95% confidence interval were calculated. Continuous variables were included after the dichotomization and the best cut-off was determined by maximizing the sum of sensitivity and specificity using the ROC curve. Analyses were conducted using SAS 9.3 (SAS Institute, Cary, NC, USA).\n\n\nResults\n\nPatient demographics are presented in Table 1 and Table 2. Of the airway evaluations performed using a comprehensive airway assessment tool 69 LMAs were utilized as a primary airway device (Table 3). Of these, 67 were successful (97.1%) and 2 were unsuccessful (2.9%), with 17 (24.6%) uLMA placements considered as difficult (Table 4). Multivariate logistic regression models identified two independent predictors of difficult airway: gender and neck circumference (Table 5). The risk of difficult LMA placement was significantly higher for female patients and patients with a neck circumference (≥44 cm). The model’s c-statistic score is 0.69 (Table 6). When at least one of two identified risk factors as a cut-off for predicting difficult LMA placement is present, the sensitivity is 87.5% and the specificity is 50%. If we use two risk factors as a cut-off, the specificity increases to the level of 98% and sensitivity is 63% (Table 5).\n\n1N=51; 2N=14; 3N=49; 4N=16; NR: not reported due to zero cells; p-values are obtained by two sample t-test for continuous variables and Chi-square test or Fisher’s exact test as appropriate for categorical variables\n\nExpec: predicted, expected, at airway assessment; DMV: difficult mask ventilation; DLMA: difficult Laryngeal Mask Airway; DL: Difficult Laryngoscopy; DI: Difficult Intubation; DSA: Difficult Surgical Airway\n\nLikelihood ratio positive=Sensitivity/(1-Specificity)\n\nLikelihood ratio negative=(1-Sensitivity)/Specificity\n\nThe table displays the sensitivity and specificity if we use the given value of the number of risk factors possessed by patients as a cut-off to classify LMA difficult. For example, when we use number of risk factors at 1 as a cut-off, i.e., any patients with >=1 risk factors will be classified as LMA Diff=1 and any patients with <1 risk factors will be classified as LMA Diff=0, the sensitivity will be 0.875 and specificity will be 0.500.\n\n\nDiscussion\n\nIn the present investigation, risk factors in 69 LMA primary airway management placements were assessed. The incidence of difficult LMA placement in our study was 24.6% and the LMA failure rate was 2.9%. Moreover, the incidence of failed LMA placement in our study is consistent with previous studies9,13,18,21,22, ranging from 0.19 to 4.7%.\n\nAlthough from a large database, the study resulted only in a few placements, which is consistent with the practice of our teaching academic center and that could give a possible explanation to the increased incidence of difficult LMA placement in our study. Beside the limited number of uLMAs utilized electively, the study provides an interesting perspective on predictive factors pertaining laryngeal mask placement: indeed, two independent risk factors were found, neck circumference ≥44 cm and female gender. A predictive score that would assist the clinician in identifying difficult LMA placement was also developed, resulting in a model with low sensitivity but specificity of 98% and a negative likelihood ratio of 95.6% (for instance, excluding difficult LMA placement in male patients with neck circumference <44 cm).\n\nThe current study supports previous findings regarding the correlation of obesity and difficult airway23–26, since increased neck circumference is also an independent risk factor for difficult mask ventilation (DMV) and difficult intubation. The most interesting finding of this study is that female gender, rather than male gender is associated with difficult LMA placement in this study population. In contrast, Ramachandran et al. found that male gender was a predictive factor for failed LMA placement13,18.\n\nAge distribution of our population was considered as a cause for this difference. Indeed, age distribution of our female population could be associated with an increased proportion of postmenopausal women. Previous studies have demonstrated that the prevalence and severity of Obstructive Sleep Apnea (OSA) is increased in postmenopausal women, as compared to pre-menauposal women, which may be related to functional changes27. However, history of OSA was not an independent predictive factor in our population. This can be attributed to the retrospective nature of our study, where OSA assessment was assessed only by patient history. Of interest, a recent but unpublished study has highlighted that the female gender was a predictor for difficult LMA placement in a study population of more than 400 patients, where LMA placement was performed by a single skilled clinician28.\n\nOf the other airway variables that were evaluated in our study, none was identified as an independent predictor of LMA failure: this finding differs from that of Ramachandran et al., who recognized the absence of teeth as an independent predictor of LMA failure, and the differences could be attributed to population included in the two studies, particularly the limited number of outcomes of our study, possible underutilization of the LMA as a primary airway device, as compared to other airway devices, the increased incidence of difficult LMA placements in our population, and the placements by trainees. Discussing the limitations of the present investigation, it is necessary to mention the retrospective nature as well the stepwise selection that may contribute to bias the study, and the subjective nature of the definition of difficult LMA placement. Additionally, we assumed that all anesthesiology residents had similar educational skills based on a previous study19, which also could have affected our findings.\n\nIn conclusion, two risk factors for LMA placement difficulty were identified: female gender and large neck circumference. Considering the airway as an entity, neck circumference is being increasingly recognized as a significant predictive factor for difficulty with airway management, especially when it is considered across the spectrum of difficulties.\n\n\nData availability\n\nData have been obtained from databases at the Memorial Hermann Hospital, Texas Medical Center, Houston, IRB approval HSC-MS-07-0144. The author can support applications to the Institutional Board to make the data accessible upon individual request. Please forward your requests to Davide Cattano.", "appendix": "Author contributions\n\n\n\nKatsiampoura Anastasia D: data analysis, data interpretation, manuscript preparation\n\nCai Chunyan: data analysis, data interpretation, manuscript preparation\n\nKilloran Peter V: study design, data acquisition, data interpretation, manuscript preparation\n\nCorso Ruggero M: manuscript preparation, data interpretation\n\nHagberg Carin A: study design, study monitoring, manuscript preparation\n\nCattano Davide: study design, data acquisition, data interpretation, manuscript preparation\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was sponsored by the Foundation in Anesthesia, Education and Research as the 2007 FAER Education Grant. Dr. Carin A. Hagberg was the Principle Investigator and Dr. Davide Cattano, the Co-Investigator.\n\nCai’s research was supported by the National Institutes of Health’s Clinical and Translational Science Award grant (UL1 TR000371), awarded to the University of Texas Health Science Center at Houston in 2012 by the National Center for Clinical and Translational Sciences.\n\n\nReferences\n\nBrain AI: The laryngeal mask--a new concept in airway management. Br J Anaesth. 1983; 55(8): 801–5. PubMed Abstract \| Publisher Full Text\n\nWhite PF: Ambulatory anesthesia advances into the new millennium. Anesth Analg. 2000; 90(5): 1234–5. PubMed Abstract \| Publisher Full Text\n\nSuhitharan T, Teoh WH: Use of extraglottic airways in patients undergoing ambulatory laparoscopic surgery without the need for tracheal intubation. Saudi J Anaesth. 2013; 7(4): 436–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrimacombe J: The advantages of the LMA over the tracheal tube or facemask: a meta-analysis. Can J Anaesth. 1995; 42(11): 1017–23. PubMed Abstract \| Publisher Full Text\n\nApfelbaum JL, Hagberg CA, Caplan RA, et al.: Practice guidelines for management of the difficult airway: an updated report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Anesthesiology. 2013; 118(2): 251–70. PubMed Abstract \| Publisher Full Text\n\nBerlac P, Hyldmo PK, Kongstad P, et al.: Pre-hospital airway management: guidelines from a task force from the Scandinavian Society for Anaesthesiology and Intensive Care Medicine. Acta Anaesthesiol Scand. 2008; 52(7): 897–907. PubMed Abstract \| Publisher Full Text\n\nApfelbaum JL, Walawander CA, Grasela TH, et al.: Eliminating intensive postoperative care in same-day surgery patients using short-acting anesthetics. Anesthesiology. 2002; 97(1): 66–74. PubMed Abstract\n\nLubarsky DA: Fast track in the post-anesthesia care unit: unlimited possibilities? J Clin Anesth. 1996; 8(3 Suppl): 70S–72S. PubMed Abstract \| Publisher Full Text\n\nVerghese C, Brimacombe JR: Survey of laryngeal mask airway usage in 11,910 patients: safety and efficacy for conventional and nonconventional usage. Anesth Analg. 1996; 82(1): 129–33. PubMed Abstract \| Publisher Full Text\n\nBuckham M, Brooker M, Brimacombe J, et al.: A comparison of the reinforced and standard laryngeal mask airway: ease of insertion and the influence of head and neck position on oropharyngeal leak pressure and intracuff pressure. Anaesth Intensive Care. 1999; 27(6): 628–31. PubMed Abstract\n\nKilloran P, Maddukuri V, Altamirano A, et al.: Use of a comprehensive airway assessment form to predict difficult mask ventilation. Anesthesiology. 2014; A442. Reference Source\n\nBrimacombe J, Keller C: Laryngeal mask airway size selection in males and females: ease of insertion, oropharyngeal leak pressure, pharyngeal mucosal pressures and anatomical position. Br J Anaesth. 1999; 82(5): 703–7. PubMed Abstract \| Publisher Full Text\n\nVan Zundert TC, Hagberg CA, Cattano D: Standardization of extraglottic airway devices, is it time yet? Anesth Analg. 2013; 117(3): 750–2. PubMed Abstract \| Publisher Full Text\n\nGoodman EJ EU, Dumas SD: Correlation of pharyngeal size to body mass index in the adult. Anesth Analg. 1997.\n\nCattano D CR, Wojtzcak J, Cai C, et al.: Radiologic Evaluation of Internal Airway Anatomy Dimensions. Anesthesiology. 2014; A1139. Reference Source\n\nGu Y, McNamara JA Jr, Sigler LM, et al.: Comparison of craniofacial characteristics of typical Chinese and Caucasian young adults. Eur J Orthod. 2011; 33(2): 205–11. PubMed Abstract \| Publisher Full Text\n\nCattano D VZT, Wojtczak J, Cai C, et al.: A New Method to Test Concordance Between Extraglottic Airway Device Dimensions and Patient Anatomy. Anesthesiology. 2014; A3148. Reference Source\n\nRamachandran SK, Mathis MR, Tremper KK, et al.: Predictors and clinical outcomes from failed Laryngeal Mask Airway Unique™: a study of 15,795 patients. Anesthesiology. 2012; 116(6): 1217–26. PubMed Abstract \| Publisher Full Text\n\nCattano D, Killoran PV, Iannucci D, et al.: Anticipation of the difficult airway: preoperative airway assessment, an educational and quality improvement tool. Br J Anaesth. 2013; 111(2): 276–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCattano D, Ferrario L, Maddukuri V, et al.: A randomized clinical comparison of the Intersurgical i-gel and LMA Unique in non-obese adults during general surgery. Minerva Anestesiol. 2011; 77(3): 292–7. PubMed Abstract\n\nGrady DM, McHardy F, Wong J, et al.: Pharyngolaryngeal morbidity with the laryngeal mask airway in spontaneously breathing patients: does size matter? Anesthesiology. 2001; 94(5): 760–6. PubMed Abstract\n\nRose DK, Cohen MM: The airway: problems and predictions in 18,500 patients. Can J Anaesth. 1994; 41(5 pt 1): 372–83. PubMed Abstract \| Publisher Full Text\n\nMohsenin V: Gender differences in the expression of sleep-disordered breathing : role of upper airway dimensions. Chest. 2001; 120(5): 1442–7. PubMed Abstract \| Publisher Full Text\n\nGuilleminault C, Quera-Salva MA, Partinen M, et al.: Women and the obstructive sleep apnea syndrome. Chest. 1988; 93(1): 104–9. PubMed Abstract \| Publisher Full Text\n\nMichael Mathis MD, Satya K. Ramachandran MD, et al.: The Failed Intraoperative Laryngeal Mask Airway: A Study of Clinical and Intraoperative Risk Factors. Anesthesiology. 2011; BOC01. Reference Source\n\nBrodsky JB, Lemmens HJ, Brock-Utne JG, et al.: Morbid obesity and tracheal intubation. Anesth Analg. 2002; 94(3): 732–6. PubMed Abstract \| Publisher Full Text\n\nDancey DR, Hanly PJ, Soong C, et al.: Impact of menopause on the prevalence and severity of sleep apnea. Chest. 2001; 120(1): 151–5. PubMed Abstract \| Publisher Full Text\n\nLeavittn O, Haisook K, Robert A, et al.: B-7 A Randomized Comparison of Laryngeal Mask Airway Insertion Methods Including a Novel External Larynx Lifting- Inflating Air (ELLIA) technique on Postoperative Pharyngolaryngeal Complications Society for Airway Management annual meeting, Seattle, WA, Sept 19–21. 2014." }	[ { "id": "8514", "date": "12 May 2015", "name": "Andrea Vannucci", "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\nI wish to thank the authors very much for the opportunity to review this interesting investigation of theirs.In this retrospective study, the authors correlated a set of prospectively collected data including a comprehensive assessment of upper airway and neck anatomy and function to the event \"difficult laryngeal mask (LMA) placement\" in 2,348 adult patients. Those patients, who underwent elective, non-obstetric procedures, received an LMA Unique™ as the primary device to control the airway during their surgery.LMA placement was generally performed by anesthesiology residents.Difficult LMA placement was defined as “… either inability to physically place a LMA device or inadequacy of ventilation, oxygenation, or airway protection after placement that required conversion to an alternative technique.”The authors identified 69 patients that met their criteria for “difficult LMA placement”.By logistic regression analysis, they came to the conclusion that neck circumference larger than 44 inches and female sex were independently associated with difficult LMA placement. The hypothesis of the study that a comprehensive preoperative assessment of the airway may help predicting difficulties at the placement of an LMA is an interesting one, certainly worth of an exploration.The main issue I see in the abstract, title, methods and conclusion of the study is an ambiguous definition of “difficult LMA placement” that likely includes two separate entities: a) failure to position an LMA (2 cases in the study, as per results presented in Table 4); and b) failure of the LMA during the use (15 cases, again as per Table 4 after removing the two above mentioned failures). If my above interpretation of the study premises is correct, it is unlikely we can understand the causes of intraoperative failure of the LMA if intraoperative factors are not explored.In particular, I believe that it would be important to consider what procedures the patients were undergoing when the LMA failure was detected, how much after the induction of the anesthesia and the start of the surgery the failure occurred, and other intraoperative and anesthetic factors like position of the operating table (flat, Trendelemburg, reverse Trendelemburg, etc.), the type of anesthesia (inhalational, intravenous, balanced technique, etc.), and so on.In fact, intra-operative factors may have had a more relevant role in determining the intra-operative failure of the LMA than the baseline patient anatomical characteristics.This additional information could provide more insight on the mechanisms of LMA failure and could also facilitate a comparison of the results of this study with the ones obtained by Dr. Ramachandran and colleagues at the University of Michigan.Therefore, my advice to the authors is to add as much information as they can retrieve in their database on intraoperative factors that may have had a role in determining LMA failure.Besides this significant limitation, the study approaches the problem of \"LMA failure\" (either initial failure to position or later failure during surgery) from an interesting perspective and provides some new and interesting data that can be of interest to many clinicians. A few additional questions:Table 1: last row title “Thyroid”. What do the authors mean here? Table 3: Can the authors provide the specific references for each of the categories: “ExpecDMV”, “ExpecDLMA”, “ExpecDL”, “ExpecDI”, “ExpecDSA”? Methods: did the authors retrieve (part of) their data from an electronic or a paper anesthetic records? This information could be reported in the methods section. Discussion, page 6. Can the authors further clarify the following point (in particular what they intend by \"stepwise selection\" and how this relates to the bias of the study) “Discussing the limitations of the present investigation, it is necessary to mention the retrospective nature as well the stepwise selection that may contribute to bias the study, and the subjective nature of the definition of difficult LMA placement.”?", "responses": [ { "c_id": "1349", "date": "12 May 2015", "name": "Davide Cattano", "role": "Author Response F1000Research Advisory Board Member", "response": "We are very grateful to Dr Vannucci for his timely and well thought review. We appreciate the opportunity to respond to some of his comments and clarify few of the work's points.The study we extracted the LMA information was designed to evaluate the prediction of the difficult airway (ref 19). It was based on a paper-based evaluation form that contained all 11 of the predictive factors the ASA and other professional societies recommended (based on the practice management guidelines from 2003) as well as others. As airway management progressed during the study, different airway options populated our database (paper form collected), including LMA placements. We agree that anatomy is not the only factor contributing to difficult or failed placement of an EAD (proper relaxation, proper technique) and others may related to anatomy though, like sizing (see in the tables the estimation of used size versus optimal size). Other than placement, other factors contributes to overall failure of an EAD, which aim to be a ventilation conduit and patent airway assistance. So ventilatory mechanical and non mechanical issues can arise. Dr Vannucci correctly suggest that such factors (intraoperative) would be of great interest. Because of the scope and information available we limited to the factors contributing to the difficulty or failure of initial positioning and ventilation. The anesthesiologist attending decided and reported whether or not the LMA was satisfactory and the assessment of success (yes, no, i.e. failed) and easiness (yes, no, i.e. difficult and easy). So of the 69 placements, 17 were considered difficult, but not failed, and of the 17 also 2 were considered ultimately a failure (that is the meaning of the 2 separate tables). Expected DMV, DI etc, are based on the preoperative definition of the airway assessment, meaning the airway evaluation resulted in a predicted difficult bag mask ventilation, difficult laryngoscopy, difficult intubation etc. They not necessarily meant a difficult airway, but the predictive portion. The Thyroid in the first table was the comment of resident evaluation or acknowledging a patient condition significant for \"thyroid pathology\" (could have been goiter as well as clinical diagnosis of hyper or hypothyroidism, aspecifically).Last, the stepwise regression was performed by our statistician taking into account the primary univariate and multivariate analysis. Considering the small sample we acknowledged that study limitations need to be taken into account when interpreting the results.In conclusion the current results propose that there may be unrecognized anatomical factors, maybe related to sizing methods, which the authors are also evaluating in other investigations (13, 17). However Dr Vannucci's points pertaining the value of post placement onset ventilation and seal failures are in need of further exploration." }, { "c_id": "1350", "date": "13 May 2015", "name": "Andrea Vannucci", "role": "Reviewer Response", "response": "Thank you to the authors for the clarifications and additional information they provided. I am fine with their response." } ] }, { "id": "8512", "date": "23 Jun 2015", "name": "Massimiliano Carassiti", "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\nThe major limitation of the study is the small sample size and that the degree of experience of the operators is not classified.The study identifies the circumference of the neck and the female gender as independent risk factors for the positioning of the LMA. The \"female risk\" is in contrast to the study of Ramachandran et al., which had a larger sample size and included the experience of operators, and this must be stressed more effectively in discussion", "responses": [ { "c_id": "1431", "date": "24 Jun 2015", "name": "Davide Cattano", "role": "Author Response F1000Research Advisory Board Member", "response": "We want to thank Dr Carassiti comments. The experience of the operators is definitely important and a major factor determining the success of a device or technique. In the specific case of the current investigation, which was derived retrospectively from a larger database, all airway manipulation/management were carried out by trainees (CA-1-CA3) with a minimum experience of six month (meaning the CA1 class was enrolled only after their first 6 months of training were elapsed), and one to one supervised by an attending anesthesiologist, which ultimately was responsible. We believe the point raised are extremely important, yet relying on the fact that tailoring for every single case to the experience based, would require a significant larger sample. While we evaluated the cases by experience of the operators, in general, we did not find any skewed distribution, yet a case by case was not deemed important, because of the number of cases. As we mentioned in our discussion the results have to be carefully considered observationally, with the possibility of selection bias due to the nature of the \"mother\" study to start with, to the limited number of cases, that would include a selected group of individuals (half of the residents, because they were assigned to a full airway assessment on multiple testing), yet not for any particular reason, different. Ramachandran et al., accessed a single institution, residency program database, were, otherwise, it is reasonable to think the majority of EGA placements occurred by experienced staff. Yet the sample is much larger to question our findings. An interesting hypothesis, based on both studies, is that a differential complexity is determined by oro-pharyngeal anatomy vs laryngeal structures and neck morphology. That may explain that in males, a larger LMA would have chosen based on weight, while oropharyngeal or laryngeal structures would not accommodate for instance a size 5. In our overweight/obese population that may also explain why females patients (maybe largely selected for LMA because not as \"morbidly obese\", or accidentally clustered in our sample) resulted in more failures in our cohort.It is a great opportunity, as suggested by Dr Vannucci, to identify new frontiers of EAD research." } ] }, { "id": "9937", "date": "08 Sep 2015", "name": "Massimo Micaglio", "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 read with interest this investigation and I wish to thank the Authors for the request of my review.With this retrospective study they aimed to identify a simple, objective list of predictive factors for difficult laryngeal mask airway placement. As they underlined, at present such a list is not available.The topic is up-to-date, remarkable and well represents a growing body of research on EGA.Because LMA placement was performed by residents, some well known factors contributing to difficult placement of a LMA (type of anesthesia induction, dose of hypnotic agents, proper “waiting time” before LMA insertion, NMBA usage or not, etc.), could have been considered. But some limitations of the study are correctly stated by authors.The conclusions that large neck circumference and female gender were independently associated with difficult LMA placement definitely provide data potentially useful to clinicians.", "responses": [] } ]	1	https://f1000research.com/articles/4-102
https://f1000research.com/articles/4-101/v1	28 Apr 15	{ "type": "Opinion Article", "title": "How difficult is the validation of clinical biomarkers?", "authors": [ "Jan Voskuil" ], "abstract": "Recent developments of introducing stratified medicine/personal health care have led to an increased demand for specific biomarkers. However, despite the myriads of biomarkers claimed to be fit for all sorts of diseases and applications, the scientific integrity of the claims and therefore their credibility is far from satisfactory. Biomarker databases are met with scepticism. The reasons for this lack of faith come from different directions: lack of integrity of the biospecimen and meta-analysis of data derived from biospecimen prepared in various ways cause incoherence and false indications. Although the trend for antibody-independent assays is on the rise, demand for consistent performance of antibodies (both in choice of antibody and how to apply it in the correct dilution where applicable) in immune assays remains unmet in too many cases. Quantitative assays suffer from a lack of world-wide accepted criteria when the immune assay is not ELISA-based. Finally, statistical analysis suffer from coherence both in the way software packages are being scrutinized for mistakes in the script and remaining invisible after small-scale analysis, and in the way appropriate queries are fed into the packages in search for output that is fit for the types of data put in. Wrong queries would lead to wrong statistical conclusions, for example when data from a cohort of patients with different backgrounds are being analysed, or when one seeks an answer from software that was not designed for such query.", "keywords": [ "biomarkers", "antibodies", "validation" ], "content": "Introduction\n\nClinical biomarkers have been around for a long time now, and the field is moving rapidly. In addition to genetic and protein markers, we now also have microRNAs, epigenetic markers, lipids, metabolites, and imaging markers. Some are extremely useful as a (companion-) diagnostic; others may serve as a mere indicator. However, there are problems. There is confusion on the nomenclature and on the way how biomarkers are meant to be validated and used. A proposal published in 2006 was meant to create some clarity and consistency in the matter1. The biggest obstacle by far is that Biomarker validation and qualification depend on confirmation at different locations (different labs). There are issues with consistency in the preparation of the biological material used in the different studies, and with consistency in the choice of antibody when required. It should also be noted that in quantitative immunohistochemistry (IHC) one needs a standard in the quantification method2. A recent opinion paper reveals yet another layer of complexity: The statistical analysis is prone to wrong conclusions down to coding errors in the software3. It may not be a surprise then that one another led to the observation that only about 11% of preclinical research papers demonstrated reproducible results4. It is time to take stock and to address the different levels of disturbance complicating the process of biomarker validation and qualification.\n\n\nBiological material\n\nThe integrity of the tissue specimens will determine the quality of the biomarker’s measurements, especially when biomarkers are instable. Post-mortem samples in particular will never represent samples from living individuals because of the post-mortem delay. As the post-mortem delay will differ from individual to individual, the level of decay will vary dramatically per sample. For this reason, post-mortem samples are best fit for qualitative analysis. Quantification of any biomarker in post-mortem samples should be interpreted with extra care5.\n\nPlasma samples can be prepared in different ways: they can be prepared either by citrate, by ethylenediaminetetraacetic acid (EDTA) or by heparin. In addition, biomarkers can be tested in serum and in whole blood. It is clear that levels of biomarkers will need to be compared between equally treated samples in order to avoid variations in noise from the different ways the samples were prepared6. Since this principle is universal, it will be true for any other tissue types.\n\nFor microscopy, tissue slides and cell suspensions have to be prepared in line with the required assay before they can be investigated. Fixatives (alcohols, aldehydes), embedding materials (paraffin, LR White, etc) and temperatures (frozen vs heated) have profound effects on the integrity of the tissues and cells and they will determine the success of the assay. Again, consistency in the tissue preparation, tissue sections and cells to be analysed is paramount7,8. Mega-data analysis may get skewed when data are collated from samples treated in different ways.\n\nA systematic approach to record and keep biospecimen has been proposed and is aimed to become the new standard: Biospecimen Reporting for Improved Study Quality (BRISQ) guidelines provide a tool to improve consistency and to standardize information on the biological samples9.\n\n\nAntibody choice\n\nMass-spec and RT-PCR quantifications will be robust by the consistency of the assay material. However, the robustness of immune assays depends highly on the choice of antibodies used in the assay. Once an antibody has been successfully validated in one assay, this assay is defined by this antibody. Change of antibody will potentially change the outcome altogether as demonstrated in the past10,11. When an antibody needs changing, the assay is no longer validated and the validation procedure will have to be repeated with the new antibody. For this reason the preference goes to monoclonal antibodies. The rationale behind this preference is that the clone number of the antibody would define its characteristics: the expectation then is that the assay will remain validated because the antibodies remain identical when using antibodies from the same clone number, no matter which vendor they are from. Unfortunately this is a myth. Depending on the vendor (and sometimes depending on the catalogue number) the formulations, all with the same clone number, will differ: the antibody may be purified from ascitic fluid, from culture media, or not purified at all (just ascitic fluid or just culture supernatant). These different formulations will have an effect on the way the antibody needs to be diluted to avoid non-specific background12. Therefore, the monoclonal antibody needs to be revalidated in the same assay when the original formulation is no longer available. But even subsequent batches from the same formulation show some level of differences, thus undermining the main argument of preference to use monoclonal antibodies in standard assays. A peptide-generated polyclonal antibody from a larger animal than rabbit (for large size batches) may serve as a cost-effective alternative because the batch-to-batch variation of such antibody is limited by the size of the immunizing peptide unlike other polyclonal antibodies12.\n\n\nAssay development\n\nWhen a new assay is being developed a monoclonal antibody may not be always readily available. Then a peptide-generated polyclonal antibody may serve as a good and cost-effective alternative. However, peptide polyclonal antibodies need a new round of validation when a new batch from a different animal arrives, just like different formulated monoclonal antibodies.\n\nDuring assay development it is essential to dilute the antibody far enough to avoid non-specific background, but it needs to be strong enough to allow measuring a dynamic range, especially when the assay is quantitative. When the assay is dependent on a secondary antibody, this antibody needs validation as well (with and without primary) so to assess its non-specific signals (noise)12.\n\nSpecificity needs to be addressed by comparing specimen spiked and un-spiked with the intended protein of interest (analyte) at various quantities. The signals need to be proportionate to the spiked quantities. In addition, specimen known not to have any of the analyte needs to be compared with specimen known to have the analyte at natural levels13.\n\n\nDetection and cut-off values\n\nSensitivity is commonly attributed to the antibody used in an assay, but this is a misunderstanding. Sensitivity is determined by the detection method of which the antibody/or primary and secondary antibodies may take part in. If levels of the analyte are low, a higher sensitivity is required. This increased sensitivity is usually not accomplished by increasing the antibody concentration, although using an antibody with higher affinity will help to some extent. But in general the change of detection method (fluorophore, isotope, PCR, etc.) is the appropriate step to take. Together with the increase of sensitivity, the noise and background will also increase. When a change to a higher sensitivity is required, the validation should focus on a more stringent regime for keeping noise and background at bay12.\n\nWhen quantification is a requirement, cut-off values need to be put in place. Both the Lowest Levels Of Quantification (LLOQ) and Highest Levels Of Quantification (HLOQ) must be determined. Often the detection limits are determined as well, but this is only relevant for qualitative work. In IHC these values become tricky, because the intensity of signal is not just a number generated by a detector; the density of signal is combined with the location in the tissue. In addition, the surface area of quantification needs well defined boundaries. And even when all these measures are in place, the quality of the tissue and the quality of the slides can potentially jeopardize these measures and skew the results14. Diagnostics by IHC is therefore prone to misinterpretation when for one specific test consistency at all levels (same antibody at same dilution, identically prepared tissue samples, identical area surface, identical staining analysed, etc.) is not followed in all laboratories in the world.\n\n\nStatistics and jumping to conclusions\n\nStatistical analysis is notoriously used to provide the convenient evidence required by the author(s). No matter what method of statistics is used, when the input data have been selected from a larger set, any outcome will be biased and flawed by default. Only analysis of ALL data (non-selected) would yield proper results, but then they might be inconclusive or inconvenient. The pressure to publish in peer-reviewed papers force authors to present statistics in the most incomprehensible way possible, knowing that their peers will not admit their confusion and likely take the author’s word for it15. Even when the statistic results are sound, they may get over-interpreted. Thus original claims were made based on prejudice and weak statistics and only over time, when more scientific details become available, a more complex picture emerged. For example how cholesterol levels are linked to cardiovascular disease16,17, how cancer is not merely caused by mutations18,19, how obesity is not a choice of lifestyle20,21 etc. Simplified claims can be (and has been) driven by apparent conflicts of interest as suggested in a study22. The reputation of biomarkers has suffered dramatically from lack of scientific integrity and as a result many scientists lost faith in the usefulness of biomarker databases. New guidelines have been introduced by publishers in order to introduce a new standard on how statistics are presented23.\n\nThere are several statistical packages on the market for scientists and clinicians to use. However, these packages are quite advanced and need expertise handling, very much like a driver’s licence is required in order to safely use a motorised vehicle on the public road. Vendors of such packages admit that their products are not always properly used (personal communications). The chosen algorithms need to be appropriate for the type of data to be analysed: some algorithms are designed for decision making, and they are not necessarily fit for scientific fact finding. In addition, the same data entered in the same system may result in different output on different occasions simply because the wrong type of results is being asked for (personal communications with statistic analysts). Finally, subtle coding errors in the software cannot always be identified in small tests on script integrity, only to skew results when large scale data are being processed3.\n\n\nProject design and personalized medical care/stratified approaches\n\nWhen all the above hurdles have been successfully taken, we are not quite there yet. Each individual is different from the next, and therefore each individual has different tolerance or sensitivity to toxins and medicines. This makes the assessment of biomarkers to follow the progress of a disease, or to follow the efficacy of a therapy, difficult to analyse when a group of patients have been treated all in the same way but the individuals in the groups are so diverse in genetic and/or ethnic background that the data can still be all over the place. Only when a group is defined by a certain genetic or environmental background, would there be sufficient homogeny to assess a biomarker for this particular defined group. For example, only recently it was found that HER2-type breast cancer patients do not benefit as well from therapies when they carry PICK3CA mutations compared to those who do not24. It is like the chicken-egg (catch-22) paradigm: one has to start clinical trials in order to identify the non-responsive patients and only then one can leave them out for proper validation of a new biomarker. However, proper validation demands positive and negative controls and not allowing to select the convenient data only. Although this paradox can be dealt with properly, it is no surprise that the search for proper clinical biomarkers remains very challenging for some time to come.", "appendix": "Competing interests\n\n\n\nThe author is the Chief Scientific Officer of Everest Biotech Ltd, a research antibody manufacturer specialised in peptide-generated reagents from goat. Although the author highlights the value of peptide-generated antibodies in animals larger than rodents or rabbits as cost-effective alternative to monoclonal antibodies under specific circumstances, this notion should not be deemed as sole advertisement for Everest antibodies, since goat and other large animals are being used by other manufacturers.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSpecial thanks go to the communities of LinkedIn and ResearchGate who have been helpful in their feedback on the troubles on commercial antibodies, on statistical issues around clinical research and subsequent claims and on sample preparation/preservation issues before their use in analysis.\n\n\nReferences\n\nLee JW, Devanarayan V, Barrett YC, et al.: Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res. 2006; 23(2): 312–28. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "8494", "date": "06 May 2015", "name": "Bjorn LDM Brücher", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review the paper from Jan Voskuil. I enjoyed reading the manuscript “How difficult is the validation of clinical biomarkers?” by Jan Voskuil and I agreed to review because of the following three aspects: I appreciate the open review process of F1000Research and that such are published, as reviewers should stop hiding behind anonymity. There is – at least to me – no criterion justifying such if science increasingly wants transparency which we all know science needs.The article provided is a must read. I would assume especially within the Biotech community but also scientists and clinicians should do so.After I read the article the 2nd time, I decided to write a review but not in the usual way of reviewing a manuscript for a journal, because the necessary aspects have already been included in a comprehensive manner. My intent was including comments as well as additional aspects which may be of importance from the aspect of a clinician, surgeon and scientist for helping to see the subject biomarker from additional and different aspects. GeneralThe author provides important aspects by critically evaluating the use of biomarkers. This is necessary as the author reminds us about reality and wishes in science as well as in clinical practice.The different headlines are well thought through and chosen critically while questioning issues surrounding standardization. This is even more important as reports published for biomarkers under investigation are not standardized and make the same mistakes which had been made in the 70s and 80s in terms of tumor markers. Despite the necessity for being critical, it should not be viewed as being a synonymous with negative behavior. The author takes the responsibility by addressing major obstacles and missing data and that is without doubt highly appreciated and needed.Biomarkers in diagnosis and treatment of diseases are measured characteristics and reflect a biological state of a disease. In terms of cancer, there is hope that biomarkers will provide a detection and screening tool for diagnosis, treatment with an influence on outcome orientated patient stratification as well as on predicting and monitoring multimodal treatment. The ideal biomarker is objectively measured in a comprehensible way independent of which laboratory it is investigated in, is easily measurable, cost-effective, and evaluated as an indicator for pathological and/or biological processes and of consistent value across differences in age, gender or ethnicity. Therefore, there is a necessity to remind us to not repeat history by including nearly any protein as a biomarker. Where are we?Many biomarkers are already declared by many companies to determine or diagnose a disease, although no data of half-life, metabolism or different interaction by different pathways are known nor provided. Again, it seems that history repeats itself as we get into the same discussions and situations as during the 70s and 80s in regard to tumor markers and cancer. Therefore, it is of importance that the author attempts to structure this theme into the headlines biological material, antibody choice, assay development, detection of cut-off values, statistics and project design. Project DesignLogistically I would have thought that having the sub-headline Project Design earlier in the paper as this would indicate the direction. Biological materialOf course there is hope that biomarkers help detecting a disease or serving as a screening tool for making a diagnosis. So far, it is not clear which biological material should be used and also it is not clear if this changes occur during different stages of diseases. Further, there is no standardization on how which biological material is stored and which variables influence the quality of the assay. There is another underestimated variable which needs to be taken into account and using cancer as an example may reveal further problems: Cancer is not one disease and contains a heterogeneous set of dysfunctions such that the information available for biomarkers are also heterogeneous. This gets worse if we remind ourselves about the following: Igarashi, et al., observed in 93 specimens investigated for tumor microvessel density (MVD) and thymidine phosphorylase (dThdPase), that tumor cells strongly stained for TP were “…often observed as a rim in the periphery of the tumor nest”1. On the other hand, biomarkers are not just expressed within tumor cell nests. Takebayashi, et al., revealed in 1998 that normal esophageal tissue showed a TP expression rate of 12.3% compared to 50.9% in the tumor cell area of 163 investigated resected ESCC, although the percentage rate of cells expressing TP was less than 5% in 85.9% of non-neoplastic tissues2. Additionally even histomorphological tumor-negative lymph nodes (pN0) showed a TP expression rate of 27.9%. These examples illustrate that use of biomarkers needs a standardization in many aspects and by this scientists and clinicians need to be involved both trying to bring together the necessary aspects for future use of biomarkers, because the examples used could also mean that the gene expression is different in terms where a biopsy is taken as well as where apart of the specimen is cut for investigating biomarkers. Now, this may even be more complicated using fluids and under which condition they had been sampled, stored: was it during an operation? Do we know if drugs influenced those biomarkers of investigation with a short half-lives? Methods (antibody choice, ..)To my knowledge there is no standardization of methods in use for different biomarkers. Again, we had that during the 70s and 80s in terms of tumor markers in use and it took long to resolve. Is there a standard protocol in use for determination of microRNA? Is it not that some use RT-PCR, Northern blotting, oligo-based arrays, in situ hybridization, different assays (together with different samples)? Are these different techniques comparable? Statistics (detection of cut-off values, statistics, …)Cut-offs can be determined of course. How many papers do you know in which a group determines all available necessary variables, such as sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy? The decision-making for a cut-off is ultimately a clinical decision, as the clinician determines what is most important to know. For example, if we do not want to overlook a patient then it can be assumed that it makes sense having a high sensitivity in terms of using response. How is this in terms using a ROC analysis (receiving operator curve analysis)? Using a ROC analysis for better determination of a threshold, we need to compare the different measurements of a biomarker against a gold standard. As addressed below, it gets even more complicated: if there is a standard in use, but we know that it is not justified declaring it as a gold standard, what should we do? Furthermore,as the author points out clearly how correct observations during the past resulted in wrong conclusions, which even increased dogma behavior in cholesterol levels associated with cardiovascular disease, lifestyle and obesity and also in terms of vitamin intake and health, as well as the somatic mutation theory being the cause for cancer. An apple found in a car is not synonymous with the proof that apples grow in cars.\n\nCritical thinking and re-thinking is continuously needed for excellence in science as well as for useful approaches in daily clinical work. Someone may enjoy reading the recent critical remarks about the wear and tear of guidelines 3. I would argue that these views are also a must reading for the future implications of biomarkers. Clinical Response ClassificationThis section is of course a huge one and has multiple aspects and due to this we cannot expect that all aspects are addressed by the author. However, this aspect is extremely important, as response is in daily clinical use and therefore I would assume, there is a must as a scientific and clinical reviewer addressing some points. One major question is: Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment? This is a major problem now because if a biomarker is measured against the gold standard. But, is it justified declaring clinical response evaluation serving as gold-standard? The readers need to make up their own minds about the following which have recently been addressed as well 4. The response classification is in use since 1971 since the publication by Miller 5. What no-one wants to see is the fact, that this response classification is based on one experiment only which was conducted by Moertel and Hanley in 1976 6. Experiment16 experienced oncologists (be aware that, in 1976, there was no definition of an oncologist and there wasn’t one of an experienced oncologist either) in which they covered solid wooden spheres with a layer of rubber foam and placed them on a soft mattress. The colleagues had to measure the diameter of these spheres in a random order using rulers or calipers. The analysis showed that there was an error of 25% in the measurement of the size of identical spheres in 25% of the measurements, and that an error of at least 50% occurred in 6.8% of the measurements. This means that the clinical response classification in use was based on a single experiment, and is still in use since some 35 years. Only some minor modifications were done: the US National Cancer Institute, together with the European Association for Research and Treatment of Cancer, proposed ‘new response criteria’ for solid tumors; a replacement of 2D measurement with measurement of one dimension was made 7. Tumor response was defined as a decrease in the largest tumor diameter by 30%, which would translate into a 50% decrease for a spherical lesion 8. However, no subsequent standardized of this recommendation was carried out, and 35 years after the primary experiment, no additional studies with a structured logistical way of accurate objective assessment of treatment response have been conducted or proposed. This opens an important question: If a biomarker is measured and analyzed according to the clinical response classification above, will this reflect biology as needed? Immune ResponseAnother important variable to address contains immunological biomarkers in terms of response: Immunologists might just declare a response if immune-competent cells have been decreased and, possibly, without clinical signs of improvement of patient condition, or decrease of tumor size. But is it appropriate to declare a decrease of an immune-competent cell as a biomarker of any utility? Disease stageThe 5 year survival rates for non-metastasized localized esophageal carcinoma according to the American Cancer Society (ACS) in stage I (IA and IB) range between 71 and 57& while they drop in Stage IIA on 46% and range in Stage III (IIIA, IIIB, IIIC) between 20 and 9% 9. Do we know if different disease stages in cancer are associated with different metabolism of different biomarkers? Influence of paths which may influence quality of biomarker measurementsLet us take an example growth factor-beta (TGF-beta). It is known that inhibitors of this pathway, especially TGF-β1, block the proliferation and trigger apoptosis in malignant as well as in benign tumor cells, but with increasing tumor growth development as TGF-β1 resistance to targeted therapy develops 10 - 12. So, does this mean, that we are not aware that the function of a biomarker quality is independent from the disease, its stage how strong or weak the biomarker under investigation influence different paths? Do different situations in which biomarkers are measured mean different necessary views and if so, how can we judge those?Another example: Epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands 13. EGFR plays a critical role in tumor progression by stimulating cell cycle progression, invasion, and metastasis 14. Response measured by the EGFR-antibody Cetuximab in anticancer-treated patients does not correlate with the observed degree of EGFR expression in tumor tissue in patients with metastatic colorectal cancer 15, 16.How many of the declarations of so-called breakthroughs in measuring a biomarker are justified? I have no doubt, that it is important to measure different biomarkers, but the marketing and promotional one should not be goal of scientists. This may be seen as an ethical aspect as well, but it is unfortunate but necessary as increasingly observations are reported and determined correctly within publications, but afterwards statements with journalists implicate having a breakthrough result, as nearly every scientific finding these days are declared as such, which from my perspective is very unfortunate. This inflationary marketing way should not be followed. ConclusionTaken the aspects reviewed above together, I repeat my statement: the article by Jan Voskuil provided is a must read. Many more aspects are necessary taking into account for future evaluation and standardization of biomarkers under investigation.", "responses": [] }, { "id": "8491", "date": "11 May 2015", "name": "Ijaz S Jamall", "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\nThe paper “How difficult is the validation of clinical biomarkers?” by Jan Voskuil is timely in that as the author points out the burgeoning growth of biomarker assays particularly in chronic diseases and notably in cancer has led to the misinterpretation of both research and clinical data for all of the reasons pointed out by the author.As correctly noted by the author, there are huge variations in sample collection, storage, preparation, assay used, antibodies involved, and analysis—all of which can lead to wide variability in results and confound the end user of such data. The author correctly points out steps that can be taken early on in the process that can minimize or preclude the accuracy of the results so obtained. There is a need for standardization. Furthermore, the statistical analyses of biomarker data need to be stipulated before the data are collected and not afterwards, using statistical software in a black-box manner to see what results might show statistical significance, even though the significance may be serendipitous or not clinically relevant (Zapf et al., 2015). The slavish adherence to standard software packages by many physicians and biomedical scientists would be amusing if the wider implications of their innumeracy were not so dire (Partin et al., 2013; Tuppin et al., 2012).In order for a biomarker to be useful, it must reflect a change in concentration in the media sampled with a change in disease status. It is frequently assumed that serum or blood are the best media for the study of biomarkers but because of the number of potentially confounding variables in serum or blood, tears and saliva, because they reflect intracellular fluids, might serve as better indicators of intracellular events long before these are reflected in the blood (Pieragostino et al. 2015; Salvisberg et al., 2014).I recommend acceptance of this very interesting paper for indexation.", "responses": [] } ]	1	https://f1000research.com/articles/4-101
https://f1000research.com/articles/3-256/v1	28 Oct 14	{ "type": "Research Article", "title": "Interaction of growth hormone receptor/binding protein gene disruption and caloric restriction for insulin sensitivity and attenuated aging", "authors": [ "Oge Arum", "Jamal Saleh", "Ravneet Boparai", "Jeremy Turner", "John Kopchick", "Romesh Khardori", "Andrzej Bartke", "Jamal Saleh", "Ravneet Boparai", "Jeremy Turner", "John Kopchick", "Romesh Khardori", "Andrzej Bartke" ], "abstract": "The correlation of physiological sensitivity to insulin (vis-à-vis glycemic regulation) and longevity is extensively established, creating a justifiable gerontological interest on whether insulin sensitivity is causative, or even predictive, of some or all phenotypes of slowed senescence (including longevity). The growth hormone receptor/ binding protein gene-disrupted (GHR-KO) mouse is the most extensively investigated insulin-sensitive, attenuated aging model. It was reported that, in a manner divergent from similar mutants, GHR-KO mice fail to respond to caloric restriction (CR) by altering their insulin sensitivity. We hypothesized that maximized insulin responsiveness is what causes GHR-KO mice to exhibit a suppressed survivorship response to dietary (including caloric) restriction; and attempted to refute this hypothesis by assessing the effects of CR on GHR-KO mice for varied slow-aging-associated phenotypes. In contrast to previous reports, we found GHR-KO mice on CR to be less responsive than their ad libitum (A.L.) counterparts to the hypoglycemia-inducing effects of insulin. Further, CR had negligible effects on the metabolism or cognition of GHR-KO mice. Therefore, our data suggest that the effects of CR on the insulin sensitivity of GHR-KO mice do not concur with the effects of CR on the aging of GHR-KO mice.", "keywords": [ "Longevity regulation", "endocrinology & metabolism", "insulin sensitivity", "growth hormone hormonal signaling", "caloric restriction", "(neuro)endocrinology of senescence" ], "content": "Introduction\n\nImprovements in insulin sensitivity or blood glucose homeostatic management are hallmarks of many slow-aging mutant and dietarily restricted animals, supporting the conjectures that these endocrine and metabolic phenomena may be positive regulators of (or simply indicators of interventions that might promote) longevity [Arum et al., 2009; Bartke, 2008; Bonkowski et al., 2006; Lawler et al., 2008; Longo & Finch, 2003; Masoro, 2003; Masoro, 2005; Mattison et al., 2007; Piper & Bartke, 2008]. A recently proffered approach to biomedical ventures endeavoring to delay aging, and thus increase healthspan (the period of life during which an organism is free of substantial morbidity or physiological disability/inability), begins with studying interventions that increase lifespan [Kenyon, 2010; Miller, 2009; Olshansky et al., 2007; Warner & Sierra, 2009]. Therefore, it is of high gerontological interest to study causal associations between longevity and physiological correlates that might result in anti-aging healthspan therapies based on engendering those physiological correlates, or that might serve as useful biomarkers for pharmacological or lifestyle interventions to delay the onset and/or decelerate the rate of senescence.\n\nThe growth hormone receptor/binding protein (Ghr/bp) gene-disrupted (knockout) (GHR-KO) mouse is homozygous for a targeted disruption (knock-out, KO) of the growth hormone (GH) receptor (GHR)/binding protein gene, and is thus GH-resistant, resulting in decreased GH hormonal signaling. GHR-KO mice were generated by insertional mutagenesis that disrupted the Ghr/bp gene; this results in decreased hepatic production of insulin-like growth factor 1 (IGF-1), which leads to markedly reduced levels of circulating IGF-1, a reduced growth rate, an approximately 20% reduction in adulthood length, and an approximately 40% reduction in adult body weight [Zhou et al., 1997].\n\nOf particular note, the GHR-KO mouse outlives its littermate control by approximately 40% [Coschigano et al., 2000; Coschigano et al., 2003].\n\nProduced in the β-cells of the pancreatic Islets of Langerhans, the hormone insulin regulates metabolism and energy homeostasis, partly by inducing the tissue uptake of glucose from blood. The GHR-KO mouse exhibits markedly decreased plasma insulin levels, due partly to decreased proliferation of β-cells [Liu et al., 2004]. As blood insulin concentration inversely mediates the systemic insulin sensitivity, insulin sensitivity is greater in the GHR-KO mouse than in its littermate control [Liu et al., 2004]. This is also the case with multiple other long-lived mice [Brown-Borg et al., 1996; Conover & Bale, 2007; Conover et al., 2008; Dominici et al., 2002; Selman et al., 2008].\n\nResults from survivorship studies reveal that aging-retarding (and thus, lifespan-increasing) dietary restriction (DR), including yet not limited to caloric restriction (CR), further increases insulin sensitivity and survivorship for some long-lived mutants, the Ames (Prop1df/df) Dwarf mouse [Bartke et al., 2001] and the growth hormone releasing hormone KO (Ghrh-/-) mouse (data not shown). However it has been reported that CR doesn’t influence insulin sensitivity, and only modestly increases the survivorship of females, in the GHR-KO mouse [Bonkowski et al., 2006].\n\nAs an initial hypothesis, if CR fails to exert much effect on one senescence-associated trait (longevity) of the GHR-KO mouse because its level of insulin sensitivity is already as great as permissible for a viable animal, then a GHR-KO mouse on CR should not vary from a GHR-KO mouse on an ad libitum (A.L.) diet in other aging-associated characteristics (namely, metabolism and cognition).\n\nTherefore, we attempted to investigate whether insulin sensitivity is sufficient to explain the severely attenuated response to CR of slow-aging associated phenotypes in GHR-KO mice. Surprisingly, in the course of our experiments we discovered that CR actually increases blood insulin concentration and starkly reduces insulin sensitivity in GHR-KO mice. These results question the assertion that CR has no effect on GHR-KO mouse blood glucose homeostatic management and the relationship, if any, between insulin sensitivity and slowed senescence in GHR-KO mice.\n\n\nMaterials and methods\n\nEthics statement. Animal Protocol #178-02-001 was approved by the Laboratory Animal Care and Use Committee of Southern Illinois University-School of Medicine.\n\nGhr/bp gene-disrupted (GHR-KO) mice were generated by inserting a neomycin cassette replacing the 3′-end of the fourth exon and the 5′-end of intron 4/5 of the genomic sequence [Zhou et al., 1997]. The founder population of GHR-KO mice was provided by Dr. John J. Kopchick (Ohio University, Athens, OH). GHR-KO and GHR-N (heterozygous littermate controls for GHR-KO mice) mice were generated by mating of GHR-KO males with females heterozygous for the Ghr/bp-disrupted allele (GHR-N). These breeding schemes produce littermate control mice that have the same genetic background and are subject to the same intra-uterine and post-natal environment as the mutants.\n\nAbiding by service provider’s instructions for sample collection and shipping, genotyping was conducted via quantitative polymerase chain reaction (q-PCR)-based technologies (Transnetyx, Inc., Cordova, TN).\n\nThe resulting mice had elements of a 129/Ola, a Balb/c, two C57Bl/6J, and two C3H/HeJ stocks; therefore, although lacking the methodological benefits of “reproducible genetic heterogeneity” [Miller, et al., 1999], this stock possesses considerable genetic variation, and thus the results are likely applicable to other mouse populations.\n\nThe animals were maintained in shoebox-type cages in light- (12 hours light to 12 hours darkness) and temperature- (22 ± 2ºC) controlled rooms with constant access to Lab Diet Formula 5001 (23% protein, 4.5% fat, 6% fiber) (Nestlē Purina, St. Louis, MO) and tap water. Littermate control pups were weaned at the age of 21–23 days, and GHR-KO pups two weeks later or at the time of weaning control pups from the next litter.\n\nAll experiments were performed in female mice, as GHR-KO stock male littermate controls are insensitive to the common dosage of insulin (0.75 U.S.P.U./kg B.W.) during insulin tolerance testing [Arum et al., 2009; Bonkowski et al., 2006].\n\nMice were 4–8 months of age at inception of restriction.\n\nThe amount of food allotted each cage of mice designated for caloric restriction was determined based on (weekly calculated) ad libitum food consumption for entire cages of gender-, genotype-, and birth date-matched controls; these values were averaged over the number of cages within each such group. Two hundred grams of the above-described food was placed in each A.L. cage-hopper on a weekly basis. After six days of food consumption, the remaining food was weighed on a Scout Pro Balance (Ohaus Co., Pine Brook, NJ) calibrated to weight standards on a monthly basis; food consumption values were calculated as follows: {(200 g.) – [food remaining after six days (in g.)]}/six (days)/number of subjects in cage.\n\nAs a protection against dissimilar food consumption within CR cages, part of the food was broken into pieces small-enough to pass through the hopper-grate (but not crumbs). Our observations confirmed that this method allowed every restricted mouse to feed ad libitum during the initial surge of food consumption. Considering the valid concerns related to differential restriction resulting from a dominant cage-mate consuming more than their fair proportion, we paid particular attention to any individual mouse weight loss and health (e.g. fight wounds indicative of physical conflicts with a cage-mate) throughout our studies. It is also worth noting that our chosen level of restriction (30%) is moderate compared to the 40% level that causes considerable concerns [Liao et al., 2010; Mattson, 2010]. This moderate level does not lead to an extinguishment of food supply after the initial gorge (thus, even subordinate mice have ample, albeit possibly delayed, access to food) and does not result in substantial weight loss for any sub-cohort of animal-subjects within our stocks (Figure 1A and B).\n\nA. 30% caloric restriction represses body weight gain (absolute or normalized-to-initial) in female GHR-KO mice and their littermate controls. B. 30% caloric restriction reins change in body weight (absolute or normalized-to-initial) in GHR-KO females and their littermate controls.\n\nOf relevance to obviating unintended interactions between experimental factors, which might produce confusing or obfuscating variation within the data, mice were housed in genotype-, age-, and diet-specific cages.\n\nMice were weighed weekly on a Scout Pro Balance (Ohaus Co., Pine Brook, NJ) that was calibrated to weight standards on a monthly basis. All mice were weighed in the late afternoon, approximately 20 hours after the restricted mice had been fed.\n\nMice were young-adults in all experiments except for the indirect calorimetry trials involving CR, the spontaneous locomotor activity experiments and the behavior (anxiety & memory) experiments, where mice had to be middle-aged in order to address gerontological queries.\n\nAge-staging was based on a combination of 1) quantitative extrapolation from prior stock-specific survivorship data [Bonkowski et al., 2006], 2) presence/appearance of aging-associated wizening (as represented quantitatively by declining body weight), and 3) spontaneous, testing-independent, (and presumably) aging-resultant mortality. Thus, young-adulthood is marked by at least 90% of reproductively competent negative control subjects being alive; middle-age is the period between when approximately 90% of the control subjects are still alive and median survivorship; old-age is the period between median survivorship and when approx. 10% of the subjects are alive; and oldest-old age is designated as the period when ≤ 10% of the controls remain.\n\nAll animals underwent home-cage assessments of gross health (Supplemental Table I) and any animal exhibiting questionable health by these criteria, or which was aberrantly hypoglycemic at the inception of a test, was excluded from the testing and/or data analysis. In addition, all animals were given at-least two weeks of recuperation in-between tests.\n\nGlucose tolerance testing [ad libitum (A.L.)-fed or fasted]\n\nFor A.L.-fed tests, animals had access to food for at least 16 hours before the test. For fasted tests, animals were fasted for 16 hours, although CR animals were A.L.-fed the day before the 16-hour fast commenced. Thirty minutes prior to beginning the test, each animal was weighed, had a small nick placed at the tip of its tail with a razor, and re-housed without access to food. After 30 minutes to recover from the handling stress of the weighing and tail-nicking, blood glucose concentration was assessed in each animal. Blood was obtained by applying a gentle pressure to the tail-tip, with a blood glucose monitoring system (glucometer and testing strips) (OneTouch Ultra 2, Lifescan, Inc., Milpitas, CA). Without releasing the grasp on the animal, it was manually repositioned to a nearly supine pose, and injected inter-peritoneally with 2 g. D-(+)-glucose (Sigma-Aldrich Co., St. Louis, MO) per kg of body weight. [The powdered glucose was dissolved in 0.9% sodium chloride (Sigma-Aldrich Co., St. Louis, MO)]. Subsequent blood glucose measurements were at 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes after the injection. Animals were given A.L. access to food immediately after completion of the test.\n\nInsulin tolerance testing\n\nAnimals had access to food for at least 16 hours before the test. Animals were prepared for injection as described for glucose tolerance testing (above). Animals were injected inter-peritoneally with 0.75 U.S.P.U. of porcine insulin (Sigma-Aldrich Co., St. Louis, MO) per kg of body weight. (The lyophilized insulin was dissolved in 0.9% sodium chloride). Subsequent blood glucose measurements were at 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes after the injection. Animals were given A.L. access to food immediately after completion of testing.\n\nPyruvate conversion testing\n\nAnimals were fasted for 16 hours and CR animals were A.L.-fed the day before the fast commenced. Animals were prepared for injection as described for glucose tolerance testing (above). Animals were injected inter-peritoneally with 2 g of sodium pyruvic acid (Sigma-Aldrich Co., St. Louis, MO) per kg of body weight. (The lyophilized sodium pyruvate was dissolved in 0.9% sodium chloride). Subsequent blood glucose measurements were at 15, 30, 45, 60, and 120 minutes after the injection. Animals were given A.L. access to food immediately after completion of testing.\n\nNon-stimulated blood glucose comparisons [ad libitum (A.L.)-fed or fasted]\n\nUn-stimulated blood glucose values were obtained from young-adult mice at the beginnings of A.L.-fed and fasted glucose tolerance tests, drawn from a small nick at the tip of the tail and measured with a blood glucose monitoring system (glucometer and testing strips) (OneTouch Ultra 2, Lifescan, Inc., Milpitas, CA). A.L.-fed blood glucose values were collected after an overnight (~16 hrs.) period of A.L. feeding for all subjects; fasted blood glucose values were gathered after equivalent overnight fasting (~16 hrs.) for all subjects.\n\nA.L.-fed blood glucose values recorded immediately preceding a sacrifice and tissue harvesting from middle-aged mice were consistent with the results obtained as above. As a control against inferences drawn from the possible effects of short-term fasting, CR mice were noted to have stomachs freighted with foodstuff upon the sacrifice that followed the blood glucose assessment.\n\nIndirect calorimetry was conducted as previously described by Westbrook et al., (2009) (Accuscan Instruments, Inc., Columbus, OH). Acclimation day testing, A.L.-fed day testing and fasted day testing were all conducted in one longitudinal stretch. Data were normalized per unit of lean body weight [Butler & Kozak, 2010] as determined by fat depot sub-dissection [Berryman et al., 2010; Muzumdar et al., 2008]. The values at the 17:00 hour were excluded from the statistical analyses, as this time was used for maintenance activities (e.g. removal of food, weighing of remaining food, and weighing of mice) during longitudinal testing paradigm. Parameters assessed are annotated in Supplemental Table II a.\n\nSpontaneous locomotion was assessed using the same equipment and the indirect calorimetry procedure described above (Accuscan Instruments, Inc., Columbus, OH). The values measured at the 17:00 hour were excluded from the statistical analyses, as this time was used for maintenance activities (e.g. removal of food, weighing of remaining food, and weighing of mice) during longitudinal testing paradigm. Parameters assessed are annotated in Supplemental Table II b.\n\nBlood cell counting was accomplished using a VetScan HM2 Hematology System (Abaxis, Union City, CA) and ≥ 25 μL of whole blood [collected in EDTA-coated Microvette 100 μL capillary tubes (Sarstedt AG & Co., Nümbrecht, Germany)] drawn from ad libitum-fed subjects. The following 18 parameters were assessed: concentration of leukocytes/white blood cells (W.B.C.), concentration of lymphocytes (LYM), concentration of monocytes (MON), concentration of granulocytes (GRA), proportion of leukocytes that are lymphocytes (LYM%), proportion of leukocytes that are monocytes (MON%), proportion of leukocytes that are granulocytes (GRA%), concentration of erythrocytes/red blood cells (R.B.C.), concentration of hemoglobin (g/dL) (HGB), hematocrit (%) (HCT), mean (erythrocytic) cell volume (M.C.V.), mean corpuscular hemoglobin (pg.) (M.C.H.), mean corpuscular hemoglobin concentration (g/dL) (M.C.H.C.), red cell (erythrocytic) distribution width (%) (R.D.W.), concentration of thrombocytes/platelet cells (PLT), mean platelet volume (M.P.V.), plateletocrit/platelet hematocrit (%) (PCT), platelet distribution width (%) (P.D.W.).\n\nPlasma insulin was measured with the multiplexed Mouse Endocrine Lincoplex ELISA kit (LINCO Research, St. Charles, MO).\n\nAll animals underwent home-cage assessments of gross health, locomotor ability and activity (Supplemental Table I). Animals exhibiting questionable health based on these criteria were excluded from the testing.\n\nDuring the light-phase of their day, the mice were individually placed in the center of a lid-less, opaque, white, 44 × 44 × 40 cm. (length × width × height) polymer box with the floor divided into 16 11 × 11 cm2. The number of squares entered within the allotted time was noted per mouse per trial; as the experiment is contingent upon the novelty of the aberrant context, each subject was only tested once. The methods used derived from standard methodologies previously used “to analyze general activity and exploratory drive” [Crawley, 2007; Selman et al., 2009].\n\nAll animals underwent home-cage assessments of gross health, and locomotor ability & activity (Supplemental Table I). Animals exhibiting questionable health based on these criteria were excluded from the testing.\n\nDuring the light-phase of their day, mice were individually placed in the center of a lid-less, opaque, white, 44 × 44 × 40 cm (length × width × height) polymer box with the floor divided into 16 11 × 11 cm2. The number of squares entered within one minute was noted per mouse per trial; 24 hours after the initial evaluation (acquisition),the mice were re-tested (retention). Memory index values were calculated per mouse as follows: (Retention activity/Acquisition activity); these reflect the degree to which the subject remembered the context presented 24 hours prior (with enhanced memory putatively resulting in more movement due to less anxiety). Final location scores evaluate the ultimate (after the 60-second testing interval) position of a mouse on the retention day, with a more-ensconced placement being indicative of greater anxiety (and, thus, worse memory of prior context) than a more-exposed positioning. The methods derived from standard methodologies used by other investigators [Crawley, 2007].\n\nGraphs were generated with Excel (Microsoft, Redmond, WA) and IrfanView Image Viewer (Irfan Skiljan, Wiener Neustadt, Austria; http://www.irfanview.com/). The measures of central tendency are arithmetic means, and all depictions of variation (error bars) represent the standard deviations (S.D.) [Glantz, 2002].\n\nPre-hoc statistical measures\n\nIn brief, experimental design approaches were taken to maximize robustness while lessening the potential need to increase sample size; utilizing 1) an a priori specification of a limited number of well-defined hypotheses, 2) refinement of experimental techniques, and 3) grouping of animals so that the effect of unit variability on the treatment was minimized.\n\nPost-hoc statistical analysis\n\nLevene’s tests (to investigate scedasticity) and Kolmogorov-Smirnov tests (to determine deviations from Gaussian distribution) were conducted to guide the choice of statistical algorithms for analysis of differences amongst groups. The combined parameters of effect size and Type 1 error probability were considered when determining phenomena meriting presentation and discussion.\n\nMost data were contrasted with unpaired, homoscedastic Student’s t-test, Analysis of Variance, or Analysis of Variance for Repeated Measures (ANOVA or ANOVA-R.M., resp.), as appropriate; followed by the Tukey’s Honestly Significant Difference (H.S.D.) or the Dunnett’s t-test post-hoc tests for multiple pairwise comparisons, as appropriate.\n\nFor repeatedly measured blood glucose regulatory assessments, the p-value for a given pairwise comparison at a given time-point represents the result of testing all of the time-points, up-to-and-including that time-point, within the repeated measures analysis; this permits testing whether both groups have experienced similar excursions in blood glucose (the null hypothesis) relative to their initial values and with consideration of all intermediate values. This mode of analysis poses more discrete and descriptive inquiries than analyzing the area under respective curves or utilizing isolated, independent blood glucose values/percentages at lone time-points. The data that are normalized to initial blood glucose values were used for the precise, time-point-specific p-values reported, yet the inferences of differences amongst groups do not depend on the use of these normalized data. For a particular pairwise comparison within a particular assay, the p-value reported in the text is the most conservative (i.e. highest) sub-0.05 p-value from the series of repeated measures analyses.\n\nIn instance of considerable variation in data confounding inferences, the data outside of 1 S.D. might have been equilaterally excluded from the data used for statistical analysis.\n\nStatistical comparisons were conducted with PSPP for Windows (Free Software Foundation, Inc., http://www.gnu.org/software/pspp/get.html).\n\n\nResults\n\nProbing parameters pursuant to proliferation\n\nA 30% CR resulted in the standard body weight (B.W.) gain attenuation, whether represented in absolute grams or in percentage-of-initial (Figure 1A) or in body weight change in grams or in percentage-of-initial (Figure 1B).\n\nWhen scrutinizing proliferation on a cellular level, hematocytometric analyses of various blood cell parameters (e.g. erythrocytes, leukocytes, and platelets) in late-middle-aged (~25 months-of-age) revealed no effect of CR on either GHR-N mice or GHR-KO mice (Table 1).\n\nBlood glucose homeostatic regulation experiments\n\nImportantly, there was no effect of the 30% CR diet on B.W. measured immediately preceding the testing for any of the blood glucose homeostasis regulation assays (Figure 2A).\n\nA. 30% caloric restriction does not affect body weight immediately preceding a tolerance or conversion test for GHR-N or GHR-KO females. B. 30% caloric restriction partially corrects the glucose intolerance of female GHR-KO mice under A.L.-fed conditions (including repeated-measures statistical analysis table). C. 30% caloric restriction does not affect the glucose intolerance of GHR-KO females under fasted conditions (including repeated-measures statistical analysis table). D. 30% caloric restriction corrects the enhanced insulin sensitivity of female GHR-KO mice (including repeated-measures statistical analysis table). E. 30% caloric restriction does not significantly alter the heightened de novo hepatic glucose production of GHR-KO females (including repeated-measures statistical analysis table). F. 30% caloric restriction increases plasma insulin in female GHR-KO mice.\n\nIn relation to our hypothesis, CR increased A.L.-fed glucose incorporation in fed female GHR-KO mice during glucose tolerance testing [(p = 0.0467), (Figure 2B)], but had no effect in fasted GHR-KO mice (Figure 2C). As for the insulin tolerance tests, CR attenuated the sensitivity of GHR-KO females to 0.75 U.S.P.U./kg B.W. of insulin [(p = 0.0483), (Figure 2D)]. CR did not alter the pyruvate conversion potential in female GHR-KO mice (Figure 2E). Additionally, CR increased the plasma insulin content in GHR-KO mice [(p < 0.05, (Figure 2F)].\n\nTherefore, our data show additive or synergistic effects of CR with the GHR-KO gene disruption on blood glucose homeostasis.\n\nIndirect measures of metabolism\n\nMeasurements estimating the general rate of metabolic processes have long been correlated with ultimate survivorship, and have been proffered as sufficient to explain the rate of senescence [Rubner, 1908]. Whether the mechanisms by which CR retards senescence include alterations (particularly, decreases) in metabolism has been an active research hypothesis for some time [Ramsey et al., 2000].\n\nIndirect (gas exchange) calorimetric measurements of metabolism have been reported to be increased [Westbrook et al., 2009], as well as decreased [Carrillo & Flouris, 2011; Mookerjee et al., 2010], in animals with extended longevity. Identifying metabolic phenotypes that transcend one particular genetic background or mode of delaying and/or decelerating aging would be important for proposing or testing mechanisms of extended lifespan and healthspan.\n\nDuring our analyses of oxygen consumption (VO2), respiratory quotient (R.Q.)/respiratory exchange ratio (R.E.R.), heat production (Calories/hr), and energy expenditure (E.E.) in A.L.-fed and fasted female GHR-KO mice on CR, no genotype- or diet-based differences were detected for food consumption, changes in body weight induced by either acclimation or fasting, or thermogenesis (as crudely measured with an ambient thermometer in each chamber) while the subjects were in the indirect calorimetry chambers from the acclimation day through the A.L.-fed day to the fasted day (Table 2).\n\nn.b.: 25 g. of food placed in each chamber at beginning of acclimation day\n\nCR did not affect these A.L.-fed indirect calorimetry-based measures of metabolism effects of female littermate controls, nor that of female GHR-KO mice (Table 3).\n\nSpontaneous locomotor activity late in life, as it can serve as a measure of the multi-factorial syndrome of frailty [Walston et al., 2006] (i.e. frail mice would presumably be disinclined to move, or cover less area when trying) is often used as a behavioral marker of delayed and/or decelerated senescence [Ingram, 2000; Manini, 2010; Minor et al., 2011; Neff et al., 2013; Wilkinson et al., 2012; Zhang et al., 2014].\n\nSimilarly, CR had no germane effect on the spontaneous locomotion of female littermate control mice, or female GHR-KO mice (Table 4).\n\nCognitive assessments\n\nThe retention of cognitive capability (in particular, memory function) into middle-age and beyond in the GHR-KO mice further supports that the effects of the Ghr/bp disruption extend beyond increasing survivorship, to include ameliorating senescence and its resultant functional decrements [Bartke, 2005; Kinney et al., 2001a; Kinney et al., 2001b; Kinney-Forshee et al., 2004; O. Arum & A. Bartke, (unpublished data)]. With noteworthy exceptions, which are partly due to varied methodologies of caloric restriction or cognitive assessment amongst scientists, CR is broadly considered to be beneficial for retarding aging-resultant cognitive decline [Arslan-Ergul et al., 2013; Joseph et al., 2009].\n\nRegarding the cognitive assessments, CR had no effect on the anxiety of either littermate control or GHR-KO females (Figure 3A). The memory index results derived from open-field activity (Figure 3B) did not support the initial hypothesis that the greater insulin sensitivity of GHR-KO mice precludes their full benefits from CR (as the GHR-KO mice on A.L. and the GHR-KO mice on CR, which have differing insulin responsiveness, did not differ in memory performance). Similar inferences were concluded for the open-field activity-based memory tests regarding the final location of the mice (Figure 3C).\n\nA. Neither Ghr/bp disruption nor 30% caloric restriction alters anxiety-betraying activity in an open field for female mice. B. 30% caloric restriction does not change the (memory index) performance of female GHR-KO mice in the open-field paradigm. C. 30% caloric restriction does not change the (final location) performance of female GHR-KO mice in the open-field paradigm.\n\n\nDiscussion\n\nThe initial aim of this study was to investigate if the very limited response of the GHR-KO mouse to a 30% CR diet in terms of longevity [Bonkowski et al., 2006] is related to the inability of these mutants to respond to a 30% CR diet with regards to insulin sensitivity [Bonkowski et al., 2006]. This was based on the hypothesis that it is the maximization of the response to CR in the insulin sensitivity test that acts as a “ceiling/floor” effect limiting the survivorship response to CR [Bonkowski et al., 2006]. Our insulin sensitivity results in GHR-KO mice on 30% CR differed from those obtained in a previous study showing that caloric restriction promotes euglycemia in GHR-KO mice (Figure 2C). These differences might have been due to the difference in ages of the animals {12 months in [Bonkowski et al., 2006] vs. 8–13 months in the present report}, or different durations of CR (10 or 12 months vs. 4–6 months, respectively). Those caveats emptor notwithstanding, that blood insulin content is increased by CR in GHR-KO mice (Figure 2E) dovetails with the improved performance in glucose bolus assimilation (Figure 2A), decreased insulin sensitivity (Figure 2C), and (statistically indistinguishable) decreased gluconeogenic capability (Figure 2D) of GHR-KO mice on CR relative to their A.L. counterparts. Moreover, data from macromolecular analysis of insulin signaling in GHR-KO mice on CR, including decreased insulin receptor (INSR) and thymoma viral proto-oncogene 1/protein kinase b (AKT1/PKB) concentrations in the skeletal musculature of GHR-KO’s on CR, and decreased phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) subunits content in the livers of GHR-KO’s on CR (relative to GHR-KO’s on AL), also corroborate and portend decreased insulin sensitivity in GHR-KO mice on CR [Bonkowski et al., 2009]. Additionally, it is worth noting that a tight regulation of euglycemia would be more consistent with health and survival than a predilection for hypoglycemia [Tan & Flanagan, 2013], thus “improving health”, as CR has been broadly documented as doing, might mean preventing the innate endocrinological/metabolic derangements that are merely coincident with the longevity of the GHR-KO mouse. Finally, to the best of our knowledge, published reports on CR-mediated induction of insulin sensitivity (vis-à-vis increased blood glucose assimilation dynamics) using insulin tolerance tests or hyperinsulinemic-euglycemic clamping assays on healthy mice are either lacking or are not consistently reproduced. This is an important limitation, and caveat emptor, given that mutant mice with abnormal growth and adult body composition have been documented to have insulin tolerance testing results in disagreement with the molecular biology-based assumptions of their insulin sensitivity [Boparai et al., 2010].\n\nWe also investigated the effects of CR on the performance of GHR-KO mice in other gerontologically associated measures. We discovered that CR did not alter the metabolism or spontaneous activity of GHR-KO mice and also revealed that CR has no effect on the anxiety or memory function of GHR-KO mice. This documentation of lacking amenability of GHR-KO mice to effects of CR further underscore a seeming epistasis of the genetic effect of Ghr/bp disruption to the environmental effect of dietary restriction.\n\nIn summary, our results question the notion of maximized insulin sensitivity obviating further lifespan increase in GHR-KO mice. Future studies aimed at elucidating concordant physiological, and ultimately (macro)molecular, underpinnings of disparate instances of longevity would benefit from heeding analyses that reduce or eliminate the likelihood of suspected mechanisms.\n\n\nData availability\n\nDataset 1. Experimental data showing the effect(s) of growth hormone (GH) receptor (GHR)/binding protein (Ghr/bp) gene disruption and/or caloric restriction on the various outcomes, http://dx.doi.org/10.5256/f1000research.5378.d37530 [Arum et al., 2014].", "appendix": "Author contributions\n\n\n\nO.A., R.K.K., and A.B. acquired funding for this study; O.A. and A.B. conceived and designed this study; J.G.T. provided accommodations for the blood glucose regulatory dynamics, and technical training for the anxiety and cognitive assessments in this study; O.A., R.K.B., & J.K.S. methodologically executed this study; O.A. statistically analyzed the data from this study; and O.A. & A.B. prepared the manuscript for this study. J.J.K. provided the founder population of the growth hormone receptor/binding protein gene-disrupted mice bred for this study. All authors approved the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Institute on Aging Grants AG19899, U19 AG023122, and 3R01AG019899-07S1, as well as a Senior Scholar Award in Aging from The Ellison Medical Foundation, and The Glenn Foundation for Medical Research.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplemental Tables\n\nCharacteristics of interest in home-cage assessments are succinctly detailed.\n\n\nReferences\n\nArslan-Ergul A, Ozdemir AT, Adams MM: Aging, neurogenesis, and caloric restriction in different model organisms. Aging Dis. 2013; 4(4): 221–32. PubMed Abstract \| Free Full Text\n\nArum O, Bonkowski MS, Rocha JS, et al.: The growth hormone receptor gene-disrupted mouse fails to respond to an intermittent fasting diet. Aging Cell. 2009; 8(6): 756–60. 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PubMed Abstract \| Free Full Text" }	[ { "id": "6555", "date": "18 Nov 2014", "name": "Subburaman Mohan", "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\nCaloric restriction prolongs longevity in experimental animals via altering insulin sensitivity. The growth hormone receptor knockout (GHR KO) mice live longer and have increased insulin sensitivity. In this study, the authors tested the hypothesis that the modest effect of caloric restriction on longevity of GHR KO mouse is because of the failure of caloric restriction to further increase the already maximally elevated insulin sensitivity in this mouse model. Surprisingly, the authors found that caloric restriction increased blood insulin concentration and reduced insulin sensitivity in GHR KO mice. These findings refute the established assertion between insulin sensitivity and and slowed senescence in GHR KO mice The paper is well written and the data analyses are appropriate. The conclusions are supported by experimental data. The number of animals per group are considerably large except for GHR-N on caloric restricted group. I recommend indexing the paper as submitted.", "responses": [] }, { "id": "7783", "date": "12 Mar 2015", "name": "Florian Muller", "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\nThe study addresses a long running question in aging research, which concerns the interaction of the two regimes known to consistently extend mammalian (mouse) lifespan: caloric restriction and manipulation of the GH/IGF1 axis.The study provides a wealth of very useful endocrinological and metabolic data and the authors must be commended their extensive work. The interpretation with regards to the effects of insulin sensitivity and caloric restriction are plausible, though ultimately, whether this is truly the mechanism by which the lifespan-extending effects are mediated (and account for the differential response to CR in GHRko mice), will require direct experimental demonstration.I would like the authors to discuss the metabolic results a little more. For example, it is indicated that the total caloric output of GHrKO mice is higher, considerably so, than WT (regardless of CR). However, the heat production is more or less the same. At the same time, CR has opposite effects on hear production in GHRko versus WT mice. It would seem to me overall, that as a whole the metabolic effects of CR in table 3, are much attenuated in the GHRko mice, i.e. WT mice have much more profound alterations in metabolic parameters in response to CR than do GHRko mice to CR.These are just suggestions. Overall, the data and paper are very valuable as is.", "responses": [] } ]	1	https://f1000research.com/articles/3-256
https://f1000research.com/articles/3-309/v1	18 Dec 14	{ "type": "Research Article", "title": "Culture and identification of Borrelia spirochetes in human vaginal and seminal secretions", "authors": [ "Marianne J. Middelveen", "Jennie Burke", "Eva Sapi", "Cheryl Bandoski", "Katherine R. Filush", "Yean Wang", "Agustin Franco", "Arun Timmaraju", "Hilary A. Schlinger", "Peter J. Mayne", "Raphael B. Stricker", "Marianne J. Middelveen", "Jennie Burke", "Eva Sapi", "Cheryl Bandoski", "Katherine R. Filush", "Yean Wang", "Agustin Franco", "Arun Timmaraju", "Hilary A. Schlinger", "Peter J. Mayne" ], "abstract": "Background: Recent reports indicate that more than 300,000 cases of Lyme disease are diagnosed yearly in the USA. Preliminary clinical, epidemiological and immunological studies suggest that infection with the Lyme disease spirochete Borrelia burgdorferi (Bb) could be transferred from person to person via intimate human contact without a tick vector. Detecting viable Borrelia spirochetes in vaginal and seminal secretions would provide evidence to support this hypothesis.Methods: Patients with and without a history of Lyme disease were selected for the study after informed consent was obtained. Serological testing for Bb was performed on all subjects. Semen or vaginal secretions were inoculated into BSK-H medium and cultured for four weeks. Examination of genital cultures and culture concentrates for the presence of spirochetes was performed using light and darkfield microscopy, and spirochete concentrates were subjected to Dieterle silver staining, anti-Bb immunohistochemical staining, molecular hybridization and PCR analysis for further characterization. Immunohistochemical and molecular testing was performed in three independent laboratories. Positive and negative controls were included in all experiments.Results: Control subjects who were asymptomatic and seronegative for Bb had no detectable spirochetes in genital secretions by PCR analysis. In contrast, spirochetes were observed in cultures of genital secretions from 11 of 13 subjects diagnosed with Lyme disease, and motile spirochetes were detected in genital culture concentrates from 12 of 13 Lyme disease patients using light and darkfield microscopy. Morphological features of spirochetes were confirmed by Dieterle silver staining and immunohistochemical staining of culture concentrates. Molecular hybridization and PCR testing confirmed that the spirochetes isolated from semen and vaginal secretions were strains of Borrelia, and all cultures were negative for treponemal spirochetes. PCR sequencing of cultured spirochetes from three couples having unprotected sex indicated that two couples had identical strains of Bb sensu stricto in their semen and vaginal secretions, while the third couple had identical strains of B. hermsii detected in their genital secretions.Conclusions: The culture of viable Borrelia spirochetes in genital secretions suggests that Lyme disease could be transmitted by intimate contact from person to person.", "keywords": [ "Lyme borreliosis", "chronic Lyme disease", "Borrelia burgdorferi", "spirochetes", "sexual transmission." ], "content": "Introduction\n\nLyme disease is the most common human tick-borne disease in the world today (Stricker & Johnson, 2014). It is transmitted by Ixodes ticks and is caused by the spirochete Borrelia burgdorferi (Bb) (Burgdorfer et al., 1982). Bb is phylogenetically related to the spirochetal agent of syphilis, Treponema pallidum (Gupta et al., 2013). T. pallidum is transmitted sexually between partners through contact of mucosal membranes, gaining access to the bloodstream through microabrasions and then disseminating systemically (Ho & Lukehart, 2011). The close phylogenic relationship of Bb to T. pallidum suggests that this mode of transmission might be possible for Bb.\n\nIn addition to theoretical considerations, evidence for non-vector transmission of Bb is based on animal models. Proof of contact transmission of Bb – without involvement of an arthropod vector – was established by two studies in mice. Burgess et al. (1986) caged uninfected deer mice with experimentally-infected deer mice and demonstrated transmission of Bb by seroconversion of contact-exposed mice from negative to positive and by the isolation of Bb from the blood of one contact-exposed mouse 42 days after initial contact. A study by Wright & Nielsen (1990) demonstrated that white-footed mice were susceptible to oral infection and transmitted infection to each other through direct contact. Furthermore, sexual transmission of Bb has been proposed in a canine model. Bb was transmitted to uninfected female dogs in estrus via semen by natural breeding with male dogs infected experimentally with Bb (Gustafson, 1993). Successful transmission of infection from male dogs to female dogs was shown by seroconversion of female dogs from negative to positive as well as the detection of Bb DNA in the tissue of fetuses from resulting pregnancies. If contact transmission of Bb occurs in mice and sexual transfer occurs in dogs, it is not unreasonable to postulate similar routes of infection in humans.\n\nWe sought to determine if viable Borrelia spirochetes could be recovered from human vaginal and seminal secretions, an important first step to investigate whether sexual transmission of these spirochetes among humans is possible.\n\n\nMaterials and methods\n\nControl subjects who were asymptomatic without a history of Lyme disease and patients with a history of Lyme disease were recruited for the study after written informed consent to collect and publish their data was obtained. Approval for sample collection was obtained from the Western Institutional Review Board, Olympia, WA (WIRB® #20141439). Further approval for sample testing was obtained from the Institutional Review Board of the University of New Haven, West Haven, CT. Serological testing of all participants was performed by IGeneX Reference Laboratories, Palo Alto, CA. Patients were considered positive for Lyme disease if they were serologically positive or if they had musculoskeletal, neurocognitive or cardiac symptoms clinically consistent with a Lyme disease diagnosis. None of the patients were taking antibiotics at the time of testing.\n\nBorrelia spirochetes were cultured as previously described (Bankhead & Chaconas, 2007; Middelveen et al., 2013b; Middelveen et al., 2014a). The inoculum for blood culture was prepared as follows: 10 milliliters of whole blood was collected by sterile venipuncture from each patient. Samples sat at room temperature for 10 to 15 minutes allowing clotting to occur. Red blood cells (RBCs) were separated by low speed centrifugation. Barbour–Stoner–Kelly H (BSK-H) complete medium was used for cultures with the addition of 6% rabbit serum (Sigma Aldrich, #B8291) and the following antibiotics: phosphomycin (0.02 mg/ml), rifampicin (0.05 mg/ml), and amphotericin B (2.5 µg/ml) (Sigma Aldrich).\n\nThe culture medium described above was inoculated for blood culture with the spun serum containing white blood cells and some RBCs, and for genital culture with either ejaculated semen or vaginal secretions collected by intravaginal swabbing with a sterile cotton-tipped swab. Blood and genital cultures were incubated at 32°C in an Oxoid anaerobic jar (Thermo Scientific) containing an AnaeroGen sachet (Thermo Scientific) to provide an anaerobic environment. Cultures were incubated for four weeks and checked weekly by light and/or darkfield microscopy for visible motile spirochetes.\n\nAll cultures were processed for microscopic imaging and PCR by centrifuging the culture fluid at 15,000 g for 20 minutes to concentrate spirochetes. The supernatant was discarded and the pellet retained.\n\nDieterle silver staining was performed using two fixation methods. In the standard method, formalin-fixed, paraffin-embedded pellets were sectioned and stained with Dieterle silver stain as previously described (Aberer & Duray, 1991; Middelveen et al., 2013a). In the newer method, culture fluid was spread and dried on a SuperFrost™ Plus microscope slide (Fisher Scientific) and fixed by incubating the slide in acetone for 10 minutes at -20°C, as previously described (Sapi et al., 2013). Dieterle silver staining was performed on the acetone-fixed slide.\n\nA. McClain Laboratories. Blood and genital culture pellets were processed for special staining at McClain Laboratories LLC, Smithtown, NY. Formalin-fixed, paraffin-embedded pellets were sectioned and stained with Dieterle silver stain or anti-Bb immunostains for spirochete detection, as previously described (Middelveen et al., 2013a; Middelveen et al., 2014a). In brief, immunostaining was performed using an unconjugated rabbit anti-Bb polyclonal antibody (Abcam ab20950), incubated with an alkaline phosphatase probe (Biocare Medical #UP536L), followed by a chromogen substrate (Biocare Medical #FR805CHC), and counterstained with hematoxylin. Positive and negative controls were prepared for comparison purposes with liver sections from Bb-inoculated mice and uninfected mice followed by Dieterle and immunostaining. Culture pellets from mixed Gram-positive bacteria and mixed Gram-negative bacteria were also prepared for comparison purposes as negative controls to exclude cross-reactivity with commonly encountered microorganisms. Staining was titrated to determine optimal antibody dilutions to achieve positive staining of spirochetes while minimizing background staining (Middelveen et al., 2013a; Middelveen et al., 2014a).\n\nB. University of New Haven. Samples were processed for Bb immunostaining as previously described (Sapi et al., 2013). Culture fluid was spread and dried on a SuperFrost™ Plus microscope slide (Fisher Scientific) and fixed by incubating the slide in acetone for 10 minutes at -20°C. Dried, fixed culture fluid was submerged under 100 μl of polyclonal FITC-labeled rabbit anti-Bb antibody (Thermo Scientific #PA-1-73005) diluted 1:50 in 1× PBS buffer with 1% BSA (Sigma Aldrich #A9418). For negative controls, the antibody was omitted and replaced with normal rabbit serum. The slides were then incubated for 1 hour at 37°C in a humidified chamber, washed with 1× PBS for 5 minutes at room temperature, rinsed twice in double distilled water and dried in a laminar air-flow hood for 10 minutes. The slides were mounted with Vectashield mounting medium with DAPI counterstain (Vector Labs) and viewed with fluorescent microscopy at 400× magnification with a Leica DM2500 microscope (Sapi et al., 2013).\n\nThe Bb molecular beacon DNA probe was generously provided by Dr. Alan MacDonald. Probe FlaB (sequence of 23 mer) was derived from the Bb open reading frame (ORF) BB0147 (approximately 1100 mer) of the flagellin B gene. A nucleotide Basic Local Alignment Search Tool (BLAST) search of the 23 mer sequence disclosed no matches in the human genome or in any other life form other than the Bb sequence of BB0147.\n\nBb detection with the molecular beacon was performed as previously described (Middelveen et al., 2014a) using the following protocol: paraffin sections were dewaxed by baking at 60°C, then immersed in serial 100% xylene baths followed by serial immersion through baths of 100% ethanol, 90% ethanol, 80% ethanol, and finally in distilled H2O, and then air-dried. Fixed sections were immersed in 20 μl of the working DNA beacon solution. The sectioned specimen was covered with a layer of plastic cut from a Ziploc® freezer bag and was heated at 90°C for 10 minutes to denature DNA and RNA. The heat was first reduced to 80°C for 10 minutes, then the slides were removed from heat and allowed to gradually cool to 24°C. The slides were washed in PBS, covered with 30% glycerol and a glass coverslip, then examined under an EPI Fluor microscope. Staining of test specimens was performed alongside staining of positive and negative controls. The positive control was prepared by embedding a known Bb strain in agarose, formalin-fixing the specimen then blocking in paraffin and staining sections as described above.\n\nThe specificity of the FlaB probe was validated in studies performed at the University of New Haven (Sapi E., unpublished observation 2014; see Supplemental Figure 1). The FlaB probe hybridized to Bb sensu stricto, yet failed to hybridize with B. afzelii, B. garinii, B. hermsii, Treponema denticola and Escherichia coli. Thus the probe appears to be specific for detection of Bb sensu stricto.\n\nBlood and genital culture pellets were first dissolved in 200 μl of Qiagen buffer, then forwarded to the University of New Haven, Department of Biology and Environmental Science, West Haven, CT, USA and Australian Biologics, Sydney, NSW, Australia for PCR detection of Borrelia.\n\nA. Australian Biologics. Detection of Borrelia by PCR was performed as previously described (Mayne et al., 2012) using the Eco™ Real-Time PCR system with primers targeted to the genes encoding 16S rRNA (Borrelia), flA (T. denticola) and fliG1 (T. pallidum) and analyzed with the software version 3.0.16.0. DNA was extracted from the dissolved culture pellets using the QIAamp DNA Mini Kit (Qiagen) and 20 μl were used for each reaction. The thermal profile involved incubation for 2 minutes at 50°C, polymerase activation for 10 minutes at 95°C then PCR cycling for 40 cycles of 10 seconds at 95°C dropping to 60°C sustained for 45 seconds. All samples were run in duplicate with positive and negative controls. Positive controls were genomic DNA samples from B. burgdorferi, B. garinii, and B. afzelii (Amplirun DNA/RNA amplification controls, Vircell S.L, Granada, Spain). Negative controls were samples of non-template DNA in molecular-grade water. The magnitude of the PCR signal generated (∆R) for each sample was interpreted as positive or negative compared to positive and negative controls.\n\nIn samples with sufficient DNA for sequencing, endpoint PCR amplification and Sanger sequencing of the Borrelia gene target from cultures was followed by BLAST comparison with known Borrelia sequences, as previously described (Mayne et al., 2012).\n\nB. University of New Haven. DNA samples were extracted from blood, vaginal or seminal cultures by lysing cells overnight in 180 µl tissue lysis buffer (Qiagen) and 20 µl Proteinase K (Qiagen) at 56°C in a shaking water bath followed by phenol:chloroform extraction the next day. The DNA was resuspended in 50–100 µl 1×TE buffer.\n\nA published TaqMan assay targeting a 139-bp fragment of the gene encoding the Borrelia 16S rRNA was used for the detection of Borrelia in DNA extracted from patient samples (O’Rourke et al., 2013). All reactions were carried out at a final volume of 20 µl and consisted of 900 nM of each primer, 200 nM of probe, and 10 µl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems) and 1 nanogram of DNA. Amplifications were carried out on a CFX96 Real-Time System (Bio-Rad), and cycling conditions consisted of 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Fluorescent signals were recorded with CFX96 Real-Time software and Cq threshold was set automatically. The reactions were performed in triplicate with positive and negative controls.\n\nNested PCR primers for the genes encoding the Borrelia 16S rRNA, fla and pyrG loci were used as previously described (Clark et al., 2013; Margos et al., 2010; Sapi et al., 2013). Reactions were carried out in a final volume of 50 µl using 10 µl template DNA. Final concentrations were 2× Buffer B (Promega), 2 mM MgCl2, 0.4 mM dNTP mix, 2 µM of each primer, and 2.5 U Taq polymerase (Invitrogen). “Outer” primers were used in the first reaction. “Inner” primers were used for the nested reaction, in which 1 µl of PCR product from the first reaction was used as template for the second. Cycling parameters were as follows: 94°C for 5 minutes followed by 40 cycles of denaturation at 94°C for 1 minute, annealing for 1 minute (temperature based on the primer set used), and extension at 72°C for 1 minute, with a final extension step at 72°C for 5 minutes. PCR products were visualized on 1–2% agarose gels. Sanger sequencing was used for gene analysis, as previously described (Margos et al., 2010).\n\n\nResults\n\nAll patient data are shown in Table 1. The control group included four asymptomatic patients (two males and two females). All four were seronegative for Bb.\n\nPatients 6 & 7 (), 8 & 9 (), 10 & 11 (†), and 12 & 13 (††) are sexual partners. Patients 8 and 11 were seronegative but clinically diagnosed with Lyme disease.\n\nThe patient group included six male subjects and seven female subjects, including four pairs of partners (Patients 6 and 7, 8 and 9, 10 and 11, and 12 and 13, respectively). Eleven of the 13 patients selected for the study were serologically positive for Lyme disease. Patient 1 was serologically equivocal and patient 8 was seronegative, although Bb plasmid DNA was detected in whole blood and serum from this patient.\n\nBlood cultures from 11 patients were incubated for four weeks and checked weekly for spirochete growth using light and darkfield microscopy. Motile spirochetes and/or motile spherules were observed in the culture fluid from all 11 patients after four weeks (Table 2). Genital cultures from the four controls were incubated for four weeks. None of the control cultures contained visible spirochetes, and the cultures were sent for PCR testing. Genital cultures from the 11 patients were incubated for four weeks and checked weekly. Motile spirochetes were observed in the culture fluid from all 11 patients after four weeks (Figure 1A). See Dataset, data file 1.\n\nSee Dataset, data file 1. ND, not done.\n\nA: Darkfield image of genital culture from Patient 1. Note numerous spirochetes. 400× magnification. See Dataset, data file 1. B: Dieterle silver stain of genital culture from Patient 12. Note darkly staining spirochete. Formalin fixed slide, 400× magnification. See Dataset, data file 2. C: Semen sample from Patient 10 showing B. burgdorferi spirochetes (left) and sperm cell (right). Dieterle silver stain of acetone fixed slide, 1000× magnification. See Dataset, data file 2.\n\nMost genital cultures grew very well and contained abundant spirochetes, but some blood cultures contained few spirochetes. Therefore, to better document the presence of spirochetes in culture, the culture fluid was concentrated into pellets by centrifugation (Table 3). Spirochetes and/or spherules were detected by sectioning and special staining of paraffin blocked pellets in all the patient blood and genital cultures concentrated by centrifugation, except for blood and genital culture pellets from Patient 1 that were lost during paraffin blocking (Table 3). Control genital culture samples were sent directly for PCR testing and were not subjected to light and darkfield microscopy.\n\nSee Dataset, data file 2.\n\nA. Dieterle silver staining. Using standard Dieterle staining, spherules and/or spirochetal forms were visible in all patient genital cultures (Figure 1B). Spirochetes were detected in all patient genital culture pellets except for Patient 1, whose pellet was lost during processing (Table 3). Using the newer fixation method, spirochetes and sperm cells were visible in semen samples and showed distinct morphology (Figure 1C). Sperm cells are known to stain with silver stains (Pathak et al., 1979; Schmid et al., 1983). Sperm cells were seen in all semen samples except for Patients 2 and 6, who had vasectomies (data not shown). Since control genital cultures had no visible spirochetes, the control samples were sent directly for PCR testing and were not subjected to Dieterle silver staining. See Dataset, data file 2.\n\nB. Anti-Bb immunostaining.\n\nI. Culture fluid – University of New Haven\n\nGenital culture fluid from Patient 1 was fixed on a SuperFrost™ Plus microscope slide and was stained with FITC-labelled polyclonal anti-Bb antibody. Staining was strongly positive, revealing well-defined spirochetes morphologically consistent with Bb (Figure 2A). The polyclonal antibody was not reactive to T. denticola (data not shown).\n\nII. Culture pellets – McClain Laboratories\n\nAnti-Bb immunostaining was positive for all genital cultures except for Patient 1, whose pellet was lost during processing (Table 4). Immunostaining revealed both spiral and globular Bb forms (Figure 2B). Since control genital cultures had no visible spirochetes, the control samples were sent directly for PCR testing and were not subjected to immunostaining. See Dataset, data file 3.\n\nSee Dataset, data files 3 and 4. ND, not done.\n\nPositive Bb immunostaining of genital culture fluid. See Results section.\n\nA: B. burgdorferi immunostaining of vaginal culture from Patient 1. Note intensely staining spiral and round forms in culture. 400× magnification. B: B. burgdorferi immunostaining of seminal culture from Patient 6. Note intensely staining spiral and round forms in culture. 400× magnification. See Dataset, data file 3.\n\nHybridization with the Fla B probe was positive for genital culture pellets from Patients 2–9 (Table 4). The culture pellet from Patient 1 was lost during processing. The molecular probe showed intense staining in vaginal secretions and less intense staining in semen samples (Figure 3A and 3B). See Dataset, data file 4.\n\nA: Molecular hybridization of B. burgdorferi-specific FlaB probe with seminal culture from Patient 6. Note intensely staining spiral and round forms in culture. 400× magnification. B: Molecular hybridization of B. burgdorferi-specific FlaB probe with vaginal culture from Patient 7. Note intensely staining spiral and round forms in culture. 400× magnification. See Dataset, data file 4.\n\nA. Australian Biologics. Borrelia 16S rRNA sequence was not detected by real-time PCR in any of the control genital culture pellets. In contrast, Borrelia 16S rRNA sequence was detected in genital culture pellets from 11 of 13 patients (Table 5A). Patient 2 had equivocal test results and Patient 3 had negative test results in seminal cultures. See Dataset, data file 5. Real-time PCR failed to detect treponemal gene sequences in any of the control or patient genital culture pellets. See Dataset, data file 5a. The 16S rRNA isolates from six patients were sequenced and subjected to BLAST analysis (see below).\n\nB. University of New Haven. PCR testing using the TaqMan assay for Borrelia 16S rRNA sequence was positive in blood culture pellets from seven of nine patients tested (Table 5B). Patients 1 and 5 had negative results in blood culture pellets using the TaqMan assay, but both were positive by nested PCR for the pyrG gene. In addition, nested PCR targeting the fla gene was performed on blood culture pellets from Patients 2, 3 and 4, and nested PCR targeting the 16S rRNA gene was performed on the blood culture pellet from Patient 6. The samples were positive, and sequencing revealed 99–100% homology with Bb sensu stricto strain B-31 (Table 5B). See Dataset, data file 7.\n\nTable 5A: Real-time PCR – Australian Biologics.\n\nTable 5B. PCR – University of New Haven.\n\nPCR testing using the TaqMan assay for Borrelia 16S rRNA sequence was negative in all four control genital culture pellets, and nested PCR targeting the pyrG and fla genes was negative in all four control samples, confirming the results of the TaqMan assay (Table 5B). In contrast, eight of nine patients were positive for TaqMan 16S rRNA sequence in the genital culture pellets. Patient 6 was negative using the TaqMan assay for 16S rRNA sequence but positive using nested PCR targeting a different portion of the 16S rRNA gene (Table 5B). Nested PCR targeting the fla gene (Patient 3) and the 16S rRNA gene (Patients 3 and 7) was also performed on genital culture pellets and was positive in those patients, confirming the results of the TaqMan assay. Patient 12 had positive PCR targeting the pyrG gene with confirmatory sequencing (see below).\n\nPCR isolates of the vaginal culture from Patient 1 (Australian Biologics) and the seminal culture from Patient 3 (University of New Haven) were subjected to Sanger sequencing and BLAST analysis and showed 97–99% homology with Bb sensu stricto strain B-31 (Table 5A and Table 5B). See Datasets, data files 6 and 7. PCR isolates of blood cultures from Patients 2, 3, 4 and 6 were subjected to Sanger sequencing and BLAST analysis at University of New Haven and showed 99–100% homology with Bb sensu stricto strain B-31 (Table 5B). See Dataset, data file 7.\n\nPCR isolates of genital cultures from three couples having unprotected sex (Patients 6-7, 10-11 and 12-13) were subjected to Sanger sequencing and BLAST analysis. Patients 6, 7, 10, 11 and 13 had sequencing done at Australian Biologics, while Patient 12 had sequencing done at University of New Haven. Sequencing revealed that the first and third couples had Borrelia strains that matched Bb sensu stricto strain B-31 (Table 6). In contrast, the second couple had PCR sequences that matched B. hermsii strain YOR. Thus the Borrelia strain shared by this couple differed significantly from the strains identified in the other couples. See Dataset, data file 6.\n\nSequencing for Patients 6, 7, 10, 11 and 13 was done at Australian Biologics. Sequencing for Patient 12 was done at University of New Haven. See Dataset, data file 6.\n\n\nDiscussion\n\nIn this study using standard and published culture, immunohistochemical, molecular hybridization and PCR techniques, we have shown that Borrelia strains are present in semen and vaginal secretions from patients with Lyme disease. Simultaneous testing for treponemal spirochetes was negative in genital secretions of all Lyme disease patients, confirming the specificity of Borrelia detection in these patients. Furthermore we have shown that couples having unprotected sex have virtually identical strains of Borrelia in their genital secretions, suggesting that Borrelia spirochetes might be transmitted from person to person without a tick vector.\n\nAs expected, PCR sequencing of cultured Borrelia from semen and vaginal secretions yielded primarily Bb sensu stricto strains, reflecting the North American origin of our study subjects. In addition, PCR sequencing of genital secretions from one couple yielded identical strains of Bb sensu stricto strains in two different laboratories. However, we were surprised to find one couple with identical strains of B. hermsii in their genital secretions. The presence of a distinct Borrelia strain in semen and vaginal secretions from a sexually active couple that differs from strains found in other couples supports the premise of Borrelia transmission via shared genital secretions. The finding is analogous to sharing distinct human immunodeficiency virus (HIV) strains, which is well recognized in sexual partners with HIV/AIDS (Shaw & Hunter, 2012).\n\nAnimal models have provided compelling evidence for contact transmission of Bb without a tick vector in mice, ducks, cats and dogs (Burgess et al., 1986; Burgess & Patrican, 1987; Burgess, 1989; Burgess, 1992; Wright & Neilsen, 1990). Bb has been shown to survive in stored semen from dogs, rams and bulls (Kumi-Diaka & Harris, 1995). Furthermore, seminal transmission of Bb has been noted in dogs, as described above (Gustafson, 1993). In contrast, contact transmission of Bb could not be demonstrated in Lewis rats and Syrian golden hamsters (Moody & Barthold, 1991; Woodrum & Oliver, 1999). Technical limitations in the study of these highly inbred rodents may have contributed to the negative results.\n\nWhile it is not possible to perform controlled sexual transmission studies in humans, several investigators have speculated that this mode of transmission is possible (Bach, 2001; Harvey & Salvato, 2003; Stricker et al., 2004). The suggestion that Bb could be transmitted sexually was initially proposed by Bach in 2001. He observed that sexually active patients had a marked propensity for antibiotic failure and speculated that re-infection occurred by intimate person-to-person contact. Bb DNA was detected by PCR technology in human breast milk, umbilical cord blood, semen and vaginal secretions taken from patients presenting at his practice (Bach, 2001).\n\nThe study of a group of chronically ill Bb-seropositive and PCR-positive patients in Houston, Texas – a non-endemic area – provided epidemiological evidence that Lyme disease could spread in the absence of a suitable vector (Harvey & Salvato, 2003). In the absence of infected ticks, intimate person-to-person transfer was implicated as the probable means of transmission (Harvey & Salvato, 2003). A study by Stricker et al. provided clinical and immunological evidence for Bb transmission from partner to partner. In heterosexual seropositive couples with Lyme disease in which only one partner had a documented tick bite, the partner with the documented tick bite tended to have more severe clinical manifestations of the disease and a lower CD57 natural killer (NK) cell level (Stricker et al., 2004). This difference in clinical severity and CD57 NK cell level was not noted in seropositive couples diagnosed with Lyme disease in which both partners had a documented history of tick bite (Stricker et al., 2004). Sexual transfer of Borrelia infection through mucosal contact therefore seems possible in humans. The fact that we have been able to culture motile, actively reproducing, viable spirochetes from human genital secretions reinforces this hypothesis.\n\nRecent reports from the Centers for Disease Control and Prevention (CDC) indicate that more than 300,000 cases of Lyme disease are diagnosed yearly in the USA (CDC, 2013). Sexual transmission of Borrelia may partly explain the large number of annual cases that is almost two times higher than breast cancer and six times higher that HIV/AIDS (Stricker & Johnson, 2014). Recognition of possible sexual transmission of Borrelia in both humans and animals is fundamentally important because of the epidemiological implications. If sexual transmission of Borrelia occurs in both animals and humans, this mode of transmission is a possible means of introducing Borrelia infection into areas not considered endemic and of introducing the spirochete to new reservoirs. Borrelia would also join the list of other spirochetes that are either proven or postulated to be sexually transmitted, including the spirochetal agents of syphilis and leptospirosis (Harrison & Fitzgerald, 1988; Maatouk & Moutran, 2014).\n\nLyme disease diagnosis is based largely upon serological testing using CDC-sanctioned two-tier surveillance criteria supported by FDA-approved commercial test kits. While most patients in this study did have positive serological test results for Lyme borreliosis, some were considered serologically negative, and the majority of our study subjects did not meet the positive standard as defined by the CDC surveillance criteria (CDC, 2014a). We were able to detect Borrelia spirochetes in the blood and/or genital secretions of all patients who were clinically diagnosed with Lyme disease, demonstrating that the CDC surveillance protocol is inadequate diagnostically. Inadequate diagnostic methodology undoubtedly results in under-reporting of Lyme disease, and at least one group has speculated that this substandard methodology is considered acceptable because Borrelia is not sexually transmitted (Lange & Sayyedi, 2002). In addition, if Borrelia spirochetes were transmitted sexually, then patients with false-negative results may unknowingly spread the infection to sexual partners.\n\nThe 2011 CDC case definition for Lyme disease states that a positive Bb culture confirms the diagnosis of the disease (CDC, 2014b). Although culture of Borrelia genital isolates may be a useful diagnostic laboratory methodology in the future, detecting and characterizing cultured Borrelia isolates is not straightforward, and both false-positive and false-negative results could occur. In our experience, human clinical isolates from genital secretions frequently propagate prolifically in culture, but on occasion they do not. In such instances, the culture must be concentrated and specific staining should be conducted to ascertain the presence of spirochetes. Once detected, spirochetes must be characterized genetically for specific identification. PCR is currently the most reliable means for correctly identifying cultured isolates, but even this methodology has drawbacks and limitations (Lange & Sayyedi, 2002; Nolte, 2012).\n\nThere are currently no standardized FDA-approved PCR protocols or kits available for Bb detection, so commercial PCR testing constitutes an array of “home brew” assays using different methodologies such as real-time PCR and nested PCR, with various primers targeting different genes, yielding wide differences in sensitivity and specificity (Nolte, 2012; Schmidt, 1997; Yang et al., 2012). False negatives can result because primers may be strain-specific and may not detect all Borrelia genotypes, and fluids such as blood, semen and vaginal secretions may contain substances inhibitory to the PCR process (Lange & Sayyedi, 2002; Nolte, 2012; Yang et al., 2012). The potential for false-positive PCR testing may also arise if there is DNA contamination in the laboratory, and appropriate positive and negative controls must be included in the assay (Nolte, 2012; Lange & Sayyedi, 2002). We experienced differences in primer specificity in our clinical isolates and also found that inhibition occurred, particularly in semen cultures.\n\nAnother complicating factor in Borrelia isolation is the morphological variation of the spirochete, which includes spherical, granular or cystic forms. Morphological variants of Bb, some of which are not culturable, are well documented in the medical literature (Barthold et al., 2010; Hodzic et al., 2014; Kurtti et al., 1987; Preac Mursic et al., 1996). These variants may play a role in infection, enabling Bb and other pathogenic spirochetes to evade the immune system (Döpfer et al., 2012; Menten-Dedoyart, 2012; Preac Mursic et al., 1996). Limited Bb growth and non-spiral morphology are thought to be induced by unfavorable environmental conditions (Brorson et al., 2009), and these features appear to be consistent with our observations. We found that Borrelia growth was more vigorous with more long slender morphological variants in cultures of genital secretions compared to cultures of blood, and we speculate that the human circulatory system is a more hostile environment for Borrelia than the human reproductive system.\n\nSeveral questions have been raised about the likelihood of Borrelia sexual transmission (Craig, 2014). First, according to the CDC surveillance system Lyme disease occurs most commonly in children and older adults. However, the CDC surveillance system only captures about 10% of Lyme disease patients, and the other 90% may have a different demographic distribution consistent with sexual transmission, as shown in a recent study from Australia (Mayne, 2015). Second, while sexually transmitted diseases like herpes simplex virus (HSV) and gonorrhea show an urban predominance, Lyme disease has a more rural distribution (Craig, 2014). However, Lyme disease is acquired in more ways than HSV and gonorrhea, and the rate of sexual transmission is unknown at present. Thus the epidemiology of Lyme disease may differ from other sexually transmitted diseases based on these undefined variables. Third, the transmission of HIV can be traced from one sex partner to another using HIV strain typing. Based on our study, a similar transmission pattern using Borrelia strain typing may be seen once larger studies are performed among couples having unprotected sex. In summary, sexual transmission of Borrelia is plausible in light of our limited knowledge about the risk of acquiring Lyme disease.\n\nIn conclusion, we have shown that Borrelia spirochetes are present in semen and vaginal secretions of patients with Lyme disease. Furthermore, virtually identical strains of Borrelia are present in couples having unprotected sex, suggesting that transmission via intimate contact without a tick vector may occur. The epidemiology and clinical risk of Borrelia sexual transmission remain to be determined.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data of Borrelia spirochetes in human vaginal and seminal secretions, 10.5256/f1000research.5778.d40491 (Middelveen et al., 2014b).\n\n\nConsent\n\nWritten informed consent to publish clinical details and study results was obtained from each participant.", "appendix": "Author contributions\n\n\n\nMJM recruited patients, performed the spirochete cultures and wrote the original manuscript. CB, KRF, AT and ES performed the IFA and PCR studies. JB, YW and AF performed the PCR studies. HAS and PJM provided patient samples and edited the manuscript. RBS recruited patients, coordinated all studies, revised the manuscript and edited it for publication. All authors approved the manuscript for publication.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to declare. Preliminary results of the study were presented at the Western Regional Meeting of the American Federation for Medical Research, Carmel, CA, on January 25, 2014, and published in abstract form (J Invest Med 2014;62:280–1).\n\n\nGrant information\n\nSupported in part by a grant to MJM from the Lindorf Family Foundation, Newark, OH. This work is dedicated to the memory of Dr. Willy Burgdorfer.\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 thank Drs. Stewart Adams, Gordon Atkins, Robert Bransfield, George Chaconas, Douglas Demetrick, Dorte Dopfer, Christopher Hardy, Nick Harris, Doug Kahn, Alan MacDonald, Steve McClain, Kary Mullis, Jyotsna Shah, Leo Shea and Janet Sperling for helpful discussion. We are grateful to Dr. Robert B. Allan, Joel Israel and Anita Vieyra for technical support, and we thank Lorraine Johnson for manuscript review.\n\n\nSupplementary material\n\nFlaB hybridization is shown in green, while DAPI counterstain of bacterial targets is shown in blue. Note specific hybridization of FlaB probe with B. burgdorferi sensu stricto and lack of hybridization with other Borrelia strains, T. denticola or E.coli. 400× magnification. See Dataset, data file 4.\n\n\nReferences\n\nAberer E, Duray PH: Morphology of Borrelia burgdorferi: structural patterns of cultured borreliae in relation to staining methods. J Clin Microbiol. 1991; 29(4): 764–72. PubMed Abstract \| Free Full Text\n\nBach G: Recovery of Lyme spirochetes by PCR in semen samples of previously diagnosed Lyme disease patients. International Scientific Conference on Lyme Disease. 2001. 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PhD thesis, University of Wisconsin, Madison. 2014. Reference Source\n\nHarrison NA, Fitzgerald WR: Leptospirosis--can it be a sexually transmitted disease? Postgrad Med J. 1988; 64(748): 163–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarvey WT, Salvato P: ‘Lyme disease’: ancient engine of an unrecognized borreliosis pandemic? Med Hypotheses. 2003; 60(5): 742–59. PubMed Abstract \| Publisher Full Text\n\nHo EL, Lukehart SA: Syphilis: using modern approaches to understand an old disease. J Clin Invest. 2011; 121(12): 4584–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHodzic E, Imai D, Feng S, et al.: Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. PLoS One. 2014; 9(1): e86907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumi-Diaka J, Harris O: Viability of Borrelia burgdorferi in stored semen. Br Vet J. 1995; 151(2): 221–4. PubMed Abstract \| Publisher Full Text\n\nKurtti TJ, Munderloh UG, Johnson RC, et al.: Colony formation and morphology in Borrelia burgdorferi. J Clin Microbiol. 1987; 25(11): 2054–8. PubMed Abstract \| Free Full Text\n\nLange R, Sayyedi S: Evidence of Lyme borreliosis infection from the viewpoint of laboratory medicine. Int J Med Microbiol. 2002; 291(Suppl 33): 120–4. PubMed Abstract \| Publisher Full Text\n\nMaatouk I, Moutran R: History of syphilis: between poetry and medicine. J Sex Med. 2014; 11(1): 307–10. PubMed Abstract \| Publisher Full Text\n\nMargos G, Hojgaard A, Lane RS, et al.: Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species, Borrelia kurtenbachii. Ticks Tick Borne Dis. 2010; 1(4): 151–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayne PJ: Investigations of Borrelia burgdorferi genotypes in Australia obtained from erythema migrans tissue. Clin Cosmet Investig Dermatol. 2012; 5: 69–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayne PJ: Clinical determinants of Lyme borreliosis, babesiosis, bartonellosis, anaplasmosis and ehrlichiosis in an Australian cohort. Int J Gen Med. 2015; 8: 1–12.\n\nMenten-Dedoyart C, Faccinetto C, Golovchenko M, et al.: Neutrophil extracellular traps entrap and kill Borrelia burgdorferi sensu stricto spirochetes and are not affected by Ixodes ricinus tick saliva. J Immunol. 2012; 189(11): 5393–5401. PubMed Abstract \| Publisher Full Text\n\nMiddelveen MJ, Mayne PJ, Kahn DG, et al.: Characterization and evolution of dermal filaments from patients with Morgellons disease. Clin Cosmet Investig Dermatol. 2013a; 6: 1–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddelveen MJ, Burugu D, Poruri A, et al.: Association of spirochetal infection with Morgellons disease [v1; ref status: indexed, http://f1000r.es/8g]. F1000Res. 2013b; 2: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddelveen MJ, McClain SA, Bandoski C, et al.: Granulomatous hepatitis associated with chronic Borrelia burgdorferi infection: a case report. Res Open Access. 2014a; 1: 875. Publisher Full Text\n\nMiddelveen MJ, Burke J, Sapi E, et al.: Raw data of Borrelia spirochetes in human vaginal and seminal secretions. F1000Research. 2014b. Data Source\n\nMoody KD, Barthold SW: Relative infectivity of Borrelia burgdorferi in Lewis rats by various routes of inoculation. Amer J Trop Med Hyg. 1991; 44(2): 135–9. PubMed Abstract\n\nMursic VP, Wanner G, Reinhardt S, et al.: Formation and cultivation of Borrelia burgdorferi spheroplast-L-form variants. Infection. 1996; 24(3): 218–26. PubMed Abstract \| Publisher Full Text\n\nNolte O: Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis - Lost in the Jungle of Methods, Targets, and Assays? Open Neurol J. 2012: 6(Suppl 1–M7): 129–139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Rourke M, Traweger A, Lusa L, et al.: Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR. PLoS One. 2013; 8(5): e63968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPathak S, Lau YF, Drwinga HL: Observations on the synaptonemal complex in Armenian hamster spermatocytes by light microscopy. Chromosoma. 1979; 73(1): 53–60. PubMed Abstract \| Publisher Full Text\n\nSapi E, Pabbati N, Datar A, et al.: Improved culture conditions for the growth and detection of Borrelia from human serum. Int J Med Sci. 2013; 10(4): 362–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt BL: PCR in laboratory diagnosis of human Borrelia burgdorferi infections. Clin Microbiol Rev. 1997; 10(1): 185–201. PubMed Abstract \| Free Full Text\n\nSchmid M, Müller H, Stasch S, et al.: Silver staining of nucleolus organizer regions during human spermatogenesis. Hum Genet. 1983; 64(4): 363–70. PubMed Abstract \| Publisher Full Text\n\nShaw GM, Hunter E: HIV transmission. Cold Spring Harb Perspect Med. 2012; 2(11): a006965. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStricker RB, Johnson L: Lyme disease: Call for a “Manhattan Project” to combat the epidemic. PLoS Pathog. 2014; 10(1): e1003796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStricker RB, Moore DH, Winger EE: Clinical and immunologic evidence for transmission of Lyme disease through intimate human contact. J Investig Med. 2004; 52: S151.\n\nWoodrum JE, Oliver JH Jr: Investigation of venereal, transplacental, and contact transmission of the Lyme disease spirochete, Borrelia burgdorferi, in Syrian hamsters. J Parasitol. 1999; 85(3): 426–30. PubMed Abstract \| Publisher Full Text\n\nWright SD, Nielsen SW: Experimental infection of the white-footed mouse with Borrelia burgdorferi. Am J Vet Res. 1990; 51(12): 1980–7. PubMed Abstract\n\nYang J, Liu Z, Guan G, et al.: Evaluation of molecular methods for detection of Borrelia burgdorferi senso lato in ticks. Diagn Microbiol Infect Dis. 2012; 73(1): 80–3. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7090", "date": "05 Jan 2015", "name": "Sam T. Donta", "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\nThere are a number of issues that mitigate against the authors' conclusion that Lyme disease can be transmitted sexually. While there are conflicting reports from animal studies that there can be transmission by contact between animals and other studies that appear to better controlled that do not provide such evidence, there is no obvious epidemiological evidence in humans that this is a likely possibility. Apart from the sociologic implications of claiming that intimate or even casual transmission is possible, there needs to be more compelling evidence that this might be the case than is offered in this report.Specific issues in this report:No evidence that samples were blinded. The numbers of patients were too small from which to draw meaningful conclusions. The actual serologic data on patients should be presented in order to be more properly assessed. PCR-DNA analyses should have been done on the original specimens; again in a blinded fashion, and weekly during the culture period. And, as PCR-DNA testing is much more sensitive than cultures, without this information, the validity of the presented information remains in question. Concurrent samples of other body fluids, i.e. blood, saliva, should have been included. Even if presuming the presence of the Lyme borrelia in vaginal secretions or semen, the numbers present would have not been sufficient to cause any transmission of infection, as, with any infectious process, there needs to be a critical inoculum to establish infection. This is the case with transmission by ticks. Borrelia may be spirochetes as are Treponemes and Leptospira, but the transmission of the latter are obviously through body fluids, and not by tick or other vectors. It is not clear that infections transmitted by ticks are also transmitted by intimate or casual contact. If it was true that 8/11 samples were positive, one would expect a much more obvious clinical picture of transmission by intimate or casual contact, which is not the case. PCR-DNA analyses and long-term cultures can be subject to contamination, making the data here more difficult to interpret.", "responses": [ { "c_id": "1179", "date": "20 Jan 2015", "name": "Raphael Stricker", "role": "Author Response", "response": "Co-written with Marianne J. MiddelveenThere are a number of issues that mitigate against the authors' conclusion that Lyme disease can be transmitted sexually. We did not conclude that “Lyme disease can be transmitted sexually”. Based on preliminary editorial comments, we were careful to state that our microscopy, immunochemistry, molecular hybridization and PCR analysis showing live, culturable Borrelia in semen and vaginal secretions suggests that these spirochetes could be transmitted in that manner. Our study does not prove this form of transmission, and we do not make this claim anywhere in the text. While there are conflicting reports from animal studies that there can be transmission by contact between animals and other studies that appear to better controlled that do not provide such evidence, there is no obvious epidemiological evidence in humans that this is a likely possibility. Apart from the sociologic implications of claiming that intimate or even casual transmission is possible, there needs to be more compelling evidence that this might be the case than is offered in this report.The statement that “better controlled” studies do not provide support for contact transmission of Borrelia in animals is contrary to the examples in mice and dogs described in the Introduction and Discussion sections of our article. We have pointed out that the two studies on highly inbred rodents that allegedly showed lack of such transmission did not use PCR techniques and therefore may have missed this transmission. The fact that the CDC now admits to more than 300,000 new cases of Lyme disease each year in the USA (and perhaps as many as one million new cases, as outlined in Stricker & Johnson, 2014) is suggestive that other forms of transmission occur, as noted in the Discussion section on page 10. 1. No evidence that samples were blinded. In response to the referee’s comment, we have noted that the laboratory testing was performed on coded samples in a blinded fashion. This has been noted in the Abstract and reiterated throughout the Methods section. 2. The numbers of patients were too small from which to draw meaningful conclusions.The “meaningful conclusions” are that  microscopy, immunochemistry, molecular hybridization and PCR analysis demonstrates live, culturable Borrelia in semen and vaginal secretions from Lyme disease patients. Although the numbers are relatively small, we feel that our detailed study supports this conclusion. 3.The actual serologic data on patients should be presented in order to be more properly assessed.We can include the actual serologic data as an original Dataset, but we don’t see how that would alter the experimental findings in our study. The serologic data is presented in Table 1 and the Results section. In response to the referee’s comment, we have added more detail about the serologic and diagnostic criteria in the Methods section on page 4, with supporting references. 4. PCR-DNA analyses should have been done on the original specimens; again in a blinded fashion, and weekly during the culture period. And, as PCR-DNA testing is much more sensitive than cultures, without this information, the validity of the presented information remains in question.PCR-DNA analysis was done on the “original specimens” in a blinded fashion  in conjunction with the microscopy, immunochemistry and molecular hybridization analysis. Repeated testing at weekly or other intervals is beyond the scope of this pilot study, and this type of testing should certainly be explored in future Lyme disease studies. 5. Concurrent samples of other body fluids, i.e. blood, saliva, should have been included.As shown in Table 5, we did do PCR testing on concurrent blood samples in some patients. Saliva testing for Borrelia would have been of interest, but this form of Borrelia testing requires further investigation and is beyond the scope of our study.  6. Even if presuming the presence of the Lyme borrelia in vaginal secretions or semen, the numbers present would have not been sufficient to cause any transmission of infection, as, with any infectious process, there needs to be a critical inoculum to establish infection. This is the case with transmission by ticks.In response to the referee’s comment, we have researched the number of spirochetes necessary for transmission of B. burgdorferi infection in mice and T. pallidum  infection in humans. The results show that a very small number of spirochetes (as little as 18 organisms) are required for transmission of infection, and we have included this information in the Discussion on page 11. We have also noted that seminal plasma and the female genital tract may provide a relatively permissive environment for spirochetes compared to blood, skin and other immune sites, making transmission theoretically easier via the genital route. Borrelia may be spirochetes as are Treponemes and Leptospira, but the transmission of the latter are obviously through body fluids, and not by tick or other vectors. It is not clear that infections transmitted by ticks are also transmitted by intimate or casual contact.In response to the referee’s query, we have provided examples of other agents (Babesia, Chlamydia, Coxiella) that are proven or postulated to be transmitted by both tickbite and intimate contact. This information is included in the Discussion on page 11 with supporting references. If it was true that 8/11 samples were positive, one would expect a much more obvious clinical picture of transmission by intimate or casual contact, which is not the case.As noted above, the substantial numbers of  new Lyme disease cases each year suggests additional forms of transmission beyond a tickbite. At this time, the true epidemiology of Lyme disease is unknown because the CDC surveillance system only captures less than 10% of Lyme disease cases, as noted in the Discussion on page 11. Other epidemiological studies have suggested that some infected patients may be relatively asymptomatic (Harvey & Salvato, 2003), so transmission via intimate contact resulting in less obvious infection is plausible. The risk of this form of transmission and correlation with symptoms merits further study. Our report simply raises the possibility, and rejecting the report will shove this issue under the rug to the detriment of Lyme patients. 9. PCR-DNA analyses and long-term cultures can be subject to contamination, making the data here more difficult to interpret.PCR-DNA analysis is subject to contamination. That is why we did blinded testing that always included negative controls in three different laboratories using microscopy, immunochemistry and molecular hybridization to confirm the PCR findings. Although PCR testing alone might be “difficult to interpret”, the combination of experimental techniques done in different laboratories makes interpretation much more reliable.We hope that the referee will change his opinion after reading the revised manuscript and our responses to his comments." } ] } ]	1	https://f1000research.com/articles/3-309
https://f1000research.com/articles/4-80/v1	26 Mar 15	{ "type": "Research Note", "title": "Funding source and primary outcome changes in clinical trials registered on ClinicalTrials.gov are associated with the reporting of a statistically significant primary outcome: a cross-sectional study", "authors": [ "Sreeram V Ramagopalan", "Andrew P. Skingsley", "Lahiru Handunnetthi", "Daniel Magnus", "Michelle Klingel", "Julia Pakpoor", "Ben Goldacre", "Andrew P. Skingsley", "Lahiru Handunnetthi", "Daniel Magnus", "Michelle Klingel", "Julia Pakpoor" ], "abstract": "Background: We and others have shown a significant proportion of interventional trials registered on ClinicalTrials.gov have their primary outcomes altered after the listed study start and completion dates. The objectives of this study were to investigate whether changes made to primary outcomes are associated with the likelihood of reporting a statistically significant primary outcome on ClinicalTrials.gov.Methods: A cross-sectional analysis of all interventional clinical trials registered on ClinicalTrials.gov as of 20 November 2014 was performed. The main outcome was any change made to the initially listed primary outcome and the time of the change in relation to the trial start and end date.Findings: 13,238 completed interventional trials were registered with ClinicalTrials.gov that also had study results posted on the website. 2555 (19.3%) had one or more statistically significant primary outcomes. Statistical analysis showed that registration year, funding source and primary outcome change after trial completion were associated with reporting a statistically significant primary outcome.Conclusions: Funding source and primary outcome change after trial completion are associated with a statistically significant primary outcome report on clinicaltrials.gov.", "keywords": [ "clinical trials", "funding", "primary outcome" ], "content": "Introduction\n\nClinical trials provide the principal method with which to assess the effectiveness of therapeutic strategies1. An important principle in the good conduct of clinical trials is that a summary of the trial protocol, with a pre-defined primary outcome, should be freely available before the study commences1. In February 2000, the United States (US) Food and Drug Administration (FDA) created an online clinical trials registry named ClinicalTrials.gov2. We and others have shown a significant proportion of interventional trials registered on ClinicalTrials.gov have their primary outcomes altered after the listed study start and completion dates3,4. In this extended analysis, we sought to investigate whether changes made to primary outcomes are associated with the likelihood of reporting a statistically significant primary outcome on ClinicalTrials.gov.\n\n\nMethods\n\nWe used R (http://cran.r-project.org/web/packages/rclinicaltrials/vignettes/basics.html) to download data from all completed interventional clinical studies registered with ClinicalTrials.gov as of 20th November 2014, as previously described3. New to this study, we also downloaded data concerning study results for these trials; specifically the ‘p value’ fields from the ‘study results’ tab for primary outcomes.\n\nChanges in primary outcomes were defined as previously described3. Probable funding source was derived using the algorithm previously described3.\n\nA trial having a statistically significant primary outcome was defined as a trial having a P value less than 0.05 in the p value field in the study results tab for any primary outcome.\n\nWe used logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (95% CI) for comparisons between significant primary outcome and non-significant primary outcome groups, using registration date, primary outcome change after study completion and funding source as explanatory variables. P-values <0.05 were interpreted as significant. Statistical analyses were conducted using the STATA 12.0.0 software.\n\n\nResults\n\nAs of 20 November 2014, 13,238 completed interventional trials were registered with ClinicalTrials.gov that also had study results posted on the website. The trials were registered between 1999 and 2014 and 2555 (19.3%) had one or more statistically significant primary outcomes. There were 3934 (29.7%) trials classed as non-industry funded, 1569 (11.9%) as mixed and 7735 (58.4%) as industry funded. 12632 (95.4%) trials had a change in the primary outcome reported at initial registration; 12243 (92.5%) of these occurred after the trial completion date.\n\nStatistical analysis showed that registration year, funding source and primary outcome change after trial completion were associated with reporting a statistically significant primary outcome (Table 1).\n\n\nConclusions\n\nWe found that the reporting of statistically significant outcomes on ClinicalTrials.gov was more likely for trials with primary outcomes that had been changed and also those funded by industry. Previous studies have documented these associations5,6, and we confirm these using ClinicalTrials.gov data. There are limitations to our analyses- we have not investigated in any detail the nature of the primary outcome change and the potential effect this would have on the statistical analysis/outcomes. As discussed previously3, some primary outcome changes that we have identified may be typographical/semantic and may not reflect actual changes to the nature of the outcome. We also did not look specifically to see whether a changed primary outcome was the one with a statistically significant finding, just whether a statistically significant finding was found for any primary outcome for the study. The vast majority of studies with results reported on ClinicalTrials.gov had a primary outcome change. This suggests that these trials are ones where the registrations have more diligent data updating. Nevertheless, this should be seen in equal measure for trials with and without statistically significant primary outcomes. In summary, funding source and primary outcome changes are associated with the reporting of statistically significant primary outcomes on ClinicalTrials.gov.\n\n\nData availability\n\nF1000Research: Dataset 1. Dataset of funding source, primary outcome changes and statistical significance of clinical trials registered on ClinicalTrials.org, 10.5256/f1000research.6312.d450567", "appendix": "Author contributions\n\n\n\nSVR and BG conceived and designed the study. SVR, JP, LH, APS, MK and DM analysed the data. SVR and BG interpreted the data. SVR drafted the article. All authors revised the article and gave final approval for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPocock SJ: Clinical trials: a practical approach. Wiley; 1983; 286. Reference Source\n\nZarin DA, Tse T, Williams RJ, et al.: The ClinicalTrials.gov results database -- update and key issues. N Engl J Med. 2011; 364(9): 852–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamagopalan S, Skingsley AP, Handunnetthi L, et al.: Prevalence of primary outcome changes in clinical trials registered on ClinicalTrials.gov: a cross-sectional study. F1000Res. 2014; 3: 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuić M, Marušić M, Marušić A: Completeness and changes in registered data and reporting bias of randomized controlled trials in ICMJE journals after trial registration policy. PLoS One. 2011; 6(9): e25258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhandari M, Busse JW, Jackowski D, et al.: Association between industry funding and statistically significant pro-industry findings in medical and surgical randomized trials. CMAJ. 2004; 170(4): 477–80. PubMed Abstract \| Free Full Text\n\nMathieu S, Boutron I, Moher D, et al.: Comparison of registered and published primary outcomes in randomized controlled trials. JAMA. 2009; 302(9): 977–84. PubMed Abstract \| Publisher Full Text\n\nRamagopalan SV, Skingsley AP, Handunnetthi L, et al.: Dataset 1 in: Funding source and primary outcome changes in clinical trials registered on ClinicalTrials.gov are associated with the reporting of a statistically significant primary outcome: a cross-sectional study. F1000Research. 2015. Data Source" }	[ { "id": "8112", "date": "30 Mar 2015", "name": "Deborah Korenstein", "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\nRamagopalan and colleagues have expanded on their previous work to assess the relationship between changes in the primary endpoint on clinicaltrials.gov and both funding source and a “positive” trial result. The authors found that changes to the primary listed endpoint were associated with both industry funding and with a positive outcome. Their sample included completed interventional clinical trials listed on clinicaltrials.gov. They defined trials as having a positive result if they had a listed p-value <0.05. This may be problematic since it appears that their sample included non-inferiority trials (though it is not clear how many) and for these trials a non-significant p-value may indicate a “positive” (or at least non-inferior) result. Since there have been growing numbers of non-inferiority trials published in recent years, this may be a substantial issue. The authors may want to consider identifying non-inferiority trials and considering their results differently, or at least reporting the prevalence of non-inferiority trials if possible. Aside from this methodologic weakness the other methods are rather straightforward and clear. However, the authors found that 95.4% of trials had changed the primary outcome at some point during the registration period. In contrast, in their previous work the same authors found that 32% of trials registered with clinicaltrials.gov had changed the primary endpoint. The reason for this dramatic difference is not clear, and the authors do acknowledge that the vast majority of studies changed their primary endpoint and that many of the changes may have been trivial. Further, in spite of this surprising finding the authors still found significant associations. However, the near-total prevalence of changes to the primary endpoint certainly suggests that changing a primary endpoint in the registry is highly routine and likely does not reflect fundamental change to the study. This weakens the relevance of the findings.", "responses": [] }, { "id": "8116", "date": "02 Apr 2015", "name": "Janet Wale", "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 Research Note is an extension of the authors' 2014 article (reference 3). It has a very clearly defined question, whether changes made to the primary outcomes are associated with statistically significant primary outcomes. The present data therefore includes only completed interventional studies on clinicaltrials.gov.The second paragraph of the Methods section refers to the 2014 article. This is unhelpful, particularly as it is not clear from the 2014 article how 'changes in primary outcomes' are defined. The final paragraph of the Results section states that registration year, funding source and primary outcome change after trial completion were associated with a significant primary outcome - yet these are in opposite directions; and registration year is complex (looking at the data and 2014 article). That is brevity has taken over from clarity.Some of the limitations are included in the Conclusions: what exactly the changes were ('semantics' versus actual change; whether the changed outcome was the statistically significant outcome reported). The authors have not gone on to analyse their results by phase of trial; if the trials are randomised controlled trials, or otherwise. Another important question is how many of the completed trials have reported their results within a set timeframe (one year/two years), that is what about the trials that have not reported their results?Has the number of industry funded trials increased over time compared with mixed and public funded trials? In plain language, what is the extent of the problem?", "responses": [] } ]	1	https://f1000research.com/articles/4-80
https://f1000research.com/articles/4-99/v1	24 Apr 15	{ "type": "Research Article", "title": "Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma aggressiveness", "authors": [ "Christi L. French", "Fei Ye", "Frank Revetta", "Bing Zhang", "Robert J. Coffey", "M. Kay Washington", "Natasha G. Deane", "R. Daniel Beauchamp", "Alissa M. Weaver", "Christi L. French", "Fei Ye", "Frank Revetta", "Bing Zhang", "Robert J. Coffey", "M. Kay Washington", "Natasha G. Deane", "R. Daniel Beauchamp" ], "abstract": "A key question in cancer systems biology is how to use molecular data to predict the biological behavior of tumors from individual patients. While genomics data have been heavily used, protein signaling data are more directly connected to biological phenotype and might predict cancer phenotypessuch as invasion, metastasis, and patient survival. In this study, we mined publicly available data for colorectal adenocarcinoma from the Cancer Genome Atlas and identified protein expression and signaling changes that are statistically associated with patient outcome. Our analysis identified a number of known and potentially new regulators of colorectal cancer. High levels of insulin growth factor binding protein 2 (IGFBP2) were associated with both recurrence and death, and this was validated by immunohistochemical staining of a tissue microarray for a secondary patient dataset. Interestingly, GATA binding protein 3 (GATA3) was the protein most frequently associated with death in our analysis, and GATA3 expression was significantly decreased in tumor samples from stage I-II deceased patients. Experimental studies using engineered colon cancer cell lines show that exogenous expression of GATA3 decreases three-dimensional colony growth and invasiveness of colon cancer cells but does not affect two-dimensional proliferation. These findings suggest that protein data are useful for biomarker discovery and identify GATA3 as a regulator of colorectal cancer aggressiveness.", "keywords": [ "Proteomics", "Reverse Phase Protein Array", "TCGA", "Colorectal Cancer", "Bioinformatics", "Prognosis", "Cancer Biology" ], "content": "Abbreviations\n\nCK, Cytokeratin\n\nCRC, Colorectal Cancer\n\nHPA, Human Protein Atlas\n\nIGFBP2, Insulin-like Growth Factor Binding Protein 2\n\nIHC, Immunohistochemistry\n\nRPPA, Reverse Phase Protein Array\n\nTCGA, The Cancer Genome Atlas\n\nTGF-β, Transforming Growth Factor Beta\n\nTMA, Tissue Microarray\n\n\nIntroduction\n\nHigh throughput data from the Cancer Genome Atlas (TCGA, https://tcga-data.nci.nih.gov/tcga/) and other publically available datasets are becoming widely available and are a rich resource for data mining and biological discovery. A challenge for the field is to identify innovative approaches to identify both biological drivers and strong prognostic markers. Gene expression datasets have been commonly used to classify tumors, due to their wide availability. However, additional types of high throughput datasets are now available and may provide a different starting point for molecular analysis of tumors. Protein expression datasets generated by mass spectrometry or reverse phase protein array (RPPA) are becoming widely available for many TCGA tumors1. Since gene expression frequently does not correlate well with protein levels2, such datasets may give additional insight into molecular mechanisms that drive tumor behaviors. In addition, phospho-protein levels may identify activation of specific signaling pathways.\n\nA common approach to the analysis of tumor data is to first classify patients by molecular characteristics, such as KRAS mutation status or gene expression clusters, and then determine prognosis or treatment differences3–5. Alternatively, one can directly identify molecular differences that are statistically associated with patient outcome characteristics. We previously used the latter approach with RPPA data from head and neck squamous cell carcinoma to identify a phosphoinositide 3-kinase high, protein kinase C α low signaling state that drives invasive behavior6. Although it is limited by the availability of patient follow-up data, this type of bioinformatics approach is potentially powerful for identifying novel molecular drivers of tumor aggressiveness.\n\nIn this study, we analyzed publicly available data from TCGA to identify proteins that are predictive of poor prognosis in colorectal adenocarcinoma (CRC)7. We analyzed RPPA data, which includes protein and phospho-protein expression levels. Our analysis identified both known and novel candidate CRC drivers statistically associated with tumor recurrence or patient survival. Of these, we characterized two molecules in more detail. IGFBP2 was associated with both death and recurrence. Validation in an independent patient dataset by immunohistochemical (IHC) staining of a tissue microarray (TMA) demonstrated that high levels of IGFBP2 are associated with poor patient prognosis. Interestingly, low protein levels of the transcription factor GATA3 were highly associated with death of CRC patients in the TCGA data set. Experimental studies in colon cancer cell lines indicate that GATA3 expression acts to suppress invasive, aggressive CRC behavior. Since GATA3 protein and RNA levels are not correlated with each other, this association would not have been detected using RNA expression data.\n\n\nExperimental procedures\n\nAntibodies and reagents – We used three GATA3 antibodies: catalog number 558686 from BD Biosciences (GATA3 BD), catalog number sc-265 from Santa Cruz (GATA3 SC), and catalog number LS-B4163 from LifeSpan Biosciences (GATA3 LS). IGFBP2 antibody was catalog number LS-C138280 from LifeSpan Biosciences and β-actin antibody was catalog number A2228 from Sigma Aldrich. Transwell invasion chambers were from Corning.\n\nTCGA Data – RPPA level 3 and clinical information was downloaded from the TCGA data portal. All primary data analyses were performed in R 1.3.18.\n\nBioinformatics Statistical Analyses – A univariate Cox’s proportional hazard’s model analysis was performed for each protein (survival package in R)9,10. Patients with <30 days of follow-up information were excluded. The Wilma algorithm works in a greedy forward strategy and optimizes a combination of the Wilcoxon and Margin statistics for finding clusters of predictor variables (supclust package in R)11. Regsubsets (Leaps package)12 is a model selection method that carries out an exhaustive search for the best subsets of independent variables that predict the dependent variable in linear regression. Nvmax was set to 5 and nbest was set to 10. The RPPA data were median-centered and scaled to one standard deviation before performing analyses. For the Wilma and Regsubsets analyses, patients were divided into good prognosis (living patients or patients with recurrence-free survival were only included if they had ≥ 3 years of follow-up data) or poor prognosis (all patients with a recurrence or death were included regardless of follow-up time).\n\nHeatmaps – Heatmaps were created with unsupervised clustering of patients and proteins, using the package “heatmap.plus” in R 1.3.1 based on Euclidian distance and complete linkage13.\n\nSurvival plots – For each protein, patients were divided into high-expressing (at or above median RPPA expression) and low-expressing (below median RPPA expression). Using SPSS, multivariable cox proportional hazard model was used to estimate overall survival and recurrence-free survival, adjusting for patient stage, and Kaplan-Meier curves were generated to compare survival and recurrence-free survival between high-expressing and low-expressing groups.\n\nCell culture: Cells were grown in previously published optimal media for each cell line (for DLD1 and KM12c, DMEM + 10% FBS and non-essential amino acids)14,15. DMEM was purchased from Corning, FBS was purchased from Denville Scientific, and non-essential amino acids were purchased from Sigma. To create GATA3-OE cells, DLD1 or KM12c cells were transduced with retrovirus created by transfecting Phoenix packaging cells with pBabePuro-GATA3 (plasmid 1286 from Addgene). Pooled transduced cells were selected by puromycin treatment and used for experiments16. Empty vector pBabePuro was used as a control.\n\n3D Matrigel growth assay: Embedded three-dimensional culture was carried out as previously published17. Briefly, 35 mm glass-bottomed Mat-tek dishes (Mat-tek Corporation) were coated with 60 µL Matrigel (Corning). 4,000 cells were plated in each dish in 200 µL 90% Matrigel, 10% growth medium. 2 mL of growth media was added to each dish after 30 minutes and replaced every four days. Cells were imaged at 10× magnification every two days starting at day 3; eight random fields from each dish were imaged and the diameter of each in-focus colony was quantitated.\n\nProliferation: 1500 cells/well were plated in triplicate in the presence or absence of 10% serum in 96 well plates and grown for five days. Each day the plates were imaged on a Cellavista automated microscope after the addition of Calcein to identify live cells, Propidium iodide to identify dead cells, and Hoechst to identify nuclei (all from Invitrogen). Data were quantitated with Cellavista imaging software to determine the number of live cells for each day.\n\nTranswell invasion assay: 50,000 cells/well were plated in triplicate on Matrigel-coated Transwell inserts in serum-free DMEM. Normal growth media was used on the bottom as a chemoattractant. Cells were allowed to invade for 48 hours and then fixed with a three-step stain (Thermo Scientific). Five random fields from each Transwell insert at 10× magnification were taken on an EVOS microscope for quantitation.\n\nTissue microarray construction and IRB information: All use of human tissue samples was conducted under IRB-approved protocols. The colorectal cancer tissue microarray (TMA) was constructed with 99 cases of colorectal cancer, using duplicate 1-mm cores of each colorectal cancer in the GI SPORE Tissue Core facility (IRB # 020338). All samples in the TMA are from formalin-fixed paraffin-embedded blocks in the pathology archives, and are from tissue removed during the course of routine clinical care. Associated outcome and demographic data are extracted from the Colorectal Carcinoma Data and Virtual Archival Specimen Repository (IRB# 101531), and are stripped of all identifiers when released to investigators. The array is enriched for special histologic subtypes of CRC such as mucinous, signet ring cell, and medullary carcinoma, and contains the full spectrum of histologic grades and tumor stages. Twelve control cases of histologically normal colorectal mucosa from surgical resections for non-neoplastic disease such as diverticulosis coli are included.\n\nTMA staining: Antigen retrieval was performed in pH 6.0 citrate buffer, by using a pressure cooker at 104°C for 20 minutes with a 10 minute bench cool down, followed by quenching with 0.04% H2O2 w/sodium azide for 5 minutes. After blocking in a serum-free protein block for 20 min, primary antibody was incubated with the samples for an hour, followed by detection with Dako Envision + HRP Labeled Polymer for 20 minutes followed by incubation with chromogen DAB+ for 5 minutes.\n\nTMA analysis: To be included in the survival or recurrence-free curves, patients needed to have the following information: stage, days until event (if deceased or recurrent), and a follow-up time of at least 30 days (if living or nonrecurrent). Through the Vanderbilt University Digital Histology Shared Resource in the Epithelial Biology Center, immunostained TMA slides were imaged at 20× magnification to a resolution of 0.5 µm/pixel with the Leica SCN400 Slide Scanner (Leica Biosystems). Tissue cores were analyzed with Ariol® Review software SL-50. Upper and lower thresholds for brown DAB positive staining were set for color, saturation, and intensity. Tumor areas with staining that registered between these thresholds were determined to be DAB-positive in an automated analysis. Brown (DAB-positive) area of each tumor core was thus used to determine cytokeratin (tumor area), IGFBP2, and GATA3 stained area. The percent of the tumor area positive for IGFBP2 was calculated by dividing the IGFBP2- positive area by the cytokeratin-positive area and multiplying by 100.\n\nNumbers and statistics: For comparison of good and poor prognosis patients, a Fisher’s exact test was used to analyze categories with two variables (gender, M). A Chi-squared test was used to analyze categories with more than two variables (Stage, T, N). Age and gender were analyzed using a Student t-test. All analyses were performed in GraphPad. For experimental data from CRC cell lines, data from the engineered cell lines were plotted and statistically analyzed in GraphPad using a Student t-test. Data plotted in bar graphs were represented as mean+/-standard error. For growth curves, error bars represent 95% confidence intervals.\n\n\nResults\n\nTo identify molecular drivers of aggressive CRC behavior, we used statistical methods to link patient outcome data to protein and phospho-protein expression in the TCGA RPPA dataset. The RPPA dataset includes protein and phospho-protein levels from tumor biopsies taken at the time of diagnosis. The clinical information for these patients is also available, including recurrence and survival information, stage, and follow up time (Table 1, Table 2; Datafile 1).\n\nTherefore, we used a combination of univariate and multivariate approaches to identify proteins associated with recurrence or death. Univariate Cox proportional hazard regression analysis9,10 relates the time to an event to a covariate (gene or protein expression) and is a common method to identify associations of protein expression with patient outcome. We also used Wilma and Regsubsets multivariate algorithms to select groups of proteins with predictive power12,18. Patient characteristics are shown in Table 1 for the Cox regression analysis and in Table 2 for the Wilma/Regsubsets analyses. The use of all 3 methods allowed us to identify whether certain proteins were chosen independent of the statistical method used.\n\nThe Wilma and Regsubsets algorithms compare groups (clusters) of patients, which we predefined by patient prognosis, and find proteins that are able to predict these clusters. For these multivariate methods, patients were divided into “good” or “poor” prognosis groups according to survival or recurrence data. “Good prognosis” patients were classified either as living or as having no recurrence with a minimum of 3 years follow-up time. We chose 3 years as a reasonable cut-off time since the great majority of colon cancer cases (91%) have a recurrence within this time frame19. Although this did reduce our sample size for patients included in the multivariate analyses compared to the univariate Cox regression (Table 1 vs. Table 2), we felt it was necessary to ensure that our “good prognosis” group was accurate. For the “poor prognosis” patient group, recurrence or death could occur at any time point. To determine whether any proteins had stage-specific statistical associations, we performed the analyses using patient groups of stages I-II, stages I-III, or stages I-IV (\"all stages\"). However, we did not use stage, node or metastasis status as traits for identification of molecular correlates for several reasons. First, we reasoned that identifying molecular correlates of stage would not add prognostic information for clinical decision making, since stage is already gathered on every patient. Second, an initial test using the Wilma algorithm suggested that RPPA protein expression changes selected to be associated with node and metastasis negativity (e.g. N0M0 vs. N+M+) did not segregate patients well into groups. Thus, two-dimensional projections indicate that proteins selected by both recurrence and death had the ability to separate patients into distinct groups, indicating good predictive power, while N/M status at the time of diagnosis did not (Supplemental Figure 1).\n\nThe full results of the analyses for molecules statistically associated with death or recurrence are shown in Supplemental Table 1–Supplemental Table 4 (Cox hazard analyses shown in Supplemental Table 1, Supplemental Table 2, and results from all analyses summarized in Supplemental Table 3, Supplemental Table 4). Modified volcano plots of these proteins shows the number of times a protein was identified vs. the difference in RPPA expression for either death or recurrence (Figure 1a). Proteins with negative values are downregulated in patients with poor outcome (such as the well- known tumor suppressor, Rb) and proteins with positive values are upregulated (such as the oncogene c-Jun). Proteins that were identified by more than one method are shown in Table 3 and Table 4 and indicated in red in the volcano plots (Figure 1a).\n\nProteins that were identified by more than one computational method (Cox regression, Wilma, or Regsubsets) were included. Proteins identified by Cox regression and the Wilma algorithm were significantly associated with prognosis (p<0.05); proteins are included for Regsubsets if they were identified five times or more.\n\nProteins that were identified by more than one computational method (Cox regression, Wilma, or Regsubsets) were included. Proteins identified by Cox regression and the Wilma algorithm were significantly associated with prognosis (p<0.05); proteins are included for Regsubsets if they were identified five times or more.\n\na) Volcano plots were created by plotting the difference in the scaled RPPA expression for each protein vs. the number of times that protein was identified in the bioinformatics analysis. A positive value on the y-axis means that protein is upregulated in poor prognosis (recurrent or deceased) patients, while negative value on the y-axis means that protein is downregulated in poor prognosis (recurrent or deceased) patients. Proteins identified by more than one bioinformatics method (Table 3, Table 4) are shown in red, and proteins selected for further analysis are boxed and labeled. b) Heatmaps were created using unsupervised clustering of all top hits (Table 3, Table 4) in stage I-II patients. Each row is a patient; each column is a protein. Red boxes outline poor prognosis (recurrence or death) clusters. Proteins selected for further analysis (GATA3 and IGFBP2) are outlined in grey boxes.\n\nProteins associated with death included known CRC drivers, including SMAD3, SMAD4, and MSH2, which respectively regulate Transforming growth factor beta (TGF-β) signaling20 and microsatellite instability21 (Table 3). In addition, a number of apoptosis and cell cycle proteins were associated with death, including Bid, Bim, Rb, and Chk1. Interestingly, the transcription factor GATA3 was our top hit associated with patient death and was identified eight times out of a potential maximum of nine times (three stage groups analyzed by three statistical methods). GATA3 is frequently mutated in breast cancer and is known to promote luminal cell differentiation in the mammary gland22–25, but has not been previously studied in colon cancer. IGFBP2, which was linked with both patient death and tumor recurrence in our analysis, was another interesting hit, as it has been associated with a number of cancer types but few studies have addressed its role in CRC26–28.\n\nProteins associated with recurrence (Table 4) also included known CRC regulators, including the pro-inflammatory enzyme COX229,30, phospho-c-Jun31 and SMAD4 (reviewed in 32). Some proteins were identified to be statistically associated with both death and recurrence, including the cell cycle regulator Rb, the autophagy regulator Beclin1, and IGFBP2.\n\nTo visualize the expression of top hits (listed in Table 3 and Table 4) in individual patient tumor samples, we created heatmaps using unsupervised clustering. Interestingly, clustering of data from Stage I and II patient tumors gave superior segregation of prognosis groups by the proteins than using data from Stages I-III or I-IV patient tumors. For both recurrence and survival, there was a “poor prognosis” cluster that segregated away from the remaining patients (Figure 1b, red boxes). Notably, the ability of the chosen proteins to cluster patients according to poor prognosis was also superior when using death as the outcome, perhaps due to the larger number of significant proteins or the larger sample size of Stage I-II patients with that follow-up metric (Figure 1b, compare death and recurrence heat maps).\n\nOf the proteins identified in our analyses, GATA3 and IGFBP2 were the most novel as regulators of CRC. Visualization by heatmaps shows a decreased expression in GATA3 and increased IGFBP2 expression in tumors within the poor prognosis clusters (Figure 1b, grey boxes). Stage-adjusted survival plots revealed that TCGA patients with low GATA3 expression levels had a significantly increased risk of death, compared with patients whose tumors had high GATA3 levels. Patients whose tumors had high IGFBP2 expression had a trend towards decreased survival, but this did not reach statistical significance (Figure 2a). Importantly, both GATA3 and IGFBP2 had significantly altered RPPA expression in deceased patients for all stages, stages I-II, and stages I-III (Figure 2b, c). Similar trends were seen in recurrent vs. non-recurrent patients, but the data did not reach statistical significance, potentially due to the smaller number of patients with recurrence follow up data (Supplemental Figure 2).\n\n(a) Stage-adjusted survival plots for GATA3 and IGFBP2. (b) and (c) Comparison of RPPA-determined expression in living and deceased patients for GATA3 (b) and IGFBP2 (c). IGFBP2 expression is significantly increased in deceased patients in Stages I-II, I-III, and I-IV, while GATA3 is significantly decreased in deceased patients in Stages I-II, I-III, and I-IV. p<0.05, p<0.01, p<0.001\n\nTo validate our findings in an independent tumor cohort, we obtained a tissue microarray (TMA) that contained 61 CRC samples with available patient follow-up data (Datafile 2). Patient characteristics are shown in Supplemental Table 5. Note that some clinical information, such as age or gender, was not available for all patients. We stained the TMA slides with antibodies against IGFBP2 as well as with the epithelial marker cytokeratin in order to identify tumor cells (Figure 3a, b). We quantified the areas of both IGFBP2 staining and cytokeratin staining (representing total tumor area), and calculated the percent IGFBP2 positive area per tumor area in order to normalize to the amount of tumor present in each sample (Datafile 2). This metric was used to divide patients into high or low IGFBP2 by median expression, and their survival or recurrence-free survival was compared. The results revealed that patients with IGFBP2 staining at or above the median had a significant reduction in both survival and recurrence-free survival time, independent of tumor stage (Figure 3a, b, lower panels). Staining of normal colon tissue also revealed strong staining in the bottom of the crypts (Figure 3c), consistent with a previous report28.\n\nIHC immunostaining of a CRC tissue microarray for IGFBP2 and cytokeratin (epithelial marker) was performed. a) Representative IGFBP2 staining in living and deceased patients and Kaplan-Meier curve comparing survival of patients with low (below median) vs. high (at or above median) IGFBP2 staining. b) Representative IGFBP2 staining in non-recurrent and recurrent patients and Kaplan-Meier curve comparing recurrence-free survival of patients with low (below median) vs. high (at or above median) IGFBP2 staining. %IGFBP2-positive area of tumor was calculated using IGFBP2 area and cytokeratin area to identify tumor. Survival and recurrence-free survival plots are adjusted for stage. c) Representative IGFBP2 and cytokeratin staining in a representative normal colon sample. Scale bars indicate 100 µm.\n\nGATA3 is a transcription factor that was originally identified as a T-cell differentiation factor33,34. However, recent data indicates that GATA3 is also expressed in some epithelia (reviewed in 35). In breast cancer, GATA3 is frequently mutated23,25. In addition, low levels of GATA3 correlate with decreased breast cancer patient survival36–40. To determine whether GATA3 was expressed in CRC cells or only in T-cells, we stained CRC TMAs as well as matched normal and colon cancer tissue (Figure 4; Datafile 3). Antibodies to cytokeratin (CK) and CD3 respectively marked the epithelial tumor cell and T-cell compartments. We found variable staining patterns with two different anti-GATA3 antibodies. Using the same antibody that was used to probe the TCGA RPPA samples (Figure 4a , GATA3 BD), there was weak cytoplasmic and occasional nuclear staining in the tumor cells and a small amount of nuclear staining in cells in the stromal compartment. It should be noted that this antibody had not been validated for IHC. Furthermore, we noticed variable staining of TMA sections from normal colon tissue, suggesting high sensitivity of this antibody to fixation conditions. We therefore tested two more antibodies that were validated for IHC. Using an antibody that has successfully been used for breast cancer stratification36, we detected very light cytoplasmic staining of epithelial cells with some nuclear staining of stromal cells in normal colon samples, but no staining of epithelial or stromal cells in paired colon cancer samples (GATA3 SC, Figure 4b). Using a second validated IHC antibody (GATA3 LS), we found strong staining of the epithelial component of both normal colon tissue and colon cancer (Figure 4b). Interestingly, with both the SC and LS antibodies, it appeared that in normal colon tissue there was increased staining in epithelial cells at the mucosal surface with nuclear localization, compared to the deep crypts (Figure 4b). Staining of the TMA with GATA3 LS gave strong staining in both the nuclei and cytoplasm of tumor cells. However, there was a high background in many of the samples with apparently nonspecific staining throughout both the tumor and stromal compartment (Figure 4a), which made the samples unsuitable for quantitation. This high background may be due to overfixation of some of the TMA blocks, since it was not apparent on separate fixed tissues that were not part of the TMA (compare Figure 4a to Figure 4b , GATA3 LS staining).\n\na) Representative immunostained tissue sections from two patient tumors from the CRC TMA showing staining for epithelial tumor (cytokeratin, CK), T-cells (CD3), and two different GATA3 antibodies (BD and LS). b) Representative staining of matched normal colonic tissue and colon cancer samples for two different GATA3 antibodies (LS and SC). Note the variability of GATA3 staining with different antibodies.\n\nWe also checked the Human Protein Atlas (HPA)41 for staining of colon tissues by GATA3 antibodies (Supplemental Figure 3). The HPA also used three different antibodies. One of them, CAB016217, is the same as the antibody we tested that gave little to no staining of colon tissue (GATA3 SC). Likewise, they found little nuclear staining, and weak or negative cytoplasmic staining across both normal and colon cancer samples. The other two antibodies stained the epithelial component of both normal and colon cancer samples with primarily nuclear or nuclear + cytoplasmic staining patterns. Thus, with four out of the five antibodies tested by our laboratory and the HPA, nuclear GATA3 staining was seen in colon epithelial and cancer cells. However, due to the variability in intensity and pattern of staining, we were not able to perform quantitations to obtain information about prognostic significance.\n\nTo determine if we could use a gene expression dataset for validation, we tested whether GATA3 RNA expression by RNA sequencing correlated with GATA3 protein expression by RPPA in TCGA samples that had both types of data. There was no correlation between GATA3 RNA and protein expression (Supplemental Figure 4a), so we were not able to use GATA3 RNA expression for correlative studies in a secondary tumor dataset. By contrast, IGFBP2 protein levels correlate well with IGFBP2 RNA levels (Supplemental Figure 4b). There was no correlation between IGFBP2 protein and GATA3 protein levels (data not shown), indicating there is likely no mechanistic link between these two proteins.\n\nAs an alternative to validation with tissue samples, we decided to investigate the biological role of GATA3 in colon cancer with in vitro experiments. We performed Western blot analysis of GATA3 levels in a panel of CRC cell lines with Jurkat T-cells as a positive control for GATA3 expression (Datafile 4). Using the same antibody that was used in the TCGA RPPA analyses (GATA3 BD), we detected a band of the correct 48 kDa size for GATA3. Compared with Jurkat cell expression, GATA3 was expressed at a much lower level in most CRC cell lines. GATA3 expression was undetectable in about half of the cell lines tested, including several with invasive characteristics, e.g. DLD1, SW480, and SW62042,43. Consistent with the known role of GATA3 in cellular differentiation34,44–48, the highest GATA3 expression was observed in the more differentiated cell lines, Caco-2, SK-CO-15 and HT-2949–51 (Figure 5a).\n\na) Representative Western blot (of 2 blots) showing that GATA3 is expressed in a subset of CRC cell lines. Jurkat is a T-cell line and used as a positive control. Higher expression is seen in the more differentiated cell lines Caco-2, HT-29, and SK-CO-15. b) Western blot showing engineered expression of GATA3 in DLD1 and KM12c CRC cell lines. pBabe is an empty vector control. c) Colony growth of engineered CRC cell lines in 3D Matrigel. Left: Representative images from day 9. Right: Growth curves. Data were gathered from duplicate wells from 3 independent experiments. The mean is plotted and error bars represent 95% CI. d) Invasion of CRC cell lines across Transwell filters. Left: Representative images of the bottom of Transwell filters after 48 hours invasion. Right: Quantitation of invaded cells/field. Data from five random fields per filter x triplicate filters for each of 3 independent experiments. Error bars represent +/- SEM. *p<0.001.\n\nTo investigate the role of GATA3 in CRC growth and invasion, we chose two of the invasive cell lines with undetectable GATA3 expression and stably expressed GATA3 in them using retroviral transduction (Figure 5b; Datafile 4). We first tested the ability of the GATA3-expressing cells to form colonies after seeding as single cells in an embedded 3D Matrigel growth assay. Colony growth in this assay represents a combination of growth and matrix remodeling activity, since the cells are fully embedded in 90% Matrigel52–54. Compared with control cells, GATA3-expressing cells formed smaller colonies in this 3D culture environment, an effect that was statistically significant beginning at day 5 (Figure 5c; Datafile 5). To determine whether the smaller colony size of GATA3-expressing cells was due to an intrinsic decrease in proliferation rate, we cultured them in 2D in the presence or absence of serum and used automated microscopy to follow the number of cells over a period of 5 days. GATA3 expression had no effect on cell numbers in the presence or absence of serum (Supplemental Figure 5; Datafile 6). To determine if GATA3 specifically controls CRC invasiveness, control and GATA3-expressing cells were allowed to invade for 48 h across a bed of Matrigel in a Transwell invasion assay. For both of the tested CRC cell lines, GATA3-expressing cells exhibited significantly decreased invasion compared to control cells (Figure 5d; Datafile 7). Taken together, these data indicate that GATA3 controls CRC invasiveness.\n\n\n\n\nDiscussion\n\nIn this study, we used high throughput protein and phospho-protein expression data from the TCGA to identify candidate drivers of CRC aggressiveness. By linking RPPA data to patient death or recurrence and using multiple statistical approaches, we identified both known and novel biomarkers of CRC aggressiveness. The top hit in our survival analysis was the transcription factor GATA3, for which low levels correlated with death. Follow-up experiments indicated that GATA3 is expressed in CRC and suppresses the invasive behavior of CRC cells. We also validated the prognostic value of the known but understudied molecule IGFBP2 in a secondary CRC dataset. These data indicate that RPPA and other high throughput protein datasets are useful for identifying potential biomarkers and drivers of aggressive tumor behavior, especially for proteins whose RNA expression does not correlate to protein expression, such as GATA3.\n\nGene expression signature discovery has been dominated by transcript profiling technologies. Since we previously found that a small RPPA dataset from human tumors can be useful as a biological discovery tool6, we tested its utility in a larger dataset from TCGA in this study. In addition to identifying proteins known to drive CRC progression, we identified several novel or understudied proteins associated with recurrence or death of CRC patients. These included IGFBP2 and GATA3, which were identified by multiple statistical methods, and a number of additional proteins that were detected by multiple (Table 3, Table 4) or any method (Supplemental Table 3, Supplemental Table 4). Validation of IGFBP2 by TMA staining and GATA3 in vitro suggests that our bioinformatic approach has utility and biological validity. Moreover, our analysis showed that GATA3 mRNA levels were not predictive of GATA3 protein levels (Supplemental Figure 4). Consistent with recent reports showing that RNA and protein expression levels frequently do not correlate with each other2,55, these data highlight the necessity of incorporating proteomics data into gene signature studies.\n\nOur approach uses a comparison of tumor tissue between good and poor prognosis patients, which differs from previous proteomics studies that have either focused on differences between tumor and normal control tissues or on stage-specific differences56–63. These studies have given insight in to the pathophysiology of CRC progression. However, our goal was to identify markers that are independent of stage and could be potentially used in the future to predict prognosis in early stage patients. It is agreed that Stage III and IV patients universally benefit from chemotherapy64, but the treatment decision for early Stage II patients is more complicated: there is disagreement over whether Stage II patients should65–67 or should not68,69 receive additional chemotherapy. While our findings are clearly a long way away from translation to the clinic, we posit that our general approach has the potential to identify biomarkers that can be used to identify early stage patients that could benefit from additional adjuvant therapy.\n\nA limitation of our study was that the TCGA CRC patient sample set is smaller for RPPA than for more standard analyses such as RNA Seq or DNA mutations (196, compared to 244 and 224 patient samples)7. In addition, many samples either did not have clinical follow-up or had only short follow-up time, further reducing our sample size. Additionally, there were no other published RPPA datasets in CRC that contained analysis of our proteins of interest. Therefore, validation of our findings required either staining of tissue microarrays or in vitro experiments. As RPPA datasets accumulate, we anticipate that there will be larger and multiple independent validation datasets with longer follow-up times. Finally, because RPPA is an antibody-based technique, it is usually typically limited in the number of proteins detected. Higher throughput proteomic approaches may solve this problem, although they are often unsuitable for quantitation of posttranslational modifications such as phosphorylation.\n\nWe identified increased expression of IGFBP2 to be associated with CRC recurrence and death. High levels of IGFBP2 have been associated with poor prognosis in several cancer types. In breast cancer, IGFBP2 has increased expression compared to normal samples70. IGFBP2 has also been shown to promote invasion of ovarian cancer cells71. In CRC, IGFBP2 has been reported to be upregulated compared to normal colon epithelia26 with a trend towards higher expression in more advanced CRC27. Interestingly, IGFBP2 is expressed predominantly in the crypts of normal colon tissue (Figure 3a and 28), opposite to the pattern we observed with GATA3 expression and suggesting a stem-cell-like expression pattern. Notably, IGFBP2 has been connected to both hematopoietic and glioma stem cell expansion and survival72,73. In addition, IGFBP2 overexpression in CRC cell lines was recently found to promote CRC tumorigenesis and metastasis28. Those data are consistent with our finding that high IGFBP2 expression in CRC tumors is significantly associated with death and recurrence in two independent datasets of CRC patients (Table 3, Table 4; Figure 3).\n\nThe top hit in our survival analysis was GATA3, which has not previously been studied in CRC. GATA3 is a transcription factor that was originally identified in T-cells, and controls the differentiation of TH2 cells34,46–48, skin cells44, hair follicles45 and luminal cells in the mammary gland22,24. The importance of GATA3 for mammary luminal cell proliferation and differentiation is suggested by the high expression of GATA3 in luminal breast cancers and recurrent mutations in the luminal subtype that stabilize GATA3 protein expression levels23,25. Conversely, similar to our findings in CRC, low GATA3 levels are associated with poor patient prognosis in breast cancer36–40. At this point it is unclear whether that represents the overall poor outcome of non-luminal breast cancers or an active role for GATA3 in suppressing aggressive behavior. Support for the latter possibility is provided by data indicating that re-expression of GATA3 in non-luminal breast cancer cells is sufficient to induce differentiation and suppress lung metastases24.\n\nIn CRC, the mechanistic role of GATA3 still remains to be defined. One possibility is that GATA3 controls CRC differentiation, similar to its function in T-cells and luminal breast cells. Consistent with our prediction, IHC stains of normal colon tissue showed higher staining in the superficial mucosa, where the most differentiated cells should be. In addition, the most differentiated CRC cell lines in our panel had the highest GATA3 expression. Additionally, we previously identified three transcriptional subtypes of CRC and then identified subtype-specific driver networks by integrating mutation and copy number alteration data from each subtype with a protein signaling network using a random walk approach5. GATA3 was included in the driver network for the “differentiated subtype” with relatively good survival outcome, although GATA3 mRNA was not significantly up-regulated in this subtype. Another nonexclusive possibility is that GATA3 regulates TGF-β signaling, a key pathway regulating CRC aggressiveness, as reported in breast cancer74. Further work is required to determine if any of these or other mechanisms are responsible for the role of GATA3 in CRC.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data of identified protein expression and signaling changes statistically associated with patient outcome, 10.5256/f1000research.6388.d4607475", "appendix": "Author contributions\n\n\n\nCF and AW conceived of the study. CF carried out bioinformatics analyses, with guidance from BZ and FY. FR performed staining of the TMAs, under guidance of KW. CF performed cell invasion, proliferation and colony assays. DB, RC, and ND provided reagents, cell lines, and advice on the project. All authors participated in the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the following NIH grants: P50CA095103 GI Special Program of Research Excellence (SPORE), including a pilot project to AMW, main projects RDB, NGD and RJC, and Translational Pathology and Imaging Core to MKW, R01CA158472 (RDB, NGD), R01CA46413 (RJC), P30CA068485 to the Vanderbilt Ingram Cancer Center, and F31DE021619 (CLF). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\n\nI confirm that the 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 Dr. Darren Tyson for advice on bioinformatic analysis methods and Dr. Joseph Roland in the Vanderbilt University Digital Histology Shared Resource in the Epithelial Biology Center for his help with the Ariol® TMA image analysis.\n\n\nSupplementary material\n\nOnly proteins with a significant p-value (<0.05) were included.\n\nOnly proteins with a significant p-value (<0.05) were included.\n\nAll number indicate individual patients; 1’s are patients with poor prognosis (death; recurrence; N or M positive at time of diagnosis) and 0’s are patients with good prognosis (living with 3 years of follow up time; non-recurrent with 3 years of follow up time; N and M negative at time of diagnosis). The distinct populations in the death and recurrence plots, showing clear separation of the good and poor prognosis patient clusters, indicate these definitions of poor prognosis can identify groups of proteins with good predictive power. The overlap of these patient clusters in the Node/Metastasis plot indicates this definition has less predictive power.\n\nDecreased GATA3 (a) and increased IGFBP2 (b) expression are evident in recurrent patient tumors, but the data were not significant (n.s.).\n\na) Subcellular localization; b) Staining, intensity, and quantity plots from the HPA. c) Representative images from matched normal colon tissue and CRC samples with three different GATA3 antibodies, as indicated.\n\nGATA3 and IGFBP2 RPPA and mRNA expression values from TCGA datasets were plotted and analyzed on an individual tumor basis. (a) GATA3 mRNA expression does not correlate with protein expression. (b) IGFBP2 mRNA expression does correlate with protein expression. Plots were created with cBioPortal using TCGA (2012) dataset6.\n\nGrowth curves (log base 10 of the cell number) from CRC cell lines grown in the presence of 10% serum (a, \"(+) serum)\") or the absence of serum (b, \"(-) serum\"). Cells were plated in triplicate and imaged on a Cellavista automated microscope in 3 independent experiments. Mean is plotted and error bars represent 95% confidence intervals. No significant differences were observed between control and GATA3-OE cells for either cell line.\n\n\nReferences\n\nAkbani R, Ng PK, Werner HM, et al.: A pan-cancer proteomic perspective on The Cancer Genome Atlas. Nature commun. 2014; 5: 3887. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang B, Wang J, Wang X, et al.: Proteogenomic characterization of human colon and rectal cancer. Nature. 2014; 513(7518): 382–387. 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PubMed Abstract\n\nMarsoni S; International Multicenter Pooled Analysis of Colon Cancer Trials Investigators.: Efficacy of adjuvant fluorouracil and leucovorin in stage B2 and C colon cancer. International Multicenter Pooled Analysis of Colon Cancer Trials Investigators. Semin Oncol. 2001; 28(1 Suppl 1): 14–19. PubMed Abstract\n\nBenson AB 3rd, Schrag D, Somerfield MR, et al.: American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol. 2004; 22(16): 3408–3419. PubMed Abstract \| Publisher Full Text\n\nBusund LT, Richardsen E, Busund R, et al.: Significant expression of IGFBP2 in breast cancer compared with benign lesions. J Clin Pathol. 2005; 58(4): 361–366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee EJ, Mircean C, Shmulevich I, et al.: Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion. Mol Cancer. 2005; 4(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsieh D, Hsieh A, Stea B, et al.: IGFBP2 promotes glioma tumor stem cell expansion and survival. Biochem Biophys Res Commun. 2010; 397(2): 367–372. PubMed Abstract \| Publisher Full Text\n\nHuynh H, Zheng J, Umikawa M, et al.: IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells. Blood. 2011; 118(12): 3236–3243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun J, He H, Pillai S, et al.: GATA3 transcription factor abrogates Smad4 transcription factor-mediated fascin overexpression, invadopodium formation, and breast cancer cell invasion. J Biol Chem. 2013; 288(52): 36971–36982. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrench CL, Ye F, Revetta F, et al.: Dataset 1 in: Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma aggressiveness. F1000Research. 2015. Data Source" }	[ { "id": "8773", "date": "01 Jun 2015", "name": "Stanley Stylli", "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\nThe research article ‘Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma aggressiveness’ by French et al presents a solid and well-structured study examining the link between protein signaling data and potentially predicting patient survival, in particular for colorectal cancer. To perform their research, they have utilized publically available databases to detect protein expression changes in colorectal cancer. The appropriateness and robustness of their experimental design is encompassed by the validation through multiple computational methods which identified a number of known, but more importantly new potential regulators of colorectal cancer. Increased levels of IGFBP2 were shown to be associated with tumour recurrence and death. In addition, they also identified that GATA binding protein 3 (GATA3) expression was also associated with patient outcome (being significantly decreased in the lower stage colorectal cancer patients). Confirmation of the role of GATA3 was shown in their proliferation and invasion laboratory studies. The results are clearly presented and extensively rationalized in the discussion, for which the authors must be commended. It is a comprehensive study which will be of interest to many readers who wish to undertake similar approaches utilizing protein expression data in public databases as a foundation of their laboratory research. It is an excellent addition to the current literature.", "responses": [] }, { "id": "8886", "date": "04 Jun 2015", "name": "Simon Saule", "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\nThe paper of French et al. \"Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma\" utilizes publicly available RPPA data for colorectal adenocarcinoma with the objective to predict patient survival. Two factors were identified to be significantly associated with bad prognosis: high levels of IGFBP2 and low levels of GATA binding protein 3. GATA3 protein level is not correlated with its RNA level, highlighting the interest of RPPA use. a Proteins accumulation in tissue microarray was performed, and biological validation in cell culture was provided for GATA3, through retroviral delivery in colon cancer cell lines. Over expression of GATA3 specifically reduced invasion in a transwell filter assay, and reduced the size of colony formation in matrigel without effect on cell proliferation in 2D cultures. The data are sound, convincing and of interest for the community.", "responses": [] } ]	1	https://f1000research.com/articles/4-99
https://f1000research.com/articles/4-98/v1	24 Apr 15	{ "type": "Opinion Article", "title": "Algorithm for the anesthetic management of cesarean delivery in patients with unsatisfactory labor epidural analgesia", "authors": [ "Sonia Vaida", "Davide Cattano", "Debra Hurwitz", "Berend Mets", "Davide Cattano", "Debra Hurwitz", "Berend Mets" ], "abstract": "The management of a patient presenting with unsatisfactory labor epidural analgesia poses a severe challenge for the anesthetist wanting to provide safe anesthetic care for a cesarean delivery. Early recognition of unsatisfactory labor analgesia allows for replacement of the epidural catheter. The decision to convert labor epidural analgesia to anesthesia for cesarean delivery is based on the urgency of the cesarean delivery, airway examination, and the existence of a residual sensory and motor block. We suggest an algorithm which is implemented in our department, based on the urgency of the cesarean delivery.", "keywords": [ "Epidural labor analgesia", "Unsatisfactory labor epidural analgesia", "Anesthesia for cesarean delivery" ], "content": "Introduction\n\nNeuraxial blockade in obstetric anesthesia is considered the preferred method of analgesia for both vaginal and surgical deliveries. One of the major benefits of labor epidural analgesia is that it can be converted to anesthesia for a surgical delivery if necessary. However, the reported incidence of failure to convert an existing satisfactory labor epidural analgesic to epidural anesthesia for cesarean delivery varies between 1.7%–19.8%1,2. This large range may be explained by the great variety of techniques used for conversion and the different criteria used for defining failure. In a postal questionnaire of 209 obstetric anesthetists, at least 13 different choices of local anesthetic and adjuvant mixtures used for conversion have been identified3.\n\nPredicting the failure to convert labor epidural analgesia to surgical anesthesia for cesarean delivery is crucial in planning the anesthetic management. Unsatisfactory labor epidural analgesia, which has an incidence of 0.9 to 27%1,4 may predict a failure to convert to surgical anesthesia5,6. Unsatisfactory epidural analgesia can be defined as: a unilateral block, unblocked sacral segments, an inadequate block level, a patchy/spotty block, and the persistence of labor pain after the administration of additional local anesthetics or manipulation of the epidural catheter. The reasons given for epidural analgesia failure are: too slow injection of small volumes of local anesthetics, malposition of the epidural catheter, the presence of a congenital median epidural septum, acquired epidural adhesions and adhesion of the dura mater7,10.\n\nEarly recognition of unsatisfactory labor analgesia allows for replacement of the epidural catheter or additional treatment such as the administration of a supplemental mixture of local anesthetics and opioids, with or without partial withdrawing of the epidural catheter5,11–13.\n\nOther predictors of failure to convert epidural analgesia to surgical anesthesia include: young age, obesity, higher gestational age at the time of delivery, a higher visual analog scale in the two hours prior to cesarean delivery, the need for more intermittent epidural top-ups during labor, increased maternal height, prolonged labor and anesthetic care being provided by an non-obstetric anesthetist2,10,13–17. An increased failure rate is also associated with a shorter time (under 10 minutes) from decision to perform a cesarean delivery to incision. This is due to insufficient time elapsed from the administration of an epidural top-up to the onset of action of the local anesthetic16. On the other hand, unsatisfactory surgical anesthesia can occur even after satisfactory labor epidural analgesia18.\n\nThe anesthetic management of cesarean delivery in a patient found to have an unsatisfactory labor epidural anesthetic depends on the urgency of cesarean delivery. Four categories of urgency are currently defined19:\n\nCategory 1: There is an immediate threat to the life of the mother or the fetus,\n\nCategory 2: There is maternal or fetal compromise which is not immediately life threatening.\n\nCategory 3: There is a need for early delivery but there is no maternal or fetal compromise.\n\nCategory 4: The Cesarean delivery can occur at a time to suit the patient and maternity team.\n\nIn this opinion article, we suggest an algorithm to help guide anesthetic management in a situation where epidural analgesia is insufficient and anesthesia is requested for Cesarean delivery.\n\nThe first management decision is based on the airway examination and the anesthetist's assessment as to whether the in situ epidural catheter is likely to provide adequate surgical anesthesia. Due to time constraints, this assessment should be based simply on the existence or not of a discernable neuraxial block.\n\nIf there is a discernable bilateral block, a bolus dose of 15–20 mL of a rapid-acting local anesthetic should be injected though the epidural catheter (Figure 1a). According to a recent meta-analysis20, a mixture of lidocaine and epinephrine with fentanyl injected into the epidural catheter can achieve one of the fastest sensory blocks in approximately 3 minutes. Alkalinization of the epidural solution with sodium bicarbonate (1mEq/10 mL) can significantly speed the onset of epidural anesthesia21, however it requires increased preparation time22 and can lead to medication errors, especially in the stressful environment of an emergent cesarean delivery22.\n\nAdministering a bolus can be justified because administration of supplemental local anesthetic in a higher volume can facilitate spread in the epidural space into previously spared areas23, while carrying a relatively small risk of unintentional intravascular (1:5000)23 or intrathecal injection (1:2900)24. In contrast, administering general anesthesia for cesarean delivery has a much higher risk (1:238)25 of failed intubation. Therefore, especially in patients with predicted difficult airways, every effort should be made to avoid general anesthesia. Preoxygenation and preparation for general anesthesia can begin once the patient is in position, even while dosing the epidural anesthetic.\n\nGiven the time constraints, a quick reassessment of the neuraxial block should be performed at the time the surgeon is ready to make the incision (Figure 1b). Loss of sensation to light touch bilaterally at the dermatomal level of T5 or above is the most reliable method to ensure satisfactory surgical anesthesia. Assessment using other modalities such as loss of sensation to cold and pin-pricks are less reliable26,27. In addition, before incision, the surgeon should be asked to test the dermatomal level of the neuraxial block by sharp-touch.\n\nIf satisfactory epidural surgical anesthesia cannot be achieved, general anesthesia should be induced to guarantee effective surgical anesthesia and cesarean delivery (Figure 1c). In order to increase the safety of conversion to general anesthesia, the patient’s position should be optimized for intubation, efficient pre-oxygenation performed, and airway backup equipment should be available.\n\nA more thorough assessment of the neuraxial block can be performed and should include the height, density and distribution of analgesia (unilateral or bilateral). Should a block be present, fractioned doses of a mixture of local anesthetics and opioids should be administered though the epidural catheter in an attempt to rescue the epidural block (Figure 2a). Caution should be used not to exceed local anesthetic toxic levels. The epidural block level should be reassessed (Figure 2b) and if found to be unsatisfactory, an immediate conversion to general anesthesia should occur (Figure 2c).\n\nIn patients with no evidence of neuraxial blockade, spinal anesthesia may be considered (Figure 2d), as this has been described to be almost as quick as a general anesthetic in experienced hands. Kinsella et al.28 described a “rapid sequence spinal” technique; a non-touch technique allowing only limited attempts, no local skin infiltration, and necessitating 8 minutes (on average) to complete. This is a newly described and not yet universally accepted technique. Criticisms of this “rapid sequence spinal” technique include the lack of aseptic preparation and the inability to establish a prior rapport in an anxious patient29,30.\n\nAlternatively general anesthesia could be used, as this is the quickest approach to reliably anesthetize the patient for cesarean delivery. Clinical situations that would favor immediate conversion to general anesthesia include the presence of an analgesic window; neuraxial dermatomal levels below T12; a unilateral block that differs by more than two or three dermatomal levels or insufficient analgesic density with uneven distribution of numbness to soft touch.\n\nIn a situation where there is no maternal or fetal compromise, a complete evaluation of the degree of the motor and sensory block can be performed. Epidural sensory block should be assessed for the highest dermatomal level at which the patient is able to detect a change in sensation to light touch, bilateral distribution and potential analgesic windows. The degree of epidural motor block can be assessed using a modified Bromage score (1 = able to raise legs above table, 2 = able to flex knees, 3 = able to move feet only, 4 = no movement in legs or feet).\n\nIn patients with residual block there are three management options:\n\nOption A – Trial of epidural (Figure 3a). The most common situation encountered in clinical practice is a patient with some degree of sensory and/or motor block. In this situation, fractioned epidural administration of one quarter to one third of the final anticipated dose is indicated in order to ascertain whether the anesthesia will be effective before injecting the entire dose. If this trial of epidural fails, one can proceed to either a combined spinal epidural (CSE) anesthetic (Figure 3-1), continuous spinal (Figure 3-2), or general anesthesia (Figure 3-3).\n\nA CSE allows the use of a lower intrathecal dose with the additional flexibility of supplementation of the block through an epidural catheter. Portnoy and Valdhera10 recommend decreasing the dose of local anesthetic injected intrathecally by 20–30% to avoid a high spinal block. Intrathecal doses of bupivacaine as low as 4.5–6.5 mg have been used successfully for cesarean delivery31,32. A standard dose spinal anesthetic at this stage could result in an unpredictable cephalad extension of the neuraxial block (vide infra)33.\n\nAlthough controversial, a continuous spinal catheter is a viable option for cesarean delivery after failed epidural analgesia34. The advantage of a continuous spinal technique lies in the immediate confirmation of a successful block, and the ability to use careful titration of local anesthetics in boluses or as a continuous infusion. 5.0 mg of 0.5% preservative-free bupivacaine, plus 15 µg fentanyl can be initially injected intrathecally, followed by 2.5 mg boluses of 0.5% bupivacaine every 5 minutes until a T5 dermatomal level is achieved34.\n\nGeneral anesthesia would assure a reliable anesthetic for cesarean delivery.\n\nOption B - Remove the epidural catheter and perform a CSE or a de novo epidural (Figure 3b). An alternative to option A is to remove the epidural catheter as soon as the decision to perform a cesarean delivery is made and then replace the epidural catheter or administer a CSE. This option should be considered especially in patients where previous attempts to rescue labor epidural analgesia have been performed (withdrawal of the epidural catheter by 1 cm followed by top-up with local anesthetics and opioids).\n\nOption C - Allow the residual block to recede and perform a single shot spinal anesthetic (Figure 3c). This option may be recommended especially in patients presenting with unilateral block and a very high level of sensory block on one side. One should wait until the residual epidural block wears off before a spinal anesthetic is performed. It has been recommended to wait at least 30 minutes after the last epidural bolus before initiating a spinal anesthetic10. This is because there is an associated risk of a subsequent high block especially after administration of a recent epidural bolus just prior to a spinal anesthetic20,35. The described cephalad spread of the block can result from the compression of the spinal space by the previously injected epidural solution and/or the leakage of the epidural solution into the intrathecal space10.\n\nGeneral anesthesia may become the preferred choice if the mother or fetus’ condition change, the total dose of local anesthetic administered approaches the potential for toxicity, or after multiple failed attempts at neuraxial anesthesia.\n\nIn patients with no residual block a spinal anesthetic can be safely performed.\n\n\nSummary\n\nIn summary, the management of a patient with unsatisfactory labor epidural analgesia poses a severe challenge for the anesthetist wanting to provide safe anesthetic care for a cesarean delivery. Good communication with the obstetric team to anticipate a cesarean delivery will allow for adequate planning for safe conversion of unsatisfactory epidural analgesia to adequate surgical anesthesia.", "appendix": "Author contributions\n\n\n\nSV, DH, BM and DC all participated in the writing and editing of the manuscript. All authors have agreed to the final content of this article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPaech MJ, Godkin R, Webster S: Complications of obstetric epidural analgesia and anaesthesia: a prospective analysis of 10,995 cases. Int J Obstet Anesth. 1998; 7(1): 5–11. PubMed Abstract \| Publisher Full Text\n\nOrbach-Zinger S, Friedman L, Avramovich A, et al.: Risk factors for failure to extend labor epidural analgesia to epidural anesthesia for Cesarean section. Acta Anaesthesiol Scand. 2006; 50(8): 1014–8. PubMed Abstract \| Publisher Full Text\n\nRegan KJ, O’Sullivan G: The extension of epidural blockade for emergency Caesarean section: a survey of current UK practice. Anaesthesia. 2008; 63(2): 136–42. PubMed Abstract \| Publisher Full Text\n\nD’Angelo R, Foss ML, Livesay CH: A comparison of multiport and uniport epidural catheters in laboring patients. Anesth Analg. 1997; 84(6): 1276–9. PubMed Abstract \| Publisher Full Text\n\nCampbell DC, Tran T: Conversion of epidural labour analgesia to epidural anesthesia for intrapartum Cesarean delivery. Can J Anaesth. 2009; 56(1): 19–26. PubMed Abstract \| Publisher Full Text\n\nRiley ET, Papasin J: Epidural catheter function during labor predicts anesthetic efficacy for subsequent cesarean delivery. Int J Obstet Anesth. 2002; 11(2): 81–4. PubMed Abstract \| Publisher Full Text\n\nAsato F, Goto F: Radiographic findings of unilateral epidural block. Anesth Analg. 1996; 83(3): 519–22. PubMed Abstract \| Publisher Full Text\n\nGallart L, Blanco D, Samsó E, et al.: Clinical and radiologic evidence of the epidural plica mediana dorsalis. Anesth Analg. 1990; 71(6): 698–701. PubMed Abstract \| Publisher Full Text\n\nHogan Q: Epidural catheter tip position and distribution of injectate evaluated by computed tomography. Anesthesiology. 1999; 90(4): 964–70. PubMed Abstract\n\nPortnoy D, Vadhera RB: Mechanisms and management of an incomplete epidural block for cesarean section. Anesthesiol Clin North America. 2003; 21(1): 39–57. PubMed Abstract \| Publisher Full Text\n\nBeilin Y, Zahn J, Bernstein HH, et al.: Treatment of incomplete analgesia after placement of an epidural catheter and administration of local anesthetic for women in labor. Anesthesiology. 1998; 88(6): 1502–06. PubMed Abstract\n\nD'Angelo R, Berkebile BL, Gerancher JC: Prospective examination of epidural catheter insertion. Anesthesiology. 1996; 84(1): 88–93. PubMed Abstract \| Publisher Full Text\n\nHalpern SH, Soliman A, Yee J, et al.: Conversion of epidural labour analgesia to anaesthesia for Caesarean section: a prospective study of the incidence and determinants of failure. Br J Anaesth. 2009; 102(2): 240–3. PubMed Abstract \| Publisher Full Text\n\nLe Coq G, Ducot B, Benhamou D: Risk factors of inadequate pain relief during epidural analgesia for labour and delivery. Can J Anaesth. 1998; 45(8): 719–23. PubMed Abstract \| Publisher Full Text\n\nLee S, Lew E, Lim Y, et al.: Failure of augmentation of labor epidural analgesia for intrapartum cesarean delivery: A retrospective review. Anesth Analg. 2009; 108(1): 252–4. PubMed Abstract \| Publisher Full Text\n\nTortosa JC, Parry NS, Mercier FJ, et al.: Efficacy of augmentation of epidural analgesia for Caesarean section. Br J Anaesth. 2003; 91(4): 532–5. PubMed Abstract \| Publisher Full Text\n\nBauer ME, Kountanis JA, Tsen LC, et al.: Risk factors for failed conversion of labor epidural analgesia to cesarean delivery anesthesia: a systematic review and meta-analysis of observational trials. Int J Obstet Anesth. 2012; 21(4): 294–309. PubMed Abstract \| Publisher Full Text\n\nKinsella SM: A prospective audit of regional anaesthesia failure in 5080 Caesarean sections. Anaesthesia. 2008; 63(8): 822–32. PubMed Abstract \| Publisher Full Text\n\nLucas DN, Yentis SM, Kinsella SM, et al.: Urgency of Caesarean Section: a new classification. J R Soc Med. 2000; 93(7): 346–50. PubMed Abstract \| Free Full Text\n\nHillyard SG, Bate TE, Corcoran TB, et al.: Extending epidural analgesia for emergency Caesarean section: a meta-analysis. Br J Anaesth. 2011; 107(5): 668–78. PubMed Abstract \| Publisher Full Text\n\nWong C, Nathan N, Brown DL: Spinal, epidural and caulda anesthesia: anatomy, physiology and technique. In: Chesnut D, Polley L, Tsen L, Wong C. ed. Chesnut’s Obstetric Anesthesia. Principles and Practice. Mosby Elsevier Churchill Livingstone, 2009; 280. Reference Source\n\nLucas DN, Borra PJ, Yentis SM: Epidural top-up solutions for emergency caesarean section: a comparison of preparation times. Br J Anaesth. 2000; 84(4): 494–6. PubMed Abstract \| Publisher Full Text\n\nDadarkar P, Philip J, Weidner C, et al.: Spinal anesthesia for cesarean section following inadequate labor epidural analgesia: a retrospective audit. Int J Obstet Anesth. 2004; 13(4): 239–43. PubMed Abstract \| Publisher Full Text\n\nJenkins JG: Some immediate serious complications of obstetric epidural analgesia and anaesthesia: a prospective study of 145,550 epidurals. Int J Obstet Anesth. 2005; 14(1): 37–42. PubMed Abstract \| Publisher Full Text\n\nRahman K, Jenkins JG: Failed tracheal intubation in obstetrics: no more frequent but still managed badly. Anaesthesia. 2005; 60(2): 168–171. PubMed Abstract \| Publisher Full Text\n\nRussell IF: At caesarean section under regional anaesthesia, it is essential to test sensory block with light touch before allowing surgery to start. Int J Obstet Anesth. 2006; 15(4): 294–7. PubMed Abstract \| Publisher Full Text\n\nYentis SM: Height of confusion: assessing regional blocks before caesarean section. Int J Obstet Anesth. 2006; 15(1): 2–6. PubMed Abstract \| Publisher Full Text\n\nKinsella SM, Girgirah K, Scrutton MJ: Rapid sequence spinal anaesthesia for category-1 urgency caesarean section: a case series. Anaesthesia. 2010; 65(7): 664–9. PubMed Abstract \| Publisher Full Text\n\nWilliamson RM: Rapid sequence obstetric spinal anaesthesia. Anaesthesia. 2010; 65(11): 1142–3. PubMed Abstract \| Publisher Full Text\n\nMenon R: Rapid sequence spinal anaesthesia: a trainee’s viewpoint. Anaesthesia. 2010; 65(11): 1143–4. PubMed Abstract \| Publisher Full Text\n\nBen-David B, Miller G, Gavriel R, et al.: Low-dose bupivacaine-fentanyl spinal anesthesia for cesarean delivery. Reg Anesth Pain Med. 2000; 25(3): 235–9. PubMed Abstract \| Publisher Full Text\n\nVan De Velde M, Van Schaubroeck D, Jani J, et al.: Combined spinal-epidural anesthesia for cesarean delivery: dose-dependent effects of hyperbaric bupivacaine on maternal hemodynamics. Anesth Analg. 2006; 103(1): 187–90. PubMed Abstract \| Publisher Full Text\n\nCarvalho B: Failed epidural top-up for cesarean delivery for failure to progress in labor: the case against single-shot spinal anesthesia. Int J Obst Anaesth. 2012; 21(4): 357–9. PubMed Abstract \| Publisher Full Text\n\nPalmer CM: Continuous spinal anesthesia and analgesia in obstetrics. Anesth Analg. 2010; 111(6): 1476–9. PubMed Abstract \| Publisher Full Text\n\nMets B, Broccoli E, Brown AR: Is spinal anesthesia after failed epidural anesthesia contraindicated for cesarean section? Anesth Analg. 1993; 77(3): 629–31. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8455", "date": "05 May 2015", "name": "Wanda Popescu", "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\nThe article provides a very good algorithm to follow in case a patient with an unsatisfactory epidural requires anesthesia for a C-section. For completeness I would suggest that the authors be more specific in describing the medications used in different situations of blocks (exact medication, recommended concentration and dose). I would also recommend that the authors be slightly more detailed when describing the \"rapid sequence spinal\", in particular as this appears to be a rather new and controversial topic. Otherwise it's a great article.", "responses": [] }, { "id": "8960", "date": "18 Jun 2015", "name": "Matteo Parotto", "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\nThis very interesting opinion article provides a proposal from the Authors of an algorithm to help guide the anesthetic management of patients with unsatisfactory labor epidural analgesia that require cesarean delivery. It is a clear, concise, substantiated suggestion on how to approach a difficult clinical scenario.I feel it would be useful for the reader to receive more specific information/author's opinion on the definition of analgesic window and its clinical implications, and on the doses of anesthetics chosen in the various situations described.There might be a typo in the title for Figure 3, which may be changed to \"Category 3 and 4 Cesarean Delivery\".", "responses": [] } ]	1	https://f1000research.com/articles/4-98
https://f1000research.com/articles/3-301/v1	10 Dec 14	{ "type": "Research Article", "title": "Student perception about working in rural United States/Canada after graduation: a study in an offshore Caribbean medical school", "authors": [ "P Ravi Shankar", "Arun K Dubey", "Atanu Nandy", "Burton L Herz", "Brian W Little", "Arun K Dubey", "Atanu Nandy", "Burton L Herz", "Brian W Little" ], "abstract": "Introduction: Rural residents of the United States (US) and Canada face problems in accessing healthcare. International medical graduates (IMGs) play an important role in delivering rural healthcare. IMGs from Caribbean medical schools have the highest proportion of physicians in primary care. Xavier University School of Medicines admits students from the US, Canada and other countries to the undergraduate medical (MD) course and also offers a premedical program. The present study was conducted to obtain student perception about working in rural US/Canada after graduation. Methods: The study was conducted among premedical and preclinical undergraduate medical (MD) students during October 2014. The questionnaire used was modified from a previous study. Semester of study, gender, nationality, place of residence and occupation of parents were noted. Information about whether students plan to work in rural US/Canada after graduation, possible reasons why doctors are reluctant to work in rural areas, how the government can encourage rural practice, possible problems respondents anticipate while working in rural areas were among the topics studied.Results: Ninety nine of the 108 students (91.7%) participated. Forty respondents were in favor of working in rural US/Canada after graduation. Respondents mentioned good housing, regular electricity, water supply, telecommunication facilities, and schools for education of children as important conditions to be fulfilled. The government should provide higher salaries to rural doctors, help with loan repayment, and provide opportunities for professional growth. Potential problems mentioned were difficulty in being accepted by the rural community, problems in convincing patients to follow medical advice, lack of exposure to rural life among the respondents, and cultural issues.Conclusions: About 40% of respondents would consider working in rural US/Canada. Conditions required to be fulfilled have been mentioned above. Graduates from Caribbean medical schools have a role in addressing rural physician shortage. Similar studies in other offshore Caribbean medical schools are required as Caribbean IMGs make an important contribution to the rural US and Canadian health workforce.", "keywords": [ "Canada", "Caribbean", "doctors", "healthcare", "physicians", "rural", "shortage" ], "content": "Introduction\n\nA recent article mentions that rural Americans have limited access to health care1. The authors mention two reasons for this; the first being many Americans, especially in rural areas, are without health insurance, and the second being only 11.4% of the country’s doctors practiced in rural areas in 2005, serving about 20% of the country’s population (http://depts.washington.edu/uwrhrc/uploads/RHRC_FR125_Rosenblatt.pdf). Family physicians distribute themselves in proportion to the population in both urban and rural areas but doctors of other specialties are much more likely to settle in urban areas.\n\nA study published in 2009 has shown that international medical graduates (IMGs) filled the gaps in requirement of primary care doctors in many rural areas but there were wide differences between various states in America2. The authors concluded that policies to address the mal-distribution of physicians in the United States (US) should also consider the role of IMGs. Critical access hospitals are a federal Medicare (the system of health insurance in the US) category for isolated rural facilities with 15 or fewer beds and IMG physicians play an important and growing role in staffing these hospitals3. Canadian trained physicians migrate both nationally and internationally and IMGs play an important role in providing healthcare in physician losing locations in Canada4. The author concludes that IMGs will be needed in underserved locations for years to come.\n\nA study published in 2013 showed Caribbean IMGs had the highest proportion of physicians practicing in primary care specialties compared to all other categories of graduates5. They make an important contribution to the US primary care workforce. A study in Canada found that around 20% of Canadian IMGs (Canadian citizens or residents who did their medical training overseas) obtained their medical degree from the Caribbean6. A study which examined US citizens who graduated from medical schools outside the US and Canada found that the Central American and Caribbean region graduated more physicians than any other region7. Thus Caribbean medical schools may play an important role in producing primary care physicians for the US and Canada.\n\nXavier University School of Medicine (XUSOM) is an offshore Caribbean medical school located in Aruba, kingdom of the Netherlands, admitting students from the US, Canada and other countries to the undergraduate medical (MD) course. Students complete the basic sciences in Aruba and do their clinical rotations in the US. The school also offers a premedical program for high school students to prepare them for admission to the MD program. The premedical program runs for four semesters, while the preclinical MD program is of six semesters. Each semester is of 15 weeks duration. Student perception about working in rural US/Canada has not been previously studied in the institution. Hence the present study was carried out. The study was conducted among premedical and basic science (preclinical) undergraduate medical students to obtain basic demographic information, understand their perceptions regarding why doctors are reluctant to work in rural US/Canada after graduation and conditions to be fulfilled before they would consider working in a rural area, identify possible problems of working in rural areas and obtain possible solutions.\n\n\nMethods\n\nThe study was conducted among Premedical 1 to 4 semester and undergraduate medical students (semesters 1 to 5) at the Xavier University School of Medicine (XUSOM), Aruba during the month of October 2014. Students were informed about the aims and objectives of the study and invited to participate. They were informed that participation in the study was voluntary and they were free not to participate. Written informed consent was obtained from each participant. The study was approved by the Institutional Review Board (IRB) of XUSOM vide notification XUSOM/IRB/2014/05.\n\nThe questionnaire used was modified from that used in a previous study on student perceptions about working in rural Nepal after graduation8. The questionnaire was modified to the US/Canadian context by the authors. Inputs were also obtained from selected faculty members of the institution. The questionnaire used in the study is shown as Supplementary File 1. Information about the semester of study, gender, nationality, place of family residence and occupation of parents were noted. Students’ responses to different questions were noted. Common responses were tabulated. Among the topics studied was whether the strident had lived in a rural area before joining XUSOM, whether they plan to work in rural US/Canada after graduation, and if yes whether they plan to do so immediately after graduation/residency or later in their career. They were also requested to state three important reasons why doctors are reluctant to work in rural areas, and also their opinions regarding how governments can encourage doctors to work in rural US and Canada. Students were also asked whether they felt their medical curriculum prepared them adequately for rural practice and if they felt this was not the case, what modifications they felt were necessary in the medical curriculum.\n\nTheir opinion regarding whether students were aware of the problems of life in rural areas, problems which they anticipate in dealing with the local population, their knowledge of initiatives to address the shortage of doctors in rural US/Canada and in other countries and whether poor return of investment was a factor hindering doctors from working in rural areas was noted. The data was analyzed manually by the authors. The demographic information was analyzed using Statistical Package for Social Sciences (SPSS) version 20 for windows.\n\n\nResults\n\nNinety-nine of the 108 enrolled students (91.7%) participated in the study. Table 1 shows the demographic characteristics of the respondents. The number of male and female respondents was approximately equal and 28.3% of respondents were from rural areas. The majority of the student’s parents worked in non health related professions.\n\nPremed – student of the premedical program, MD- student of the medical program\n\nForty respondents (40.8%) were in favor of working in rural US/Canada after graduation. Ten respondents (mostly from the premedical course) were not sure about whether they planned to work in rural US/Canada at this initial stage. With regard to the duration for which they planned to work in a rural area, 12 stated for most of their career or indefinitely while nine respondents stated for 5 to 10 years while four respondents stated between 1 to 5 years. Eighteen respondents (18.2%) preferred to work in a semi urban or semirural area while eight preferred a rural area. However neither the authors nor the respondents defined what exactly was meant by a rural or a semirural area.\n\nTable 2 shows important conditions which may need to be fulfilled before respondents would consider working in a rural area. Respondents were concerned about housing, regular supply of electricity, internet and telecommunication facilities, and good quality water supply. Family-related factors were mentioned as important and were related to whether the doctor would be able to bring their family to the location, the place should be not very far from his/her hometown, and there should be facilities for education of children. When respondents were referring to proper working conditions they were mainly talking about adequate diagnostic and treatment facilities in the hospital or clinic where they would be working. Respondents also mentioned availability of treatment facilities for themselves and their family if they fall ill is important, and that the practice area should not be extremely cold, the community should welcome individuals from different backgrounds, absence of racism, provide opportunities for personal growth and help with repayment of student loans as necessary conditions. A respondent [Participant 83(P 83)] mentioned, “I have lived in a rural area for almost my whole life. I have seen how we have to travel to cities for basic medical services and to help make a change in this situation I would like to work in a rural area”. Forty-four respondents mentioned that they plan to work in a rural area immediately after graduation while 23 respondents mentioned that they would prefer to do so later in their careers.\n\nTable 3 shows important reasons why doctors are reluctant to serve in rural areas according to the respondents. Among other reasons mentioned were issues of safety, isolation, slow pace of rural life, racism and greater potential for career and personal growth in an urban area. A participant (P 33) stated, “Rural practice is mostly a GP job and most future doctors want to be specialists (including myself). Away from home (if you are from the city). Looks ‘boring’”. With regard to how the government can encourage doctors to work in rural US/Canada important initiatives/measures mentioned were providing greater benefits and salary to rural doctors as compared to doctors practicing in urban areas (57 respondents), loan forgiveness or help with repayment of student loans (12 respondents), improving the facilities at rural and community hospitals (10 respondents), provide opportunities for professional growth and development for rural doctors (8 respondents), and emphasize ‘educating doctors for rural areas’ (6 respondents). Four respondents mentioned making certain duration of rural service mandatory as has been done in many developing nations. A respondent [participant 98 (P 98)] stated, “Make it mandatory for graduates to serve in rural areas and have them serve at least a year or two”. Seventy-one respondents felt their medical training adequately prepared them for a career as a rural physician while 19 respondents were of the opinion that it did not do so.\n\nMany respondents did not answer the question regarding suggestions for re-focusing medical education for preparing doctors for rural practice. Among the suggestions mentioned were educating students on how to treat and manage medical conditions in a resource constrained setting, having preceptors with rural experience, initiating clinical exposure for students in rural areas and conducting sessions on cultural diversity and cultural issues in rural areas. When asked whether medical students in the institution were aware of the problems of life in rural areas 43 students (45.4%) were of the opinion that they were aware while 39 respondents stated that students were not aware. Regarding how the awareness can be created 19 respondents mentioned increasing rural exposure of students, while three respondents stated sessions (either lectures or small group sessions) by rural doctors should be conducted. Among other suggestions were sessions on this topic during the orientation program, medical humanities sessions and talks by rural community leaders.\n\nRegarding potential problems which students anticipate while dealing with the rural population fourteen respondents mentioned difficulty in being accepted by the rural community, while eight respondents mentioned that the rural population may be less educated which may lead to problems in their acceptance of treatments and advice provided by the doctor. Eight respondents opined that they did not have much awareness of and exposure to rural life which may lead to problems in adjusting to rural practice. Culture gap, fewer available resources and differences in lifestyle were also mentioned. Racism and language barriers were also mentioned.\n\nTwenty-four respondents mentioned they were aware of initiatives to address the shortage of rural doctors in the US and Canada but specific initiatives were not mentioned. Fifty-seven respondents mentioned they were not aware of these initiatives. Among the initiatives mentioned were debt forgiveness, return of service obligations, flying doctors and encouraging IMGs to practice in rural areas. Fifty-six respondents felt poor return on investment was a factor preventing doctors from working in rural areas while 17 mentioned that this was not a factor. Respondents mentioned recovery from medical school debt requires a job/position which pays well, the world is money oriented, doctors have to study for a long time before they can make money, there are fewer opportunities for making money in rural areas and urban areas provide greater opportunities. A respondent (P 12) stated, “Doctors are always in debt after med school. They do not want a poor return. They want progress financially”. Another respondent (P 21) stated, “Payment and financial stability should always be considered when making movements to and from geographical locations”.\n\nA respondent (P 63) mentioned, “This is an interesting study. It is a very important topic, but I fear many people are not as up to date with latest global developments in this area – including myself”.\n\n\nDiscussion\n\nOnly about 30% of respondents were from rural areas. Around 41% of respondents were in favor of working in rural US/Canada after graduation. Many respondents however preferred to work in a semi-rural area. Good living conditions, family related factors, welcoming community were among the conditions necessary to be fulfilled. Issues of safety, isolation, lesser potential for personal growth were among the reasons mentioned why doctors were reluctant to serve in rural areas. Difficulty in being accepted by the community, lack of exposure to rural life, cultural issues were mentioned as potential problems for rural practice.\n\nLike in the Nepalese study8, the majority of the respondents were from urban areas. Students from countries other than the US or Canada planned to work in US/Canada after graduation. Caribbean offshore medical schools offer a similar curriculum to US medical schools and students complete their clinical rotations in the US. Most offshore medical schools (OMSs) admit three groups of students a year and provide an increasing contribution to the US health workforce9. Like in Nepal, most respondents willing to work in a rural area preferred a semi urban/semirural area as their work location. The conditions that need to be fulfilled were also similar to the Nepalese study. In Tanzania, increased salaries, hardship allowance, decent housing, good infrastructure, and offering continuing education after a period of service were mentioned as factors making rural jobs more attractive to health workers10. In a survey conducted among healthcare professionals in the Scottish highlands it was found that a rural background, perceived ease of access to children’s education, access to job for spouse and healthcare were important factors influencing rural location11. Professional isolation was an important issue for those working in rural areas.\n\nIn a study conducted among occupational therapy students in Australia it was seen that rural fieldwork experience and the influence of fieldwork supervisors were important positive factors for rural practice while the desire to be a specialist, and lack of professional development opportunities in a rural area were negative factors12. The desire for specialization was also mentioned as a negative factor in the present study. In the state of North Carolina in the US incentives such as loan repayment programs, salary guarantees and practice assistance for rural physicians were important factors in attracting primary care physicians to rural areas13. Having grown up in an area with a population of less than 11000 was highly predictive of rural practice. A study conducted in the US found that debt forgiveness, financial incentives and wage guarantees may increase participants’ interest in rural practice14. This was similar to that reported in our study. In the US loan repayment and direct financial incentive programs were successful in recruiting and retaining doctors in needy local communities15.\n\nFamily-related factors were mentioned by respondents as influencing their decision regarding working in rural areas. In the US study14 it was noted that medical students with a rural background and with spouses and significant others having a rural background were more likely to practice in a rural area. In a Turkish study it was noted that respondents who were born in an underdeveloped region of the country or had lived for a significant amount of time in an underdeveloped region and with a lower background family income were more likely to work in a rural area16. Higher salaries was the most influential factor for working in these areas. In XUSOM, a large percentage of students, although they are US or Canadian citizens, are of Asian origin. A large percentage of them are from cities and have minimal exposure to rural life and they anticipate problems in being accepted by rural communities.\n\nMedical schools in developed nations with a mandate to create rural doctors are providing a greater proportion of decentralized teaching-learning to students. Students stay in small groups in rural communities and lessons are delivered using the internet and other media. At the University of Calgary in Canada, two separate preclinical programs were taught at two separate rural sites and no significant difference in examination scores were noted between students at the rural sites and students learning at the main university17. At the University of Missouri School of Medicine in the US the summer community program was developed in which second year medical students work alongside rural, community-based physician preceptors18. The program positively influenced student perspective of rural practice and lifestyle and increased their interest in rural practice.\n\nIn the US state of Kansas, the Scholars in Rural Health program is designed to attract and retain young rural Kansans with a high probability of successful careers in rural communities. Scholars accepted into and satisfactorily completing this program are admitted automatically to the School of Medicine. The program has shown success in maintaining a pipeline of doctors for rural Kansas19. At the University of Alabama in the US, fifteen years of operation of the rural health leaders pipeline was studied21. Rural students are targeted at multiple levels ranging from elementary school through residency. Puppet shows highlighting different health professions are conducted in elementary schools, a rural health schools program is offered to 11th grade students, a minority rural health pipeline program is offered to college students and a rural medical scholars program and assured admissions to family medicine residency are offered to medical students.\n\nA recent article analyzing rural medical education in the US, Canada and Australia identified three types of medical schools, mixed urban/rural schools, defacto rural schools and stand alone rural schools21. These schools all adopted a pipeline approach to meeting the need for rural doctors focusing on: early recruitment; admissions; locating clinical education in rural settings; rural health focus to curriculum; and support for rural practice. In Australia, the Broken Hill University Department of Rural Health was established to improve health care in far-western New South Wales and offers an Enhanced Rural Inter-professional Cultural Health (ENRICH) program to health science students designed to deepen the medical student experience22. The program uses community resources, professional, cultural and artistic to provide stimulating educational resources.\n\nWe have not been able to obtain studies regarding the career intentions of graduates of offshore Caribbean medical graduates. A study conducted at the University of West Indies in Jamaica had shown that two-thirds of those who planned to work in a rural health facility planned to do so immediately after graduation but most did not plan to work in these facilities for more than five years23. A study conducted in California had shown that IMG holders of temporary visas had the highest obligation to serve in health professional shortage areas24. A similar phenomenon was noted in the present study with many students from countries other than the US and Canada expressing willingness to serve in areas of physician shortage in return for residency rights. XUSOM also offers scholarships to students from Caribbean community (CARICOM) countries (http://blog.xusom.com/?p=268). Providing rural exposure on the island of Aruba with well developed infrastructure and health system is challenging. Among the affiliated US hospitals where students do their clinical training most are in urban areas but community hospitals are also included in the list. Students graduating from XUSOM may be interested in serving in rural US/Canada if appropriate incentives and opportunities for professional growth are offered.\n\nThe high response rate was the strength of the study. Also a variety of responses were obtained in response to the questions. The study also had limitations. Student perception about working in rural US/Canada was obtained using a questionnaire which had been used in a previous study in Nepal. The questionnaire was modified to suit the present context, and feedback about the modified questionnaire was obtained. The findings of the present study were not triangulated with information obtained using other modalities. Respondents’ awareness about possible changes in medical education and curricula to produce doctors for rural areas and about specific initiatives underway in the US and Canada to address the shortage of rural physicians was low. The study was limited to premedical and preclinical students and was not conducted among students during the clinical years of the course due to logistical difficulties.\n\n\nConclusions\n\nMost respondents were from urban areas. About 40% of respondents were in favor of working in rural US/Canada after graduation. Most were however considering working in a small town or a semi-rural area not far from a big city. Like in previous studies good living conditions, family related factors, incentives were among conditions necessary to be fulfilled before students would consider working in rural areas. Most respondents considered reduced opportunities for growth, their lack of exposure to rural life, and cultural issues as hindering factors. Students from offshore Caribbean schools could play a role in partly addressing the physician shortage in rural US/Canada provided the right incentives and growth opportunities are offered. Similar studies are required in other offshore Caribbean medical schools.\n\n\nData availability\n\nF1000Research: Dataset 1, 2 and 3. Demographic characteristics and responses of student respondents, 10.5256/f1000research.5927.d4041125", "appendix": "Author contributions\n\n\n\nPRS, AKD, and AN conceived the study. BLH and BWL provided inputs during the design of the study. PRS, AKD and AN conducted the study. PRS was involved in analyzing the data. AKD, AN, BLH and BWL helped in interpretation of the data. PRS wrote the manuscript with inputs from the other authors. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge all students who participated in the study.\n\n\nSupplementary file 1\n\nStudent perception about working in rural US/Canada after graduation questionnaire.\n\nModified questionnaire used in the study “Student perception about working in rural US/Canada after graduation”. Click here to access the File.\n\n\nReferences\n\nRosenblatt RA, Hart LG: Physicians and rural America. West J Med. 2000; 173(5): 348–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson MJ, Hagopian A, Fordyce M, et al.: Do international medical graduates (IMGs) “fill the gap” in rural primary care in the United States? A national study. J Rural Health. 2009; 25(2): 124–34. PubMed Abstract \| Publisher Full Text\n\nHagopian A, Thompson MJ, Kaltenbach E, et al.: The role of international medical graduates in America’s small rural critical access hospitals. J Rural Health. 2004; 20(1): 52–8. PubMed Abstract \| Publisher Full Text\n\nDauphinee WD: The circle game: understanding physician migration patterns within Canada. Acad Med. 2006; 81(12 Suppl): S49–54. PubMed Abstract \| Publisher Full Text\n\nvan Zanten M, Boulet JR: Medical education in the Caribbean: quantifying the contribution of Caribbean-educated physicians to the primary care workforce in the United States. Acad Med. 2013; 88(2): 276–81. PubMed Abstract \| Publisher Full Text\n\nSzafran O, Crutcher RA, Banner SR, et al.: Canadian and immigrant international medical graduates. Can Fam Physician. 2005; 51: 1242–3. PubMed Abstract \| Free Full Text\n\nMcAvinue MB, Boulet JR, Kelly WC, et al.: U.S. citizens who graduated from medical schools outside the United States and Canada and received certification from the Educational Commission for Foreign Medical Graduates, 1983–2002. Acad Med. 2005; 80(5): 473–8. PubMed Abstract \| Publisher Full Text\n\nShankar PR, Thapa TP: Student perception about working in rural Nepal after graduation: a study among first- and second-year medical students. Hum Resour Health. 2012; 10(1): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEckhert NL: Perspective: private schools of the Caribbean: outsourcing medical education. Acad Med. 2010; 85(4): 622–30. PubMed Abstract \| Publisher Full Text\n\nKolstad JR: How to make rural jobs more attractive to health workers. Findings from a discrete choice experiment in Tanzania. Health Econ. 2011; 20(2): 196–211. PubMed Abstract \| Publisher Full Text\n\nRichards HM, Farmer J, Selvaraj S: Sustaining the rural primary healthcare workforce: survey of healthcare professionals in the Scottish Highlands. Rural Remote Health. 2005; 5(1): 365. PubMed Abstract\n\nMcAuliffe T, Barnett F: Factors influencing occupational therapy students’ perceptions of rural and remote practice. Rural Remote Health. 2009; 9(1): 1078. PubMed Abstract\n\nDuffrin C, Diaz S, Cashion M, et al.: Factors associated with placement of rural primary care physicians in north Carolina. South Med J. 2014; 107(11): 728–33. PubMed Abstract \| Publisher Full Text\n\nRoyston PJ, Mathieson K, Leafman J, et al.: Medical student characteristics predictive of intent for rural practice. Rural Remote Health. 2012; 12: 2107. PubMed Abstract\n\nPathman DE, Konrad TR, King TS, et al.: Outcomes of states’ scholarship, loan repayment, and related programs for physicians. Med Care. 2004; 42(6): 560–8. PubMed Abstract \| Publisher Full Text\n\nMollahaliloglu S, Aydogan Ü, Kosdak M, et al.: Physician scarcity in underdeveloped areas of Turkey: what do new graduate physicians think? Rural Remote Health. 2012; 12: 2067. PubMed Abstract\n\nMyhre DL, Adamiak P, Turley N, et al.: Beyond bricks and mortar: a rural network approach to preclinical medical education. BMC Med Educ. 2014; 14: 166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKane KY, Quinn KJ, Stevermer JJ, et al.: Summer in the country: changes in medical students’ perceptions following an innovative rural community experience. Acad Med. 2013; 88(1): 1157–63. PubMed Abstract\n\nKallail KJ, McCurdy S: Scholars in Rural Health: outcomes from an assured admissions program. Fam Med. 2010; 42(10): 729–31. PubMed Abstract\n\nWheat JR, Brandon JE, Leeper JD, et al.: Rural health leaders pipeline, 1990–2005: case study of a second-generation rural medical education program. J Agromedicine. 2007; 12(4): 51–61. PubMed Abstract \| Publisher Full Text\n\nTesson G, Strasser R, Pong RW, et al.: Advances in rural medical education in three countries: Canada, The United States and Australia. Rural Remote Health. 2005; 5(4): 397. PubMed Abstract\n\nMoore M, Bolte K, Bennett P: Innovative training for rural medical students. Clin Teach. 2012; 9(4): 238–42. PubMed Abstract \| Publisher Full Text\n\nPage J, Allison M, Andrade S, et al.: Factors influencing medical interns trained at U.W.I. to work subsequently in a rural area in Jamaica. West Indian Med J. 1992; 41(2): 75–8. PubMed Abstract\n\nOgunyemi D, Edelstein R: Career intentions of U.S. medical graduates and international medical graduates. J Natl Med Assoc. 2007; 99(10): 1132–7. PubMed Abstract \| Free Full Text\n\nShankar PR, Dubey AK, Nandy A, et al.: Dataset 1, 2 and 3. Demographic characteristics and responses of student respondents. F1000Research. 2014. Data Source" }	[ { "id": "7546", "date": "04 Feb 2015", "name": "Anthony David", "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\nThe title could be changed a little. The characteristics of the students, rural or urban also was studied and that is not reflected in the title. The study as such is well researched and clear. The findings have been well analyzed and presented in the paper. The abstract reflects the paper well. The explanation of the study design & methods is clear. A little more explanation on the analysis would be useful.The conclusions are well justified by the results. The data is useful for replication.Overall this paper can be indexed with a few minor modifications as suggested.", "responses": [ { "c_id": "1295", "date": "23 Apr 2015", "name": "Pathiyil Ravi Shankar", "role": "Author Response", "response": "Dr DavidWe thank the reviewer for the comments.Even though we have studied the demographic characteristics of the student respondents the study was not designed with the objective of comparing perception about working in rural areas among different groups of students. Hence we feel a change of title may not be required as with a change in title readers may expect us to study differences in perception among subgroups of respondents.We have explained the analysis towards the end of the 2nd paragraph of page 4. We used mainly descriptive statistics and numbers and percentages.We thank the reviewer for the other comments" } ] }, { "id": "7893", "date": "07 Apr 2015", "name": "Stacey Friedman", "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\nThis paper addresses the important topic of student perceptions of rural practice, via a questionnaire for students of one Caribbean medical school.The authors might be more explicit and specific in defining how they are using the term “IMGs from Caribbean medical schools” in this paper since the study participants include students of US, Canadian, and other nationalities, as well as both “premedical” and MD students.The study’s respondents are diverse, as shown in Table 1 – including “premedical” and MD students, different nationalities, etc. It would be informative to see more comparison in the analysis, to see if demographic characteristics are related to perspectives on rural practice. There is some mention of this in the Discussion but it is not detailed in the Results.It would be helpful if the authors would clarify whether all enrolled premedical and MD students were invited to participate in this study. The description in the Methods suggests that this is the case but it is not explicitly stated how many students were enrolled at the school at the time of the study, including a breakdown by premedical and MD. This information would be helpful, as well as confirmation that the study respondents are representative of the overall student population. Also, some more background on the school, including how it is comparable to and how it differs from other Caribbean schools would be helpful for judging the generalizability of the study results. Some of this information is in the Discussion.The authors note that “Many respondents did not answer the question regarding suggestions for re-focusing medical education for preparing doctors for rural practice.” It would be helpful to know exactly how many respondents there were to this question, particularly given its importance. It is understandable that the authors would want to be cautious about stating results from this question given the limited response. However, while stating this caution, the authors may want to offer in the Discussion possible actions that Caribbean medical schools might take to begin to address rural practice needs, including direction for future research. The low question response rate (despite the high questionnaire response rate) might also be noted as a study limitation.", "responses": [ { "c_id": "1296", "date": "23 Apr 2015", "name": "Pathiyil Ravi Shankar", "role": "Author Response", "response": "We are submitting the revised version of the manuscript titled ‘Student perception about working in rural United States/Canada after graduation’. The manuscript has been revised in the light of the reviewers’ comments. Response to specific comments is as follows:S Friedman: We thank the reviewer for her comments. We have explained what we meant by ‘IMGs from Caribbean medical schools’ on page 3, paragraph 4. The studies we have mentioned from the United States (US) and Canada had defined a Caribbean IMG as either a citizen of the US or Canada who had studied in an offshore Caribbean medical school or students from other countries who had studied at these schools and eventually plan to practice in the US/Canada. Comment: The study’s respondents are diverse, as shown in Table 1 – including “premedical” and MD students, different nationalities, etc. It would be informative to see more comparison in the analysis, to see if demographic characteristics are related to perspectives on rural practice. There is some mention of this in the Discussion but it is not detailed in the Results.No important difference with regard to perspective about rural practice was noted among different subgroups of respondents. The number of students was small which may have been partly responsible for not detecting differences. Also the study was not designed to statistically examine for differences among subgroups. This has been mentioned on page 5, paragraph 1 (Results section). Comment: It would be helpful if the authors would clarify whether all enrolled premedical and MD students were invited to participate in this study. The description in the Methods suggests that this is the case but it is not explicitly stated how many students were enrolled at the school at the time of the study, including a breakdown by premedical and MD. This information would be helpful, as well as confirmation that the study respondents are representative of the overall student population. In the Methods section (page 4) we have clarified that only students enrolled during the basic science years of the MD program and in the Premedical program were included in the study. In the first paragraph of the Results section we have provided the number and percentage breakdown. Students during the clinical years were not included as they do their clinical postings in geographically diverse locations across the US and Canada. This has been mentioned as a limitation of the study on page 10, last paragraph. Comment: Also, some more background on the school, including how it is comparable to and how it differs from other Caribbean schools would be helpful for judging the generalizability of the study results. Some of this information is in the Discussion.We have provided this information on page 3, paragraph 3. As the reviewer has mentioned some of this information is also in the discussion. XUSOM has lesser number of students and a greater proportion of students of South Asian and Asian origin compared to other bigger and older schools. Comment: The authors note that “Many respondents did not answer the question regarding suggestions for re-focusing medical education for preparing doctors for rural practice.” It would be helpful to know exactly how many respondents there were to this question, particularly given its importance. Only 14 respondents answered this question. We have mentioned this in page 7, paragraph 1.Comment: It is understandable that the authors would want to be cautious about stating results from this question given the limited response. However, while stating this caution, the authors may want to offer in the Discussion possible actions that Caribbean medical schools might take to begin to address rural practice needs, including direction for future research. We have briefly mentioned some of these initiatives on page 10, paragraph 2 of the manuscript.Comment: The low question response rate (despite the high questionnaire response rate) might also be noted as a study limitation.We have mentioned this in the limitations of the manuscript on page 10, last paragraph." } ] } ]	1	https://f1000research.com/articles/3-301
https://f1000research.com/articles/4-96/v1	22 Apr 15	{ "type": "Correspondence", "title": "The “NF-ĸB interacting long noncoding RNA” (NKILA) transcript is antisense to cancer-associated gene PMEPA1", "authors": [ "Johannes M. Dijkstra", "David B. Alexander", "David B. Alexander" ], "abstract": "This correspondence concerns a recent publication in Cancer Cell by Liu et al.1 who analyzed a long noncoding RNA (lncRNA) that they designated “NKILA”. Liu et al. found that NKILA (1) is upregulated by immunostimulants, (2) has a promoter with an NF-ĸB binding motif, (3) can bind to the p65 protein of the NF-ĸB transcription factor and then interfere with phosphorylation of IĸBα, and (4) negatively affects functions that involve NF-ĸB pathways. And, importantly, they found that (5) low NKILA expression in breast cancers is associated with poor patient prognosis. However, they entirely failed to mention PMEPA1, a gene which runs antisense to NKILA, and the expression of which is associated with several tumors and which encodes a protein that participates in immune pathways.The PMEPA1 locus, including its promoter region, which Liu et al.1 only discuss in regard to NKILA, is highly conserved through evolution. Our impression is that NKILA emerged only later in evolution, possibly as an additional means of PMEPA1 regulation. Liu et al., however, only consider direct binding between NKILA and NF-ĸB as the mechanism for their in vivo observations of NKILA function, but do not provide solid evidence for their model. If in vivo observations by Liu et al. could be explained by NKILA regulation of PMEPA1, it would contribute to the establishment of PMEPA1 as an important topic of cancer research. We feel that the herein presented discussion is necessary for a correct interpretation of the Liu et al. article.", "keywords": [ "Breast cancer", "NKILA", "PMEPA1", "NF-ĸB", "long noncoding RNA", "antisense", "promoter", "evolution" ], "content": "Correspondence\n\nLiu et al.1 investigated breast cancer cell lines for possible association of known long noncoding transcripts with immunostimulation. They found that an unspliced lncRNA, which they designated “NKILA” (represented by large yellow box in Figure 1), could be upregulated by several immune agents. However, they did not mention the existence of a reported spliced form of NKILA (GenBank accession DA866558, see Figure 1), or, - our major criticism - that NKILA is divergently transcribed from prostate transmembrane protein, androgen induced 1 (PMEPA1) and antisense to some of its transcripts (see Figure 1). One of the alternative names for PMEPA1 is solid tumor associated gene 1 (STAG1)2, and its expression was found upregulated in several tumors including breast cancer (e.g., references 2 and 3). Liu et al.1 found that the NKILA promoter region contains an NF-κB binding motif (Figure 1), which according to our analysis is rather well conserved among eutherian mammals (Supplementary file S1). However, whereas the PMEPA1-001 transcript open reading frame and a part of the intergenic promoter region are highly conserved through evolution, this seems to be true to a lesser degree for NKILA equivalent transcripts (Supplementary file S1 and discussion therein). So, from the standpoint of evolution, a logical hypothesis for the function of the seemingly younger NKILA is its possible interference with PMEPA1 expression4,5.\n\nPMEPA1 expression is strongly enhanced by TGF-β6,7, something which agrees with the two SMAD binding element motifs that we found conserved in its promoter (Supplementary file S1). PMEPA1 function is not well understood, but it is believed to encode a transmembrane protein that with its cytoplasmic domain can bind SMAD proteins and can positively affect activation of Akt7. Signaling pathways involving Akt and NF-κB are known to converge8, which might be relevant for a possible indirect effect of NKILA through PMEPA1 on NF-κB functions. PMEPA1 levels were reported to be high in invasive MDA-MB-231 breast cancer cells, and low in non-invasive MCF-7 and T47D breast cancer cells, agreeing with observations for aggressive versus non-aggressive tumors9. This is exactly opposite to the expression pattern observed by Liu et al. for NKILA in these cell lines and among tumors. PMEPA1 knockdown has been found to be able to attenuate growth and motility of MDA-MB-231 breast cancer cells9, which is interesting since Liu et al.1 found that forced NKILA expression (which might knockdown PMEPA1 expression) in MDA-MK-231 cells achieves similar effects. Consistent with these findings is the observation that high PMEPA1 expression in breast cancer is associated with poor patient prognosis7, and high NKILA expression with better patient prognosis1. Although there are also published PMEPA1 reports which are harder to reconcile with such a model (e.g. reference 10), and which are hard for us to validate, at least the above selected set of data suggests that NKILA has a negative effect on PMEPA1 function. More research on both NKILA and PMEPA1 will be necessary before conclusions can be made, but for now the possibility that NKILA can downregulate PMEPA1 expression appears to be a reasonable model4,5.\n\nThe figure summarizes several data from the study by Liu et al. for NKILA and its promoter, while also showing overlapping transcripts that were neglected in that study. The NKILA transcript identified by Liu et al. roughly corresponds with transcript RP5-1059L7.1-001 as summarized in the GRCh38.p2 dataset of the Ensembl database (http://www.ensembl.org/index.html). GenBank accession DA866558 (RP5-1059L7.1-002 in Ensembl) contains an expressed sequence tag (EST) which represents the 5’ end of a spliced transcript and for which the 3’ end is not known. The depicted summary of the PMEAP1 transcripts -001, -002 and -201, is derived from the Ensembl database and agrees with GenBank reports; for additional variations of PMEAP1 transcripts we refer to the Ensembl database. Exons are indicated by boxes, with protein coding regions in black. The 3’ UTR of PMEPA1 is not drawn in correct proportion to the other exon regions. Arrows indicate the direction of transcription, and genomic regions are measured in basepairs. The figure also shows from the Liu et al. report the positions of the NF-κB binding promoter element, the NKILA hairpin-prone regions, the NKILA region that binds miR-103 and -107, the NKILA binding sites for the Northern blot probe and RT-PCR primers, and the shNK1 and shNK2 regions from which sequences were derived for cloning into shRNA constructs.\n\nLiu et al.1 concluded that NKILA interferes with pathways that involve NF-κB function, and we feel that in essence this conclusion can be believably deduced from their abundant experimental data. However, mechanistically Liu et al. only consider a direct interaction with NF-κB components and fail to consider an indirect effect through PMEPA1 regulation. Liu et al.1 did find direct binding between the NF-κB component p65 and NKILA, but whether this can be considered as evidence of physiological specificity of NKILA for NF-κB is questionable. When they analyzed proteins that they could pull down with NKILA they only compared different NF-κB pathway components using Western blot analysis1. In addition, when they quantified NKILA by RT-PCR on genetic material that co-precipitated with NF-κB factors, they did not exclude the possibility that they might be measuring genomic NKILA DNA1.\n\nLiu et al.1 mapped the NKILA interaction with p65 to hairpin-prone regions A and B, and showed that the hairpin-prone region C can interact with NF-κB pathway factor IκBα (for hairpin-prone region locations see Figure 1). By mutation analysis, Liu et al.1 found that all these three hairpin-prone regions are important for the inhibitory effect of NKILA on NF-κB activity. Although the relevance of this overlap is not clear, we point out that all three identified hairpin-prone regions, and also the region which Liu et al.1 found to confer sensitivity to miRNA induced degradation, overlap with known PMEPA1 transcript regions (Figure 1).\n\nAs an additional remark, we would like to state that the somewhat discussable manner in the way Liu et al.1 performed or described some of their experiments (see our comments in Supplementary file S2) does not help to convey the image of a study which is solid in its quantitative aspects. However, despite our criticism, it is only fair to state here that according to our judgement the very elaborate study by Liu et al.1 believably shows (1) how NKILA can bind (in vitro) to NF-κB, (2) that NKILA can interfere with functions that involve NF-κB pathways, and (3) that low NKILA expression predicts poor clinical outcome in patients with breast cancer. But they should mend the open ends, which means providing more evidence of the specificity of the NKILA binding to NF-κB, and to take the possible effects of NKILA through regulation of PMEPA1 into consideration. In regard to the more general claim by Liu et al.1 that there exists “a class of lncRNAs that regulate signal transduction at post-translational level”, we believe as before11 that such a conclusion needs more evidence than currently has been presented. We hope that our present discussion leads to an increased interest in the PMEPA1-NKILA locus, because whatever mechanism may be correct, Liu et al. did provide evidence that this locus is clinically important in breast cancer.", "appendix": "Author contributions\n\n\n\nJMD initiated the study. DBA and JMD performed the analysis. JMD wrote the article with help of DBA. Both authors critically edited the correspondence and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary file S1.\n\nAlignment of PMEPA1-NKILA promoter region sequences of representative animals and deduced PMEPA1 amino acid sequences for the species compared.\n\nClick here to access the data. http://dx.doi.org/10.5256/f1000research.6400.s45984\n\nSupplementary file S2.\n\nList of issues regarding the Liu et al. (2015). Cancer Cell 27, 370–381 paper which in our opinion need attention.\n\nClick here to access the data.\n\n\nReferences\n\nLiu B, Sun L, Liu Q, et al.: A Cytoplasmic NF-κB interacting Long Noncoding RNA Blocks IκB Phosphorylation and Suppresses Breast Cancer Metastasis. Cancer Cell. 2015; 27(3): 370–81. PubMed Abstract \| Publisher Full Text\n\nRae FK, Hooper JD, Nicol DL, et al.: Characterization of a novel gene, STAG1/PMEPA1, upregulated in renal cell carcinoma and other solid tumors. Mol Carcinog. 2001; 32(1): 44–53. PubMed Abstract \| Publisher Full Text\n\nGiannini G, Ambrosini MI, Di Marcotullio L, et al.: EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer. Mol Carcinog. 2003; 38(4): 188–200. PubMed Abstract \| Publisher Full Text\n\nVillegas VE, Zaphiropoulos PG: Neighboring gene regulation by antisense long non-coding RNAs. Int J Mol Sci. 2015; 16(2): 3251–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q, Su Z, Xu X, et al.: AS1DHRS4, a head-to-head natural antisense transcript, silences the DHRS4 gene cluster in cis and trans. Proc Natl Acad Sci U S A. 2012; 109(35): 14110–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunschwig EB, Wilson K, Mack D, et al.: PMEPA1, a transforming growth factor-beta-induced marker of terminal colonocyte differentiation whose expression is maintained in primary and metastatic colon cancer. Cancer Res. 2003; 63(7): 1568–75. PubMed Abstract\n\nSingha PK, Pandeswara S, Geng H, et al.: TGF-β induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer. Genes Cancer. 2014; 5(9–10): 320–36. PubMed Abstract \| Free Full Text\n\nMeng F, Liu L, Chin PC, et al.: Akt is a downstream target of NF-kappa B. J Biol Chem. 2002; 277(33): 29674–80. PubMed Abstract \| Publisher Full Text\n\nSingha PK, Yeh IT, Venkatachalam MA, et al.: Transforming growth factor-beta (TGF-beta)-inducible gene TMEPAI converts TGF-beta from a tumor suppressor to a tumor promoter in breast cancer. Cancer Res. 2010; 70(15): 6377–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnazawa Y, Arakawa H, Nakagawa H, et al.: Identification of STAG1 as a key mediator of a p53-dependent apoptotic pathway. Oncogene. 2004; 23(46): 7621–7. PubMed Abstract \| Publisher Full Text\n\nDijkstra JM, Ballingall KT: Non-human lnc-DC orthologs encode Wdnm1-like protein [v2; ref status: indexed, http://f1000r.es/4el]. F1000Res. 2014; 3: 160. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8429", "date": "27 Apr 2015", "name": "Peter G. Zaphiropoulos", "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\nThe Dijkstra and Alexander correspondence suggests that the noncoding NKILA RNA effects on NF-κB signaling in breast cancer cells of the recent Liu et al. Cancer Cell publication may be partly mediated via the protein coding PMEPA1 gene, which is positioned in an antisense orientation (head to head) to the NKILA locus. While the authors do not question the main thesis of the Liu paper, namely the direct interaction of the NKILA RNA to p65 that leads to inhibition of IκB phosphorylation, they highlight certain published data, which are indicative of a role of PMEDA1 in breast cancer and, interestingly, of a possible reciprocal regulation of PMEDA1 and NKILA. In my opinion, the hypothesis put forward by Dijkstra and Alexander is of interest and should be experimentally addressed to test whether there is indeed an interplay between the sense-antisense PMEDA1-NKILA gene pair, which may further expand the biological roles and mechanisms of action of the NKILA noncoding RNA. In particular, I would like to see the impact of PMEDA1 depletion in the observable NKILA biological effects and vice versa the impact of NKILA depletion in PMADA1 gene expression. Clearly, a state of validating or refuting a hypothesis by experimental means is in front of the scientific community.", "responses": [ { "c_id": "1312", "date": "27 Apr 2015", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Zaphiropoulos,Thank you for your review and your approval. Since you are an expert in RNA functions, we are very happy that you consider our hypothesis of NKILA possibly affecting PMEPA1 function to be a realistic possibility.However, you are incorrect in that we do not question the main thesis of the Liu et al. paper. We agree that for the direct NKILA to NF-kB binding mechanism, Liu et al. provided a lot of support by in vitro experiments. However, in our opinion, they did not provide the necessary conclusive in vivo experimental evidence for making a new model on lncRNA function. So we agree that they proved that NKILA can bind to NF-kB, but we say that they did not provide evidence that it does so in a specific or functional manner in vivo. Their paper is difficult to interpret because of the many experiments they did, combined with rather frequent incomplete descriptions of materials and methods (some of those issues we listed in our supplementary file S2). However, despite some uncertainties about what they actually did, a very critical flaw running through a large part of their paper appears to be the inability to distinguish cDNA from genomic DNA by their RT-PCR method.We would be interested to hear from you, as an RNA expert, whether you believe that our criticism of the Liu et al. article is correct.We are sorry that you misinterpreted our story. If the other reviewers will have a similar misinterpretation, we will have to write our criticism of the Liu et al. paper in a more explicit way.Sincerely,also on behalf of David B. Alexander,Johannes M. Dijkstra" }, { "c_id": "1321", "date": "29 Apr 2015", "name": "Peter G. Zaphiropoulos", "role": "Reviewer Response", "response": "Dear Johannes (and David), Thank you for your post. In my reviewing of your correspondence I tried to distil its essence in terms of biological significance. In this spirit of constructive refereeing, the take-home message, in my opinion, is to test the possible interplay of the antisense protein-coding gene and the NKILA noncoding RNA, and urge you to engage in such experimental approaches to push science forward. Concerning, your criticism of technical aspects of the Liu et al paper, I am not so sure. For example, your claim in supplementary file S2 and in your post that “RT-PCR amplification of an intronless sequence as done by the authors (see Figure 1) should consider the possibility of DNA contamination” is not in-line with the data of the Northern analysis depicted in the same Figure, panel A, which are consistent with the qRT-PCR data of Figure 1, panel C. Thus, my conclusion is that their qRT-PCR assay is robust enough and detects cDNA, as apparently their RNA preparations are essentially free of genomic DNA contamination. Finally, I believe that it is appropriate to contact the authors of the Liu et al paper, either directly or via Cancer Cell, to express any additional concerns that you may have. Best wishes, Peter" }, { "c_id": "1322", "date": "30 Apr 2015", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Peter,Thank you for your answer. But I like to disagree with your technical assessment of \"essentially free of DNA contamination\", since the methods they say to have used do not provide such material. For which of the assays, if for any, the DNA contamination had a substantial impact on the results can't be known of course, but the authors should have excluded such possibility. To my knowledge, many scientists facing similar issues are very serious to get rid of the DNA, and it is at least peculiar that in the Liu et al. study such was not tried. Awareness of the problem would also have urged them to explain the sequences of the primers for ACTB gene amplfication, since now the ACTB versus NKILA comparison could theoretically be a measure for the amount of isolated DNA. I interpret your statement of \"I am not so sure\" as a correct assessment of the Liu et al. paper, which in its wordings does not seem to express the normal level of scientific insecurity itself.Personally we are currently not so interested in doing lncRNA experiments ourselves, but we will start study of PMEPA1 at the protein level. At least that may be something good that has come of it.Best wishes,also on behalf of David,Johannes" } ] }, { "id": "8428", "date": "11 May 2015", "name": "Pothana Saikumar", "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\nOur laboratory (Saikumar) published two manuscripts on the PMEPA1/TMEPAI gene product that acts as a molecular switch in converting transforming growth factor-beta (TGF-β) from a tumor suppressor to a tumor promoter by suppressing canonical Smad signaling and promoting non-canonical PI3K/Akt signaling using breast cancer models. We have been invited to review and comment here on the correspondence paper by Dijkstra and Alexander. The correspondence titled \"The NF-kB interacting long noncoding RNA (NKILA) transcript is antisense to cancer-associated gene PMEPA1” by Dijkstra and Alexander, provided an alternate explanation to the results presented by Liu et al in a recent publication “A cytoplasmic NF-kB interacting long noncoding RNA blocks IkB phosphorylation and suppresses the breast cancer metastasis” in Cancer Cell (27: 370-381, 2015). Although the novel finding of the later is that the long noncoding RNA designated NKILA is upregulated by immunostimulants due to the presence of a NF-kB binding motif in the promoter region of this gene which is located in the human chromosome 20q13.31. NKILA driven by NFkB acts as a negative feedback regulator of NFkB activation through a direct interaction with NF-kB p65-IkB complex, which was the basis for the negative effects on the events mediated by NF-kB signaling pathways. Importantly, these authors either ignored or failed to notice the similarities between NKILA and PMEPA1, another gene present in the same locus of chromosome 20. A simple BLAST search would have indicated the similarities between these two genes and their locations. Interestingly, NKILA appears to fit the description of a head-to-head antisense RNA for PMEPA1.Dijkstra and Alexander rightly noticed this omission and highlighted in their correspondence the following: i) Both NKILA and PMEPA1 genes are in anti-sense and sense orientation in the same region in chromosome 20; ii) Based on the overlap, Dijkstra and Alexander proposed a provocative hypothesis that NKILA transcript may have the potential to regulate PMEPA1. The authors are correct in pointing out that Liu et al. completely overlooked the existence of PMEPA1 and did not take into account in their discussion of the mechanism involving NKILA; iii) They rightly state that indeed future experiments should verify a link between NKILA and PMEPA1. Some of the issues they raised in the supplementary file 2 were scientifically valid. This correspondence is purely theoretical and short of much needed experimental data to show that NKILA negatively regulates PMEPA1 mRNA levels. However, in defense of the authors of the original paper, there is no evidence to suggest that immuno-stimulants like TNF-α and LPS can induce PMEPA1.Overall the correspondence merits indexing with high importance because it will initiate research activity to identify the link between these two genes during inflammation.", "responses": [ { "c_id": "1343", "date": "12 May 2015", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Saikumar,Thank you for your nice and sophisticated review of our article. It is important to us that as an expert of PMEPA1 studies you deem our referencing of those studies to be correct. We also appreciate that at least in part you support our criticisms of the Liu et al. article. Thank you also for acknowledging the importance of our article. The highly conserved PMEPA1 gene and its promoter are fascinating, the possible association of the PMEPAP1-NKILA locus with cancers is evident as also shown by Liu et al., and it would be nice if our article can boost PMEPA1-NKILA research.Sincerely,also on behalf of David B. Alexander,Johannes M. Dijkstra" } ] } ]	1	https://f1000research.com/articles/4-96
https://f1000research.com/articles/4-71/v1	17 Mar 15	{ "type": "Correspondence", "title": "Ocular disconjugacy cannot be measured without establishing a solid reference", "authors": [ "Jun Maruta" ], "abstract": "This correspondence points out a need for clarification concerning the methodology utilized in the study “Eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion”, recently published in Journal of Neurotrauma. The authors of the paper state that binocular eye movements were recorded using a single-camera video-oculography technique and that binocular disconjugate characteristics were analyzed without calibration of eye orientation. It is claimed that a variance-based disconjugacy metric was found to be sensitive to the severity of a concussive brain injury and to the status of recovery after the original injury. However, the reproducibility of the paper’s findings may be challenged simply by the paucity of details in the methodological description. More importantly, from the information supplied or cited in the paper, it is difficult to evaluate the validity of the potentially interesting conclusions of the paper.", "keywords": [ "Mild traumatic brain injury", "mTBI screening", "vergence" ], "content": "Correspondence\n\nI wish to point out a need for clarification concerning the methodology utilized in the study “Eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion” by Samadani et al., 20151. The authors state that binocular eye movements were recorded using a single-camera infrared-based video-oculography technique (EyeLink 1000, SR Research, Ontario, Canada) and that binocular disconjugate characteristics were analyzed without calibration of eye orientation. The authors claim that their variance-based disconjugacy metric was sensitive to the severity of a concussive brain injury and to the status of recovery after the original injury.\n\nThe EyeLink 1000 system is an excellent eye tracker with a single high resolution camera and an infrared light source affixed to the camera. A typical recording setup consists of a computer monitor with which the visual stimuli are presented to the subject and the camera unit (placed in front of the monitor base) with which the eye movement is recorded monocularly or binocularly. The system has an option of easily outputting image-based, uncalibrated eye coordinates with hundreds of units representing 1° of eye rotation.\n\nThe concern I would raise with Samadani et al.’s paper is the unclearness of the relationship between their metric and binocular disconjugacy. Logically, for an identical amount of eye rotation, any asymmetry in the spatial relationship that the camera or the infrared light source has with the two fellow eyes would result in different extents of relocation of the images of the pupils or corneal reflections. Asymmetries exist because there is a physical separation between the two eyes as well as between the camera and the infrared light source. In addition, although the biometric characteristics of eyes are highly symmetrical within individuals, they are not perfectly symmetrical2 and a 1–2% non-conformity in corneal curvature or axial length is not uncommon, which further confounds the relationship between eye rotation and changes in pixel coordinates. Moreover, each of the two fellow eyes has its own function that maps pixel movement to the eye rotation, and this mapping is not linear. Thus, the arithmetic difference between the uncalibrated coordinates of the two eyes is quite removed from a physical representation of gaze misalignment.\n\nBeyond the factors associated with the raw data, the analytic methods in the paper also do not seem to be constructed with a clear intent. It is puzzling why the disconjugacy metric is represented by the variance of the left-right differences after independently averaging for each eye the uncalibrated coordinates over several cycles for a given stimulus position, as opposed to the straightforward variance of the left-right differences at all sample points. Furthermore, the ranges of outcome values presented in the series of figures run from 0 to at most 0.25, but how the value 0 could have been obtained is not clear. The question arises because in the two eyes’ uncalibrated coordinates there must be a constant bias related to the interocular distance. Lastly, what the high end of the outcome range represents is not clear. Since one unit in EyeLink’s uncalibrated data output is smaller than 0.01° of eye rotation, being able to report differences in 0.25 square units or less seems implausible. If the raw data were numerically centered or scaled, the procedure should have been noted in the text.\n\nThe authors discuss some valid points regarding potential pitfalls associated with calibration and phoria. However, these points can be directly addressed by implementing a calibration procedure under monocular viewing3,4. A comparison between the results from thus calibrated and uncalibrated data, and a demonstration of test-retest reliability could have improved the paper.\n\nIn summary, the reproducibility of the paper’s findings may be challenged simply by the paucity of details in the methodological description. More importantly, however, from the information supplied or cited in the paper it is difficult to evaluate the validity of the potentially interesting conclusion that deficits in conjugacy of eye movements may quantitate physiologic impact of brain injury.", "appendix": "Competing interests\n\n\n\nThe author holds stock option in SyncThink, Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSamadani U, Ritlop R, Reyes M, et al.: Eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion. J Neurotrauma. 2015. PubMed Abstract \| Publisher Full Text\n\nLi Y, Bao FJ: Interocular symmetry analysis of bilateral eyes. J Med Eng Technol. 2014; 38(4): 179–187. PubMed Abstract \| Publisher Full Text\n\nKapoula Z, Bucci MP, Eggert T, et al.: Impairment of the binocular coordination of saccades in strabismus. Vision Res. 1997; 37(19): 2757–2766. PubMed Abstract \| Publisher Full Text\n\nBucci MP, Kapoula Z, Yang Q, et al.: Binocular coordination of saccades in children with strabismus before and after surgery. Invest Ophthalmol Vis Sci. 2002; 43(4): 1040–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7999", "date": "31 Mar 2015", "name": "Johannes van der Steen", "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\nThe author makes a justified appeal for clarification of methodological issues concerning the study “Eye tracking detects disconjugate eye movements associated with structural traumatic brain injury and concussion” by Samadani et al., 2015. The arguments raised by J. Maruta are very valid. Apart from clarification there is also a need for validation before this technique can be introduced in e.g. emergency rooms to quantify the severity of a concussive brain injury and the degree of recovery.", "responses": [] }, { "id": "8105", "date": "08 Apr 2015", "name": "Christopher Tyler", "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\nOverall, this Correspondence does not offer a significant criticism of the paper of Samadani et al. (2015). The author does not specify what kinds of asymmetries exist due to the camera/source geometry. Usually the subject is centered in the Eyelink viewing aperture, so the view of the eyes would be symmetric. The attempt to argue that a 1-2% asymmetry between the eyes would significantly degrade the assessment capability does not seem plausible.The criticism of methodological unclarity may be justified in itself, but it has no bearing on the outcome of the paper since all patients would be equally subject to the same degree by the effects of asymmetry and lack of calibration. As stated, none of the criticisms suggest a systematic bias between the different patient categories. The significant differences among categories cannot therefore be attributed to any of the factors raised by the author, and controlling these factors should only improve the significance of the Samadani et al. results. The author’s challenge to the findings of the paper is thus easily refuted.Moreover, despite the fact that this Correspondence is entirely concerned with calibration, it neglects to mention the fact that a core motivation for the Samadani et al. paper is the issue of a misleading spatial calibration metric. They are making the point that restrictions of eye movement may restrict the array of calibration positions, providing a false assessment that the subsequent test movements are normal. Their goal is thus to use the uncalibrated differences between the two eyes (which they term “temporal calibration”) as a probe for deficits that would be masked by the spatial calibration procedure. It is this strategy that the author should have addressed directly.On a stylistic note, I do not favor using the English possessive “s” on the Latin phrase “et al(ia)” and recommend the form above, as in “the Samadani et al. paper”.", "responses": [ { "c_id": "1289", "date": "13 Apr 2015", "name": "Jun Maruta", "role": "Author Response", "response": "I thank Dr. Tyler for his comments and the opportunity to think through what I had written again. One revision I can make in the second paragraph of the original text is to add “a side of” to “an infrared light source affixed to the camera” so that the phrase reads “an infrared light source affixed to a side of the camera.” This revision would make it clearer that the camera may be centered in front of the subject but the infrared light source cannot be centered simultaneously, the consequence of which is an asymmetric view of the corneal reflections. I can also specify that EyeLink 1000 uses a dark pupil-corneal reflection principle for tracking eye movements. Another clarification I can offer is to point out that the physical separation of the two eyes is individually variable [1,2]. This variability contributes to variations in the extent of asymmetry in the camera view of the landmark features of the eyes. With this consideration, Dr. Tyler’s assertion, that all subjects would be equally subject to the same degree by the effects of asymmetry, may be only partially correct. A systematic bias may indeed be created when the demographic composition of the subject groups are different. For example, having a larger male-to-female ratio in one group could increase the extent of binocular asymmetry in uncalibrated data since men tend to have a larger interpupillary distance [1,2]. Incidentally, Samadani et al. note a tendency toward the positive head CT group having more males than the non-injured control, with the positive head CT group of 13 patients being 35.9% female and the control group of 64 subjects being 47.9% female. (Curiously, the percentage of female subjects times the group size does not yield a whole number in any of the four subject groups in the Samadani et al. paper.) The assessment capability claimed by Samadani et al. appears to be variability of binocular alignment on the order of 0.01° magnitude. This value seems too small in light of the two sources of asymmetries discussed above in uncalibrated gaze-associated binocular measures, and also in light of 1-2% asymmetry between the two eyes. Regarding the problems that Samadani et al. point out in terms of spatial calibration, my intent is not to dispute the existence of alternatives to eye movement assessments based on spatial calibration. Rather, my emphasis is that disconjugacy itself is spatial in nature, and thus its quantification requires a solid spatial reference frame. This intent may be made clearer by including the term “spatial” in the title so that it reads “Ocular disconjugacy cannot be measured without establishing a solid spatial reference.” Finally, in my future writing I will keep in mind your recommendation regarding mixing the English possessive on the Latin phrase “et al.” References:1. Pryor HB: Objective measurement of interpupillary distance. Pediatrics. 1969; 44(6): 973-7.2. Gordon CC, Churchill T, Clauser CE, et al.: 1988 anthropometric survey of U.S. Army personnel: methods and summary statistics. U.S. Army Natick Research, Development and Engineering Center, Natick MA. 1989." } ] } ]	1	https://f1000research.com/articles/4-71
https://f1000research.com/articles/3-176/v1	30 Jul 14	{ "type": "Research Article", "title": "Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior", "authors": [ "Julien Colomb", "Björn Brembs", "Julien Colomb" ], "abstract": "We collected five sub-strains of the standard laboratory wild-type Drosophila melanogaster Canton Special (CS) and analyzed their walking behavior in Buridan's paradigm using the CeTrAn software. According to twelve different aspects of their behavior, the sub-strains fit into three groups. The group separation appeared not to be correlated with the origin of the stocks. We conclude that founder effects but not laboratory selection likely influenced the gene pool of the sub-strains. The flies’ stripe fixation was the parameter that varied most. Our results suggest that differences in the genome of laboratory stocks can render comparisons between nominally identical wild-type stocks meaningless. A single source for control strains may settle this problem.", "keywords": [ "Genetic background", "Buridan's paradigm", "walking behavior", "wild-type" ], "content": "Introduction\n\nIn our quest for understanding gene function, we commonly manipulate gene expression and compare the phenotypes of the manipulated versus control organisms. For technical reasons and to facilitate comparison as well as reproducibility between different experiments, a limited number of control strains have been established in most model organisms. For instance, the C57BL, 129 and FVB strains are commonly used in mouse studies; N2 is the common control strain used in Caenorhabditis elegans; and Canton-Special (CS) is one of the most-used wild-type strains in Drosophila melanogaster genetics studies. The CS stock was established by C. B. Bridges1 and chosen because of its low mutation rate. S. Benzer introduced CS to what was to become neurogenetics in his landmark study in 19672, because of its strong fast-phototaxis response. The strain has been used as a control in neurogenetics studies ever since.\n\nWith time and reproductive isolation, populations of laboratory control strains can diverge, in spite of ideal breeding conditions and seemingly little selective pressure. Several studies comparing the behavior of sub-strains of mice showed that their behavior differs3. For instance, Paylor and colleagues measured that one sub-strain of C57 mice showed a higher startle amplitude after tactile stimulation than another4. Similarly, the behavior of different N2 C. elegans sub-strains was found to vary to a considerable extent5.\n\nIn this study, we tested five different CS Drosophila melanogaster sub-strains in Buridan’s paradigm6–8, where flies walk between two stripes on a platform surrounded by a water moat. We could separate the CS sub-strains into three groups according to their behavior during the experiment. In addition, we found that the between-strain variability in the stripe fixation score is particularly high. We discuss possible solutions to prevent sub-strain related problems.\n\n\nMaterials and methods\n\nFlies were kept in vials (68 ml, Art.-Nr. 217101, Greiner Bio-One GmbH, Maybachstr.2, 72636 Frickenhausen) in a controlled density9 on standard cornmeal/molasses medium10 at 25°C in a 12 h:12 h dark/light cycle for one generation before being tested. Flies were collected 0–1 day after hatching and put in new food vials for one day. Approximately ten female flies (N=11-12 in each group) were then CO2-anaesthetized and their wings were cut with surgical scissors at two thirds of their length, before being taken back to their vial to recover overnight. They were then captured individually using a fly aspirator and put in the experimental setup to be tested.\n\nFive sub-strains of CS wild-type Drosophila melanogaster were collected in the lab from 2008 to 2011. Troy Zars took his CS_TZ stock to Columbia, MO, USA in 2002 when leaving Martin Heisenberg’s lab in Würzburg, Germany. It arrived in our laboratory in Berlin in 2008. The CS_TP stock was separated from Tim Tully’s strain in Waltham, MA, USA in 1992 and moved to Paris, France. The CS_JC stock was derived from the CS_TP stock in 2007 when one of the authors (JC) was in Thomas Préat’s lab. Both strains (TP and JC) arrived in Berlin in 2009. The CS_BS stock was separated by Bruno van Swinderen from Ralf Greenspan’s stock in San Diego, CA, USA in 1999 and brought to Brisbane, Australia. From there it arrived in Berlin in 2008. Finally, Henrike Scholz received her stock from Ulrike Heberlein in San Francisco, CA, USA. The CS_HS strain arrived in Berlin in 2007. Flies of all strains were kept at 18°C until being tested in 2012 and 2013.\n\nExperimental details are described in detail elsewhere7. Briefly, two black stripes producing 11° wide landmarks were positioned 293 mm from the center of a platform with a diameter of 117 mm, surrounded by water and illuminated with bright white light from behind. The centroid position of the fly was recorded via custom software (BuriTrack, http://buridan.sourceforge.net). If flies jumped from the platform, they were taken back to the platform with a brush and the tracker was reinitialized. Each data file represents five minutes of uninterrupted walking. We measured two replicates of the same five sub-strains in consecutive years, 2012 and 2013.\n\nFor more than three decades, experiments in Buridan’s paradigm demonstrated that wild-type flies typically walk back and forth between the landmarks. We performed 5 minute long walking experiments with five different wild type Canton S (CS) sub-strains: CS_TP, CS_TZ, CS_JC, CS_BS and CS_HS. The locomotion parameters we calculated can be divided into three broad categories: temporal (activity/pause structure), spatial (stripe fixation, thigmotaxis, trajectory straightness) and mixed (speed, number of walks between stripes, distance travelled) measures (Table 1)7.\n\nA more detailed description is available at http://dx.doi.org/10.6084/m9.figshare.844624.\n\nThe experiments in 2012 were done according to the previously published setup7, while the 2013 experiments were performed in four new setups. In the new setups, illumination is slightly brighter (10–11 klx in the new setup, 7.5–8.5 klx in the old setup). We did not detect any difference in the temperature on the platforms (27°C for all machines). The platform was cleaned between flies in the 2012 replicate, while the platform was rotated between two tests in the 2013 replicate, and cleaned only after a series of five flies had been tested.\n\nThe data was analyzed using CeTrAn v.4 (https://github.com/jcolomb/CeTrAn/releases/tag/v.4). Data with a mean distance travelled smaller than 50 mm/min was excluded to avoid outliers (2 data points were excluded in the second replicate, one for CS_JC and one for CS_HS).\n\nTwelve different parameters were calculated (Table 1) and a Principle Components Analysis (PCA) was performed to visualize the results and identify potential groupings of the sub-strains. The effects of genotype and replicate were analyzed with an ANOVA (in R) using the second principal component, since the first and the third components were not normally distributed (assessed with a Shapiro test). Transition plots and the stripe deviation plot have not been tested statistically.\n\nRaw trajectory data (including outliers) of the results of the CeTrAn analysis and the PCA result table are available on figshare: http://dx.doi.org/10.6084/m9.figshare.1014264.\n\n\nResults\n\nIn Buridan’s paradigm, wild-type flies typically walk back and forth between two inaccessible landmarks and their walking behavior is then analyzed. We performed 5 minutes long walking experiments with five different Canton S (CS) sub-strains: CS_TP, CS_TZ, CS_JC, CS_BS and CS_HS. We tested them in two replicates in two consecutive years using different hardware and under slightly varying experimental details (see Materials and Methods). The locomotion parameters that we calculated can be divided into three broad categories: temporal (activity/pause structure), spatial (stripe fixation, thigmotaxis, trajectory straightness) and mixed (speed, number of walks between stripes, distance travelled) measures (Table 17). Flies’ walking behavior was also visualized in transition plots, where the frequency of passage at each platform position is indicated by a heatmap. A distinction between sub-strains, which is consistent between the two replicates, can be seen in the visualization of this purely spatial parameter (Figure 1).\n\nTransition plots represent the position of the fly on the platform, excluding the time when the fly was immobile. The scale is proportional, with red points meaning that the number of times the fly was in that position is at least 95% of the maximal score obtained for any position. A Gaussian smooth was applied to the resulting heat map. The two points outside the platform were added manually to assure orthogonal axes of the representation. Sample size is 11-12 for each plot.\n\nUsing CeTrAn 4.0, we took twelve measurements of the flies’ walking behavior and analyzed them using PCA. For simplicity of representation, we plotted the mean and standard error of the three first principal components, while pooling the replicates (Figure 2). Since the first and third principle components were not normally distributed (Shapiro test), we performed an ANOVA for the second component with the fly sub-strains and the replicates as factors. This analysis demonstrated significant effects of the sub-strain (F value = 37.315 < 2e-16), the replicate (4.155 0.04374), and the interaction sub-strain × replicate (F value = 3.891 0.00527). A post hoc test of the sub-strain effect, together with a non-parametric test for the third component of the PCA, showed three groups: CS_TZ and CS_TP together in one group, CS_BS and CS_HS together and CS_JC alone (CS_TZ and CS_JC could be separated only on PC3).\n\nA PCA was performed over the 12 measured variables capturing the flies’ locomotion. The three first principal components are plotted against each other: from the center of the axes; PC1 to the left, PC2 up and PC3 down and to the right. Since units are arbitrary, they were not indicated. For each genotype, we represent the mean and standard error of the mean for the different PCs as a colored cross (data from the two replicates were pooled). The three groups are best visualized separately on the PC2-PC3 plot (upper-right). Sample size for each group is 23-24.\n\nStrikingly, the stripe fixation behavior covered the full range from strong fixation (10° average deviation from the stripe) to almost no fixation at all (30°: a random walk generates a 44° score,7) (Figure 3). We did not perform any statistical tests on this data, as they are already included in the PCA.\n\nFor every movement of the fly, the angle between its direction and the direction toward the stripes was calculated. The median of these angles was calculated for each fly, representing a quantification of stripe fixation by the fly. The value of each sub-strain in each session is depicted in boxplots: for each group, we represent the median, 25–75% quantiles and the total spread of the values (excluding outliers) as line, box and whiskers, respectively. The version of this figure on the F1000Research website is interactive, readers can define the type of whiskers displayed as either the 10th–90th percentiles (A) or Tukey whiskers (1.5 x IQR from 1st/3rd quartile; B). The text color code used for the genotypes is analogous to that used in Figure 2. The red horizontal line corresponds to the median value for random walks: 44°. Sample size is 11–12 for each boxplot. No statistical analysis was performed.\n\nIn order to estimate the variability range of the CS behavior on a larger scale, we have set up a trajectory database to receive data from CS flies in different laboratories, using similar machines and protocols. Figure 4 currently visualizes the result of a PCA analysis over our data and one additional data set, contributed by the Botella lab in Regensburg. However, in future versions of this article, data collected from other laboratories will be continuously fed into this database, with Figure 4 providing real-time visualizations of this incoming data. Four additional labs have already agreed to run these experiments using similar machines and protocols; researchers that are interested in contributing to this project can contact the corresponding author, who will provide them with further details about how to participate.\n\nThis CS_JB strain was ordered from the Bloomington stock center (stock #1) approx. seven years ago. CS_JB falls within the range of variability seen so far, but does not appear to clearly group with any of the previously measured strains. In future versions, this figure will be updated with Canton S data from other laboratories, tested according to the same parameters as described in the Materials and Methods section. Please email BB for instructions on how to submit. The final version of this figure will have final instructions for data submission.\n\n\nDiscussion\n\nBy analyzing the trajectories of five nominally identical CS sub-strains of Drosophila melanogaster in Buridan’s paradigm, we were able to distinguish three different groups of sub-strains. All strains were treated similarly in the same laboratory conditions for many generations (4 to 6 years) before being tested. There was no difference in rearing or experimental conditions between the different groups of flies. Therefore, it is a straightforward assumption that the differences in behavior we report here are genetic in origin. The sub-strain differences were comparable in the two replicates conducted one year apart, indicating that the genetic differences between the sub-strains were stable over this time span, although the CS_BS sub-strain appeared to have been modified during that time.\n\nFrom the twelve parameters of walking that we tested, stripe deviation showed the most striking variability. Stripe fixation likely depends on multiple parameters, such as the fly’s light/dark preference, their anxiety state, visual acuity, leg motor coordination or effects of wing clipping. It was used as a determining behavioral feature of Buridan’s paradigm11. Our results call for special care with the genetic background of the tested strains when analyzing this behavioral feature.\n\nThe numerically small but statistically significant difference between the two replicates (see raw data for individual variables) may be attributed to the differences in test setups and conditions. Since the behavior of the flies did not tend to converge (at least not over the one year time-frame we covered), the different strains apparently did not evolve particular traits to cope with our particular laboratory conditions. It is therefore plausible that such micro-evolution played little role in differentiating the sub-strains in the first place; the major cause for the difference between sub-strains might therefore be founder effects produced when a new fly stock is established, or population bottlenecks in the history of each strain. This hypothesis is also supported by the fact that common descent fails to explain the grouping we found in the PCA. In particular, two strains originating from the Paris lab (CS_TP and CS_JC) showed strikingly different locomotor behavior. This suggests that founder effects or bottlenecks were leading to dramatic alterations of behavior in Buridan’s paradigm. These results raise the question of which other phenotypes might be affected in the numerous CS sub-strains present in laboratories throughout the world.\n\nInterestingly, the use of a control line may lead to inaccurate interpretation of the data. For example, crammer mutant flies were reported to either show12 or not show13 an appetitive short term memory deficit with identical memory retention scores, because the scores of the control “CS” flies were different in the two studies. Our results further emphasize the need for a more systematic scheme addressing control populations. Existing genetic background differences may indeed explain discrepancies between results obtained in different laboratories, and that the use of the “CS” as a control strain is not enough to achieve comparability or reproducibility. A homogenization of the genetic backgrounds of ‘standard’ control strains would indeed be required.\n\nFortunately, our experiments suggest that the primary cause for differences in wild-type strains come from founder effects and not laboratory selection. One possible, but logistically challenging solution might be to have a common source for lines used for out-crossing events (including control lines), kept in massive, randomly interbreeding populations, for each lab to purchase at regular intervals. Large stock centers such as the Bloomington stock center would in principle be the candidate locations to implement such a solution. However, the phenotypes of mutations can vary depending on the genetic background within which the mutation is embedded14–16. The choice of one or multiple reference wild-type strain(s) is therefore not without implications for the future of the field and should be carefully investigated.\n\n\nData availability\n\nfigshare: Buridan raw data: Sub-strains of Drosophila Canton-S differ markedly in their locomotor behaviour, doi: 10.6084/m9.figshare.101426417", "appendix": "Author contributions\n\n\n\nJC and BB conceived the study, discussed the data and co-wrote the manuscript. JC performed the experiments and analyzed the data.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank Troy Zars, Thomas Préat, Henrike Scholz and Bruno van Swinderen for generously sharing their Canton S and many other strains with us over the years.\n\n\nReferences\n\nStern C, Schaeffer EW: On Primary Attributes of Alleles in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1943; 29(11): 351–61. PubMed Abstract \| Free Full Text\n\nBenzer S: Behavioral mutants of Drosophila isolated by countercurrent distribution. Proc Natl Acad Sci U S A. 1967; 58(3): 1112–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrawley JN, Belknap JK, Collins A, et al.: Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology (Berl). 1997; 132(2): 107–24. PubMed Abstract \| Publisher Full Text\n\nPaylor R, Crawley JN: Inbred strain differences in prepulse inhibition of the mouse startle response. Psychopharmacology (Berl). 1997; 132(2): 169–80. PubMed Abstract \| Publisher Full Text\n\nGems D, Riddle DL: Defining wild-type life span in Caenorhabditis elegans. J Gerontol A Biol Sci Med Sci. 2000; 55(5): B215–9. PubMed Abstract \| Publisher Full Text\n\nBuelthoff H, Gotz KG, Herre M: Recurrent inversion of visual orientation in the walking fly Drosophila melanogaster. Springer Berlin/Heidelberg. J Comp Physiol A. 1982; 148(4): 471–81. Publisher Full Text\n\nColomb J, Reiter L, Blaszkiewicz J, et al.: Open source tracking and analysis of adult Drosophila locomotion in Buridan’s paradigm with and without visual targets. PLoS One. 2012; 7(8): e42247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGötz KG: Visual guidance in Drosophila. Basic Life Sci. 1980; 16: 391–407. PubMed Abstract \| Publisher Full Text\n\nBrembs B: Operant learning of Drosophila at the torque meter. J vis Exp. 2008; 16. pii: 731. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo A, Liu L, Xia SZ, et al.: Conditioned visual flight orientation in Drosophila: dependence on age, practice and diet. Learn Mem. 1996; 3(1): 49–59. PubMed Abstract \| Publisher Full Text\n\nPhillips AM, Smart R, Strauss R, et al.: The Drosophila black enigma: the molecular and behavioural characterization of the black1 mutant allele. Gene. 2005; 351: 131–42. PubMed Abstract \| Publisher Full Text\n\nColomb J, Kaiser L, Chabaud MA, et al.: Parametric and genetic analysis of Drosophila appetitive long-term memory and sugar motivation. Genes Brain Behav. 2009; 8(4): 407–15. PubMed Abstract \| Publisher Full Text\n\nKrashes MJ, Waddell S: Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila. J Neurosci. 2008; 28(12): 3103–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIvanco TL, Greenough WT: Altered mossy fiber distributions in adult Fmr1 (FVB) knockout mice. Hippocampus. 2002; 12(1): 47–54. PubMed Abstract \| Publisher Full Text\n\nMineur YS, Sluyter F, de Wit S, et al.: Behavioral and neuroanatomical characterization of the Fmr1 knockout mouse. Hippocampus. 2002; 12(1): 39–46. PubMed Abstract \| Publisher Full Text\n\nLipp HP, Wolfer DP: Genetic background problems in the analysis of cognitive and neuronal changes in genetically modified mice. Clin Neurosci Res. 2003; 3(4–5): 223–31. Publisher Full Text\n\nColomb J, Brembs B: Buridan raw data: Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior. figshare. 2014. Data Source" }	[ { "id": "5630", "date": "08 Aug 2014", "name": "Gregg Roman", "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\nThis manuscript does an excellent job demonstrating significant strain differences in Burdian's paradigm. Since each Drosophila lab has their own wild type (usually Canton-S) isolate, this issue of strain differences is actually a very important one for between lab reproducibility. This work is a good reminder for all geneticists to pay attention to the population effects in the background controls, and presumably the mutant lines we are comparing. I was very pleased to see the within-isolate behavior was consistent in replicate experiments one year apart. The authors further argue that the between-isolate differences in behavior arise from a Founder's effect, at least in the differences in locomotor behavior between the Paris lines CS_TP and CS_JC. I believe this is a very reasonable and testable hypothesis. It predicts that genetic variability for these traits exist within the populations. It should now be possible to perform selection experiments from the original CS_TP population to replicate the founding event and estimate the heritability of these traits. Two other things that I liked about this manuscript are the ability to adjust parameters in figure 3, and our ability to download the raw data. After reading the manuscript, I was a little disappointed that the performance of the five strains in each 12 behavioral variables weren't broken down individually in a table or figure. I thought this may help us readers understand what the principle components were representing. The authors have made this data readily accessible in a downloadable spreadsheet.", "responses": [] }, { "id": "5635", "date": "15 Aug 2014", "name": "Josh Dubnau", "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\nThis is an interesting manuscript that I hope will be read widely. In this field, we give a lot of lip service to controlling genetic background because we all know it is important. But often, \"wild type\" lines such as CS are assumed to be equivalent and this turns out to be quite problematic conceptually. Now Colomb and Brembs have put some empirical evidence forward that confirms our fears about this practice. CS is a commonly used outbred strain, but my CS and your CS are different due to founder effects, drift, and selection. In this particular study, the founder effect has a massive impact on Buridan's paradigm, a relatively simple locomotor behavior. And this would likely be true for any quantitative trait. I have one comment on the conclusion; the effects seem to be mostly due to founder effects rather than selection (drift should be considered as well). This seems like a likely explanation in this current comparison, but this might change when we deal with mutants and transgenes maintained on an outbred strain. Such mutants or transgenes may cause accumulation of modifiers, which I would consider to be selection based.Regardless of the underlying mechanism, I like the fact that the authors of this study propose a solution, in which a stock center might maintain a large population of an outbred strain such as CS, and then we all would outcross our mutants and transgenes to that line. This could work, and offers the advantage that results from different groups would be comparable. Without such a mechanism, it is important, at a minimum, that all labs at least back cross all mutants/transgenes to their own CS or equivalent w.t. line. On the other hand, I would argue that genetic screens and collections of large populations of Gal4 and other such lines should be generated on an INBRED background which eliminates or reduces founder effects. This provides a more useful reagent for the community to use because it eliminates the onerous need to outcross every single line from a large collection.Overall, this is an important issue, and this study does a nice job of shedding light with actual data.", "responses": [] }, { "id": "5633", "date": "02 Sep 2014", "name": "Hiromu Tanimoto", "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\nColomb and Brembs reported an important piece of information that highlights large differences in the walking trajectory of six substrains of a wild-type Drosophila strain (Canton S) in the so-called Bridan paradigm. Drawing conclusions from behavioral comparisons of strains could therefore be moot, if the tested strains do not share the same genetic background (at least for the Buridan paradigm).This report is presented in a clear and succinct way. In addition, the authors invite submission of data by other labs, and the addition of these new data will be plotted in Fig. 4. This is an interesting endeavor which nicely uses the function of this journal. One has yet to keep in mind that fly behavior can be dramatically affected by ‘unwritten’ lab conditions (e.g. fly food, rearing conditions), and therefore the new data from other labs might not be comparable to the current dataset. One suggestion for the contributing labs to circumvent this caveat is to use one (or more) of the strains analyzed in this study and to check the reproducibility.In addition, I have a few minor comments that may be addressed:The authors argue that the basis of the behavioral variability is differences in the genetic background, but other reasons (e.g. epigenetic differences) can conceivably contribute to the variability as well. The authors state that there was a significant effect of the replicate on Principal Component 2 (page 3, left column, third line from bottom), but later state that “(…) sub-strain differences were comparable in the two replicates conducted one year apart” (page 5, left column, line 5 from top). Either of the statements should be amended. “A” in “PCA” stands for analysis. Use “PCA” instead of “PCA analysis”. My understanding of PCA is “Principal Component Analysis” rather than “Principle Component Analysis” the authors use in the paper. Use PC1, PC2, PC3 in the axes of Fig.2 and Fig. 4.", "responses": [] } ]	1	https://f1000research.com/articles/3-176
https://f1000research.com/articles/4-93/v1	20 Apr 15	{ "type": "Research Article", "title": "Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis", "authors": [ "Claire A. Sand", "Anna Starr", "Manasi Nandi", "Andrew D. Grant", "Claire A. Sand", "Anna Starr", "Manasi Nandi" ], "abstract": "Sepsis is a systemic inflammatory response triggered by microbial infection that can cause cardiovascular collapse, insufficient tissue perfusion and multi-organ failure. The cation channel transient receptor potential vanilloid 4 (TRPV4) is expressed in vascular endothelium and causes vasodilatation, but excessive TRPV4 activation leads to profound hypotension and circulatory collapse - key features of sepsis pathogenesis. We hypothesised that loss of TRPV4 signaling would protect against cardiovascular dysfunction in a mouse model of sepsis (endotoxaemia).Multi-parameter monitoring of conscious systemic haemodynamics (by radiotelemetry probe), mesenteric microvascular blood flow (laser speckle contrast imaging) and blood biochemistry (iSTAT blood gas analysis) was carried out in wild type (WT) and TRPV4 knockout (KO) mice. Endotoxaemia was induced by a single intravenous injection of lipopolysaccharide (LPS; 12.5 mg/kg) and systemic haemodynamics monitored for 24 h. Blood flow recording was then conducted under terminal anaesthesia after which blood was obtained for haematological/biochemical analysis. No significant differences were observed in baseline haemodynamics or mesenteric blood flow. Naïve TRPV4 KO mice were significantly acidotic relative to WT counterparts. Following induction of sepsis, all mice became significantly hypotensive, though there was no significant difference in the degree of hypotension between TRPV4 WT and KO mice. TRPV4 KO mice exhibited a higher sepsis severity score. While septic WT mice became significantly hypernatraemic relative to the naïve state, this was not observed in septic KO mice. Mesenteric blood flow was inhibited by topical application of the TRPV4 agonist GSK1016790A in naïve WT mice, but enhanced 24 h following LPS injection. Contrary to the initial hypothesis, loss of TRPV4 signaling (either through gene deletion or pharmacological antagonism) did not attenuate sepsis-induced cardiovascular dysfunction: in fact, pathology appeared to be modestly exaggerated in mice lacking TRPV4. Local targeting of TRPV4 signalling may be more beneficial than global inhibition in sepsis treatment.", "keywords": [ "TRPV4", "Sepsis", "Endotoxaemia", "Mouse model", "Vascular dysfunction", "Blood flow", "Haemodynamics" ], "content": "Introduction\n\nSepsis, the systemic inflammatory syndrome that can occur in response to infection, represents an enormous global healthcare burden; it can progress to septic shock, characterised by refractory hypotension and insufficient organ perfusion, with associated mortality rates of up to 80% (Martin, 2012). However, treatment options for sepsis are currently limited, highlighting the need to refine pre-clinical septic shock models to allow more effective identification of novel drug targets. There is now increasing evidence that several cation-permeable transient receptor potential (TRP) channels, most notably of the vanilloid (TRPV) subfamily, can influence physiological systems compromised in sepsis, and may represent potential therapeutic targets.\n\nTRPV4, originally identified as an osmosensor in the kidney (Liedtke et al., 2000; Strotmann et al., 2000; Wissenbach et al., 2000), is expressed in numerous tissues, including the heart and vasculature (Wissenbach et al., 2000). Its expression in both endothelial and smooth muscle cells is well-established, and its activation in vascular tissue is associated with endothelium-dependent vasodilatation, and both direct and indirect smooth muscle hyperpolarisation (Baylie & Brayden, 2011). In addition to extracellular hypotonicity, it is gated by various physical and chemical stimuli, including shear stress, arachidonic acid metabolites (particularly epoxyeicosatrienoic acids) and endocannabinoids (Nilius et al., 2004). These chemical factors may be increased under inflammatory conditions (Kiss et al., 2010; Theken et al., 2011; Varga et al., 1998). Furthermore, previous studies have identified sensitisation of TRPV4 without direct activation by other common inflammatory mediators (Alessandri-Haber et al., 2006). Recent reports have linked excessive TRPV4 activation to lethal endothelial failure, oedema formation, hypotension and circulatory collapse (Sonkusare et al., 2012; Willette et al., 2008), suggesting it may play a key role in sepsis pathogenesis. Given that numerous endogenous agonists and sensitisers of TRPV4 are upregulated in sepsis, we hypothesised that blockade of TRPV4 activity using a pharmacological antagonist HC-067047, or genetic deletion of the channel (in global knockout mice) would be protective in a murine model of sepsis, attenuating the cardiovascular collapse associated with the development of septic shock.\n\n\nMethods\n\nLipopolysaccharide (LPS) from Salmonella typhimurium and the TRPV4 agonist GSK1016790A were purchased from Sigma-Aldrich. The TRPV4 antagonist HC-067047 was purchased from Tocris Bioscience. All other materials were obtained from Sigma-Aldrich, unless otherwise stated.\n\nMale mice, either wild type (WT) or littermates lacking functional TRPV4 (KO), were bred in-house from heterozygous breeding pairs originally derived from a colony generated by Liedtke & Friedman (Liedtke & Friedman, 2003). Mice were maintained on a 12-h light/dark cycle (7am–7pm, and 7pm–7am, respectively) and given access to food (normal chow) and water ad libitum. All animal experiments were conducted under a UK Home Office licence (PPL 70/7049), following local ethics committee approval and in accordance with the Home Office Animal (Scientific Procedures) Act, 1986. Experiments were designed and conducted in a blinded manner and in accord with the ARRIVE guidelines (Kilkenny, 2010). All treatments were randomised. Mice were used between 10 and 14 weeks of age (approx. 25–30g). Individual mice were considered to be independent experimental units. A power calculation on pilot laser perfusion imaging data (α = 0.05; β = 0.2; minimum effect size = 20% alteration in blood flux value) indicated that 6 mice in each experimental group would provide sufficient power.\n\nLPS-induced endotoxaemia was used as a model for sepsis. Endotoxaemia was induced by intravenous injection of the bacterial endotoxin LPS (12.5 mg/kg). Injections of 100 µl (pre-warmed to 37°C) were administered into the tail vein (using a 29 G needle and insulin syringe) under inhaled isoflurane anaesthesia (2% by air pump). A heating mat set to 37°C was used to dilate the tail vein for improved accessibility. Buprenorphine hydrochloride [15 µg/kg, intramuscular (i.m.)] was administered into the two hind-limbs to provide post-operative analgesia and 0.9% (w/v) saline (20 ml/kg) was administered subcutaneously to provide fluid resuscitation. Mice were placed in a recovery chamber at 27°C for up to 24 h and were monitored frequently. An arbitrary severity score of 1–5 (based on mobility, facial expression, piloerection and aversion to touch) was employed to assess animal welfare. Any mouse reaching a score of 5 was immediately euthanised. All scoring was conducted by an experimenter blind to mouse genotype and treatment.\n\nA multi-parameter approach, encompassing haemodynamic, microcirculatory and haematological assessment, was used to determine the impact of TRPV4 blockade on endotoxaemia pathophysiology, as described previously (Sand et al., 2015). A timeline summarising the sequence of experimental procedures is provided in Figure 1.\n\nMice were implanted with telemetry probes on day 1, and were left to recover for 10 days. Basal haemodynamic data were then recorded over a 48-h weekend period (BL1). The following weekend, WT mice were injected with either vehicle (10% DMSO in saline) or TRPV4 antagonist HC-067047 (10 mg/kg, i.p.) and a second 48-h baseline recording (BL2) was taken for all mice. TRPV4 KO mice were left untreated. Injections in WT mice were performed in a randomised and blinded fashion. Following the second weekend baseline recording, WT mice received a second injection of either HC-067047 or vehicle (consistent with initial treatment) and LPS (12.5 mg/kg, i.v.) was administered to all mice. Haemodynamic data were recorded over 24-h of endotoxaemia progression. Mice were then terminally anaesthetised for blood flow recording and ex vivo analysis. A separate cohort of naïve mice were included in blood flow and ex vivo studies.\n\nPA-C10 telemetry probes (Data Sciences International; DSI, USA) were implanted into 10–12-week-old mice weighing 25–30g, under inhaled isoflurane anaesthesia using aseptic technique. Mice were placed in the supine position on a homeothermically-controlled heating mat (Harvard Instruments), and the thorax was shaved and disinfected with chlorhexidine. Buprenorphine hydrochloride (15 µg/kg, i.m.) was administered into the two hind-limbs to provide post-operative analgesia, and eyes were protected with Viscotears. A small thoracic incision was made and the carotid artery was isolated by gentle blunt dissection. Telemeter catheters were advanced into the left carotid artery and secured with three silk sutures. The body of the telemetry probe was placed in a subcutaneous pocket in the flank of the animal, and the incision was closed using a 5.0 Vicryl suture. Mice were then placed in a recovery cabinet at 27°C for 4 h, before being moved to a holding room, where they were allowed to recover for at least 7 days before the start of haemodynamic monitoring. The viability of each haemodynamic trace was checked, and mice with significant dampening or loss of signal were excluded from analysis (two in this study). All implantation, monitoring and data analysis were conducted by an experimenter blind to mouse genotype and treatment, and assignment of telemetry probes was randomised.\n\nFollowing recovery, baseline haemodynamic parameters were recorded over a 48-h weekend period. Food and water intake was measured continuously, and mice were closely monitored for adverse signs. After baseline recordings, the TRPV4 antagonist HC-067047 [10 mg/kg in 500 µl saline, intraperitoneal (i.p.)] or vehicle (10% dimethyl sulfoxide, DMSO, in 500 µl saline, i.p.) was administered in a blinded and randomised fashion to WT mice only. KO mice were left untreated. Baseline haemodynamic parameters were then recorded over a second 48-h weekend period. On the following Monday, TRPV4 WT mice were re-injected with either HC-067047 or vehicle (the same as the first treatment), and endotoxaemia was induced by intravenous administration of LPS. Haemodynamic parameters were recorded for a further 24 h, and mice were then terminally anaesthetised for blood flow assessment.\n\nPA-C10 telemetry probes allow continuous and remote monitoring of blood pressure, heart rate and locomotor activity in freely moving conscious animals. Data were acquired continuously at 500 Hz using standard acquisition software (DSI, USA). Baseline recording was carried out over weekends in order to minimise disruption.\n\nIn light of evidence suggesting that microvascular circulation is more prognostic in sepsis than global haemodynamics (De Backer et al., 2002; Sakr et al., 2004), we analysed mesenteric blood flow in TRPV4 WT and KO mice, both at baseline and during endotoxaemia. The mesenteric bed was chosen for assessment of microcirculatory function for its accessibility and its significant contribution to peripheral vascular resistance.\n\nMesenteric blood flow was measured in naïve and endotoxaemic mice under inhaled isoflurane anaesthesia (2% delivered by air pump), as described previously (Sand et al., 2015). Core temperature was recorded and controlled by a rectal probe coupled to a homeothermic heating mat (Harvard Instruments); mice were maintained at the temperature at which they initially presented. The abdominal region was shaved, and a small midline incision was made. A portion of the small intestine was gently exteriorised onto a Parafilm-coated heating mat, and was pinned out through the gut wall to expose the mesenteric vasculature. The exposed vascular bed was kept moist with saline (0.5 ml, aerosolised) pre-warmed to 37°C. An additional heating mat was placed over the anaesthetised animal.\n\nMesenteric blood flow was recorded using a moorFLPI full-field laser perfusion imaging system and review software (Moor Instruments, Devon, UK) either in naïve animals, or at 6 or 24 h after the induction of endotoxaemia. The following acquisition modes and settings were used: high resolution capture (25 frames, 1 s/frame); exposure 20 ms; automatic gain; flux palette set at 0–5000; background threshold 60 flux units. Vessels within the field of view were designated as 1st, 2nd or 3rd order branches of the mesenteric tree, and regions of interest, in which flux over time was measured, were defined in each visible vessel. All assessment and analysis was performed by an experimenter blind to mouse genotype and treatment. Baseline recordings were taken over 5 min, after which the mesenteric bed was gently sprayed (two pump compressions from a distance of approximately 10 cm) with selective TRPV4 agonist GSK1016790A (1 µM) dissolved in saline containing 1% DMSO, and pre-warmed to 37°C. Responses to GSK1016790A were measured over a further 5 min. Data were analysed in moorFLPI Review software (V 4.0; Moor Instruments, UK) and GraphPad Prism (V 5.0; GraphPad Software Inc, USA), and are expressed as mean area under the curve (AUC) over time ± SEM.\n\nFollowing blood flow recording, a venous blood sample was drawn from the inferior vena cava, and animals were killed by cervical dislocation. Blood biochemistry was assessed immediately from 100 µl venous blood using a hand-held iSTAT® point-of-care analyser (Abbott Laboratories, IL, USA), with CG8+ cartridges (Abbott Laboratories).\n\nOedema formation was measured by comparing wet and dry weights of lungs, hearts, upper liver lobes, right kidneys, 1 cm sections of small intestine and whole spleens. Tissues were excised immediately after blood flow recording and wet weights were recorded. Samples were then placed in an oven at 50°C until a constant dry weight was reached (approximately 3 days). The ratio of wet weight: dry weight was used as an indication of oedema formation.\n\nStatistical analysis was conducted in GraphPad Prism. Mean data for ‘n’ number of animals were compared by 1-way ANOVA with Bonferroni post-hoc test, unless stated otherwise. The threshold for statistical significance was p < 0.05.\n\n\nResults\n\nTRPV4 WT and KO mice were singly-housed from the time of radiotelemeter implantation throughout the duration of the study. Food and water intake were monitored throughout. Recovery from telemetry surgery (measured as recovery in body weight, food and water intake and physical examination) was equivalent across all animals. All mice returned to their pre-surgery weight and food water intake within 7 days of implantation. Following recovery, TRPV4 KO mice were found to consume less water than WT counterparts, though this difference was not statistically significant (Figure 2a). Systemic administration of either HC-067047 or its vehicle to WT mice reduced water intake, though the magnitude of change was significantly greater in HC067047-treated animals (Figure 2b). Water intake was reduced in all animals over the 24 h following the induction of endotoxaemia (Figure 2c). No difference in food intake was observed between different genotypes basally (Figure 2d). Systemic administration of either vehicle or HC-067047 reduced food intake, though to a similar degree across treatment groups (Figure 2e). Induction of sepsis with LPS [12.5 mg/kg, intravenous (i.v.)] markedly reduced food intake to a similar degree in all groups (Figure 2f).\n\n(a) Mean basal water intake in TRPV4 WT and KO mice over approximately 1 week (not including 7-day recovery period from telemetry surgery). (b) Change in water intake over 24-h period following i.p. administration of either vehicle (10% DMSO) or HC-067047. (c) Water intake over 24-h period following administration of LPS (12.5 mg/kg i.v.). (d) Mean basal food intake in TRPV4 WT and KO mice over approximately 1 week (not including 7-day recovery period from telemetry surgery). (e) Change in food intake over 24-h period following i.p. administration of either vehicle (10% DMSO) or HC-067047. (f) Food intake over 24-h period following administration of LPS (12.5 mg/kg i.v.). Data for individual animals are represented as individual symbols, with group mean denoted by horizontal line. p<0.05, relative to vehicle-treated controls, 1-way ANOVA + Bonferroni post-hoc test, n = 5–12.\n\nUnder basal conditions, all animals exhibited diurnal variation in blood pressure, heart rate and ambulatory activity (Figure 3), within the expected physiological range for mice (Curtis et al., 2007). No significant difference in basal haemodynamic parameters was observed between genotypes, although TRPV4 KO mice exhibited a trend towards lower blood pressure (both daytime and night-time) and lower daytime ambulatory activity than WT counterparts [average mean arterial pressure (MAP) over 24 h: KO = 93.71 ± 2.83 mmHg vs. WT = 99.89 ± 1.87 mmHg, ns; mean daytime activity over 12 h: KO = 3.25 ± 0.43 counts/min vs. WT = 4.31 ± 0.33 counts/min, ns (n = 5–10)] (Figure 3a,c,d,f).\n\n(a) Mean arterial pressure, (b) heart rate, (c) systolic pressure, (d) diastolic pressure, (e) pulse pressure and (f) locomotor activity at baseline (BL1), following systemic treatment (WT mice only) with HC-067047 (HC) or vehicle (10% DMSO) (BL2) and after induction of endotoxaemia (at time 48 h: LPS, 12.5 mg/kg, i.v.). Boxed regions denote periods of darkness. Data were acquired by radiotelemetry in conscious, ambulatory male C57BL/6 mice, and are presented as mean ± SEM, n = 4–6.\n\nSystemic administration of the TRPV4 antagonist HC-067047 caused an increase in mean daytime blood pressure of approximately 5 mmHg, though this was reversed after 12 h, leading to small but consistent decrease in night-time pressure (Table 1). Systemic administration of vehicle (10% DMSO in saline) also caused an increase in blood pressure, though this was more transient (Table 1 & Figure 3). Neither administration of HC-067047 nor of vehicle significantly altered any other haemodynamic parameters under basal conditions, though heart rate was transiently increased relative to time-matched pre-treatment values in both groups, and subsequently declined during night-time (Table 1). In both cases (daytime tachycardia and night-time bradycardia) the magnitude of the change was greater in HC-067047-treated mice than in vehicle-treated controls, though this was not statistically significant. Mice treated with HC-067047 did exhibit significantly greater daytime ambulatory activity, relative to time-matched pre-treatment values (Table 1); this change was not observed in vehicle-treated animals, and was no longer evident during night-time hours.\n\nData are presented as mean difference (95% CI), and are divided into daytime and night-time phases. BL1 represents mean baseline values in untreated mice over 48-h weekend recording. BL2 represents mean baseline values following i.p. treatment (WT mice only) with vehicle (10% DMSO) or HC-067047; BL2 in KO mice represents a separate untreated 48-h baseline recording. LPS represents mean values following systemic treatment with LPS (12.5 mg/kg, i.v.) over 24-h recording period. p<0.05, p<0.01, p<0.001, 1-way ANOVA + Bonferroni post-hoc test.\n\nSince TRPV4 KO mice were not treated with pharmacological stimuli, baseline parameters were simply re-recorded in the same mice. Values were relatively consistent across separate baseline recording periods (Table 1), confirming the robustness of the monitoring system.\n\nSystemic administration of LPS caused a significant decline in blood pressure in all animals (Figure 3a,c,d & Table 1), that began to stabilise after approximately 12 h. No significant difference between genotypes was observed, though both TRPV4 KOs and antagonist-treated mice exhibited a trend towards more severe hypotension, particularly in systolic pressure (Figure 3a,c and Table 1). Pulse pressure was compromised in TRPV4 KO and HC-067047-treated mice, relative to vehicle-treated controls (Figure 3e and Table 1).\n\nAn immediate increase in heart rate was observed in both HC-067047- and vehicle-treated WT animals following the induction of endotoxaemia, and this tachycardia was sustained during daylight hours. TRPV4 KO mice exhibited a transient increase in heart rate immediately after LPS administration, but became bradycardic thereafter, indicating failure of the baroreceptor reflex (Figure 3b and Table 1). All mice exhibited marked bradycardia during night-time hours, relative to time-matched pre-LPS values, though the magnitude of the decrease was greatest in TRPV4 KO mice (Table 1). Ambulatory activity was almost entirely abolished in all mice following the induction of endotoxaemia (Figure 3f).\n\nAll mice telemetered for haemodynamic recording were subjected to mesenteric blood flow analysis. A group of naïve animals were included for comparison, and additional vehicle- and HC-067047-treated WT mice were used to increase statistical power.\n\nMesenteric blood flow was significantly reduced in all groups 24 h after the induction of endotoxaemia (Figure 4a–c). No significant differences were observed between genotypes or treatment groups, though endotoxaemic mice pre-treated with HC-067047 exhibited a trend towards improved flow. In order to determine whether blockade of TRPV4 may play a role earlier in pathogenesis, mesenteric flow was recorded in a separate cohort of WT mice treated for 6 h with HC-067047 and LPS. At this time-point, blood flow in HC-067047-treated mice was decreased relative to vehicle-treated controls, though these changes were not statistically significant (Figure 4e–f).\n\n(a–c) Mesenteric blood flow in TRPV4 KO mice and WT mice treated i.p. with vehicle (10% DMSO) or HC-067047 (HC) for 24 h, either under naïve or endotoxaemic (LPS 12.5 mg/kg, i.v., 24 h) conditions. Data are expressed as total area under the curve (AUC; ×103 flux units.time) over 5-min baseline recording in 1st, 2nd and 3rd order vessels, respectively. p<0.01, *p<0.001, relative to naïve counterparts, 1-way ANOVA + Bonferroni post-hoc test. (d–f) Mesenteric blood flow in WT mice treated i.p. with vehicle (10% DMSO) or HC-067047, either under naïve (24-h treatment) or endotoxaemic conditions (6 h or 24 h; drugs administered simultaneously with LPS) in 1st, 2nd and 3rd order vessels, respectively. AUC for each animal is represented as an individual symbol, with horizontal line denoting group mean, n = 6–9.\n\nAntagonism or gene ablation of TRPV4 had only minor effects on clinical phenotype, though in opposing directions. In line with marginally improved mesenteric perfusion, antagonist-treated mice exhibited a lower sepsis severity score after 24 h treatment with LPS, relative to vehicle-treated controls. On the other hand, the severity score in TRPV4 KO mice was significantly greater than in antagonist-treated counterparts (Figure 5a). The percentage weight loss over 24 h of endotoxaemia was roughly equivalent between groups, but was lower in antagonist-treated and TRPV4 KO mice, than in vehicle-treated controls (Figure 5b). Core temperature decreased significantly in all LPS-treated mice, though remained stable in all animals throughout the recording period (Figure 5c). Although no statistically significant differences in core temperature were observed between groups, there were trends towards attenuated hypothermia in HC-067047-treated mice, and exaggerated hypothermia in TRPV4 KO mice, relative to vehicle-treated counterparts.\n\n(a) Arbitrary severity score assigned in a blinded fashion after 24-h treatment with LPS (12.5 mg/kg, i.v.) based on voluntary mobility, gait, aversion to touch, facial expression and piloerection. WT mice were treated i.p. with vehicle (10% DMSO) or HC-067047 (HC) at the time of LPS administration. TRPV4 KO mice did not receive any pharmacological treatment. (b) Percentage weight loss over 24-h endotoxaemic period. Values for each animal are presented as individual symbols, with horizontal line denoting group mean. #p<0.05, relative to HC-treated animals, 1-way ANOVA + Bonferroni post-hoc test (n = 8–9). (c) Core temperature measured by rectal probe throughout blood flow recording period. Data are presented as mean ± SEM (n = 8–9).\n\nPharmacological TRPV4 antagonism and gene ablation also appeared to have divergent effects on blood biochemistry (Table 2). Although the concentration of urea was elevated in all groups 24 h post-LPS, the increase in TRPV4 KO mice was greatest, whereas that in antagonist-treated mice did not reach statistical significance. Blood pH was also lowest in LPS-treated TRPV4 KO mice, whereas acidosis was attenuated in antagonist-treated mice, relative to vehicle-treated controls. PCO2 was increased by LPS treatment, and bicarbonate decreased, indicative of respiratory and metabolic acidosis, respectively, though no significant differences were observed across treatment groups. All mice became significantly hypoglycaemic following the induction of endotoxaemia. While both vehicle- and antagonist-treated animals became significantly hypernatraemic and hyperchloraemic over the course of endotoxaemia, consistent with dehydration and loss of bicarbonate respectively, no change in plasma Na+ or Cl- levels was observed in TRPV4 KO mice. Correspondingly, bicarbonate levels decreased in both vehicle- and antagonist-treated mice, but not in TRPV4 KO mice. Both haemoglobin and haematocrit decreased slightly with endotoxaemia, though to an equivalent extent across groups.\n\nInterestingly, naïve TRPV4 KO mice also exhibited trends towards pathological blood biochemistry. Blood pH was significantly decreased in these mice, and they also showed a trend to elevated blood urea concentration, relative to vehicle-treated WT controls. Basal haemoglobin and haematocrit levels were also decreased in naïve TRPV4 KO mice, relative to WT controls, indicative of basal anaemia (Table 2).\n\nBlood gas and biochemistry were measured from venous blood samples by iSTAT point-of-care analyser in either TRPV4 KO mice, or WT mice treated i.p. with vehicle (10% DMSO) or HC-067047 (HC) for 24 h, either under naïve or endotoxaemic (LPS 12.5 mg/kg, i.v., 24 h) conditions. Data are presented as mean ± SEM. p<0.05, p<0.01, p<0.001, relative to naïve controls; #p<0.05, relative to vehicle-treated controls, 1-way ANOVA + Bonferroni post-hoc test (n = 6–9).\n\nAbbreviations: PCO2, partial pressure of CO2; TCO2, total carbon dioxide; PCV, packed cell volume; HC, HC-067047; KO, TRPV4 knockout; LPS, lipopolysaccharide\n\nThe effect of pharmacological TRPV4 antagonism was also assessed at an earlier time-point in pathogenesis. In contrast with the 24 h time-point, no significant difference in severity score, percentage weight loss, or core temperature was observed between vehicle- and antagonist-treated mice at 6 h post-LPS (data not shown). Blood biochemistry was similarly equivalent between treatment groups at the 6 h time-point (Table 3).\n\nBlood gas and biochemistry were measured from venous blood samples by iSTAT point-of-care analyser in WT mice treated i.p. with vehicle (10% DMSO) or HC-067047 (HC), either under naïve (24-h treatment) or endotoxaemic (LPS 12.5 mg/kg, i.v., 6 h or 24 h) conditions. Data are presented as mean ± SEM. p<0.05, p<0.01, p<0.001, relative to naïve controls, 1-way ANOVA + Bonferroni post-hoc test (n = 8–9).\n\nAbbreviations: PCO2, partial pressure of CO2; TCO2, total carbon dioxide; PCV, packed cell volume; HC, HC-067047; KO, TRPV4 knockout; LPS, lipopolysaccharide\n\nOedema formation was measured by comparing wet and dry weights of various tissues. Surprisingly, treatment with LPS for 24 h did not cause any appreciable oedema formation in WT mice. Endotoxaemic TRPV4 KO mice exhibited marginally elevated oedema formation in the liver and spleen, and significantly increased oedema in the kidney. No difference was observed between vehicle and antagonist-treated mice (Figure 6).\n\nOedema formation was measured by comparing wet and dry weights of (a) lungs, (b) hearts, (c) upper liver lobes, (d) right kidneys, (e) 1-cm sections of small intestine and (f) whole spleens. WT mice were treated i.p. with vehicle (10% DMSO) or HC-067047 (HC) for 24 h, either under naïve or endotoxaemic (LPS 12.5 mg/kg, i.v., 24 h) conditions. Values for each animal are presented as individual symbols, with horizontal line denoting group mean. p<0.001, relative to naïve controls, 1-way ANOVA + Bonferroni post-hoc test (n = 6–9).\n\nThe effect of TRPV4 antagonism on oedema formation was also assessed at an earlier time-point in pathogenesis. In contrast with the 24-h time-point, significant liver oedema was observed in both vehicle- and HC-067047-treated mice after 6 h, though this had returned to baseline levels by 24 h post-LPS (Figure 7). No significant difference in oedema formation was observed in any other tissues, however. No changes in the ratio of heart weight to body weight were observed following the induction of endotoxaemia in any of the animal groups.\n\nOedema formation in WT mice treated with TRPV4 antagonist or vehicle. Oedema formation was measured by comparing wet and dry weights of (a) lungs, (b) upper liver lobes, (c) hearts, (d) right kidneys, (e) 1-cm sections of small intestine and (f) whole spleens. WT mice were treated i.p. with vehicle (10% DMSO) or HC-067047 (HC) at the time of LPS (12.5 mg/kg, i.v., 6 or 24 h). Values for each animal are presented as individual symbols, with horizontal line denoting group mean. p<0.01, **p<0.001, relative to naïve controls, 1-way ANOVA + Bonferroni post-hoc test (n = 6–9).\n\nIn order to assess changes in TRPV4 activity in vivo after the induction of endotoxaemia, 1 µM GSK1016790A and vehicle (2% DMSO in pre-warmed saline) were sequentially applied to the exposed mesenteric bed by aerosolised spray. No significant responses to GSK1016790A were observed in the mesenteric beds of naive mice: both vehicle and GSK1016790A caused a non-significant decrease in blood flow, likely to reflect evaporative cooling (Figure 8a–c and Table 4). In LPS-treated mice, on the other hand, GSK1016790A caused an increase in blood flow (Figure 8d–f) that was significant in 2nd and 3rd order mesenteric vessels (Table 4).\n\nBaseline mesenteric blood flow was recorded for 5 min in naïve (a–c) and endotoxaemic (d–f) mice, 24 h after injections of LPS. Vehicle (V; 2% DMSO in saline) was then administered as an aerosolised spray, followed by administration of GSK1016790A (G; 1 µM) 5 min later. Blood flow was recorded in (a & d) 1st order, (b & e) 2nd order and (c & f) 3rd order mesenteric vessels. Data are presented as mean ± SEM. p<0.05, area under the curve relative to baseline, 1-way ANOVA + Bonferroni post-hoc test (n = 5).\n\nBaselines (BL), and responses to aerosolised vehicle (Veh; 10% DMSO in pre-warmed saline) and GSK1016790A (GSK; 1 µM) were recorded over sequential 5-min periods. Data are presented as mean percentage change in area under the curve over 5-min recording period. p<0.05, *p<0.01, relative to vehicle response in the same mouse, paired 2-tailed Student’s T-test; #p<0.05 relative to corresponding response in naïve controls, 1-way ANOVA + Bonferroni post-hoc test (n = 5).\n\n\nDiscussion\n\nBased on observations that excessive TRPV4 activation can cause profound hypotension, endothelial failure and circulatory collapse (Willette et al., 2008), we hypothesised that enhanced TRPV4 activity may contribute to haemodynamic and vascular dysfunction during sepsis. Numerous endogenous agonists and regulators of TRPV4, including anandamide (Varga et al., 1998), arachidonic acid and its metabolites (Bruegel et al., 2012), protein kinase A (Yang et al., 1997), shear stress and temperature have been shown to be upregulated or altered in sepsis. It is conceivable therefore, that inflammation-induced upregulation of endogenous factors could contribute to sepsis-associated circulatory failure via excessive TRPV4 activation. By extension, blockade of TRPV4 activity may be expected to attenuate the circulatory dysfunction that underlies sepsis pathogenesis. To test this hypothesis we used both TRPV4 KO mice and the selective TRPV4 antagonist, HC-067047.\n\nConsistent with previous reports (Nishijima et al., 2014; Zhang et al., 2009), TRPV4 KO mice exhibited lower basal arterial pressure than WT counterparts during both daytime and night-time periods. This is surprising given that TRPV4 has been implicated in both nitric oxide- (NO) and endothelium-derived hyperpolarising factor (EDHF)-mediated vasodilatory responses to physical and chemical stimuli in both conduit and resistance arteries (Adapala et al., 2011; Hartmannsgruber et al., 2007; Köhler et al., 2006; Loot et al., 2008; Ma et al., 2013; Mendoza et al., 2010; Rath et al., 2012 Sonkusare et al., 2012; Zhang et al., 2009). A possible explanation is that loss of TRPV4 leads to compensatory upregulation of alternative vasodilatory pathways during development. Although studies of isolated vessels from TRPV4 KO mice have shown impaired vasodilatation in response to a range of physiological stimuli (Earley et al., 2009; Zhang et al., 2009), the effects of isolation and ex vivo culture may have altered the mechanisms regulating vascular tone. Alternatively, it is possible that loss of TRPV4 expression in vascular smooth muscle may represent loss of a vasoconstrictor mechanism. While numerous studies have suggested that TRPV4 in vascular smooth muscle couples with hyperpolarising K+ channels to induce vasodilatation (Earley et al., 2005; Earley et al., 2009), activation of TRPV4 simultaneously across multiple cells (as opposed to very localised activation) may trigger Ca2+-dependent vasoconstriction to maintain a basal level of vascular tone. Indeed, TRPV4-mediated vasoconstriction has previously been demonstrated in the lung, where TRPV4 gene ablation suppresses the development of chronic hypoxic pulmonary hypertension (Xia et al., 2013).\n\nGiven that TRPV4 exhibits tonic activity both in heterologous expression systems (Strotmann et al., 2000) and in primary endothelial cells (Sonkusare et al., 2012), its blockade may be expected to cause acute hypertension. While systemic administration of HC-067047 did induce a transient increase in blood pressure under basal conditions, a similar effect was observed in vehicle-treated animals, suggesting that the transient hypertension may have been a stress response to manual handling. In accordance with these data, previous studies have shown that administration of a different TRPV4 antagonist – GSK 2193874 – does not significantly alter blood pressure after either acute intravenous administration or repeated oral gavage (Thorneloe et al., 2012).\n\nThe lack of any dramatic effect on blood pressure is consistent with a previous ex vivo study showing that blockade of the TRPV4-cytochrome P450 epoxygenase axis only inhibits flow-induced vasodilatation in the presence of nitric oxide synthase (NOS) and prostacyclin (PGI2) antagonists (Loot et al., 2008). In other words, blockade of one endothelium-dependent vasodilatory pathway can lead to an acute increase in activity of a compensatory pathway. Elevated activity in an alternative (and perhaps more potent) vasodilatory pathway following loss of TRPV4 activity may also explain the subsequent night-time decreases in blood pressure following HC-067047 administration, which were not evident in vehicle-treated mice (as well as the trend towards hypotension in TRPV4 KO mice). A compensatory increase in, for example, NOS activity and PGI2 production could also account for the night-time decreases in heart rate following TRPV4 antagonism, since both are known to produce negative chronotropy (Chiba & Malik, 1980; Feron et al., 1998). Further experiments are necessary to determine whether the activity of other vasorelaxant pathways is altered in TRPV4 knockout animals.\n\nContrary to expectations, neither TRPV4 gene deletion nor pharmacological antagonism attenuated LPS-induced haemodynamic dysfunction. In fact, both HC-067047-treated and TRPV4 KO mice exhibited a trend towards exaggerated hypotension that was most evident in measurements of systolic pressure and pulse pressure. The exaggerated systolic hypotension and greater decrease in pulse pressure observed in both antagonist-treated and KO mice may be indicative of reduced cardiac output and left ventricular dysfunction, though these parameters were not measured in this study. Reduced water intake in these animals prior to the induction of sepsis may have contributed to hypovolaemia, decreasing cardiac output. Alternatively, it is possible that elevated vascular NO production (in response to loss of other TRPV4-dependent vasodilator mechanisms) could have increased aortic compliance in these animals, leading to greater systolic hypotension and a narrower pulse pressure.\n\nOverall, the haemodynamic consequences of endotoxaemia appeared to be worse in mice lacking TRPV4: hypotension was slightly exaggerated, and the baroreceptor reflex (compensatory tachycardia in response to a fall in blood pressure) was almost entirely absent in TRPV4 KO mice. Given that NO is thought to antagonise the positive chronotropic effects of increased sympathetic drive (Feron et al., 1998), it is possible that excessive NO production in TRPV4 KO mice could account for the bradycardia following LPS administration.\n\nLow mesenteric flow is known to correlate strongly with multiple organ failure and mortality, both in animal models (Baykal et al., 2000) and in human patients (Takala, 1996). Clinically, gut ischaemia is known as the ‘motor of multiple organ failure’ (Carrico et al., 1986), probably because impaired mesenteric blood flow is associated with intestinal hyperpermeability. This breakdown in barrier function facilitates the leakage of endotoxins and microorganisms into the lymphatic and cardiovascular circulation, which can exacerbate the inflammatory response (Sautner et al., 1998). Furthermore, gastric perfusion is known to correlate well with sublingual perfusion (Marik, 2001) – a robust indicator of outcome in patients (De Backer et al., 2010). Despite numerous reports of an important role for TRPV4 in mediating agonist- and flow-induced vasoactive responses in small resistance vessels (Adapala et al., 2011; Earley et al., 2009; Köhler et al., 2006; Ma et al., 2013; Mendoza et al., 2010; Sonkusare et al., 2012; Zhang et al., 2009), we did not observe any marked changes in mesenteric blood flow in antagonist-treated or TRPV4 KO mice. There was, however, a trend towards improved flow in 1st and 2nd order vessels in endotoxaemic HC-067047-treated mice. Of note, perhaps, is the observation that antagonist-treated mice exhibited a trend towards exaggerated hypotension following LPS administration, but marginally better mesenteric perfusion; these results are consistent with the notion that stabilisation of arterial pressure during sepsis may occur at the expense of microcirculatory flow (Sand et al., 2015).\n\nThe exact half-life of HC-067047 has not been reported, though plasma levels are known to exceed the IC50 for some hours after i.p. administration (Everaerts et al., 2010), so we also investigated the effect of TRPV4 antagonism on mesenteric blood flow at an earlier time-point. At 6 h post-LPS, there was a trend towards lower blood flow in antagonist-treated mice, consistent with a vasodilatory role for TRPV4 in the mesenteric vasculature, though this was not statistically significant.\n\nWhile TRPV4 is known to dilate small resistance vessels (such that its blockade may be expected to decrease mesenteric blood flow) its activation has also been associated with increased endothelial permeability and oedema formation, which would impair perfusion (Willette et al., 2008). These divergent effects on microvascular blood flow may account for the reverse in trends between 6 h and 24 h treatment with LPS. Nonetheless, our data do not support a prominent role for TRPV4 in regulating mesenteric blood flow in this model, either under basal or endotoxaemic conditions. It is likely that activity in compensatory pathways is increased both acutely and chronically to compensate for its loss.\n\nIn contrast to numerous studies demonstrating TRPV4-mediated dilatation of mesenteric arteries (Adapala et al., 2011; Earley et al., 2009; Köhler et al., 2006; Ma et al., 2013; Mendoza et al., 2010; Sonkusare et al., 2012; Zhang et al., 2009) topical application of GSK1016790A did not induce any marked changes in blood flow in naïve mice. Despite the relatively high concentration used (1 µM), it is possible that owing to the aerosolised delivery method (which may be prone to evaporation) insufficient concentrations of the agonist contacted vascular receptors. While TRPV4 is expressed in vascular smooth muscle (Earley et al., 2005), the compound may not have reached the luminal side of the vessel to induce endothelium-dependent vasodilatation. Additional experiments using higher concentrations or a different delivery method are required to elucidate this further. The observation that GSK1016790A caused an increase in blood flow in LPS-treated mice may indicate inflammatory TRPV4 sensitisation, with small amounts of luminal GSK1016790A now able to induce vasodilatation, in line with our overall hypothesis.\n\nInterestingly, naive TRPV4 KO mice exhibited signs of pathology. Blood pH was significantly lower in these animals compared with WT counterparts; base excess was correspondingly reduced, and partial pressure of CO2 elevated, indicative of respiratory acidosis. These data are consistent with a potential acid-sensing role for TRPV4 reported previously (Suzuki et al., 2003). The observation that blood pH was also slightly reduced in naive HC-067047-treated mice relative to vehicle-treated controls, suggests that TRPV4 may be involved in maintaining acid-base homeostasis, though the mechanism by which this occurs is unclear.\n\nFollowing the induction of endotoxaemia, TRPV4 KO mice exhibited a more dramatic increase in plasma urea concentration than WT mice, consistent with a role for TRPV4 in regulating renal function (Liedtke & Friedman, 2003). Although not measured in this study, it is possible that loss of TRPV4 activity may result in impaired renal circulation. TRPV4 KO mice also exhibited the lowest blood pH of all groups during sepsis, but this was not significantly different from the mildly acidotic basal pH in these animals. Both antagonist- and vehicle-treated WT mice, in contrast, exhibited a significant decline in pH between basal and septic conditions, though the magnitude of the decrease was less pronounced in HC-067047-treated animals. This suggests that blockade of TRPV4 activity may attenuate the development of metabolic acidosis. Consistent with this notion, base excess was slightly lower and bicarbonate levels slightly higher in both antagonist-treated and TRPV4 KO mice, relative to vehicle-treated controls.\n\nBoth vehicle- and antagonist-treated WT animals became severely hyperchloraemic over the course of sepsis. Fluid loss, and perhaps the administration of saline resuscitation at the time of sepsis, could have contributed to this finding. Hyperchloraemic metabolic acidosis (in which serum Cl- levels are elevated, bicarbonate reduced and blood pH is low) is also indicative of kidney dysfunction (Walker, 1990). Surprisingly, given the elevated plasma urea levels, TRPV4 KO mice did not exhibit LPS-induced hyperchloraemia or hypernatraemia.\n\nFollowing reports linking excessive TRPV4 activation to endothelial failure and oedema formation (Alvarez et al., 2006; Willette et al., 2008) we aimed to establish the role of TRPV4 in oedema formation during endotoxaemia – a condition characterised by increased endothelial permeability (Bridges & Dukes, 2005). No marked changes in the ratio of tissue wet-to-dry weight were observed in WT mice following 24 h treatment with LPS; only the lung and the kidney exhibited trends towards increased plasma extravasation, and values were not different between vehicle- and antagonist-treated mice. Although these data indicate that oedema is not present at this time-point in endotoxaemia pathogenesis, it is possible that the method used is insufficiently sensitive to detect small changes in plasma extravasation and interstitial fluid volume. We did observe evidence of oedema formation in the kidneys of LPS-treated TRPV4 KO mice (there were also trends towards increased wet-to-dry weight ratios in the livers and spleens of these animals). This was surprising given that these mice did not exhibit an altered ionic balance, and furthermore, because activation of TRPV4 has been associated with increased vascular permeability (Alvarez et al., 2006; Willette et al., 2008). Nonetheless, it appears that deletion of the TRPV4 gene is associated with slightly exaggerated oedema formation in the kidney, liver and spleen following LPS administration, though it is not clear what mechanism may be involved.\n\nThe dose of HC-067047 used in this study (10 mg/kg) was chosen based on the only two previous reports of its administration in vivo (Everaerts et al., 2010; Materazzi et al., 2012). While the effects of systemic TRPV4 antagonism were not dramatic, use of higher concentrations was not possible: intraperitoneal injection of 100 mg/kg HC-067047 has been shown to cause obvious adverse effects, including hunching and piloerection (Everaerts et al., 2010), and higher concentrations of DMSO could potentially be toxic. Repeated dosing may represent a more viable option for blood flow recording or biochemical analysis, though repeated manual handling and intraperitoneal injections would certainly preclude meaningful haemodynamic recording. Supplementation of drinking water with orally available TRPV4 antagonists (Thorneloe et al., 2012) would be required to further elucidate the effects of TRPV4 blockade on cardiovascular function in endotoxaemia or sepsis.\n\nNonetheless, plasma levels of the compound have been shown to remain above the IC50 for more than 2 h after intraperitoneal administration of 10 mg/kg HC-067047 (Everaerts et al., 2010), suggesting that any prominent role for TRPV4 in sepsis-associated cardiovascular dysfunction would at least have been evident in the early phase of sepsis. The fact that no major changes in haemodynamics, mesenteric blood flow, blood biochemistry or tissue oedema were observed at 6 h post-LPS, suggests either that TRPV4 does not play a major role in cardiovascular regulation during endotoxaemia, or that other pathways can be upregulated acutely to compensate for its loss. However, further studies in which research conditions are optimised may yet uncover evidence of a vasoregulatory role for TRPV4 in sepsis.\n\nOverall, our data do not support a major role for TRPV4 in sepsis-associated cardiovascular dysfunction. It is possible that upregulation of alternative vasodilatory pathways may compensate for its loss both acutely and chronically, and that changes would only be evident on blockade of, for example, NO- and PGI2-dependent pathways. Alternatively, since plasma osmolality is known to increase in sepsis (Jochberger et al., 2009), and hypertonicity inhibits TRPV4 activity (Strotmann et al., 2000), it is possible that under septic conditions, TRPV4 activity is inhibited such that gene deletion or pharmacological antagonism will produce no further effect. Regardless of underlying mechanisms, based on data obtained in this study, TRPV4 does not appear to be a viable target in the treatment of sepsis-associated cardiovascular dysfunction.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw Data for Figure 2, 10.5256/F1000Research.6298.d45231 (Sand et al., 2015a).\n\nF1000Research: Dataset 2. Raw Data for Figure 3, 10.5256/F1000Research.6298.d45232 (Sand et al., 2015b).\n\nF1000Research: Dataset 3. Raw Data for Figure 4, 10.5256/F1000Research.6298.d45233 (Sand et al., 2015c).\n\nF1000Research: Dataset 4. Raw Data for Figure 5, 10.5256/F1000Research.6298.d45234 (Sand et al., 2015d).\n\nF1000Research: Dataset 5. Raw Data for Table 2, 10.5256/F1000Research.6298.d45237 (Sand et al., 2015e).\n\nF1000Research: Dataset 6. Raw data for Table 3, 10.5256/F1000Research.6298.d45238 (Sand et al., 2015f).\n\nF1000Research: Dataset 7. Raw data for Figure 6, 10.5256/F1000Research.6298.d45235 (Sand et al., 2015g).\n\nF1000Research: Dataset 8. Raw Data for Figure 7, 10.5256/F1000Research.6298.d45236 (Sand et al., 2015h).", "appendix": "Author contributions\n\n\n\nCAS, MN and ADG planned and designed the study. CAS, AS and ADG acquired the data. CAS, MN and ADG analysed and interpreted the data. CAS, AS, MN and ADG prepared the manuscript. All authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the British Pharmacological Society (IPF Pump Priming award 13-14) and the British Heart Foundation (grant FS/10/51/28677).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAdapala RK, Talasila PK, Bratz IN, et al.: PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells. Am J Physiol Heart Circ Physiol. 2011; 301(3): H757–H765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlessandri-Haber N, Dina OA, Joseph EK, et al.: A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. J Neurosci. 2006; 26(14): 3864–3874. PubMed Abstract \| Publisher Full Text\n\nAlvarez DF, King JA, Weber D, et al.: Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res. 2006; 99(9): 988–995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaykal A, Kavuklu B, Iskit AB, et al.: Experimental study of the effect of nitric oxide inhibition on mesenteric blood flow and interleukin-10 levels with a lipopolysaccharide challenge. World J Surg. 2000; 24(9): 1116–1120. PubMed Abstract \| Publisher Full Text\n\nBaylie RL, Brayden JE: TRPV channels and vascular function. Acta Physiol (Oxf). 2011; 203(1): 99–116. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiedtke W, Friedman JM: Abnormal osmotic regulation in trpv4 -/- mice. Proc Natl Acad Sci U S A. 2003; 100(23): 13698–13703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoot AE, Popp R, Fisslthaler B, et al.: Role of cytochrome P450-dependent transient receptor potential V4 activation in flow-induced vasodilatation. Cardiovasc Res. 2008; 80(3): 445–452. PubMed Abstract \| Publisher Full Text\n\nMa X, Du J, Zhang P, et al.: Functional role of TRPV4-KCa2.3 signaling in vascular endothelial cells in normal and streptozotocin-induced diabetic rats. Hypertension. 2013; 62(1): 134–139. PubMed Abstract \| Publisher Full Text\n\nMarik PE: Sublingual capnography: a clinical validation study. Chest. 2001; 120(3): 923–927. PubMed Abstract \| Publisher Full Text\n\nMartin GS: Sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. Expert Rev Anti Infect Ther. 2012; 10(6): 701–706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaterazzi S, Fusi C, Benemei S, et al.: TRPA1 and TRPV4 mediate paclitaxel-induced peripheral neuropathy in mice via a glutathione-sensitive mechanism. Pflugers Arch. 2012; 463(4): 561–569. PubMed Abstract \| Publisher Full Text\n\nMendoza SA, Fang J, Gutterman DD, et al.: TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress. Am J Physiol Heart Circ Physiol. 2010; 298(2): H466–H476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNilius B, Vriens J, Prenen J, et al.: TRPV4 calcium entry channel: a paradigm for gating diversity. Am J Physiol Cell Physiol. 2004; 286(2): C195–C205. PubMed Abstract \| Publisher Full Text\n\nNishijimam Y, Zheng X, Lund H, et al.: Characterization of blood pressure and endothelial function in TRPV4-deficient mice with l-NAME- and angiotensin II-induced hypertension. Physiol Rep. 2014; 2(1): e00199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRath G, Saliez J, Behets G, et al.: Vascular hypoxic preconditioning relies on TRPV4-dependent calcium influx and proper intercellular gap junctions communication. Arterioscler Thromb Vasc Biol. 2012; 32(9): 2241–2249. PubMed Abstract \| Publisher Full Text\n\nSakr Y, Dubois MJ, De Backer D, et al.: Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock. Crit Care Med. 2004; 32(9): 1825–1831. PubMed Abstract\n\nSand CA, Starr A, Wilder CDE, et al.: Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis. J Appl Physiol (1985) 2015; 118(3): 344–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSand CA, Starr A, Nandi M, et al.: Dataset 1 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015a. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 2 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015b. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 3 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015c. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 4 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015d. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 5 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015e. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 6 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015f. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 7 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015g. Data Source\n\nSand CA, Starr A, Nandi M, et al.: Dataset 8 in: Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis. F1000Research. 2015h. Data Source\n\nSautner T, Wessely C, Riegler M, et al.: Early effects of catecholamine therapy on mucosal integrity, intestinal blood flow, and oxygen metabolism in porcine endotoxin shock. Ann Surg. 1998; 228(2): 239–248. PubMed Abstract \| Free Full Text\n\nSonkusare SK, Bonev AD, Ledoux J, et al.: Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function. Science. 2012; 336(6081): 597–601. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStrotmann R, Harteneck C, Nunnenmacher K, et al.: OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol. 2000; 2(10): 695–702. PubMed Abstract \| Publisher Full Text\n\nSuzuki M, Mizuno A, Kodaira K, et al.: Impaired pressure sensation in mice lacking TRPV4. J Biol Chem. 2003; 278(25): 22664–22668. PubMed Abstract \| Publisher Full Text\n\nTakala J: Determinants of splanchnic blood flow. Br J Anaesth. 1996; 77(1): 50–58. PubMed Abstract\n\nTheken KN, Deng Y, Kannon MA, et al.: Activation of the acute inflammatory response alters cytochrome P450 expression and eicosanoid metabolism. Drug Metab Dispos. 2011; 39(1): 22–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorneloe KS, Cheung M, Bao W, et al.: An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure. Sci Transl Med. 2012; 4(159): 159ra148. PubMed Abstract \| Publisher Full Text\n\nVarga K, Wagner JA, Bridgen DT, et al.: Platelet- and macrophage-derived endogenous cannabinoids are involved in endotoxin-induced hypotension. FASEB J. 1998; 12(11): 1035–1044. PubMed Abstract\n\nWalker HK, Hall WD, Hurst JW: Clinical Methods: The History, Physical, and Laboratory Examinations. Boston. 3rd ed., 1990. PubMed Abstract\n\nWillette RN, Bao W, Nerurkar S, et al.: Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2. J Pharmacol Exp Ther. 2008; 326(2): 443–452. PubMed Abstract \| Publisher Full Text\n\nWissenbach U, Bödding M, Freichel M, et al.: Trp12, a novel Trp related protein from kidney. FEBS Lett. 2000; 485(2–3): 127–134. PubMed Abstract \| Publisher Full Text\n\nXia Y, Fu Z, Hu J, et al.: TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension. Am J Physiol Cell Physiol. 2013; 305(7): C704–715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang SL, Hsu C, Lue SI, et al.: Protein kinase a activity is increased in rat heart during late hypodynamic phase of sepsis. Shock. 1997; 8(1): 68–72. PubMed Abstract\n\nZhang DX, Mendoza SA, Bubolz AH, et al.: Transient receptor potential vanilloid type 4-deficient mice exhibit impaired endothelium-dependent relaxation induced by acetylcholine in vitro and in vivo. Hypertension. 2009; 53(3): 532–538. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8381", "date": "13 May 2015", "name": "Scott Earley", "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\nSand et al. examinesd the hypothesis that activity of the Ca2+-permeable the transient receptor potential (TRP) channel TRPV4 contributes to vascular dysfunction in an animal model of sepsis. The study utilized selective pharmacology and TRPV4 knockout animals to determine if loss of TRPV4 expression would be protective against sepsis induced by injection of lipopolysaccharide (LPS). Contrary to expectations, loss or blockade of TRPV4 had very little effect on LPS-induced hypotension. Similarly, reduction in mesenteric blood flow following LPS administration did not differ between treatment groups. LPS (24 hours) did not induce significant edema formation in control mice, but did cause edema in the kidneys of TRPV4 knockout mice. Edema was present in some tissues at an earlier (6 hours) time point but this response was not affected by TRPV4 block. Administration of the TRPV4 agonist GSK1016790A caused an increase in mesenteric blood flow in 2nd and 3rd order mesenteric arteries in LPS but not control animals. This study is clearly written and the work was well designed and properly executed. The guiding hypothesis - block of TRPV4 channels would diminish vascular effects of sepsis - was based on an important study that showed circulatory collapses following systemic administration of a TRPV4 activating drug. This idea is entirely logical and of tremendous interest. Indeed, TRPV4 block appears to improve pulmonary edema associated with heart failure in rodents (Thorneloe et al., 2012). However, the current findings do not indicate that block of TRPV4 is beneficial during sepsis. These disparate observations may reflect important differences between the pulmonary and systemic endothelium.", "responses": [] }, { "id": "8382", "date": "14 May 2015", "name": "David X. Zhang", "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\nSand et al. examined the hypothesis that loss of TRPV4 function prevents cardiovascular dysfunction in sepsis. Using TRPV4-knockout mice and animals treated with TRPV4 inhibitor (HC-067047), the authors did not find a protective role of TRPV4 deletion/blockade in the model of LPS-induced endotoxaemia. Although the results are not supportive of the original hypothesis, the study is well-designed and provides further insight into the role of TRPV4 in vivo in the regulation of cardiovascular function.Consistent with previous reports, TRPV4 KO animals (a different colony generated by Liedtke & Friedman) exhibited lower basal arterial pressure than WT controls during both daytime and night-time periods while acute administration of TRPV4 antagonist HC-067047 did not significantly alter blood pressure as compared to vehicle. The minor impact of TRPV4 activity on the regulation of blood pressure is in contrast to the important role of this channel in vasodilatory response to physical and chemical stimuli in conduit and resistance arteries. The authors proposed that this discrepancy could be due to the activation of compensatory pathways in the absence of TRPV4 function. It also remains to be explored whether TRPV4 function in the vascular tissue could be counteracted by its activity in other tissues such as those involving in baroreceptor reflex or in long-term sodium/water balance by using tissue-specific KO.The endotoxaemic mice used in this study showed only small decrease in blood pressure over baseline, e.g. an average reduction in MAP of 7.6 mmHg during daytime and 12.9 mmHg at night-time in WT. The animals also exhibited reduced mesenteric blood flow and lower core body temperature. Together, these signs seem to indicate a hypodynamic state of sepsis, and the obtained results might underestimate or overlook hemodynamic effects of TRPV4 activation occurred during an early hyperdynamic stage of sepsis. This potential limitation could be discussed in the manuscript.It was also noted that endotoxaemic animals have very low ambulatory activity throughout 24-h period after LPS and a marked reduction in blood glucose (by as much as 10-fold over baseline). Are these alterations also found in other similar septic murine models? A reduced ambulatory activity at night-time could contribute to observed greater reduction in blood pressure during this period after LPS challenge.Other pointsTable 1, column 1. It seems that the comparison should be “BL2-BL1”, “LPS-BL1”, and “LPS-BL2”. Figures 4 and 8, mesenteric blood flow. A large variation in AUC values is noted between the two figures.", "responses": [] } ]	1	https://f1000research.com/articles/4-93
https://f1000research.com/articles/4-75/v1	23 Mar 15	{ "type": "Method Article", "title": "Follow-up: Prospective compound design using the ‘SAR Matrix’ method and matrix-derived conditional probabilities of activity", "authors": [ "Disha Gupta-Ostermann", "Yoichiro Hirose", "Takenao Odagami", "Hiroyuki Kouji", "Jürgen Bajorath", "Disha Gupta-Ostermann", "Yoichiro Hirose", "Takenao Odagami", "Hiroyuki Kouji" ], "abstract": "In a previous Method Article, we have presented the ‘Structure-Activity Relationship (SAR) Matrix’ (SARM) approach. The SARM methodology is designed to systematically extract structurally related compound series from screening or chemical optimization data and organize these series and associated SAR information in matrices reminiscent of R-group tables. SARM calculations also yield many virtual candidate compounds that form a “chemical space envelope” around related series. To further extend the SARM approach, different methods are developed to predict the activity of virtual compounds. In this follow-up contribution, we describe an activity prediction method that derives conditional probabilities of activity from SARMs and report representative results of first prospective applications of this approach.", "keywords": [ "compound design", "SAR matrix", "structure activity" ], "content": "Introduction\n\nIn recent years, graphical methods have substantially expanded the medicinal chemistry repertoire for analyzing Structure-Activity Relationships (SARs)1,2. The development of computational techniques to visualize SAR patterns and identify key compounds has in part been catalyzed by increasing volumes and complexity of activity data in medicinal chemistry. Going beyond a purely descriptive nature of graphical SAR exploration, as exemplified by activity landscape representations1, the SAR Matrix (SARM) approach3 was conceptualized to combine large-scale graphical SAR analysis and compound design. SARM calculations generate many virtual compounds (VCs) that populate chemical space around structurally related series. In order to prioritize virtual candidate compounds from SARMs in a target/assay-specific manner, activity prediction methods have been developed including local Quantitative SAR (QSAR) models utilizing compound neighborhood information in SARMs4 and an approach that derives conditional probabilities of activity from SARMs5.\n\nIn a previous Method Article6, the SARM methodology and extensions have been described including matrix-based QSAR4 and navigation of multi-target activity spaces7. In this follow-up contribution, we focus on a conditional probability-based approach to activity prediction, which is distinct from QSAR analysis, and report results of first prospective applications. While we are currently unable to disclose the structures of active compounds (due to patent issues of PRISM Biolab Corporation), the prediction statistics and exemplary results we report for an actual drug discovery project should be helpful to put SARM-based predictions into perspective, beyond computational benchmarking, and might spark the interest of practitioners in this field.\n\n\nMethods\n\nSince details of the SARM methodology and matrix-based QSAR modeling have been presented in the accompanying article6, we initially provide only brief summaries of these methods, followed by a detailed description of the conditional probability approach.\n\nTo generate SARMs compounds are subjected to a systematic two-step fragmentation procedure yielding matched molecular pairs (MMPs)8. An MMP is defined as a pair of compounds that only differ at a single site. In the first step, compounds are fragmented into core structures and substituents. In the second step, resulting core structures are subjected to fragmentation. This two-step fragmentation protocol identifies series of compounds with related core structures (forming “core MMPs”). Series of compounds with cores forming MMP relationships are organized in individual SARMs, as illustrated in Figure 1. Each matrix cell defines a unique combination of a core and substituent (reminiscent of yet distinct from R-group tables). Following MMP terminology, the core is called key fragment and the substituent value fragment8. Each filled cell represents an actual compound color-coded by activity or potency and each empty cell a VC representing a previously unexplored core-substituent (key-value) combination. Accordingly, VCs are thought to generate a “chemical space envelope” around structurally related compound series. Depending on the structural relationships that are present within a given compound set, varying numbers of SARMs are obtained that systematically organize available analog series and provide many VCs for further consideration. The more similar data set compounds are to each other, the more SARMs are typically obtained.\n\nA schematic representation of a SARM is shown. Compound fragmentation (indicated by thick lines in matrix cells) yields three analog series with structurally related cores (keys). Each series consists of analogs that share a core and differ by a single substitutent (value, blue). Structural differences between the cores of the three series are highlighted in red. Each SARM combines all analog series with structurally related cores available in a compound set. Rows and columns represent compounds sharing the same core and substituent, respectively. In each cell, the combination of a core and a substituent defines a unique molecular structure. Compounds present in the data set are represented by filled cells that are color-coded according to activity. In addition, empty cells represent virtual compounds (i.e., previously unexplored key-value combinations resulting from MMP fragmentation).\n\nA compound neighborhood (NBH) approach was developed for potency prediction of VCs based on known potencies of structural analogs4, as illustrated in Figure 2. A qualifying NBH consists of two known active compounds that contain the key and value fragment of a given VC, respectively (D and G in Figure 2a), and a third active compound (E) that consists of the key of D and value of G. The potency of a VC can then be predicted from its neighbors by applying the additivity assumption underlying Free-Wilson analysis9 using the equation shown in Figure 2a. For a given VC, all qualifying NBHs are identified across all SARMs, as illustrated in Figure 2b, and for each NBH, an individual potency prediction is carried out using a local “mini-QSAR” model. The average potency over all NBHs is then calculated to yield the final prediction.\n\n(a) A NBH of virtual compound X is marked in blue in a model SARM and compounds forming this NBH are displayed. Compounds D and G share the same substituents and core with X, respectively, and the third neighbor E consists of the core of D and substituent of G. At the lower left, the equation to predict the potency of X from the values of D, E, and G is shown. (b) The process of NBH mining is illustrated. For X, the set of all qualifying NBHs (marked in blue) in a given SARM are identified and potency values are predicted for individual NBHs (indicated by color-coded squares). “act” stands for activity (in this case, numerical potency values are used).\n\nThe NBH approach is based upon numerical values and thus well suited for potency prediction during compound optimization considering multiple analog series. Principal limitations of QSAR modeling also apply to the NBH methodology, given its Free-Wilson foundation. Hence, meaningful potency predictions can only be expected in the presence of SAR continuity (when small structural changes are accompanied by gradual changes in potency). By contrast, SARMs capturing discontinuous SARs or activity cliffs10 fall outside the QSAR applicability domain. Because potency predictions are carried out over multiple NBHs in different SARMs, standard deviations of predictions provide a simple yet effective indicator of prediction reliability. High and low standard deviations indicate the presence of SAR discontinuity and continuity, respectively, for compound subsets involved in the predictions. When standard deviations are low, accurate SARM-based potency predictions can be expected4.\n\nA conceptually different approach was developed for hit expansion from screening data based upon conditional probabilities of activity derived from SARMs, as outlined in Figure 3. In contrast to NBH-based prediction of numerical potency values, the conditional probability method can utilize approximate potency measurements (e.g., primary screening data) leading to a binary classification of inactive vs. active data set compounds and ensuing prediction of a probability of activity for VCs.\n\nSteps and equations required to derive probabilities of activity from SARMs for prediction of virtual compound X are shown using a model SARM with nine compounds (A-I) that contain three cores (c1-c3) and four values (v1-v4). Matrix cells are color-coded according to compound activity (red, inactive; green, active). In the first step, initial class probabilities are calculated for all cores and values using equation 2 and 1, respectively. Value- and core-weighted matrices are then derived via equations 4 and 3. The class contribution of core c3 is obtained from the value-weighted matrix using equation 5. Analogously, the class contribution of value v1 is obtained from the core-weighted matrix using equation 6. The value and core class contributions are then normalized using equations 7 and 8. Finally, the activity probability px of 0.13 is obtained for X by combining the normalized core c3 and value v1 class probabilities using equation 9.\n\nThe conceptual basis of the approach is provided by the following ideas: based on the observed frequency of occurrence of given core and value fragments in active versus inactive compounds (in the following referred to as the active versus inactive class), probabilities of activity and inactivity can be derived for cores and values. Importantly, the contributions of cores and values are thought to be influenced by each other because compounds are represented in SARMs as combinations of individual core and value fragments. Considering the conditional nature of core and value contributions to activity, initial probabilities are weighted to derive class probabilities for any core and value. For a given VC, probabilities of its core and value are then combined to yield a final probability of activity.\n\nKey steps of the methodology are summarized in Figure 3 (and for each step, the respective equation is provided). To illustrate the approach in an intuitive manner, we will go through an exemplary probability calculation for a given VC, guided by Figure 3.\n\nThe SARM in Figure 3 contains nine compounds (A-I) that comprise three cores (c1-c3) and four values (v1-v4). The probability of activity will be predicted for virtual compound X that shares core c3 with compounds G, H, and I and value v1 with compounds A and D.\n\nGiven the distribution of individual values v and cores c in active and inactive compounds, probabilities of activity and inactivity are calculated using equation 1 and equation 2. In case of value v1, both class probabilities are equal (i.e., 1/2) because v1 is contained in one active and one inactive compound. By contrast, the probability of inactivity is two times higher for core c3 than its probability of activity (2/3 vs. 1/3).\n\nThese initial estimates are further refined by taking information from all SARM compounds into account. The underlying idea is to statistically assess if a core or value contributes more to activity or inactivity. Therefore, in the next step, core probabilities using value-weighted matrices and value probabilities using core-weighted matrices are derived. The value-weighted matrix results from the assignment of a weight to each compound using equation 3 and the core-weighted matrix is obtained using equation 4. For example, the class probability of core c3 is updated by considering information from values v2, v3, and v4 in compound G, H, and I, respectively. The inverse of the value class probability yields a weight. All compounds containing value v2 are active (2/2); hence, the core class probabilities of compounds B and G are assigned a weight of 1.0 (through value-weighting). For value v3, the compounds show equal class frequency of (in)activity (1/2); thus, both active and inactive compounds are assigned the same weight of 2.0. Finally, two of three compounds containing value v4 are inactive. Accordingly, inactive compound I receives a lower weight of 1.5 indicating that its inactivity is more likely due to v4. It follows that with increasing frequency of inactivity for a given value, core weights of inactive compounds decrease (and vice versa), indicating that the value is likely to be responsible for inactivity. Analogous considerations apply to assess probabilities of activity.\n\nWeighted core class contributions are calculated with equation 5. For core c3, contributions of 0.34 and 1.12 to activity and inactivity are obtained, respectively, using a smoothing factor of α=0.1 (this factor is applied to prevent zero probabilities when no compound is available to represent a possible core or value class):\n\n\n\n\n\nThrough normalization using equation 7 core class probabilities between 0 and 1 are obtained; for c3 values of 0.23 (activity) and 0.76 (inactivity).\n\nAnalogously, value class probabilities are refined using the core-weighted matrix (generated using equation 4). For example, class probabilities of value v1 are adjusted by considering information from cores c1 and c2 in compounds A and D that contain v1. Compound A is active and belongs to the majority class of c1 and is thus assigned a lower weight than D, which is inactive and belongs to the minority class of c2. The higher weight assigned to compound D means that its inactivity is statistically more likely to result from value v1 than core c2. Weighted value class contributions calculated using equation 6 give activity and inactivity contributions of 0.72 and 1.40, respectively, for value v1 (applying a smoothing factor of α=0.1):\n\n\n\n\n\nNormalization using equation 8 then yields updated v1 class probabilities of 0.34 (activity) and 0.66 (inactivity).\n\nFinally, the normalized core and value probabilities are combined via equation 9 yielding an activity probability px (ranging from 0 to 1) for any core-value combination representing a VC. Increasing px values indicate an increasing probability of activity. For classification, a threshold value of activity must be set (e.g., 0.5). In our example, the normalized core and value class probabilities for c3 and v1 result in an activity probability px of 0.13 for virtual compound X representing this core-value combination. Thus, given the low probability of activity, this VC is predicted to be inactive. In benchmark calculations on sets of known active and inactive compounds, conditional probability calculations yielded reasonably accurate predictions of activity, at least comparable to current state-of the-art machine learning approaches5.\n\nBecause the conditional probability approach is statistically grounded, prediction accuracy is expected to increase with sample sizes and matrix density6. Therefore, it makes sense to exclude SARMs from the calculations that contain only a small number of data set compounds or have limited row overlap (accounting for shared substitution patterns among structurally related series)6. Accordingly, SARMs with more than 50% row overlap are typically considered informative and prioritized for probability calculations.\n\nDifferent from the NBH approach, the conditional probability method is generally applicable and not confined to compound subsets representing continuous SARs. Thus, QSAR applicability domain restrictions do not apply in this case.\n\n\nApplication\n\nThe conditional probability method has been used for activity predictions (hit expansion) starting from the results of a screen of the PRISM library of alpha helical turn mimetics11,12 carried out in search of new inhibitors of the Wnt/β-catenin protein-protein interaction and pathway13,14. The Wnt pathway is implicated in a variety of disease states including several forms of cancer. Consequently, inhibitors of the Wnt/β-catenin interaction are thought to have high therapeutic potential13,14. PRISM’s current helix mimetics library contains more than 10,000 small molecules with closely related scaffolds11,12 suitable for SARM analysis. The library screen was carried out using a luciferase reporter gene assay of the Wnt pathway15,16 and the stably transfected cell line Hek-293, STF1.111. Figure 4 summarizes SARM analysis of the library and activity predictions. The library contained a total of 10,540 compounds that yielded 11,033 stereochemistry-sensitive SARMs (i.e., matrices explicitly accounting for all stereoisomers) with a total of 231,143 VCs. This matrix distribution was solely determined by structural relationships between library compounds.\n\nSAR matrix statistics for a library of alpha helical turn mimetics are provided and activity predictions for virtual compounds are summarized. For these predictions, conditional probabilities of activity were derived from library screening data.\n\nScreening of the library in the reporter gene assay yielded 64 active and 10,476 inactive compounds (applying a threshold of less than 50% residual luciferase activity). Hence, only a limited number of compounds were classified as active applying this threshold. Active and inactive compounds were then mapped to SARMs and a subset of 1,049 informative matrices (with at least 50% row overlap) was selected that contained 10,504 VCs. Probability calculations predicted 28 VCs to be active. Twenty candidates were synthesized, re-screened, and tested in confirmatory assays, leading to the identification of five novel hits with activities in the low-micromolar range.\n\n\nConcluding remarks\n\nIn this contribution, we have discussed methodological advances for activity prediction on the basis of SARMs, which systematically account for structural/analog relationships in compound sets of any source, organize structurally related compound series, and yield virtual candidate compounds. In combination with the SAR matrix method, compound neighborhood analysis based upon Free-Wilson principles and derivation of conditional probabilities of activity are applicable to predict novel active compounds at different stages of chemical optimization efforts. The conditional probability approach detailed herein is particularly suitable for hit expansion and can be applied to raw screening data. Going beyond benchmark calculations, first prospective applications have yielded promising results. For example, screening data of the PRISM library of helix mimetics made it possible to prioritize a small number of candidate compounds for synthesis from a pool of ~10,000 pre-selected VCs on the basis of only 64 preliminary screening hits. These predictions ultimately resulted in the identification of five new active compounds by considering only 20 candidates. These compounds provide new starting points for chemical optimization efforts. Of course, further prospective validation studies will need to be performed to better understand the performance of SARM-based activity predictions for different compound classes, targets, and screening assays. However, considering the well-defined scaffold-substituent patterns of compounds representing alpha helical turn mimetics and the systematic design of the library, which plays into the strength of the SARM approach, successful activity predictions are also anticipated for library screens using assay systems and targets engaged in other therapeutically relevant protein-protein interactions.", "appendix": "Author contributions\n\n\n\nJB conceived the study and DGO carried out SARM analyses and activity predictions. YO and TO synthesized candidate compounds and collected assays data. YO, TO, and HK analyzed the experimental data. JB wrote the manuscript, JB and DGO designed and generated display items, and all authors examined the manuscript and agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors thank Dr. Anne Mai Wassermann, Dr. Dilyana Dimova, Dr. Preeti Iyer, and Jenny Balfer for valuable contributions to the development of the SARM approach and activity prediction methods. DGO gratefully acknowledges support of doctoral studies from Boehringer Ingelheim.\n\n\nReferences\n\nWassermann AM, Wawer M, Bajorath J: Activity landscape representations for structure-activity relationship analysis. J Med Chem. 2010; 53(23): 8209–8223. PubMed Abstract \| Publisher Full Text\n\nStumpfe D, Bajorath J: Methods for SAR visualization. RSC Adv. 2012; 2(2): 369–378. Publisher Full Text\n\nWassermann AM, Haebel P, Weskamp N, et al.: SAR matrices: automated extraction of information-rich SAR tables from large compound data sets. J Chem Inf Model. 2012; 52(7): 1769–1776. PubMed Abstract \| Publisher Full Text\n\nGupta-Ostermann D, Shanmugasundaram V, Bajorath J: Neighborhood-based prediction of novel active compounds from SAR matrices. J Chem Inf Model. 2014; 54(3): 801–809. PubMed Abstract \| Publisher Full Text\n\nGupta-Ostermann D, Balfer J, Bajorath J: Hit expansion from screening data based upon conditional probabilities of activity derived from SAR matrices. Mol Inf. 2015; 34(2–3): 134–146. Publisher Full Text\n\nGupta-Ostermann D, Bajorath J: The ‘SAR Matrix’ method and its extensions for applications in medicinal chemistry and chemogenomics [v2; ref status: indexed, http://f1000r.es/3rg]. F1000Res. 2014; 3: 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta-Ostermann D, Hu Y, Bajorath J: Systematic mining of analog series with related core structures in multi-target activity space. J Comput Aided Mol Des. 2013; 27(8): 665–674. PubMed Abstract \| Publisher Full Text\n\nHussain J, Rea C: Computationally efficient algorithm to identify matched molecular pairs (MMPs) in large data sets. J Chem Inf Model. 2010; 50(3): 339–348. PubMed Abstract \| Publisher Full Text\n\nKubinyi H: Free wilson analysis. Theory, applications and its relationships to hansch analysis. Quant Struct-Act Relat. 1988; 7(3): 121–133. Publisher Full Text\n\nStumpfe D, Bajorath J: Exploring activity cliffs in medicinal chemistry. J Med Chem. 2012; 55(7): 2932–2942. PubMed Abstract \| Publisher Full Text\n\nKouji H, Kogami Y, Odagami T: Alpha Helix mimetic compositions for treating cancer and other CBP/catenin-mediated diseases and conditions. US 8691819 B2, 2014. Reference Source\n\nOdagami T, Kogami Y, Kouji H: Alpha helix mimetics and methods thereto. WO 2010128685 A1, 2010; US 20120088770 A1, 2012. Reference Source\n\nMoon RT, Kohn AD, De Ferrari GV, et al.: WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet. 2004; 5(9): 691–701. PubMed Abstract \| Publisher Full Text\n\nKlaus A, Birchmeier W: Wnt signalling and its impact on development and cancer. Nat Rev Cancer. 2008; 8(5): 387–398. PubMed Abstract \| Publisher Full Text\n\nMolenaar M, van de Wetering M, Oosterwegel M, et al.: XTcf-3 transcription factor mediates beta-catenin-induced axis formation in xenopus embryos. Cell. 1996; 86(3): 391–399. PubMed Abstract \| Publisher Full Text\n\nVeeman MT, Slusarski DC, Kaykas A, et al.: Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol. 2003; 13(8): 680–685. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8159", "date": "31 Mar 2015", "name": "Hans Matter", "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\nThis interesting contribution by Bajorath et al. nicely extends the idea of graphical methods for SAR analysis in computational medicinal chemistry. The SARM method was shown earlier to capture SAR information from larger collections by matched molecular pairs (MMPs) and to present it in an intuitive way. Furthermore the combination of large-scale SAR analysis with virtual compounds allows guiding synthesis to explore straightforward ideas as direct outcome of SAR interpretation. Therefore this approach is attractive to rapidly identify activity trends and cliffs.The paper reports a conditional probability-based approach to activity prediction from SAR knowledge. Such a conditional probability measures the probability of activity for one compound given that a structurally related compound was active. Individual probabilities are extracted from rows and columns in the underlying SARMs. While such a probabilistic approach only works for SARMs, which are sufficiently populated and have shared substitution pattern, the approach is not restricted to compound subsets representing continuous SAR only.The prospective application of this interesting concept suffers from the lack of chemical structures, so that the degree of similarity between actives and follow-up design cannot be assessed. Furthermore the description of the HTS assay, substructure alerts, additional filtering, assay validation and retesting rates, compound QCs for actives is missing. This makes it difficult to evaluate the true HTS outcome using potentially noisy data for such a challenging PPI target.To illustrate the value of the novel activity estimation approach from matrices, it might be useful constructing a standard 2D-QSAR model and check is for predictivity of the synthesized top-20 design proposals in comparison to the matrix-derived conditional probability. It might be of interest to see, how robust both approaches work with noisy primary screening data.The manuscript title and abstract cover the content well. The chemoinformatics approach is clearly described and can most likely be reproduced. As this is not the case for the HTS actives and the assays for this study, the results will be difficult to reproduce. The authors might also want to mention, whether software tools from their study are available to the public.", "responses": [ { "c_id": "1287", "date": "09 Apr 2015", "name": "Jürgen Bajorath", "role": "Author Response F1000Research Advisory Board Member", "response": "A conventional QSAR model has been difficult to derive in this case because of the rather approximate nature of activity annotations obtained from raw screeing data. Instead, a cross-validated binary QSAR model has been generated from the screening data using the Molecular Operating Environment (version 2013.08; Chemical Computing Group Inc., Montreal, Canada) and applied to predict the activity state of the 20 test compounds, producing an accuracy of 0.65 for active and inactive compounds." } ] }, { "id": "8069", "date": "31 Mar 2015", "name": "Georgia B. McGaughey", "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\nGupta-Ostermann's \"follow-up\" manuscript is well written and clearly laid out. I only have a few (minor) recommendations, which I believe would help readers more easily replicate their work.The added value of this manuscript lies in figure 3 where \"conditional probabilities of activity\" are explained. The authors have explained conditional probabilities with figures, text and associated mathematical equations and have even gone so far as to carry out the math for the weighted core class contributions. For interested readers who want to implement the conditional probabilities concept in their own research, I highly suggest that real (or toy) data be included, in the very least, as supplemental material with all the data completely worked out, not just the weighted core class contributions. This would allow one to implement the concept, carry out the math and compare the results to the published results more easily. Additionally, although text is included to explain conditional probabilities, I found myself having to read this section a few times to fully understand the clear impact this method could have. I think this section needs to be expanded with more text.Finally, although it is understandable that the work carried out herein with PRISM BioLab Corporation, is proprietary, it is unfortunate that more information regarding the \"twenty synthesized candidates\" can not be elaborated upon. Any information regarding the similarity of these compounds to the actives (or even the similarity range of the actives themselves) would be informative.", "responses": [] }, { "id": "8160", "date": "31 Mar 2015", "name": "Stefan A Laufer", "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\nThis Method article mostly describes an extension of the SAR Matrix approach to predict active compounds from many virtual candidates that are contained in matrices derived from compound libraries. The new activity prediction method is generally applicable to screening data to facilitate hit expand and can make use of approximate activity measurements such as % inhibition. This would be attractive in practice. The conditional probability method, which was first published in an informatics journal, is not trivial and probably not easy to understand for many medicinal chemists.Therefore, the authors were obviously motivated to make this prediction methodology accessible to wider audience in screening and medicinal chemistry. They have done so by going step by step through exemplary calculations that illustrate ideas behind this approach and show how active compounds are predicted.In addition, they report first practical applications that should make this method in combination with the SAR matrix structure attractive to many.Although the application on a library of helix mimetics is essentially proprietary (structures of active compounds cannot be shown), the statistics of the predictions are interesting. Of thousands of virtual compounds the SAR matrix approach generates for this library, only 28 were predicted to be active using the new method when processing reporter gene assay data probing the Wnt pathway. Twenty of these compounds were synthesized and tested and 5 new hits were identified with low-micromolar potency. Clearly, if the combined SAR matrix / activity prediction approach produces similar results in additional applications of this library or other screening libraries, it will berather useful for hit expansion.Taken together, the authors have attempted to make a relatively complex computational approach easier to appreciate by a screening or chemistry audience by providing easy to follow examples and practical applications. This could hardly be accomplished in a specialized computational journal.", "responses": [ { "c_id": "1286", "date": "09 Apr 2015", "name": "Jürgen Bajorath", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank the reviewer for pointing at a primary motivation for this publication and for emphasizing accessibility to a wider than an expert computational audience." } ] }, { "id": "8067", "date": "08 Apr 2015", "name": "Dragos Horvath", "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 interesting upgrade of the SARM methodology, now endeavored with a probability-driven activity prediction tool described in this paper. Unfortunately, I cannot recommend indexation as is, because the methodology is not comprehensively described: some formulae embedded in a Figure are never rigorously explained, except by means of some hand-waiving example. Therefore, I (hope I) got the principle of the method - looks very much like naive Bayes to me. If so - does it do better than standard naive Bayes, with some fragment count descriptors? Honestly, I could not write a piece of code implementing it on the basis of what is said in the paper. Don't understand cryptic annotations like 1(v(x)=v) - suppose it's some Kronecker delta symbol 'sum only over lines with v(x)=v'... but, by the way, what is 'x'? Molecules? Matrix columns? Rows? This is the best-kept secret of the publication...", "responses": [ { "c_id": "1288", "date": "10 Apr 2015", "name": "Jürgen Bajorath", "role": "Author Response F1000Research Advisory Board Member", "response": "The SARM-based probability approach and Naïve Bayesian (NB) classification are both probabilistic in nature and attempt to estimate the posterior probability of a given compound x to belong to a class y, i.e. P(y \| x). However, following Bayes’ theorem, NB modeling derives the posterior from the prior and class likelihood. The class likelihood, P(x \| y), is estimated from the data. By contrast, the SARM-based approach assigns weights to the cores (keys) based on substituents (values) and vice versa. This is done under the assumption that either cores, values, or their combination might be responsible for the activity. Thus, the approach estimates the posterior from the data and then applies a re-weighting (refinement) scheme by calculating core and value class contributions.All methodological details of the SARM-based probability of activity approach are provided in reference 5 of the paper." } ] } ]	1	https://f1000research.com/articles/4-75
https://f1000research.com/articles/4-92/v1	13 Apr 15	{ "type": "Data Note", "title": "Portrayed emotions in the movie \"Forrest Gump\"", "authors": [ "Annika Labs", "Theresa Reich", "Helene Schulenburg", "Manuel Boennen", "Gehrke Mareike", "Madleen Golz", "Benita Hartigs", "Nico Hoffmann", "Sebastian Keil", "Malú Perlow", "Anne Katrin Peukmann", "Lea Noell Rabe", "Franca-Rosa von Sobbe", "Michael Hanke", "Annika Labs", "Theresa Reich", "Helene Schulenburg", "Manuel Boennen", "Gehrke Mareike", "Madleen Golz", "Benita Hartigs", "Nico Hoffmann", "Sebastian Keil", "Malú Perlow", "Anne Katrin Peukmann", "Lea Noell Rabe", "Franca-Rosa von Sobbe" ], "abstract": "Here we present a dataset with a description of portrayed emotions in the movie ”Forrest Gump”. A total of 12 observers independently annotated emotional episodes regarding their temporal location and duration. The nature of an emotion was characterized with basic attributes, such as arousal and valence, as well as explicit emotion category labels. In addition, annotations include a record of the perceptual evidence for the presence of an emotion. Two variants of the movie were annotated separately: 1) an audio-movie version of Forrest Gump that has been used as a stimulus for the acquisition of a large public functional brain imaging dataset, and 2) the original audio-visual movie. We present reliability and consistency estimates that suggest that both stimuli can be used to study visual and auditory emotion cue processing in real-life like situations. Raw annotations from all observers are publicly released in full in order to maximize their utility for a wide range of applications and possible future extensions. In addition, aggregate time series of inter-observer agreement with respect to particular attributes of portrayed emotions are provided to facilitate adoption of these data.", "keywords": [ "Emotional episodes", "Emotional processing cues", "fMRI", "Audio-visual stimulus", "Forrest Gump" ], "content": "Background\n\nRecently, we have published a dataset with high-resolution functional magnetic resonance (fMRI) data of 20 participants listening to a two-hour audio-movie version of the Hollywood feature film “Forrest Gump”1. Using prolonged complex naturalistic stimuli, such as this one, is one approach to study cognitive processing in situations that resemble real-life contexts more closely than controlled laboratory settings typically employed to investigate individual cognitive functions in isolation2. However, multidimensional stimuli and the resulting lack of experimental control can make it harder to isolate the intervening parameters2. The goal of this study was to extend the information about this movie stimulus in order to enable further analysis of the already published brain imaging data as well as to investigate the utility of this particular stimulus for future studies and additional data acquisitions. To this end, we focused on a highly relevant aspect of social cognition that is, at the same time, difficult to infer from an audio-visual stimulus by means of computer algorithms: portrayed emotion (the expression of the emotional state of a movie character by an actor).\n\nThe presentation of movies (clips) to elicit emotional responses is an established component of emotion research, in particular with respect to more differentiated emotional states, such as remorse or pride, that go beyond primary emotions, like fear, in complexity and time-scale3. Emotion cues in movies can be manifold: facial expressions (e.g. smiling)4, verbal cues (e.g. swearing), voice characteristics (e.g. trembling voice)5, or even context cues that can be used to reason about emotional aspects of a situation based on abstract causal principles rather than direct perceptual cues6. Moreover, the emotional response to a stimulus in a real-life setting is further dependent on additional factors. For example, observing a facial expression of sadness may yield an emotional response of pity or satisfaction, depending on whether the person is a friend or a punished offender of social norms. A smiling face is not an expression of happiness when the larger context identifies it as a strategy to avoid unpleasant social interactions (a fake smile). Consequently, labeling portrayed emotions in feature films is a task that requires human observers to perform complex judgments.\n\nTwo groups of frameworks for systematic description of emotions are distinguished in the literature: a) schemes with discrete emotions (labels) and b) dimensional models7. Models using discrete emotion labels vary considerably in the number of differentiated emotion states. Many theories assume few basic, innate emotions8; others discriminate up to 36 affective categories9. Dimensional models, for example the circumplex model, locate different emotional states in a two-dimensional space, commonly using the axes arousal and valence10.\n\nCurrent emotion assessment tools, like the Facial Action Coding System (FACS) or the Specific Affect Coding System (SPAFF)11, attempt to combine both approaches. However, they typically involve following complex instructions regarding the interpretation of facial expressions or other physical and cultural indicators of emotion (11, p. 281) and thus require an intensive training of observers. While these tools provide reliable procedures for rating emotions, they are not very intuitive and are consequently only accessible to experts.\n\nIn this study, the primary goal was not to generate an objective labeling of portrayed emotions in the Forrest Gump movie. Congruent with our goal to enable further studies of already published brain imaging data, we rather aimed at producing a description of the emotional content of the Forrest Gump movie stimulus as it was likely perceived by the participants in our past and future brain imaging studies (potentially biased by age, native language, or education). Therefore, our approach was to collect data from multiple observers that stem from the same student population, using a simplified procedure that does not require extensive training.\n\nPortrayed emotions were independently annotated for the audio-only version of “Forrest Gump” (used in Hanke M et al.1) and for the original audio-visual movie in order to obtain information about which aspects of portrayed emotions are congruent between both stimulus variants.\n\nThe resulting dataset of annotations of portrayed emotions combines a dimensional rating with a categorical labeling of emotions and a description of their associated perceptual evidence. In the following, we provide evidence that our procedure yielded a reliable description that can be used to segment the movie into episodes of portrayed emotions. Moreover, we show that the time course of inter-observer agreement (IOA) with respect to many aspects of portrayed emotion is a reliable measure of their perceptual ambiguity throughout the movie.\n\nIn combination with the already publicly available brain imaging data, these annotations form a two-hour high resolution fMRI measurement for auditory emotion cue processing from 20 participants. With the addition of a future publication of brain imaging data recorded from a stimulation with the audio-visual movie, the full dataset will enable comparative studies investigating the processing of rich emotional stimuli via different sensory pathways. Moreover, these new annotations of portrayed emotions are another step towards a comprehensive description of this reproducible movie stimulus1 and improve its general utility for independent studies on social cognition with a focus on the perception of emotions in real-life situations.\n\n\nMaterials and methods\n\nThe annotated stimulus was a slightly shortened version of the movie “Forrest Gump” (R. Zemeckis, Paramount Pictures, 1994). Two different variants of this movie were annotated separately. The first was a German audio-description as broadcast as an additional audio track for visually impaired listeners on Swiss public television (Koop, Michalski, Beckmann, Meinhardt & Benecke produced by Bayrischer Rundfunk, 2009). This audio-only stimulus was the same as the one used in Hanke M et al.1, and is largely identical to the dubbed German soundtrack of the movie except for interspersed narrations by a male speaker who describes the visual content of a scene. These descriptions take place when there is no dialog, off-screen speech, or other relevant audio-content in the movie. The second variant is the audio-visual movie with the original dubbed German soundtrack (without additional narrations) and the exact same timing (and cut scenes) as the audio-only version (1, contains instructions on how to reproduce the stimulus from the DVD release).\n\nAnnotations were created by the authors themselves as part of a practical course on scientific observation. A total of 12 students at the Otto-von-Guericke-University in Magdeburg, Germany participated in this effort and received course credit. Based on personal preference, students assigned themselves to one of two groups tasked with the annotation of portrayed emotion in either the audio-visual movie, or the audio-only version. The audiovisual group consisted of 9 students (all female), the audio-only group comprised 3 students (all male). This gender bias could have had an impact on the perceived emotions. None of the observers participated in the previous brain imaging study1.\n\nAnnotations were performed with the help of Advene12, a dedicated open-source video annotation tool that offers convenient navigation of structural units of the stimulus and provided uniform position information with subsecond precision. Each observer annotated all of 205 previously identified movie scenes in an individual random order to minimize “carry-over” effects and help observers focus on current indicators of portrayed emotions in contrast to more slowly changing characteristics, such as mood, or other biases introduced by the movie plot. Scenes were used as units for annotation, because transitions between them typically involve a change of location or fast progression of time that make persistence of the nature of portrayed emotions across scenes less likely. Observers used a scene annotation of the movie imported into Advene to aid navigation within the movie. They only had to click on the visual representation of a scene (a numbered box) in a time line display to start the playback at the start of a scene. Advene could also be configured to play a selected scene in a continuous loop. For composing the annotation content, observers used a separate spreadsheet application and not the data input features of Advene.\n\nObservers were instructed to work alone in a setting free of distractions and to use headphones for optimal saliency of the stimulus. There was no strict guideline regarding the length of an annotation session, but observers were informed to stop working for any number of breaks whenever they could no longer guarantee a high level of attention for the task. Sessions were typically spread over multiple days over the course of three weeks. On average, observers reported that it required around 30 hours each to complete the annotation.\n\nEach observer collected annotations in a spreadsheet with columns for start time and end time of an emotion; a label of the movie character portraying the emotion; variables for valence, arousal, and direction of the emotion as well as an optional emotion category label; and a list of identified indicators for the start and end of an emotion. Each of these variables are described in more detail in the remainder of this section. In addition, observers also recorded the index of the scene containing a respective emotion. This variable was only used for error detection (mismatch of emotion duration with the start and end time of a scene) and is not included in the published dataset.\n\nStart and end time The start time identifies the onset of the first observed indicator of an emotion. Likewise, the end time corresponds to the offset of an emotion — defined as the time where no evidence for an emotion is present anymore. Observers were instructed to aggregate short segments of uniform emotional episodes into a single annotation. For example, if a character was happy and smiling throughout a long period in a scene, the episode would span all the time from the beginning to the end of the smile even if the character’s face was not visible the entire period, as long as no evidence of a change of emotion was found. All times are reported in seconds. Although Advene offers time stamps with the precision of the movie frame rate (25 frames per second), pre-tests revealed that the temporal accuracy of human observers for complex emotion indicators, like a developing facial expression, is much coarser. In order to reduce the chance of unrecoverable typos in the time stamps, observers were instructed to record timing information with second-precision only.\n\nOnset and offset cues Onset indicators describe what kind of evidence for an emotion was detected in the movie stimulus. We distinguished facial expressions, gestures or body language, context information, verbal, and non-verbal audio cues. Observers could record multiple onset indicators. Despite the term “onset”, these indicators did not all have to be present at the very beginning of an emotional episode. For example, an extended period of sadness could start with a facial expression and later on add a congruent body language cue. In that case, the respective labels were aggregated into a list. In the audio-only stimulus, the narrator was frequently the only source of information regarding the emotional state of a character; thus, we included a dedicated category for this scenario.\n\nIn addition to onset indicators, observers also recorded the type of evidence for the end of an emotional episode. Four conditions were distinguished: 1) changing from one emotion to another 2) entering a neutral emotional state 3) a character leaving an ongoing scene with no further evidence for its emotional state 4) the end of a scene. Table 1 describes all distinguished cue categories.\n\nCharacter label This label identifies which movie character is portraying an emotion. Observers were provided with a list of character labels that was derived from an annotation of the movie’s dialog in order to achieve a consistent labeling. However, some characters portray emotions but have no lines of dialog, and, in other instances, observers chose inconsistent labels. During data curation, character labels were therefore manually consolidated into a set of 35 categories that uniformize labeling across observers. The full list of character labels is shown in table 2 and was derived using the following rules: a) main characters and famous/familiar characters receive individual labels as do the two narrator voices b) relatives of main characters are labeled according to their relation c) all other characters are consolidated into generic categories that preserve gender, age (children, adults, or seniors), and number (individual or group).\n\nAny annotation of a portrayed emotion is associated with exactly one of these 36 labels. While main characters, their relatives, and famous characters have individual labels, all remaining characters in the movie are aggregated into generic categories that preserve information on gender, age group, and number.\n\nIt is important to note that only emotions portrayed by one or more movie characters were recorded. This excludes other potentially emotion-inducing stimuli (e.g, music in the soundtrack) or inanimate stimuli (e.g., the depiction of a beautiful sunset or chirping birds).\n\nValence, arousal, and direction Binary variables were used to record the valence of an emotion (positive vs. negative), the state of arousal of the character portraying it (high vs. low), and whether the emotion is directed towards the character itself or another one (e.g. feeling pity for somebody). Together, these three indicator variables offer a coarse categorization of observed emotions in the spirit of dimensional models of emotion.\n\nArousal was considered a global indicator of the emotional status of a character, i.e., for any given time in the movie annotations, indicate either a high or low level of arousal for a character — but never both at the same time. Unlike arousal, valence and direction indicators were considered non-exclusive, i.e., a character could show signs of both positive and negative emotions at the same time. Likewise, a character could simultaneously exhibit self-directed emotions and emotions directed towards others. In order to annotate such situations, observers had to create records for two or more portrayed emotions with overlapping timing.\n\nEmotion labels Observers were instructed to assign one of 22 labels for discrete emotion categories. These categories were derived from a schema proposed by Ortony13 and concrete definitions were developed based on the Specific Affect Coding System11. The complete list of emotions is shown in table 3. Pre-tests using a larger and more detailed set of emotion categories revealed weak agreement between observers for a forced categorization of emotional episodes. Based on these results, we decided to make this part of the annotation optional, and observers were instructed to assign an emotion label only if they saw a “perfect” match with any of the categories.\n\nWe used an automated procedure to check annotation records of individual observer for errors or potential problems. Observers submitted their annotations in tabular form to a script that generated a list of error and warning messages. Using this feedback, observers double-checked their annotations as often as necessary until no objective errors were found and all warning messages were confirmed to be false positives. The following annotation properties were tested:\n\nstart time had to precede end time (error)\n\nno character label (error)\n\nno onset cue label (error)\n\nmissing arousal, valence, or direction value (error)\n\nconflicting simultaneous arousal annotation for the same character (error)\n\nemotion portrayed for longer than a minute (warning)\n\nannotation starts prior to a scene start or ends after a scene end (warning)\n\nunrecognized code/label for any variable (warning)\n\nno offset cue label (warning)\n\nThe released data comprises four components: 1) raw annotations 2) aggregate inter-observer agreement (IOA) time series 3) emotion episode segmentations 4) the source code to generate all data derived from the raw annotations and all summary statistics presented in the Data Note.\n\nRaw annotations In order to maximize the utility of the annotations, they are released in full rather than only as aggregate information. The annotations from each observer are available in an individual plain text file with comma-separated value (CSV) markup in the raw directory. This file format facilitates import and further processing in virtually any software application that supports data in tabular form. The file names indicate the annotated stimulus type (prefix av for the audio-visual movie and ao for the audio-only movie) as well as the observer identity.\n\nEach file contains nine columns that correspond to the annotation properties described above. start, end) start and end time of an emotion episode reported as seconds from the start of the movie; character) a single character label from the set shown in table 2 indicating the character portraying the emotion; arousal) state of arousal indicated with the labels LOW or HIGH; valence) emotional valence indicated with the label POS or NEG; direction) direction of the emotion indicated with the labels SELF or OTHER; emotion) a space-separated list of emotion labels from the set shown in table 3; oncue) a space-separated list of onset indicators for a portrayed emotion from the set shown in table 1; and offcue) a space-separated list of offset indicators from the set also shown in table 1. With the exception of the columns emotion and offcue, all variables are considered mandatory and are present for all annotations. The remaining columns contain optional attributes and can be empty.\n\nInter-observer agreement (IOA) time series IOA time series reflect the consistency of observations at any particular time in the movie. The time series amplitude corresponds to the fraction of observers indicating the presence of a particular attribute of an emotion episode (interval [0, 1]). Time series for the three bipolar attributes arousal, valence, and direction were computed by subtracting the IOA with respect to the presence of the both extremes from each other. For example, the time series for arousal was computed by subtracting the time series of the IOA for low-arousal episodes from the time series of high-arousal episodes. This results in values from an interval of [−1, 1] — where −1 corresponds to perfect agreement with respect to the presence of a low-arousal episode and +1 indicates perfect agreement regarding high arousal.\n\nIOA time series were computed for three different segment durations (i.e., agreement is considered when observers indicate the presence of a particular attribute anywhere in a segment): 1 s (temporal precision of the annotations), 2 s (corresponding sampling rate of the available brain imaging data), and the actual location and duration of individual shots in the movie (median duration ≈5 s). Individual time series for the three bipolar attributes, all 22 emotion categories, and the six emotion onset cues are available in the timeseries directory. The file names are encoded as follows: prefix ioats, followed by a label for the segment duration (e.g. 1s), followed by the stimulus label (av or ao), and a character label (e.g. forrest, see table 2) or allchar for an aggregate time series across all characters. All files are in plain text format with CSV markup. The column headers indicate the corresponding emotion attribute.\n\nEmotion episode segmentation Based on IOA time series, a segmentation of the movie into episodes of portrayed emotion was performed for each of the 35 character categories individually. IOA time series for 33 attributes (arousal (bipolar), valence and direction (separate for each state), 22 emotion category labels, and six onset cues) were binarized by thresholding with a minimum absolute IOA of 50%. An episode of portrayed emotion was defined as a time window with at least one super-threshold emotion attribute. The duration of an episode was determined by the number of consecutive movie segment without a change in the unique combination of super threshold attributes.\n\nEmotion episodes for all characters are available in the segmentation directory in plain-text files with tab-separated value (TSV) markup. The file format follows the conventions required by the Advene software for “simple-structured content”, but are also compatible with generic spreadsheet applications. Each file contains three columns (without a header). The two columns contain the start and end time of an episode in seconds. The third column contains a space-separated list of key/value pairs. Five such properties are present for each episode: char) the label of the character portraying the emotion; tags) a comma-separated list of tags corresponding to all super-threshold emotion attributes; arousal, val_pos, and val_neg) these three properties each correspond to the median IOA value across the entire episode for arousal (bipolar) and positive and negative valence (each unipolar). The names of all tags typically correspond to the lower-case name of the attribute, with the exception of the three bipolar attributes where each extreme is coded with a dedicated label (arousal: ha/la (high/low), valence: pos/neg, direction: self/other).\n\nEmotion episode segmentation was performed separately for the audio-visual and the audio-only movie—once using IOA time series sampled with a 1 s segment size and a second time sampled with the actual duration and location of shots in the audio-visual movie. File names encode these conditions as follows: prefix emotions followed by the stimulus label (av or ao) followed by the segment size label (1s or shots).\n\nSource code The full source code for all descriptive statistics and figures included in this paper is available in descr_stats.py (Python script). Moreover, this script also contains all functionality to generate the IOA time series as well as the emotion episode segmentation from the raw annotations in the data release. The required information on the timing of movie scenes and shots is contained in two additional CSV files: movie_scenes.csv and movie_shots.csv.\n\nTo be able to use the provided emotion annotations for an analysis of functional data (fMRI, cardiac trace, etc.) acquired using the previously described procedure1, timing information has to be converted because data were recorded in eight sessions using partially overlapping segments of the movie. Table 4 specifies the location of these segments with respect to the timing of the unsegmented movie used for emotion annotation. Subtracting movie segment start times from annotation time stamps yields valid relative time stamps with respect to the time series of the available functional data.\n\nThe last column lists the position of the scene boundary used as a reference for the segment transition. All times are in seconds and refer to the full (unsegmented) stimulus, which is a shorted version of the original movie1.\n\n\nDataset validation\n\nCharacters portraying emotions Annotations for a total of 34 and 27 different character categories were created for the audio-visual movie (AV) and the audio-only movie (AO) respectively. In the AV case, these comprise all possible characters except the narrator of the audio-description. There are 9 (AV) and 8 (AO) characters for which there is a 50% minimum inter-rater agreement on the presence of a portrayed emotion for at least five episodes of one second or longer throughout the entire movie (arbitrary thresholds). The number of emotion episodes for the AV/AO union of these characters sets is shown in figure 1 (top). As expected, the majority of all portrayed emotions are associated with the main movie characters Forrest, Dan, and Jenny.\n\nThe minimum criterion for counting emotion episodes is a 50% inter-observer agreement. Only categories/characters with a minimum of five such episodes are shown. The left panels show the number of episodes for both movie variants; the right panels show the cumulative duration of emotion display across all considered episodes. The top panels show the annotation frequency by movie character; the bottom panels show the corresponding frequencies for emotion categories.\n\nPortrayed emotion categories For the audio movie, 97.5% of all annotations include at least one of the 22 explicit emotion labels (see table 3), whereas only 68.4% of annotations for the audio-visual movie contain such a label. This is an indication that emotion cues in the visual movie are more diverse and ambiguous in comparison to the audio-only stimulus. Figure 1 (bottom) shows the frequency and display duration for individual emotions. For both stimulus types, the majority of all emotional episodes involve the five categories anger/rage, fear, happiness, love, and sadness. Some of our pre-determined emotion categories were not attributed to any emotion episodes in the movie, were used very infrequently, or were used only by a small subset of observers. Such emotions include resentment, gratification, fears confirmed, or satisfaction (see table 5). It remains unknown whether these emotions are not portrayed in the movie or if the respective categories were not appropriately defined.\n\nAll values are Spearman correlations for 1 Hz modulations of the fraction of IOA (as, for example, depicted in figure 3) with respect to a particular emotion attribute across the entire duration of the movie. The specified range corresponds to the width of the 95% confidence interval of the mean correlation for all possible combinations of partitioning observers into two sub-groups (audio-visual 4 vs. 5; audio-only 1 vs. 2). Higher correlations indicate higher consistency of agreement modulations across observer sub-groups. Correlations higher than 0.5 (arbitrary threshold) are depicted in bold for visualization purposes. The all characters column indicates the agreement for a particular emotion attribute over time irrespective of the annotated character. The columns on the right show the corresponding correlations for the two main characters. n/a fields indicate an insufficient number of annotations to compute the consistency measure.\n\nEmotion onset cues Figure 2 shows the frequency of individual emotion onset cues for all six distinguished cue types. When only considering annotations with a minimum inter-rater agreement of 50% with respect to the type of onset cue, the two dominating emotion cues are facial expressions (AV only) and verbal cues (for both stimulus types), followed by gestures and body language (AV only). Figure 3 (bottom row) shows a rather uniform distribution of verbal emotion cues across the entire duration of the movie for both stimulus types. This suggests that a comparative analysis of verbal emotion cue processing during AV vs. AO stimulation, as suggested above, may be feasible.\n\nExtreme values indicate perfect agreement with respect to presence or absence of a particular emotion attribute at a given time. IOA time series for the two movie types are overlaid on top of each other. For the purpose of visualization, the depicted time series reflects IOA computed using a 10 s segment size — in contrast to 1 s used for all other statistics presented in this paper.\n\nIt can be assumed that observers in this study have used individual intensity thresholds for determining the start, end, or quality of an emotional episode. Therefore, the IOA with respect to a particular aspect of portrayed emotions at any given time in the movie could be seen as a measure of the relative probability of perceiving an emotion feature. In order to assess the reliability of such a measure, we generated time series for the IOA with a sampling rate of 1 Hz, as described above, and evaluated their reliability and validity in terms of their correlation across observers, stimulus types, and with each other.\n\nIntra-stimulus inter-observer consistency The reliability of IOA time courses was estimated by repeatedly splitting the set of observers into two groups of approximately equal size. Within each group, IOA time courses where computed and correlated across the observer subgroups. Split-half correlations were computed for all possible ways of combining observers into two groups (without replacement). Table 5 shows the average time series correlation across all splits and the width of the 95% confidence interval of the mean.\n\nIn general, IOA reliability is larger for the AV movie annotations than for the AO movie. This finding is likely a result of the lower number of observers for the latter. Intra-stimulus IOA reliability estimates suggest that a subset of emotion attributes or categories is not contained in the AO movie stimulus. The most prominent example is the direction of an emotion. Emotion categories like hate, shame, or relief also show large reliability differences between stimulus types. However, in these cases the small number of emotion episodes may multiply the negative effect of the small number of observers on the interpretability of this estimate.\n\nUniformly high reliability estimates were found for the two main dimensional emotion attributes arousal and valence, as well as for the five most frequently annotated emotion categories anger/rage, fear, happiness, love, and sadness.\n\nIntra-stimulus inter-variable correlations In order to assess possible confounds in the annotations, we computed the correlation of intra-stimulus IOA time courses (across all observers) for all pairwise combinations of emotion attributes and categories. The correlation matrices for the AV and the AO movie stimulus are depicted in figure 4. The results reveal an expected correlation pattern between individual emotion categories and the two main dimensional attributes arousal and valence — with stronger correlations being observed for more primary and more frequently portrayed emotions. Interestingly, audio and verbal cues in the AV stimulus seems to be more associated with a high state of arousal and emotions directed towards other people than in the AO stimulus. In the AV stimulus, facial expressions seem to have a tendency to communicate negative emotions rather than positive ones.\n\nThe lower triangular matrix depicts the correlations between the IOA time courses for the three primary bipolar emotion attributes (arousal [high/low], valence [pos/neg], and direction [self/other]), the 22 emotion categories, and the six emotion onset indicators for the audio-visual movie. The upper triangular matrix shows the corresponding correlations for the audio-only movie. There were no observations for the emotion “fears confirmed”, or the facial expression onset cue in the audio-only movie. Likewise, there were no observations for the “narrator” onset cue in the audio-visual movie. The corresponding undefined correlations are depicted as zeros.\n\nInter-stimulus consistency To estimate the similarity of the two stimulus types, we computed the correlation of IOA time courses for the AV and the AO movie with respective to emotion attributes and categories. Table 6 shows the respective correlations and their 95% confidence intervals. Again, the three dimensional attributes as well as the five most frequent emotion categories show significant correlations. Notably, verbal cues are the most consistent indicator of emotions across both types of stimuli. This is evidence for an expected substantial semantic similarity with respect to the emotional content in the two variants of the “Forrest Gump” movie.\n\nAll values are Spearman correlations of 1 Hz modulations of the fraction of IOA (as, for example, depicted in figure 3) for the two stimulus types (audio-visual and audio-only movie). The specified range corresponds to the width of the 95% confidence interval of the correlation (computed via Fisher transformation). Correlations higher than 0.5 (arbitrary threshold) are depicted in bold for visualization purposes. n/a fields indicate an insufficient number of annotations to compute the consistency measure.\n\n\nData availability\n\nF1000Research: Dataset 1. Annotations of portrayed emotions in the movie “Forrest Gump”, 10.5256/f1000research.6230.d4532814", "appendix": "Author contributions\n\n\n\nAKP, AL, BH, FRS, HS, LNR, MB, MGE, MGO, MP, NH, SK, and TR designed the data collection procedure, performed the annotation, and contributed to the manuscript; MH conceived the study, aggregated data from individual observers into the released form, performed the data analysis, and contributed to the manuscript. All authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was, in part, supported by the German Federal Ministry of Education and Research (BMBF) as part of a US-German collaboration in computational neuroscience (CRCNS; awarded to James Haxby, Peter Ramadge, and Michael Hanke), co-funded by the BMBF and the US National Science Foundation (BMBF 01GQ1112; NSF 1129855). Michael Hanke was supported by funds from the German federal state of Saxony-Anhalt, Project: Center for Behavioral Brain Sciences.\n\n\nAcknowledgements\n\nWe are grateful to Roland Nigbur for his advice during all stages of this study. Additionally, we thank Alex Waite and Florian Baumgartner for their feedback on the manuscript.\n\n\nReferences\n\nHanke M, Baumgartner FJ, Ibe P, et al.: A high-resolution 7-Tesla fMRI dataset from complex natural stimulation with an audio movie. Scientific Data. 2014; 1. Publisher Full Text \| Free Full Text\n\nHasson U, Honey CJ: Future trends in Neuroimaging: Neural processes as expressed within real-life contexts. Neuroimage. 2012; 62(2): 1272–1278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGross JJ, Levenson RW: Emotion elicitation using films. Cognition & Emotion. 1995; 9(1): 87–108. Reference Source\n\nEkman P: Facial expressions of emotion: New findings, new questions. Psychological Sci. 1992; 3(1): 34–38. Publisher Full Text\n\nEthofer T, Van De Ville D, Scherer K, et al.: Decoding of emotional information in voice-sensitive cortices. Curr Biol. 2009; 19(12): 1028–1033. PubMed Abstract \| Publisher Full Text\n\nSkerry AE, Saxe R: A common neural code for perceived and inferred emotion. J Neurosci. 2014; 34(48): 15997–16008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGray EK, Watson D: Assessing positive and negative affect via self-report. In J. A. Coan and J. J. B. Allen, editors, Handbook of emotion elicitation and assessment. Oxford University Press, Oxford. 2007; 171–183. Reference Source\n\nEkman P: An argument for basic emotions. Cognition & Emotion. 1992; 6(3–4): 169–200. Publisher Full Text\n\nScherer KR: What are emotions? And how can they be measured? Social Sci Inf. 2005; 44(4): 695–729. Publisher Full Text\n\nRussell JA: A circumplex model of affect. J Personality Social Psychol. 1980; 39(6): 1161–1178. Publisher Full Text\n\nCoan JA, Gottman JM: The specific affect coding system (SPAFF). In J. A. Coan and J. J. B. Allen, editors, Handbook of emotion elicitation and assessment. Oxford University Press, Oxford. 2007; 267–285. Reference Source\n\nAubert O, Prié Y: Advene: active reading through hypervideo. In Proceedings of ACM Hypertext. 2005; 235–244. Publisher Full Text\n\nOrtony A: The cognitive structure of emotions. Cambridge university press, 1990. Reference Source\n\nDataset 1. Annotations of portrayed emotions in the movie “Forrest Gump”. F1000Research. 2015. Data Source" }	[ { "id": "8300", "date": "21 Apr 2015", "name": "Dylan Wagner", "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\nThis data note describes a dataset consisting of time indices and annotations for the emotional content of scenes from the movie Forest Gump. The primary intent of this dataset is to encourage analyses of real-world cognition during the viewing of naturalistic stimuli and, as such, it is meant to accompany a previously released dataset of functional neuroimaging data using the same movie stimulus as used here. I have only minor quibbles:The authors may wish to indicate which observers were familiar with the movie prior to annotation. I recommend adding a section in which the authors discuss some of the potential issues that may limit the correspondence between the emotion annotation dataset and the neuroimaging dataset of the same movie. Specifically, the use of random scene order during annotation (but not during movie viewing) or the gender split among observers who annotated the audio movie and those that annotated the audiovisual movie. At the expense of looking a gift horse in the mouth, an ipython notebook capable of generating all figures and descriptive data would have been marginally preferable to a script. Finally, I would like to note that the release of this dataset along with the associated neuroimaging data previously released by members of this group represents an extremely generous sharing of resources. I commend the effort and look forward to seeing what emerges from researchers looking into this treasure trove of data.", "responses": [ { "c_id": "1301", "date": "22 Apr 2015", "name": "Michael Hanke", "role": "Author Response", "response": "Thanks a lot for the swift and positive feedback!re familiarity: I think it is fair to say that by the time when observers started the actual annotation, all of them were somewhat familiar with the movie, due to the pre-tests we ran on the feasibility of particular annotation approaches -- independent of whether they saw the movie before. We'll add a corresponding statement.re correspondence limitation: we could prepare a dedicated summary of aspects that may have an impact. However, other than the two aspects mentioned by the reviewer it may only be a question of \"detection probability\" which should be reflected in the inter-observer agreement. The issue of scene order randomization should emphasize the portrayed emotions as opposed to lower-frequency mood modulations induced by the movie (see procedure description). The gender split is an issue, but its impact is unclear. Most likely it causes the annotations to be less representative for the opposite gender. However, the annotation procedure encouraged a repeated, rather analytic dissection of the stimulus, in contrast to a single-pass introspection of how a scene \"felt\". This may compensate for an initial gender bias in the perception of the stimulus.re ipython notebook: great suggestion! We'll convert the script into a notebook, and will provide both.Thanks again!" } ] }, { "id": "8301", "date": "27 Apr 2015", "name": "Ariel Rokem", "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\nThis paper provides a description and analysis of a publicly released data-set: an annotation of the emotions expressed by characters in different variants of the movie Forrest Gump, in either audio or audio-visual formats. The data-set is interesting in its own right, and will be eminently useful in conjunction with the fMRI data that this group has previously released, and will continue to release. This data paper goes well beyond a standard description of the data-set, by providing analysis and interpretation of the data.I have just one comment with regards to providing direction for eventual interpretation of the data by users: The conflation of gender differences in perceiving emotions with audio vs. visual distinctions is a real concern, and the authors acknowledge this, but the current description does not provide enough direction on how to deal with this potential confound. I think that it would be helpful if a few sentences about gender differences in perceiving emotion would be added, and some evaluation of what the effects of these differences might be in this data. For example, do differences between AO and AV annotations coincide with predictions from the gender difference literature?It is commendable (and still sadly unusual) that that authors make software that is so easy to use, which reproduces pretty much every single statement they make about the data and produces all the figures. That said, I do have a few comments for improvements of this software:Though not strictly necessary, in this day and age, it would be nice to make the scripts provided compatible with python 3 (from a quick look, it looks like the addition of some parentheses should do the trick, and see more below). Making the code PEP8 compatible (e.g. line-wrapping) would make it easier to read. The authors might disagree, but pymvpa seems like an onerous dependency just to supply some utility functions used in the analysis. I would add `plot_bars`, `xunique_combinations` and `unique_combinations` to the code distributed with the data. Here's a gist of the file I used when my computer refused to compile libsvm: https://gist.github.com/arokem/cd4270a78cbad58c2cd4 As a conclusion from the previous point, a (github) repository with the code used here would be good, for future users of this data/code to be able to make their proposals for improvements to the code via pull requests. The state of the code for this publication could be a tag/release in this repo.Minor:Table 3, the description of HAPPYFOR is: 1) not clear and 2) not capitalized (in contrast to the other descriptions). I am guessing you meant something like: \"Like HAPPINESS, but as reflected off another character\", or some-such.", "responses": [] } ]	1	https://f1000research.com/articles/4-92
https://f1000research.com/articles/4-91/v1	10 Apr 15	{ "type": "Case Report", "title": "Case Report: A case of neurogenic bladder in the setting of Behçet's disease after an initial diagnosis of multiple sclerosis", "authors": [ "Alexander W Pastuszak", "Timothy B. Boone", "Timothy B. Boone" ], "abstract": "Behçet’s disease (BD) is an autoimmune vasculitis with an unclear etiology presenting with a classic triad of symptoms including oral and genital ulcers as well as iridocyclitis. A subset of BD patients exhibit neurological symptoms including psychiatric disturbances, balance problems, and voiding dysfunction, and the symptoms of BD can mimic other neurological diseases, including multiple sclerosis (MS). Differentiating between potential diagnoses is challenging due to the lack of specific tests for these disorders and the overlap between clinical symptoms and radiological findings. We describe the case of a 52 year old woman initially diagnosed with and treated for MS. From the urologic standpoint, she was treated for neurogenic detrusor overactivity with detrusor-sphincter-dyssynergia utilizing ileocecal augmentation cystoplasty with a continent stoma for intermittent catheterization. The patient was later diagnosed with BD in light of additional clinical findings.", "keywords": [ "Neurogenic bladder", "Voiding dysfunction", "Micturitional disturbance", "Bladder dysfunction", "Detrusor-sphincter-dyssynergia", "Multiple sclerosis", "Behçet’s disease", "Multiple sclerosis" ], "content": "Background\n\nBehçet’s disease (BD) was first described by the Turkish dermatologist Hulusi Behçet in 1937 as a triad of clinical symptoms consisting of recurrent uveitis and aphthous ulcers of the mouth and genitalia1. BD is now known to be a multi-system autoimmune inflammatory vasculitis with an unclear etiology and a strong link to the HLA-B51 haplotype2. Patients with Behçet’s disease may present with a variety of symptoms, most commonly with mucocutaneous and ocular involvement thought to be vasculitic sequelae. In addition, BD may also involve the joints as well as the gastrointestinal and nervous systems3.\n\nApproximately 5–15% of patients with BD demonstrate neurological symptoms including dementia, psychiatric disturbances, cranial nerve palsy, cerebellar ataxia and pyramidal tract signs4. Micturitional disturbances have been found in 5–67% of patients with neurologic BD and these have been linked to pontine lesions affecting the pontine micturition center (PMC), pontine urine storage center (PUSC), and the nucleus reticularis pontis oralis (PoO)1,3,5. Generally, nervous system lesions in neurologic BD have been observed primarily in the brain stem and spinal cord1. Post-mortem examination has demonstrated degeneration of the pyramidal tracts as well as focal necrosis of the upper tegmentum pontis, subthalamic area, and internal nucleus of the basal ganglia6,7. Generally, neurological BD is progressive in 85% of patients with remission and relapse occurring in the remaining 15%8.\n\nGiven that the etiology of BD is incompletely elucidated, its diagnosis remains an inexact science and relies on magnetic resonance imaging (MRI), analysis of evoked potentials and cerebrospinal fluid (CSF), and clinical symptoms. CSF analysis typically yields pleocytosis with polymorphonuclear predominance in the absence of oligoclonal bands1. Clinical presentation is usually with an acute or subacute attack followed by remission with or without sequelae, or with progression after an initial attack. Clinically, patients can exhibit a variety of symptoms, many of which overlap with other neurological and autoimmune conditions. The spectrum of symptoms includes, but is not limited to, arthralgia and arthritis, gastrointestinal tract inflammation, deep venous thrombosis, superficial thrombophlebitis, inflammation within the cardiovascular system, inflammatory problems in chest and lungs, auditory disturbances, balance difficulties, fatigue, and psychiatric disturbances9,10. As a result of the lack of specificity of the above findings, serologic testing to narrow the spectrum of differential diagnoses is typically undertaken, but there is currently no sensitive or specific serologic test for diagnosis of BD itself.\n\nMultiple sclerosis (MS), like BD, is regarded as an autoimmune disorder involving the central nervous system (CNS) and resulting in axonal demyelination. Symptoms of MS are variable in spectrum and presentation and can involve almost any aspect of the nervous system. As a result, clinical data alone may be insufficient to make the diagnosis, and supporting data come from neuroimaging, CSF analysis, and evoked potentials. MRI findings can demonstrate plaques, which, in contrast to those found in BD, can be located in white matter of the optic nerve, the basal ganglia, and close to the ventricles of the cerebellum in addition to in the brain stem and spinal cord11. CSF analysis may show pleocytosis, but in contrast with the polymorphonuclear nature of that seen in BD, pleocytosis in MS is typically lymphocytic. In addition, oligoclonal bands of IgG may be seen on electrophoresis of CSF in 75–85% of patients with MS12. Analysis of evoked potentials demonstrates decreased amplitudes secondary to demyelination13. In contrast to BD, which can either present with an initial attack with or without sequelae or in a secondary progressive manner, MS is always progressive, and can follow either a relapsing remitting, relapsing progressive, or primary or secondary progressive pattern.\n\nWe describe the case of a 52 year old female who was initially diagnosed with relapsing remitting MS. This diagnosis was amended to Behçet’s disease after further workup 10 years later. In the interim, the patient was diagnosed with a neurogenic detrusor overactivity and detrusor-sphincter-dyssynergia and was treated initially with anticholinergic medications, intermittent catheterization, and subsequently with ileocecal augmentation cystoplasty.\n\n\nCase presentation\n\nThe patient is a 52 year old Hispanic female with a history of obsessive compulsive disorder and anorexia since adolescence who developed depression and insomnia while in her forties. In addition, she exhibited progressively worsening bilateral hand tremors, worsening dexterity, anorexia, and balance difficulties. A single-photon emission computed tomography (SPECT) scan of the brain on initial presentation in 1998 demonstrated hypoperfusion of the basal ganglia and frontal lobes. The patient was treated with steroids at the time, resulting in transient improvement of her insomnia. A MRI of the brain performed in June of 2000 demonstrated white matter lesions within the subcortical regions including the corpus callosum and periventricular areas, as well as posterior fossa atrophy. Visual and somatosensory evoked responses, as well as auditory brainstem responses, were normal. The patient declined a lumbar puncture, and thus cerebrospinal fluid was not available for analysis. Immune panels, anti-nuclear antibody (ANA), anti-neutrophil cytoplasmic antibody (ANCA), angiotensin converting enzyme (ACE), and chest X-ray were all within normal limits. Per report from the patient, an echocardiogram performed in Mexico in 2000 demonstrated minimal mitral valve prolapse, but neither the echocardiogram report nor images were available for review. At this time, her physical exam demonstrated myokymia in a cranial nerve VII distribution, right upper extremity pyramidal drift, postural and action tremors in bilateral upper extremities, and balance difficulties when asked to stand on one foot. In light of these findings, which met the Barkhoff and Tintoré criteria for diagnosis of MS, and despite the presence of a significant psychiatric component, a diagnosis of relapsing-remitting MS was made14,15. Notably, the literature supports a diagnosis of MS in the setting of psychiatric disease16.\n\nAs a result of her neurological symptoms and findings on imaging, the patient was started on glatiramer acetate and intermittently remained on this medication until 2002. She was not treated with interferons given their potential to worsen depression17. Initially, her symptoms improved markedly with near-resolution of her hand tremor. However, she continued to complain of balance difficulties and memory problems. In addition, in October 2000, she experienced vertigo and confusional episodes as well as worsening depressive symptoms, all of which responded to pulsed intravenous solumedrol. In January 2002, she demonstrated worsening right hand dexterity with no changes in memory and operational judgment when compared with her prior visit. Repeat MRI at that time demonstrated decreased burden of disease per report. She underwent neuropsychological evaluation at this time as well, which demonstrated organic memory deficits with intact intellect. In March 2002, the patient became mentally disorganized and was admitted to the hospital for treatment and observation. At this time, her glatiramer acetate was stopped and the diagnosis of MS questioned. Repeat MRI in June 2002 demonstrated no increase in disease burden. On physical examination at this time, she continued to exhibit minimal tremor in bilateral upper extremities but no longer had pyramidal drift in her right upper extremity. As a result of her symptomatic improvement, glatiramer acetate was not restarted.\n\nIn October 2003, a MRI of the brain evidenced an increase in disease burden and symptomatically the patient exhibited worsening memory deficits. At this time, however, the patient was complaining of urgency and urge incontinence and was evaluated by other urologists. She underwent a Marshall-Marchetti-Krantz bladder neck suspension in late 2003 and subsequently developed immediate urinary retention requiring placement of a Foley catheter. As a result, she was referred to our practice for evaluation and further management. Video urodynamic evaluation in January 2004 demonstrated neurogenic detrusor overactivity with some loss of compliance, detrusor-sphincter dyssynergia, elevated voiding pressures, poor flow, and a post-void residual of 350ml. Her first sensation occurred at 90ml bladder volume with first urge to void at 270ml and a functional bladder capacity of 340ml (Figure 1). She did not leak with cough or Valsalva maneuver. Fluoroscopy and electromyogram (EMG) demonstrated mid-urethral obstruction at the sphincter and no vesicoureteral reflux.\n\n“EMG” – electromyogram “Pves” – intravesical pressure, measured in centimeters of water; “Pabd” – intra-abdominal pressure, measured in centimeters of water; “Pdet” – detrusor pressure, measured in centimeters of water; “Flow” – urinary flow, measured in milliliters per second. The arrow corresponds to the functional bladder capacity of 340 milliliters.\n\nThe patient desired to be free of urethral catheter drainage of her bladder, which was inconvenient and difficult given her limited manual dexterity. As a result, the patient underwent ileocecal augmentation cystoplasty with continent stoma in February 2004 without complications18. Her post-operative course was unremarkable and on subsequent office visits, she was doing well with much improved bladder symptoms consisting primarily of infrequent bladder spasms and occasional urinary tract infections with no urethral incontinence. These were successfully treated with intravesical anticholinergic medication and antibiotics. On subsequent video urodynamic evaluation performed in June 2010 an augmented bladder with normal compliance and 500ml capacity was demonstrated. There was no evidence of uninhibited contractions, urgency, vesicoureteral reflux, or leakage with cough or Valsalva.\n\nIn addition to the above symptoms, the patient was noted to be complaining of intermittent gastric upset, joint pain and swelling, muscle aches, oral, vaginal, and rectal ulcers, and intermittent fevers over the past several years. As a result, she was referred for rheumatologic workup in early 2010 which was remarkable for an elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), but with all other tests, including ANA, anti-cardiolipin antibody, rheumatoid factor, ACE, HLA-B27, antithrombin III, and complement studies within normal limits. Given the symptoms that she was exhibiting in addition to the neurological symptoms she initially presented with, the patient was diagnosed with fibromyalgia and Behçet’s disease and started on aziathioprine and vitamin D supplementation, on which she remains to this day with stable symptoms.\n\n\nDiscussion of treatment and management\n\nThe above patient represents a diagnostic dilemma resulting in a change in diagnosis during the course of her treatment. Initially, the patient was diagnosed with MS on the basis of imaging findings as well as clinical symptoms that were consistent with the diagnosis, including CNS plaques in a distribution typically associated with MS, cranial nerve palsy, balance difficulties, and upper extremity tremors. However, CSF could not be obtained for evaluation, limiting the ability to differentiate between MS and other neurologic disorders. Furthermore, the patient initially presented with psychiatric symptoms that are atypical in the setting of MS, though an association between MS and psychiatric symptoms has been described16. Finally, additional serologies demonstrated no evidence for autoimmune disease or other findings that would suggest an alternate diagnosis. In addition, evaluation of evoked potentials was normal, which is unusual in the setting of MS, as 85% of MS patients have abnormal visual evoked potentials, 77% have abnormal somatosensory evoked potentials, and 67% have abnormal brainstem auditory evoked potentials. Nevertheless, it was not unreasonable to make an initial diagnosis of MS given the clinical and laboratory evidence at the time.\n\nDuring the course of the patient’s treatment, additional symptoms became manifest that resulted in a change in diagnosis from MS to BD. These symptoms included oral and anogenital ulcers, gastrointestinal upset, and joint pain and swelling. Serologies continued to be unremarkable save for a transient increase in erythrocyte sedimentation rate and CRP. Together with the previously described symptoms and findings on imaging, this led to a change in diagnosis to neurologic BD. Lamentably, no evaluation of HLA-B51 haplotype was undertaken in this patient, as the Bw51 allele is closely associated with BD19. In addition, the absence of CSF for analysis further hindered an early, accurate diagnosis in this patient.\n\nThe patient’s initial treatment, based on the MS diagnosis, included pulsed steroids and glatiramer acetate. It is likely that the patient derived benefit from steroids, as these were used over short periods during symptom exacerbations and have demonstrated efficacy in treatment of both MS and BD symptoms20,21. In contrast, it is less clear whether the patient derived significant benefit from the glatiramer acetate, as there appear to be no studies evaluating its use in BD, and while its use in MS is established, the relapsing/remitting nature of the patient’s symptoms and the possibility of a wrong diagnosis calls into question the efficacy of the drug as administered to this patient.\n\nFrom a urologic standpoint, the patient was not properly diagnosed by her urologists. She carried a clear neurologic diagnosis and her complaints were “overactive” in nature, not centered on stress urinary incontinence. The retropubic suspension aggravated her retention until she sought further medical care. She presented with neurogenic detrusor overactivity and detrusor-sphincter-dyssynergia along with symptoms that may be found in patients with both MS as well as BD22. Few studies exist examining micturitional disturbances in BD patients and the incidence of micturitional disturbances in patients with BD ranges between 5–67%23–25. In one study comprising eight patients with BD evaluated for lower urinary tract symptoms, seven demonstrated bladder dysfunction. The most common urodynamic finding was detrusor overactivity demonstrated in six patients, which is consistent with our findings. Bladder biopsies from these patients demonstrated blood vessel wall thickening and inflammatory infiltration of the lamina propria, consistent with prior findings by the same authors26, 27. Unfortunately, no tissue samples from our patient were available for pathologic analysis, preventing a comparison with the findings in the literature. This reported pathologic finding could result in loss of detrusor compliance.\n\nA larger study evaluating 24 subjects with neurological BD demonstrated a 50% incidence of detrusor overactivity and 12.5% incidence of detrusor-sphincter-dyssynergia3. These patients generally had high post-void residuals, early first sensation, and low-normal bladder capacities, which mimic the findings in our patient. In addition, the authors mapped the presence of CNS lesions in their subjects, demonstrating the majority in the brain stem with a subset in the cerebrum, which is consistent with other studies1. Our review of the images revealed no lesions in areas typically affected by BD or in areas that may affect micturition as described above. The high incidence of overactive bladder symptoms demonstrated in our patient is also echoed in several other studies5,23,24,28, as are our findings on urodynamics1.\n\nOur patient initially received anticholinergic medications for her overactive bladder symptoms with incomplete relief. Recommendations in the literature suggest an initial trial of anticholinergic therapy with the goals of preventing uninhibited contractions and lowering intravesical pressure, parameters which have been shown to improve with anticholinergic treatment in up to 75% of patients3,26. Our patient developed significant dry skin with oral anticholinergic medication and asked to be taken off the medication. While intravesical instillation of anticholinergic medications is an option that has been shown to be effective in patients with bladder symptoms due to neurological BD, this was not attempted in our patient24. Given the incomplete response to oral medications in our patient, as well as the desire to be free of an indwelling catheter in the setting of limited dexterity, ileocecal augmentation cystoplasty with a continent stoma was performed with a notable decrease in urgency symptoms and increase in bladder capacity postoperatively. Several studies have discussed the use of augmentation cystoplasty in patients with neurological BD resulting in improvements in bladder symptoms, continence status and renal function3,23,26. In agreement with these studies, we found our patient to have no uninhibited contractions as well as improved bladder compliance and increased bladder capacity after augmentation cystoplasty.\n\n\nConclusions\n\nIn summary, we describe a case of BD initially diagnosed as MS in a female with a neurogenic bladder secondary to her neurologic disease. The treatment of her neurologic disease was in line with the accepted standard of care for her diagnoses at the time they were made, as was the treatment of her neurogenic bladder. This case demonstrates the current challenges to accurate diagnosis of certain neurologic conditions and highlights the need for development of further sensitive and specific assays to facilitate these diagnoses, along with prudent use of urodynamic testing. Furthermore, this case demonstrates the efficacy of bladder augmentation with a continent catheterizable stoma in the setting of neurogenic detrusor overactivity in a patient with neurologic BD.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nAWP reviewed all patient information, performed a literature review, participated in intellectual discussion pertinent to the conception of the report, and drafted the manuscript. TBB is the physician of the patient presented herein, provided all patient information, and participated in intellectual discussion pertinent to the conception of the report. Both authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nA.W.P. is a K12 scholar supported by a Male Reproductive Health Research Career Development Physician-Scientist Award (grant HD073917-01) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Program (to Dolores J. Lamb).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nYesilot N, Mutlu M, Gungor O, et al.: Clinical characteristics and course of spinal cord involvement in Behçet’s disease. Eur J Neurol. 2007; 14(7): 729–737. PubMed Abstract \| Publisher Full Text\n\nDurrani K, Papaliodis GN: The genetics of Adamantiades-Behçet’s disease. Semin Ophthalmol. 2008; 23(1): 73–79. PubMed Abstract \| Publisher Full Text\n\nErdoğru T, Koçak T, Serdaroğlu P, et al.: Evaluation and therapeutic approaches of voiding and erectile dysfunction in neurological Behçet’s syndrome. J Urol. 1999; 162(1): 147–153. PubMed Abstract \| Publisher Full Text\n\nInaba G: Behçet’s Disease. In: Handbook of Clinical Neurology. edn. Edited by Vinken P. Amsterdam: Excerpta Medica; 1989; 593–610.\n\nSakakibara R, Hattori T, Boku K, et al.: Micturitional disturbance in neuro-Behçet’s syndrome. Auton Neurosci. 2000; 83(1–2): 86–89. PubMed Abstract \| Publisher Full Text\n\nRubinstein LJ, Urich H: Meningo-encephalitis of Behçet’s disease: case report with pathological findings. Brain. 1963; 86: 151–160. PubMed Abstract \| Publisher Full Text\n\nIshino H, Higashi H, Otsuki S: “Neuro-Behçet’s syndrome”. Case report with pathological findings. Folia Psychiatr Neurol Jpn. 1971; 25(1): 27–36. PubMed Abstract \| Publisher Full Text\n\nMotomura S, Tabira T, Kuroiwa Y: A clinical comparative study of multiple sclerosis and neuro-Behçet’s syndrome. J Neurol Neurosurg Psychiatry. 1980; 43(3): 210–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerkeni N, Zaraa I, Ayachi J, et al.: Behçet’s disease: A profile of mucocutaneous features. Acta Dermatovenerol Alp Panonica Adriat. 2010; 19(2): 11–15. PubMed Abstract\n\nAlpsoy E, Donmez L, Onder M, et al.: Clinical features and natural course of Behçet’s disease in 661 cases: a multicentre study. Br J Dermatol. 2007; 157(5): 901–906. PubMed Abstract \| Publisher Full Text\n\nCompston A, Coles A: Multiple sclerosis. Lancet. 2008; 372(9648): 1502–1517. PubMed Abstract \| Publisher Full Text\n\nLink H, Huang YM: Oligoclonal bands in multiple sclerosis cerebrospinal fluid: an update on methodology and clinical usefulness. J Neuroimmunol. 2006; 180(1–2): 17–28. PubMed Abstract \| Publisher Full Text\n\nGronseth GS, Ashman EJ: Practice parameter: the usefulness of evoked potentials in identifying clinically silent lesions in patients with suspected multiple sclerosis (an evidence-based review): Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology. 2000; 54(9): 1720–1725. PubMed Abstract \| Publisher Full Text\n\nBarkhof F, Filippi M, Miller DH, et al.: Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis. Brain. 1997; 120(Pt 11): 2059–2069. PubMed Abstract \| Publisher Full Text\n\nTintoré M, Rovira A, Martínez MJ, et al.: Isolated demyelinating syndromes: comparison of different MR imaging criteria to predict conversion to clinically definite multiple sclerosis. AJNR Am J Neuroradiol. 2000; 21(4): 702–706. PubMed Abstract\n\nLyoo IK, Seol HY, Byun HS, et al.: Unsuspected multiple sclerosis in patients with psychiatric disorders: a magnetic resonance imaging study. J Neuropsychiatry Clin Neurosci. 1996; 8(1): 54–59. PubMed Abstract \| Publisher Full Text\n\nHepgul N, Mondelli V, Pariante CM: Psychological and biological mechanisms of cytokine induced depression. Epidemiol Psichiatr Soc. 2010; 19(2): 98–102. PubMed Abstract\n\nSutton MA, Hinson JL, Nickell KG, et al.: Continent ileocecal augmentation cystoplasty. Spinal Cord. 1998; 36(4): 246–251. PubMed Abstract \| Publisher Full Text\n\nOhno S, Ohguchi M, Hirose S, et al.: Close association of HLA-Bw51 with Behçet’s disease. Arch Ophthalmol. 1982; 100(9): 1455–1458. PubMed Abstract \| Publisher Full Text\n\nMilligan NM, Newcombe R, Compston DA: A double-blind controlled trial of high dose methylprednisolone in patients with multiple sclerosis: 1. Clinical effects. J Neurol Neurosurg Psychiatry. 1987; 50(5): 511–516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerma KK, Tejasvi T, Verma K, et al.: Severe mucocutaneous Behçet’s disease treated with dexamethasone pulse. J Assoc Physicians India. 2005; 53: 998–999. PubMed Abstract\n\nNakipoglu GF, Kaya AZ, Orhan G, et al.: Urinary dysfunction in multiple sclerosis. J Clin Neurosci. 2009; 16(10): 1321–1324. PubMed Abstract \| Publisher Full Text\n\nTheodorou C, Floratos D, Hatzinicolaou P, et al.: Neurogenic bladder dysfunction due to Behçet’s disease. Int J Urol. 1999; 6(8): 423–425. PubMed Abstract \| Publisher Full Text\n\nSaito M, Miyagawa I: Bladder dysfunction due to Behçet’s disease. Urol Int. 2000; 65(1): 40–42. PubMed Abstract \| Publisher Full Text\n\nPorru D, Pau AC, Scarpa RM, et al.: Behçet’s disease and the neuropathic bladder: urodynamic features: case report and a literature review. Spinal Cord. 1996; 34(5): 305–307. PubMed Abstract \| Publisher Full Text\n\nCetinel B, Akpinar H, Tüfek I, et al.: Bladder involvement in Behçet’s syndrome. J Urol. 1999; 161(1): 52–56. PubMed Abstract \| Publisher Full Text\n\nCetinel B, Obek C, Solok V, et al.: Urologic screening for men with Behçet’s syndrome. Urology. 1998; 52(5): 863–865. PubMed Abstract \| Publisher Full Text\n\nKarandreas N, Tsivgoulis G, Zambelis T, et al.: Urinary frequency in a case of Neuro-Behçet disease involving the brainstem - clinical, electrophysiological and urodynamic features. Clin Neurol Neurosurg. 2007; 109(9): 806–810. PubMed Abstract \| Publisher Full Text" }	[ { "id": "19760", "date": "30 Jan 2017", "name": "John T. Stoffel", "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 interesting report on a patient with Behcet’s disease and urological symptoms. The neurological description of presentation, progression, and treatment is outstanding. The case report nicely reviews Behcet’s disease for the reader in a clear and concise way. The authors should be commended for highlighting this uncommon disease. The presentation of the urological symptoms and the relationship to Behcet’s disease in this patient are not as clear. My comments:\n\nThe introduction notes that the patient was treated with an ileocecal augment/continent stoma for neurogenic detrusor overactivity and DSD.The description, however, notes that she was in retention and could not void after a MMK procedure.Presenting fluro images would be helpful for the reader to better understand how the diagnosis of DSD was reached versus post procedural obstruction. By the history, she could not void after the MMK making it more likely that this is contributing to her retention. The discussion notes that the patient was not properly diagnosed by her urologists.Is it possible that she did have mixed incontinence prior to MMK and then developed complications from this procedure rather than a missed diagnosis of neurogenic DO? More data could be presented to highlight educational opportunities on what the authors feel the work up could have included prior to MMK to avoid the complication and to better work up neurogenic bladder patients. The authors could also touch on the role of Botox in treating neurogenic DO.", "responses": [] }, { "id": "23007", "date": "05 Jun 2017", "name": "Fereydoun Davatchi", "expertise": [ "Reviewer Expertise Behcet'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\nFirst, the authors have to prove that this patient has Behcet’s Disease. For that, they have give the exact medical history of the patient. We know approximately when the patient started to have Neuro-psychiatric manifestations, but we don’t know when the oral ulcers started and how long after the genital ulcers appeared.\nSecond, we have to know how long each attack of oral ulcer took to disappear. Then we have to know how long the duration between the two attacks was. Then after, we have to know how many ulcers appeared in each attack. Finally we have to know the exact clinical manifestations of the ulcers and their progression until their disappearance.\nThe same has to be given for genital ulcers.\nIt is primordial to remember that not any oral or genital ulcer is an aphthous ulcer, and only an aphthous ulcer can be used as a diagnostic criterion. There are many oral or genital ulcers that may resemble an aphthous lesion, to the eyes of a non-expert. It is why for case reports like this, a high definition picture of the lesion is essential to be sure of the nature of the lesion.\nOnce it is accepted that the oral lesion is an aphthous lesion, the authors have to prove that the genital ulcers were also aphthous ulcers. Once the presence of oral and genital aphthous ulcers is proved, one can say that the patient may have a Behcet’s Disease, because the patient fulfills the International Criteria for Behcet’s Disease (the ICBD). However, as said before, the patient may not have Behcet’s Disease. To be sure, one has to not find any other reason for the presence of the symptoms together.\nWhen it is sure that the patient has Behcet’s Disease, one has to show that the neurological manifestations are related to Behcet’s Disease. A patient can have Behcet’s Disease and another neurological disease like Multiple Sclerosis. In this case, the patient refused an examination of the Cerebrospinal fluid (CSF).\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": [] }, { "id": "23008", "date": "06 Jun 2017", "name": "Bertil Blok", "expertise": [ "Reviewer Expertise urinary incontinence", "neurourology" ], "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 'Case Report: A case of neurogenic bladder in the setting of Behçet's disease after an initial diagnosis of multiple sclerosis' is an interesting paper. However, I have some comments addressed below.\nThe case report is a readable report on an interesting topic. However, the authors do not report on any vaginal child delivery nor do they mention the BMI of the patient. Both are risk factors for stress urinary incontinence. It is very possible that before the MMK a mixed urinary incontinence was present and in retrospect it is always easy to say that the previous physicians did not do a good job.\nThe blaming distracts from the main important message that patients with a neurogenic bladder are different from patients without a neurogenic bladder. Both referring physicians and physicians who provided the irreversible surgical treatment were responsible for the patient. This means that also the general practitioner and neurologist should be informed and know to whom they send their patients to. On a regular basis we observe maltreatment because the referring physician did not care to refer his or her patient specifically to an expert in the field.\nSome attention should be given to treatment with botulinum toxin and midurethral tapes, which were also around when the bladder augmentation was given.\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/4-91
https://f1000research.com/articles/4-90/v1	10 Apr 15	{ "type": "Study Protocol", "title": "Design and conduct of Xtreme Everest 2: An observational cohort study of Sherpa and lowlander responses to graduated hypobaric hypoxia", "authors": [ "Edward Gilbert-Kawai", "Adam Sheperdigian", "Thomas Adams", "Kay Mitchell", "Martin Feelisch", "Andrew Murray", "Mark Peters", "Grace Gilbert-Kawai", "Hugh Montgomery", "Denny Levett", "Rajendra Kumar", "Michael Mythen", "Michael P. Grocott", "Daniel Martin", "Edward Gilbert-Kawai", "Adam Sheperdigian", "Thomas Adams", "Kay Mitchell", "Martin Feelisch", "Andrew Murray", "Mark Peters", "Grace Gilbert-Kawai", "Hugh Montgomery", "Denny Levett", "Rajendra Kumar", "Michael Mythen", "Michael P. Grocott" ], "abstract": "Objective: Oxygen availability falls with ascent to altitude and also as a consequence of critical illness. Because cellular sequelae and adaptive processes may be shared in both circumstances, high altitude exposure (‘physiological hypoxia’) assists in the exploration of the response to pathological hypoxia. We therefore studied the response of healthy participants to progressive hypobaric hypoxia at altitude. The primary objective of the study was to identify differences between high altitude inhabitants (Sherpas) and lowland comparators.Methods: We performed an observational cohort study of human responses to progressive hypobaric hypoxia (during ascent) and subsequent normoxia (following descent) comparing Sherpas with lowlanders. Studies were conducted in London (35m), Kathmandu (1300m), Namche Bazaar (3500m) and Everest Base Camp (5300m). Of 180 healthy volunteers departing from Kathmandu, 64 were Sherpas and 116 were lowlanders. Physiological, biochemical, genetic and epigenetic data were collected. Core studies focused on nitric oxide metabolism, microcirculatory blood flow and exercise performance. Additional studies performed in nested subgroups examined mitochondrial and metabolic function, and ventilatory and cardiac variables. Of the 180 healthy participants who left Kathmandu, 178 (99%) completed the planned trek. Overall, more than 90% of planned testing was completed. Forty-four study protocols were successfully completed at altitudes up to and including 5300m. A subgroup of identical twins (all lowlanders) was also studied in detail.Conclusion: This programme of study (Xtreme Everest 2) will provide a rich dataset relating to human adaptation to hypoxia, and the responses seen on re-exposure to normoxia. It is the largest comprehensive high altitude study of Sherpas yet performed. Translational data generated from this study will be of relevance to diseases in which oxygenation is a major factor.", "keywords": [ "Hypoxia", "Critical care", "Sherpa", "High altitude", "Microcirculation", "Nitric Oxide", "Mitochondria" ], "content": "Introduction\n\nA wide range of pathologies can lead to deterioration of a patient such that they require admission to a critical care unit. Critically ill patients are therefore a heterogeneous group of severely ill individuals, in whom hypoxaemia (a lack of oxygen in arterial blood) is common and may subsequently lead to cellular hypoxia1. The resulting cellular dysfunction may cause organ damage, and in some cases death2. Traditional management of established critical illness, based on efforts to increase convective oxygen delivery through augmented cardiac output, haemoglobin concentration and oxygenation, does not appear to improve outcomes3. Additionally excessive oxygen therapy may cause harm4,5. Consequently the role of alternative potential therapeutic targets such as the microcirculation, mitochondrial activity and nitric oxide (NO) are becoming increasingly more popular as alternative strategies6–9. Permissive hypoxaemia has also been proposed as a viable option for critically ill patients, to avoid the harms associated with invasive methods of restoring normoxaemia in the critically ill10.\n\nUnderstanding the mechanisms of human hypoxic adaptation in the context of critical illness is difficult. The wide range of underlying diseases and the complexity of treatment interactions with physiology make structured experiments challenging. The study of human responses to hypoxia occurring as a consequence of hypobaria at high altitude may be used as an alternative method of exploring elements of the pathophysiology of critical illness1,11,12. Studying healthy individuals progressively exposed to hypobaric hypoxia defines beneficial adaptive responses and may identify candidate pathways in the critically ill. Animal models, often proposed as being a suitable alternative to large-scale field studies, fail to match the complexity of human physiology in the critically ill patient13, and discordance between multiple models has been described14.\n\nIn 2007, the University College London (UCL) Centre for Altitude, Space and Extreme Environment Medicine (CASE Medicine) conducted the largest study of human volunteers at high altitude, Caudwell Xtreme Everest (CXE)12,15. Resulting data have emphasized the need for studying the microcirculation16–18, NO formation and metabolism19, and mitochondrial biology20–22. A further research programme (Xtreme Everest 2 (XE2)) was therefore proposed to address this23. XE2 was designed to study the physiological (especially microcirculatory, mitochondrial, NO-related metabolic and epigenetic) responses to hypobaric hypoxia in native lowlanders, and compare them to those in Sherpas23. Sherpas are descended from an ancestral high altitude population resident on the Tibetan plateau for over 500 generations24. Such high altitude populations show evidence of genetic selection25–29 that may underpin their anecdotally reported extraordinary tolerance to hypoxia. Their phenotype may therefore hold the key to successful hypoxic adaptation in humans.\n\nWe describe the design and conduct of XE2, our approach to high altitude translational research, and discuss the strengths and weaknesses of this programme of investigation.\n\n\nProtocol\n\nXE2 (December 2012 to May 2013) was undertaken by the Xtreme Everest Oxygen Research Consortium (XE-ORC), which comprises a partnership between the UK’s UCL CASE Medicine, the University of Southampton Centre for Human Integrative Physiology (CHiP), and Duke University Medical Centre (DUMC) in the USA.\n\n\nEthical approval\n\nThe study design, risk management plan and protocols were approved (in accordance with the declaration of Helsinki) both by the UCL Research Ethics Committee and the Nepal Health Research Council (NHRC).\n\n\nEnrolment and informed consent\n\nAll potential participants, recruited through word of mouth and advertisement, were given written information about the study. Our Nepali Research Leader (RKBC) translated this locally in Nepal. Opportunities for questions, in person or over the telephone, were offered at multiple stages in both countries, and all participants submitted written consent for participation in the studies. At all stages throughout the expedition, independent translators were present to allow for communication between Sherpas and investigators.\n\n\nMedical screening\n\nEligible participants were lowland children aged 8 to 17 years, or adults (aged 18 years or above) of either lowland or Sherpa origin. For the lowland participants, an independent expert experienced in mountain medicine identified those fit to travel to altitude by reviewing a health-questionnaire (supplementary material) and telephone interviews as required. The forms of those selected were then screened by the expedition Chief Medical Officer (GGK) to confirm fitness both to travel to altitude, and to participate in research. Potential participants considered ‘at risk’ were either telephoned or reviewed in person by GGK. Where appropriate, and with permission, the participant’s medical practitioner was contacted. Those with significant cardiac or respiratory disease (e.g. severe chronic obstructive airway disease, ischemic heart disease with angina, or symptomatic heart failure) were excluded. For the Sherpa subjects, two doctors performed a pre-recruitment screening interview in Nepali based on the health-questionnaire. One doctor was a local Nepali (RKBC) and one an expert experienced in mountain medicine and XE2 investigator (DL/MG) who confirmed fitness for travel to altitude and research. As all participants would be undergoing cardiopulmonary exercise testing (CPET), additional exclusion criteria based on the American Thoracic Society/American College of Chest Physicians guidelines for clinical exercise testing were also adhered to30.\n\n\nMedical safety\n\nAt each laboratory, an independent Medical Officer (MO) was responsible for decisions relating to the participants involvement with research protocols, the administration of medication and ascent or descent from their current altitude. Participants were recommended not to self-medicate, but to consult either the site MO, or their trek leader when between laboratory sites. In order to standardize medical care, clear guidelines for common altitude and non-altitude related conditions were formulated prior to departure, and adhered to. Medication use was recorded by the individual (in a standardized daily diary) and by the trek leader or MO.\n\n\nStudy participants\n\nIn total, 187 participants were selected for inclusion in the study and underwent baseline testing. Sherpas were defined as direct descendants (for two generations) of Nepali Sherpas, drawn from communities in the Solukhumbu and Rowaling valleys. Sherpa participants were recruited by word of mouth through local community contacts and were required to have evidence of two generations of all Sherpa ancestors (parents and grand-parents). A detailed altitude history was then taken from all Sherpa participants including their altitude in utero, at birth, during childhood, in adulthood, and for the 12 months preceding XE2. Lowlanders were recruited in the UK; all were born and lived below 1000m, and were not from a native high altitude population (e.g. Tibetan, Andean, Ethiopian). They included European, American and South African participants. Lowlanders were divided into four cohorts:\n\nCore - eligible for all studies; eight of these participants were monozygotic twins (four pairs), for a pilot epigenetics study.\n\nNitrate metabolism - allocated only to be involved in a study of whole body NO production.\n\nInvestigators - who conducted the studies and remained at altitude for the duration of the expedition permitting studies of exposure to chronic hypoxia.\n\nChildren - who took part in the Young Everest 2 Study (YES2) expedition linked to XE2 in which children ascended to Namche Bazaar (NB) to participate in a programme of non-invasive studies. YES2 will not be discussed further in this manuscript.\n\nPotential investigators were sourced through word of mouth at CASE Medicine events and interviewed prior to their appointment (EGK, DM, KM).\n\nAll participants were required to provide baseline information that included details of previous altitude exposure and the occurrence of any altitude-related illnesses. Importantly, within the Core and Investigator cohorts, some selected participants had previously taken part in CXE (2007), thereby allowing the evaluation of consistency in the individual response to an identical high altitude exposure.\n\nOf the 187 participants that were selected for the study and underwent baseline testing, 180 departed for high altitude investigations (Figure 2). Of the seven participants that withdrew from the study prior to ascent to altitude, six lowlanders withdrew in London (LDN) (three for medical reasons), and one Sherpa withdrew in Kathmandu (KTM). Additionally, in LDN one person did not meet the American Thoracic Society/American College of Chest Physicians guidelines for clinical exercise testing, and was withdrawn from CPET prior to departure. Baseline characteristics for each cohort are listed in Table 1.\n\nOf the 104 lowlanders (children excluded), and 64 Sherpas leaving KTM, 102 (98%) and 64 (100%) reached Everest base camp (EBC) respectively. Of the two who did not reach EBC, one dropped out at NB, and the other at Pheriche due to gastrointestinal and cardiovascular problems respectively. All the Investigators reached their allocated laboratories, however, one left EBC early for medical reasons, and two for personal reasons. Two investigators left NB early for personal reasons, and one investigator left NB early to move to EBC. One investigator also arrived late at EBC.\n\n\nStudy setting\n\nBaseline ‘normoxic’ (35m) data for lowlander Core and Investigator groups were collected at The London Clinic Hospital, from 3rd December 2012 to 25th January 2013. Sherpa baseline testing was performed in KTM (1300m; 4th March to 16th April 2013). Between baseline testing and trek ascent, participants remained below 3000m in order to avoid hypoxic exposure prior to the expedition.\n\n\nAscent profile\n\nParticipants trekked in groups of up to 14. All lowlanders flew to KTM and spent one night there prior to flying to Lukla (2800m). Similarly, Sherpas flew from KTM to Lukla at the beginning of the trek. In order to ensure that study participants were exposed to an identical pattern of hypoxia exposure, all participants followed an identical ascent and descent profile (Figure 1). High altitude field laboratories were situated at NB (3500m) and EBC (5300m). The KTM laboratory was also used for descent testing for participants following their return from EBC. Barometric pressure, temperature, and humidity data were recorded twice daily at each laboratory and are summarized in Table 2.\n\nKey: LDN = London, KTM = Kathmandu, NB = Namche Bazaar, EBC = Everest Base Camp.\n\nKey: NB = Namche Bazaar, EBC = Everest Base Camp.\n\nKey: NB = Namche Bazaar, EBC = Everest Base Camp.\n\nKey: LDN = London, KTM = Kathmandu, NB = Namche Bazaar, EBC = Everest Base Camp.\n\nThe ascent to EBC from KTM was completed over 11 days with incorporated rest days built in to the schedule to reduce the likelihood of acute mountain sickness (AMS). This ascent profile was identical to the CXE profile both because it was proven safe with minimal participant dropouts and because it would allow comparison of data between the two expeditions12.\n\nThe Investigator cohort underwent a similar ascent profile to the rest of the participants, but then remained in situ at their relevant laboratories for six weeks prior to descent. By using repeated testing at serial intervals, we were able to study the effects of both acute and chronic hypoxia on these participants. It should also be noted that upon their arrival at EBC, the Investigators were required to construct the laboratory and set up equipment, and consequently testing was started two days after arrival.\n\n\nLaboratory testing and studies\n\nUpon arrival at each laboratory participants all were given a camp safety and science brief, and individualized timetables. Within these, in an attempt to minimize diurnal variations in physiological responses, participants were tested for each study at the same time at each site. Furthermore as many studies required abstinence from caffeine and food for specific periods, fasting periods and meal times were clearly highlighted for each individual. Within each laboratory, the timetable was adhered to and subjects were tested for particular studies on either day one or day two. These specific days were kept constant throughout the trek so as to control for the effects of acute acclimatization and responses over time. Because of the potential interaction between some studies, not every participant underwent all of the studies. The XE2 research portfolio, detailing core studies, additional studies and those in common with CXE, are highlighted in Table 3 and Table 4.\n\n^ = Also studied on CXE\n\nConducted in: Ψ = Sherpas, Φ = Lowlanders (Core), Ω = Investigators, ξ = Nitrate\n\n^ = Also studied on CXE\n\nConducted in: Ψ = Sherpas, Φ = Lowlanders (Core), Ω = Investigators\n\n\nBiological sample storage and transport\n\nBlood, urine, saliva and exhaled breath condensate samples were all kept in -40°C freezers at each site. Muscle biopsy specimens (LDN, KTM and EBC) were stored in liquid nitrogen (-196°C), and then shipped to the UK on dry ice. All samples were brought from the field to KTM by helicopter and then repatriated to the UK on dry ice (-78°C).\n\n\nOutline of analysis plan\n\nData analysis will follow a predetermined strategy of:\n\ni) Description of phenotypes for each of the studies as outlined in Table 3 and Table 4 including plasma biomarkers and metabolomic analyses. This will include comparison with data obtained during a matched ascent in the CXE 2007 study (e.g. intra-individual comparison of the phenotypes from individuals who were participants in XE2 and CXE) as well as sub-group analyses (e.g. twins).\n\nii) Comparison of phenotypic adaptations between Sherpas and lowlanders during ascent and descent. We hypothesise that Sherpas will have a phenotype at baseline that is better adapted to hypoxia in comparison to lowlanders, that lowlanders ascending to altitude will tend to converge on the Sherpa phenotype, and that the Sherpa baseline phenotype will be less perturbed by altitude exposure than the lowlander baseline phenotype.\n\niii) Integrative analysis of genotype-epigenome-transcriptome-phenome across multiple datasets (XE2, CXE). The XE2 dataset will contribute to the accumulated Extreme Everest BioResource acquired during a number of high altitude research expeditions. Data will be incorporated into our bespoke comprehensive database, which enables linkage of all data elements with meta-data describing the provenance of each data item through the use of semantic web technology. Key questions will be raised in an iterative manner, driven both by a priori hypotheses and subsequently by data mining focused on the results obtained by unbiased outputs from individual ‘omics analyses.\n\nResults from this study will be disseminated in peer-reviewed journals, on the Xtreme Everest website (www.xtreme-everest.co.uk) at scientific conferences and at public meetings.\n\n\nDiscussion and conclusions\n\nWe have characterized many features of the human response to progressive hypobaric hypoxia in a cohort of 180 individuals; 44 individual studies being safely conducted over four locations up to 5300m. In addition, the response to relative re-oxygenation was studied. The standardized ascent protocol ensured that differences between participants reflected inter-individual variability in hypoxic adaptation, as opposed to variability in hypoxic exposure. In matching the 2007 CXE ascent profile, the data from the two studies may also be combined to create a single cohort. The parallel study of lowlanders and highlanders will permit the identification of beneficial phenotypic adaptations and genetic alteration (and their relationships) in these groups. The study of investigators exposed to sustained (six weeks) hypoxia facilitated study of immediate and longer-term adaptive processes.\n\nThe slow ascent profile minimized symptoms of AMS, increasing the number of participants successfully reaching EBC and available for the study. Despite good medical care, gastrointestinal illness may have occasionally confounded results. However, the application of standardized medical protocols, with detailed documentation of illness and medication, will ensure this can be accounted for. Whilst the expedition was promoted through word of mouth and public advertisement, the very nature of the trek itself meant that participants were of a self-selected nature as enthused to undertake a rigorous trek. They may thus not be representative of the general population. Laboratories in Nepal were temporary as opposed to permanent structures. Temperature and pressure fluctuations, both having the potential to confound data obtained, were recorded on a daily basis (Table 2). Whilst we attempted to maintain a constant temperature between all laboratories through the use of heaters, we accept that any measured differences may confound the results.\n\nWe believe that the wealth of data obtained from XE2 will aid our understanding of the human adaptive response to hypoxia, offering insights that may be of value to patients suffering from sub-acute hypoxemia as a result of critical illness.", "appendix": "Author contributions\n\n\n\nDM, MG, MM, KM and DL conceived the study. EGK, DM, TA, AS, MF, AM, MP, HM, DL and MG designed the experiments. EGK, AS, TA, KM, AM, MP, GGK, DL, RK, MM and DM carried out the research. EGK and DM prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNo grant funding supported this work; financial contributions were provided by the following organisations: Xtreme Everest 2 was supported by the Royal Free Hospital NHS Trust Charity, the Special Trustees of University College London Hospital NHS Foundation Trust, the Southampton University Hospital Charity, the UCL Institute of Sports Exercise and Health, The London Clinic, University College London, University of Southampton, Duke University Medical School, the United Kingdom Intensive Care Society, the National Institute of Academic Anaesthesia, the Rhinology and Laryngology Research Fund, The Physiological Society, Smiths Medical, Deltex Medical, Atlantic Customer Solutions and the Xtreme Everest 2 volunteer participants who trekked to Everest Base Camp.\n\nSome of this work was undertaken at University College London Hospital- University College London Biomedical Research Centre, which received a proportion of funding from the United Kingdom Department of Health’s National Institute for Health Research Biomedical Research Centres funding scheme. Some of this work was undertaken at University Hospital Southampton-University of Southampton Respiratory Biomedical Research Unit, which received a proportion of funding from the United Kingdom Department of Health’s National Institute for Health Research Biomedical Research Units funding scheme.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nMembers of the Xtreme Everest 2 Research Group are as follows: S Abraham, T Adams, W Anseeuw, R Astin, B Basnyat, O Burdall, J Carroll, A Cobb, J Coppel, O Couppis, J Court, A Cumptsey, T Davies, S Dhillon, N Diamond, C Dougall, T Geliot, E Gilbert-Kawai, G Gilbert-Kawai, E Gnaiger, M Grocott, C Haldane, P Hennis, J Horscroft, D Howard, S Jack, B Jarvis, W Jenner, G Jones, J van der Kaaij, J Kenth, A Kotwica, R Kumar BC, J Lacey, V Laner, D Levett, D Martin, P Meale, K Mitchell, Z Mahomed, J Moonie, A Murray, M Mythen, P Mythen, K O’Brien, I. Ruggles-Brice, K Salmon, A Sheperdigian, T Smedley, B Symons, C Tomlinson, A Vercueil, L Wandrag, S Ward, A Wight, C Wilkinson, S Wythe.\n\nMembers of the Xtreme Everest 2 Research Scientific Advisory Board: M Feelisch, E Gilbert-Kawai, M Grocott (chair), M Hanson, D Levett, D Martin, K Mitchell, H Montgomery, R Moon, A Murray, M Mythen, M Peters.\n\nXtreme Everest 2 is a research project coordinated by the Xtreme Everest Oxygen Research Consortium, collaboration between the UCL Centre for Altitude, Space, and Extreme Environment Medicine, the Centre for Human Integrative Physiology at the University of Southampton and the Duke University Medical Centre.\n\n\nReferences\n\nGrocott M, Montgomery H, Vercueil A: High-altitude physiology and pathophysiology: implications and relevance for intensive care medicine. Crit Care. 2007; 11(1): 203. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAldenderfer MS: Moving up in the world; archaeologists seek to understand how and when people came to occupy the Andean and Tibetan plateaus. Am Sci. 2003; 91(6): 542–550. Publisher Full Text\n\nBigham A, Bauchet M, Pinto D, et al.: Identifying signatures of natural selection in Tibetan and Andean populations using dense genome scan data. PLoS Genet. 2010; 6(9): e1001116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalacrida S, Katsuyama Y, Droma Y, et al.: Association between human polymorphic DNA markers and hypoxia adaptation in Sherpa detected by a preliminary genome scan. Ann Hum Genet. 2007; 71(Pt 5): 630–638. PubMed Abstract \| Publisher Full Text\n\nPeng Y, Yang Z, Zhang H, et al.: Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas. Mol Biol Evol. 2011; 28(2): 1075–1081. PubMed Abstract \| Publisher Full Text\n\nXu S, Li S, Yang Y, et al.: A genome-wide search for signals of high-altitude adaptation in Tibetans. Mol Biol Evol. 2011; 28(2): 1003–1011. PubMed Abstract \| Publisher Full Text\n\nYi X, Liang Y, Huerta-Sanchez E, et al.: Sequencing of 50 human exomes reveals adaptation to high altitude. Science. 2010; 329(5987): 75–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nATS/ACCP Statement on cardiopulmonary exercise testing. American Thoracic Society; American College of Chest Physicians. Am J Respir Crit Care Med. 2003; 167(2): 211–277. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8288", "date": "05 May 2015", "name": "James E. Clark", "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\nThis article described the planning and undertaking of the study design for the Xtreme Everest 2 research expedition which looks at Sherpa and lowlander responses to graduated hypobaric hypoxia.The authors are experienced in this kind on study having already worked on Xtreme Everest 1 study some years ago. The objectives are stated clearly and the rationale for the study described with the underlying physiology. Part of the difficulty of this kind of study is the recruitment process. It would be hard to recruit widely for this kind of expedition as the subjects are usually self-selecting, those people who are keen to explore and ascent to altitude. However the authors have explained their recruitment methods and the study participant information has been shown.The study itself is fairly straightforward and includes a gradual, measured ascent to altitude and a series of tests undertaken prior, during and after ascent and the descent following the expedition. Within the basic study there is a cohort of subjects taking part in the \"nitrate\" study, to investigate whether dietary nitrate supplementation has an impact on hypoxic tolerance and adaptation to altitude. A comprehensive table of outcomes and measurements is given which shows the magnitude of the study and the way in which the investigators aimed to separate the workload between cohorts. The measures taken are widespread and range from skeletal muscle mitochondrial function to blood composition and numerous cardiovascular and respiratory functional analysis for which this group of investigators have considerable experience. The data from this study will be beneficial to the field of hypoxia, altitude performance and will give us a better understanding into the adaptations found in those living at altitude. This will translate into patient care in the clinical scenario as hypoxia and oxygen delivery can affect many in intensive care.", "responses": [] }, { "id": "8809", "date": "28 May 2015", "name": "Jean-Paul Richalet", "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\nThe article by Gilbert-Kawai and co-workers presents the design of Xtreme Everest 2 expedition, which looks like a continuation of the first Caudwell Xtreme Everest expedition led in 2007.They present a complete description of protocols and groups of subjects. Part of the experiments concern classical domains already explored by many scientific expeditions (exercise capacity, hemglobin, ventilation, heart rate variability, etc..). Some aspects are more interesting and original, such as epigenetics, microcirculation, study of twins and Sherpas, although some important information are lacking concerning, for example the tissue studied for epigenetics, if Sherpas are involved in the epigenetic study, etc.Altogether, we can hope that a lot of new data will be available for a better understanding of physiological responses to hypoxia, and a greater number of publications than for the first Xtreme Everest expedition is expected.The argument that was often put forward to justify this extremely complex and costly type of experimental design is that it might be of great value for patients suffering from critical illnesses associated with hypoxia. I think this argument is unnecessary. The study of normal persons exposed to hypoxia is, by itself, very interesting and worthwhile, and does not need any justification. Applications to patients of what will be found is probable but impossible to predict.", "responses": [] } ]	1	https://f1000research.com/articles/4-90
https://f1000research.com/articles/4-88/v1	08 Apr 15	{ "type": "Research Article", "title": "Sex differences in cortical and subcortical human brain anatomy", "authors": [ "Timothy J. Herron", "Xiaojian Kang", "David L. Woods", "Xiaojian Kang", "David L. Woods" ], "abstract": "Previous research has reported many sex differences in cortical and subcortical anatomy, but only a subset of findings is consistent across studies. Here, we used improved Freesurfer-based automated methods to analyze the properties of the cortex and seven subcortical structures in young, right-handed subjects (69 male and 69 female), carefully matched in age and education. Significant sex differences were observed. Females had greater gyral complexity (i.e., greater bending energy). In contrast, males had greater unadjusted cortical surface area (+10.3%), but area differences were reduced (to +2.8%) when area was adjusted for total intracranial volume (ICV). There were no significant omnibus sex differences in cortical thickness. Males showed larger unadjusted subcortical gray matter structural volumes, as well as larger ICV-adjusted volumes in the amygdala. These results help to resolve some of the inconsistencies in previous studies of sex differences in brain anatomy.", "keywords": [ "Gender", "Frontal", "Temporal", "Parietal", "Occipital", "Limbic", "Normalization", "Scaling" ], "content": "Introduction\n\nBrain structure has been reported to differ between male and female brains (Bostan et al., 2013; Inano et al., 2013; Kim et al., 2012; Koolschijn & Crone, 2013; Ruigrok et al., 2014), but locations showing sex differences have varied across studies. In the present study, we used novel, improved automated methods to analyze sex-related differences in brain structure in 69 right-handed males and 69 right-handed females, who had been carefully matched in age and education. By establishing consistent sample characteristics, image scan quality, segmentation, and corrections for overall intracranial volume, we were able to identify sources of inconsistency in previous studies, and provide a clearer picture of sex differences in brain anatomy. We use two independently collected and previously analyzed, high-quality anatomical image sets to increase confidence in our results.\n\nSex differences in overall brain size are consistently observed in all studies: adult males have 7.4%–11.5% greater gray matter brain volume than females and show a 10.4%–13.6% increase in intracranial volume (ICV) (Good et al., 2001; Ruigrok et al., 2014). Differences between the sexes of roughly this magnitude are evident by age five (Brain Development Cooperative, 2012; Koolschijn & Crone, 2013), or even earlier in development (Dekaban, 1977; Luders & Toga, 2010). Substantial differences in volume remain even when accounting for body size difference (Sacher et al., 2013), although it should be noted that no body size covariate has been yet found to be fully adequate to account for overall brain size within most human adult populations (Peters et al., 1998).\n\nWhen considering regional sex differences however, results have been less consistent across studies. As seen in Table 1, classical results report inconsistent sex differences throughout in the cortex, but a recent meta-analysis of several voxel-based morphometry (VBM) studies shows suggested reliable regional sex-related variations (Good et al., 2001; Ruigrok et al., 2014). However, most of the VBM differences have not been replicated in more accurate surface-based measurements of thickness and area/volume (Luders et al., 2006; Sowell et al., 2007). Considering surface-based studies, the results in Table 1 suggest that females may hold relative size advantages in superior temporal cortex, parietal lobe, and frontal lobe, but there appears to be little agreement on which hemisphere and/or intra-lobar regions are affected, suggesting that the results may vary with the analytical procedure used (Fischl et al., 1999b; Im et al., 2010; Jiang et al., 2013; Leonard et al., 2008; Luders et al., 2006; Sowell et al., 2007; Van Essen, 2005). Critically, there is no single widely-used standard for normalizing brain volumes to ICV, an issue contributing to the inconsistency in results, with some groups asserting that sex differences largely disappear when the overall gray matter (GM) difference noted above is removed with proper scaling covariates (Im et al., 2008).\n\nMagenta; Female > Male; Cyan: Male > Female, L Left Hemisphere, R Right Hemisphere (a) Center column: Consistent VBM results from a recent meta-analysis (Ruigrok et al., 2014): (b) Right Columns: Area and thickness cortical measurements and subcortical volume measurements of N: Luders et al., 2006, G: Luders et al., 2009, I: Im et al., 2006. B: Brain Development Cooperative Group, 2012; J: Jiang et al., 2013; T: Inano et al., 2013; F: Fjell et al., 2009, K: Koolschijn Crone, 2013; S: Sowell et al., 2007; Z: Im et al., 2008; Y: Lentini et al., 2013. X: Neufang et al., 2009.\n\nStudies of sex differences in subcortical structures have also produced conflicting results (Table 1). For example, VBM studies show greater relative hippocampal volume in men than women, while other studies show the opposite result (Inano et al., 2013; Sowell et al., 2007; Szabo et al., 2006). Similar inconsistences are found when examining sex differences in the basal ganglia (Filipek et al., 1994; Inano et al., 2013) and the thalamus (Bramen et al., 2011; Inano et al., 2013). Ultimately, age-related changes may be playing a crucial role in these discrepant results (Sowell et al., 2004). However, the amygdala is one structure that appears consistently larger in men in most studies (Goldstein et al., 2001; Good et al., 2001) (Inano et al., 2013).\n\nThese inconsistencies in the literature on sex differences may be caused by many factors, including differences in sample characteristics, image scan quality, segmentation approaches, and statistical procedures. In the present study, we address some of these inconsistencies by using a well matched, young, healthy population of male and female subjects, imaged by one operator on a single scanner, and analyzed with automated segmentation and coregistration procedures using multiscale data analysis. Studying a well-matched young adult population avoids the potential problem of differential age-related changes in brain size rates of change for males and females (Crivello et al., 2014; Thambisetty et al., 2010) (Fjell et al., 2009). The young population also avoids as well the issue of aging-driven brain water content loss (Bansal et al., 2013) that affects image contrast and may influence cortical thickness measurements.\n\nMultiscale analysis identifies the spatial scales at which sex differences manifest themselves. Isolating the scale at which we find differences or the lack thereof can help with confirming possible mechanisms of sex differences: e.g. an overall brain size difference without further relative differences on finer scales is consistent with a single-factor that may influence early development such as sex hormones or genetic sex differences. Also, multiscale analysis is important for proper interpretation of the results in cases where size differences are roughly similar across the cortex; e.g., it avoids claiming that some areas that are just above a statistical threshold show significant differences while others, just below threshold, do not. Rather, a multiscale (nested) analysis might rather suggest that there are sex differences at a coarse spatial scale but no further significant residual differences between areas at finer spatial scales.\n\nThe examination of sex differences faces an additional challenge mentioned previously: defining the appropriate scaling coefficients to correct for the overall difference in brain size of male and female subjects. Therefore, we briefly review the literature examining the relative size of brain structures in different mammalian species, and then empirically define the coefficients that best relate human cortical and subcortical structures to overall brain size when different metrics (e.g., cortical volume, area, and thickness) are used. Finally, we use the optimal scaling coefficients computed from a secondary dataset to characterize sex-related differences in brain structure that are independent of overall differences in brain size.\n\n\nMethods\n\nThe anatomical images of 138 young, right-handed subjects were processed by FreeSurfer (http://surfer.nmr.mgh.harvard.edu), whose automated segmentation procedures also parcellated the cerebral cortical surface (Desikan et al., 2006) and subcortical structures (Fischl et al., 2002; Fischl et al., 2004). Interhemispheric asymmetries and sex differences in the cerebral cortex were examined using three cortical surface metrics: surface curvature bending energy, surface area, and cortical thickness. Differences in subcortical structure were evaluated with volume measures. Sex differences and sex by hemispheric interactions in cortical and subcortical structure were analyzed using male and female subjects carefully matched in both age and education.\n\nWe studied the cortical and subcortical anatomy of 138 young subjects recruited from several colleges and military veteran sources in the northern California area, a cohort that we have used in a previous method manuscript (Kang et al., 2012). All subjects were right handed by self-report. There were 69 male and 69 female subjects, who were carefully matched in age (females: 18–38 years, mean 26.3 years; males: 18–38 years, mean 26.1 years) and education (approximately 15 years for both females and males). All subjects gave informed written consent following procedures approved by Institutional Review Board #1 (IRB00000615; Federal Wide Assurance #FWA00001687) of the Northern California Health Care System of the U.S. Department of Veterans Affairs. We also used an auxiliary group of high quality T1 images contained in a public repository, the OASIS dataset (http://www.oasis-brains.org; (Marcus et al., 2007)). This independent dataset was used to parameterize our volume normalizations using estimated intracranial volume as detailed below. In particular, we used a young, healthy subset of right-handed OASIS subjects containing 65 males (mean age 22.9; range 18–32) and 65 females (mean age 22.8, range 18–32) and processed them identically to images from our primary dataset.\n\nTwo high-resolution T1 anatomical images (TR = 15 ms, TE = 4.47 ms, Flip Angle = 35o, voxel size 0.94 × 1.30 × 0.94 mm) were acquired on a 1.5 T Philips Eclipse scanner. These anatomical images were re-sampled to 1 × 1 × 1 mm resolution, averaged, segmented and then inflated to the cortical surface using FreeSurfer (Dale et al., 1999; Fischl et al., 1999a). The inflated cortical surfaces of the left hemisphere (LH) and right hemisphere (RH) were then co-registered to a spherical coordinate system (Fischl et al., 1999b). Figure 1A–D show the inflated cortical surfaces and spheres of LH averaged across all the 138 subjects. Six anatomical areas were identified (Kang et al., 2012) on the cortical surfaces based on the neuroanatomical parcellation (Desikan et al., 2006): Front Lobe (FL), Insular Cortex (IC), Limbic Cortex (LC), Occipital Lobe (OL), Parietal Lobe (PL) and Temporal Lobe (TL). The spherical view of the averaged LH was further projected to a flat map, as shown in Figure 1E, using Mollweide projection to visualize the entire 3D cortical surface in two dimensions (Kang et al., 2012).\n\nLateral (A) and medial views (B) of the inflated left hemisphere (LH) averaged across 138 subjects. (C) and (D) are the two views of the averaged sphere of LH when co-registered to the spherical coordinate system by FreeSurfer. (E) shows the Mollweide projection map of the averaged spherical surface of LH. The temporal and occipital lobes were positioned in the front/central area of the map. Anatomical areas were shown by the color contours. FL: Front Lobe; IC: Insular Cortex; IHC: Inter-Hemispheric Connection; LC: Limbic Cortex; OL: Occipital Lobe; PL: Parietal Lobe; TL: Temporal Lobe.\n\nFreeSurfer also provides the labels and statistical analysis of the segmented subcortical structures (Abe et al., 2010; Fischl et al., 2002; Fischl et al., 2004). Figure 2 shows the seven FreeSurfer-segment subcortical structures discussed herein: i.e., the cerebellum, thalamus, caudate, putamen, pallidum, hippocampus, and amygdala.\n\nThe labels indicate the structures on the right hemisphere.\n\nAnatomical features on the cortical surface obtained in FreeSurfer, like surface curvature, bending energy, surface area, and cortical thickness, were extracted and resampled from each individual subject into the hemispherically unified coordinate system on the Mollweide projection map. Cortical surface bending energy is the area-weighted square of mean curvature after subtracting hemispheric average mean curvature. Bending energy better reflects the number of gyri and sulci in a region compared to other common cortical folding measures (Pienaar et al., 2008). Here we use the Bending Energy Density (BED), dividing out by the total area of a region, in order to use a quantity that is not highly correlated with surface area. The surface area associated with a vertex is the averaged area of all the triangles associated with that vertex on the surface (Fischl et al., 1999a). The total surface area of a region or hemisphere is the summation of the area of all vertices that it includes (Winkler et al., 2012). The above anatomical parameters for LH and RH were compared without correction for intra-cranial volume (ICV) or age. The volumes of the automatically segmented subcortical structures were also compared using linear regressions, with ICV as a covariate.\n\nSeveral methods have previously been used to normalize gray matter volume results for both cortical and subcortical structures, most commonly either by using simple normalization (division) by a total brain volume, or by normalizing brain volumes in stereotaxic (MNI or Talairach) space. However, it is well-established that gray matter scales sublinearly, and white matter (WM) supralinearly, with respect to overall cranial or brain volume across mammals in general (Zhang & Sejnowski, 2000), and across primates in particular (Herculano-Houzel et al., 2010; Ventura-Antunes et al., 2013). Because the relationship between ICV and size may vary from structure to structure (Bush & Allman, 2003), more sophistical scaling is required (Im et al., 2008). Therefore, we used the auxiliary OASIS dataset to establish the fractional powers by which subcortical gray matter structures (e.g. putamen, amygdala, etc.) and total cortical area and thickness vary with respect to total ICV, as computed using FreeSurfer (Buckner et al., 2004). This was accomplished by using linear regression over logarithmically transformed ICV and log-transformed structure volumes (or cortical area or thickness). ICV to the estimated power was then used as a covariate within subsequent normalized regression analyses to examine sex differences.\n\nThe sex differences of the cortical surface parameters and the volumes of the subcortical structures were analyzed both without ICV correction and with ICV correction using empirically optimized regression coefficients. We compared our data to published results (Leonard et al., 2008; Li et al., 2014; Luders et al., 2004; Luders et al., 2006; Ruigrok et al., 2014; Sowell et al., 2007). Unlike most previous studies, the male and female subgroups were balanced in age and education.\n\nMultivariate linear regression (using R’s lm function v. 2.15.3 and mle4 library: r-project.org) over sex, age, ICV and a dependent variable of overall subcortical structure volume or cortical area/thickness/bending, either hemispherically averaged or differenced, were used to analyze overall sex and hemispheric differences. Purely categorical within-subject factors were analyzed using repeated measures ANOVA (using CLEAVE: www.ebire.org/hcnlab) with Geisser-Greenhouse non-sphericity corrections uniformly applied. Correlations were computed using MATLAB (v7.14) statistics toolbox (www.mathworks.com), with Pearson correlations used to analyze pairwise relationships and with partial correlations to control for covariates. Because we were looking simultaneously at many effects, we adjusted statistical thresholds so that p<0.01 would indicate a trend, p<0.001 weak statistical significance, and p<0.0001 to indicate a strong statistical relationship. We also report effect size estimates (η) in ANOVA results, and regression coefficients to clarify effect magnitude.\n\n\nResults\n\nThe results are presented as follows: we first give a whole-cortex overview of how our anatomical quantities are related to each other and to the relevant demographic variables. Next, we characterize how ICV is best related to anatomical quantities as well as test ICV’s relationship to local cortical asymmetry. Then we record the sex difference results for the whole cortex, cortical regions, and subcortical anatomical structures.\n\nWe characterized the relationships between whole cortex area, thickness, and BED mean values using full partial Pearson correlations, i.e. pairwise correlations factoring out all other considered quantities. In addition to relating age, sex, and ICV to mean area, thickness, and BED, we also add the three quantities of mean left hemisphere minus mean right hemisphere area, thickness, and BED for concurrent consideration. The most significant of the Bonferroni-corrected partial correlations was found between ICV and mean area (r=+0.89, p<0.0001). The second most significant global correlation was between ICV and mean BED (r=-0.39, p<0.0001), reflecting the constraint that smaller topologically spherical 2D surfaces must have greater mean bending than do larger ones. A slightly weaker positive partial correlation (r=+0.36, p<0.0001) was found between mean cortical area and mean BED. This makes sense as greater cortical area (with ICV held constant) requires greater cortical folding. The weakest partial correlation of significance (r=-0.31, p<0.001), was found between areal and thickness asymmetries. We do not know if this negative correlation reflects a true tradeoff in cortical volume configuration or is an artifact of FreeSurfer’s estimation methods, e.g. that FreeSurfer’s delineation of the WM/GM boundary is used in estimating both surface area and cortical thickness.\n\nTwo additional trend-level cortical relationships were found. Mean thickness (holding ICV and area constant) correlated negatively with BED (r=-0.29, p<0.01), i.e. greater thickness was associated with less cortical folding, implying that thicker cortex has fewer folds within a given volume. We also found a weak relationship between age and mean cortical thickness (r=-0.29, p<0.01) which is not surprising, even within this young-adult cohort. We obtained similar whole-cortex results using the OASIS dataset as well (Supplemental Table S3).\n\nTable 2 shows the estimated exponent β of ICV that correlated best with our measured size quantities Q, using the model Q=α×ICVβ with the auxiliary OASIS dataset and employing the current dataset for comparison. All subcortical volumes had sublinear relationships with ICV (β<1). However, of cortical quantities only area had a substantial positive, but still sublinear relationship with ICV. Moreover, cortical thickness as measured by FreeSurfer was uninfluenced by brain size. Thus, typical methods of normalizing gray matter volumes based on linear ICV correction would significantly overcorrect cortical values, especially for thickness.\n\nPowers of intracranial volume (ICV) estimated to correlate best with volume (subcortical) and cortical area and thickness quantities. All log-log regressions are significant p<0.0001 except for thickness and bending energy density (BED). Results are shown with 95% confidence intervals for the primary (right column) and OASIS (left column) datasets.\n\nWe first describe the results of regression over the demographic variables of sex and age for thickness, BED, and area, averaged across the entire cortex of both hemispheres. The independent variable age failed to reach significance in any of the three regressions, with the highest significance being in the thickness regression with an estimated loss of thickness of 0.004 mm/yr (t135=2.4, p=0.017). Sex also did not impact mean cortical thickness or mean BED. However, as expected from previous studies, sex correlated strongly with mean area: men had 10.4% (93 cm2) more area than women (t135=7.9, p<0.0001), similar to their 10.2% increase in ICV (157 cm3) [t136=7.1, p<0.0001].\n\nHowever, when ICV0.87, the strongest predictor of cortical area (t134=17.8, p<0.0001; α=1.33 [ICV cm3, area cm2]), was added to the analysis, the omnibus sex effect was reduced to a trend (t134=3.0, p<0.01; males +2.8% (23 cm2) greater than females). We also regressed the overall mean differences between the left and right hemispheres in thickness and area over the factors sex, age, and combined mean thickness, area, or BED as independent variables (and for area, also ICV0.87). The only trend to be found in those three regressions was that subjects with larger ICVs tended to show a small increase in relative left hemisphere cortical area (t134=2.6, p=0.01; every +10% ICV change implies a +0.7% LH area change).\n\nWe used ANOVAs with sex, lobe, and parcel factors to look for regional cortical differences between men and women. We used ICV corrected area for all area comparisons, using the estimated ICV0.87 parameter from Table 2. This assumes that the ICV exponent applies across the entire cortex (Im et al., 2008), a stance which has some support from cortical area genetic heritability studies (Eyler et al., 2011) (see also Supplementary Table S2).\n\nThere were no clear sex differences in regional BED or cortical thickness. BED values did not differ between men and women at the lobar level, with the maximum difference found in the occipital lobe (F1,136=4.1, p>0.01). Nor were there significant parcel level differences (p<10-6) in BED. For thickness, the only lobar trend was found in the limbic cortex, where females had 0.05 mm thicker cortex (F1,136=7.1, p<0.01, η=0.05).\n\nIn contrast, significant sex differences were found in ICV-corrected area measures as shown in Figure 3. As might be expected given the fact that males had greater overall area, even after adjusting for ICV, males had modestly significant greater area in the frontal lobe (+3.3%, F1,136=13.6, p<0.001, η=0.09), and a trend toward greater area in the occipital lobe (+3.8%, F1,136=7.2, p<0.01, η=0.05), and the temporal lobe (+2.4%, F1,136=7.1, p<0.01, η=0.05). There were no residual sex differences at the parcel level in any lobe, and this result held even when the p value was lowered to trend levels (p<10-4). Overall (Figure 3) the corrected area differences across the sexes appeared to be symmetric, though there was a trend in the hemisphere × temporal lobe interaction with sex (Sex × Hemi × Parcel, F6,816=3.3, p=0.015, η=0.02) that might support lateral temporal areal asymmetries specific to just one sex. Finally, we saw no significant sex by hemisphere interactions in any parcel in any lobe in thickness, area or BED.\n\nRed areas indicate males > females (%) and blue females > males, ICV adjusted. Gyral and sulcal structures are shown by the light and dark gray in the background. LH left hemisphere, RH right. Anatomical structures (white labels): AG, angular gyrus; CG, cingulate gyrus; CalcS, calcarine sulcus; CS, central sulcus; FG, fusiform gyrus; HG, Heschl’s gyrus; IFG, inferior frontal gyrus; IPS, intraparietal sulcus; ITG, inferior temporal gyrus; LOS, lateral occipital sulcus; MedFG, medial frontal gyrus; MFG, mid-frontal gyrus, MTG, middle temporal gyrus; OFC, orbitofrontal cortex; PreCun, precuneus; SF, Sylvian fissure; SFG, superior frontal gyrus; SMG, supramarginal gyrus; SPL, superior parietal lobule; STG, superior temporal gyrus; TOS, transverse occipital sulcus.\n\nThe ANOVA in Table 3 also shows that males had 3–6% larger subcortical volumes on average than did females (η=0.12 to 0.30); i.e., an increase substantially less than the sex difference in ICV. Table 3 also records the results of linear regression analyses using ICV as a covariate (with exponents shown in Table 2) to identify sex differences not accounted for by overall brain size. With ICV corrections, only the amygdala remained substantially relatively larger in men (4.0%) though there was also a trend for the male cerebellum to be larger (1.8%) as well. Finally, Table 3 shows that in this uniformly young subject group, there was little detectable volume loss likely due to age, with trends seen only in the cerebellum (-0.3%/yr) and the putamen (-0.4%/yr).\n\nT values and mean % difference for the multifactorial regression where the dependent variable was structure mean volume across hemispheres; with age and sex as factors as well as ICV (to the power estimated in Table 2) as an independent variable. An ANOVA over sex (rightmost column) was used to identify unadjusted volume value quantities associated with sex. * p<0.01, p<0.001, * p<0.0001, Bonferroni corrected. Significant results in boldface.\n\n\nDiscussion\n\nIt is clear from Table 2 that gray matter in both the cerebral cortex and major subcortical structures increases sublinearly with respect to intracortical volume (ICV). Supplementary Table S1 and Supplementary Table S2 show that in addition, both sexes generally have similar estimated ICV exponents, although the appropriate exponent to use with cortical surface area may vary from lobe to lobe (we used a fixed exponent above). These facts reaffirm that the use of affine normalized brains or the use of simple ICV-based normalization overestimate gray matter volumes for subjects with smaller ICVs, e.g. in females as a group (Im et al., 2008). This is particularly true for cortical thickness which is well-conserved across different mammalian species (Karbowski, 2014); e.g., ranging from 0.8 mm in the mouse to 3.0 mm in the elephant, consistent with an ICV exponent of approximately 0.1 (Zhang & Sejnowski, 2000).\n\nA previous study found ICV exponents relating to thickness of 0.24 and to area of 0.74 (Im et al., 2008). The lack of correlation of ICV with thickness in our results might be due to FreeSurfer's conservative method for measuring thickness using minimum distances from pial to WM/GM junction surfaces, or might reflect the fact that our magnetic resonance imaging (MRI) resolution of ~1.0 mm was small compared to the mean human cortical thickness of 2.5 mm. On the other hand the estimate of Im et al. (2008) of 0.24 estimated with the CIVET automated surface rendering/measurement tool is likely too high: a separate estimate in that same study showed that cortical GM volume correlated best with ICV0.85. Given that Im et al.’s regressions for volume and area with ICV were much more reliable (as are ours) than those for thickness (Im et al., 2008), and assuming that volume roughly equals thickness times area, the optimal exponent for correlating ICV with thickness should have been much closer to 0.1 (i.e., 0.85 minus 0.74). Thus, it may be that current MR imaging or thickness estimation tools lack adequate precision for estimating proper thickness to ICV relationships within humans.\n\nWith the exception of the well-known differences in overall cortical surface area (Luders & Toga, 2010), intracranial volume (Ruigrok et al., 2014) and to a lesser extent subcortical volumes (Li et al., 2014), sex differences were generally small in the current study. The sex difference of 10% in cortical surface area that we found was similar to the results of most recent MRI studies (Im et al., 2008; Nopoulos et al., 2000; Pakkenberg & Gundersen, 1997; Ropele et al., 2009; Ruigrok et al., 2014; Van Essen et al., 2012) and post-mortem results (Pakkenberg & Gundersen, 1997), but is somewhat greater than the areal differences reported by others (Acer et al., 2010; Barta & Dazzan, 2003; Ronan et al., 2006; Salat et al., 2004). When we adjust overall cortical surface area for ICV, males had modest but significantly greater surface area, particularly in the frontal lobe. In order to confirm this result, we did a follow up analysis of a subset of our subject group consisting of 43 pairs of men and women matched for ICV, as was done by Luders et al. (2009). We found the same small difference in surface area (F1,42=7.5, p=0.009, 2.5% M>F, η=0.15) plus an even weaker, opposite one in thickness (F1,42=4.0, p=0.05, 1.5% F>M, η=0.09) and none in BED (F1,42=0.5, p=0.50) between men and women (see also Supplementary Table S5). Thus, the ICV adjustment of area appears to be unbiased with respect to our data when used to analyze relative sex differences in this moderate brain sized subgroup.\n\nThe effect sizes for other significant sex-related differences were small (η<0.1). The only lobar level thickness difference we found was a trend for females having greater limbic cortex values. Such a result is consistent with observations that the entire limbic cortex contains many estrogen/androgen receptors during development (Goldstein et al., 2001). In fact, when 33 cortical parcels are ranked as having either a high or low density in estrogen and androgen receptors during early development (Goldstein et al., 2001), there was a significant relationship to those Desikan parcels having significant (t score) percentile thickness (but not area) advantages favoring females across our 138 subjects [Thickness: ANOVA F1,32=10.8, p<0.01, η=0.25; Area: F1,32=0.3, n.s.]. Thus, though regional sex differences in thickness are small, the overall pattern appears to support a classic sex steroid mechanism for cortical thickness (but not area) differentiation.\n\nOn the other side, males had small relative areal advantages, even after ICV correction, in the frontal and occipital lobes, as well as in patchy regions of the temporal and parietal lobes. In particular, we failed to find evidence of sex differences in volume favoring females in either the parietal or occipital lobes as reported in several recent studies (Im et al., 2006; Luders et al., 2006; Lv et al., 2010; Savic & Arver, 2014). Overall, the sex differences that we observed were much smaller in magnitude and effect size than those of hemispheric asymmetries (Supplemental Table S5; Kang et al., 2015). However, when we take the overall pattern of parcel level area differences between the sexes, and correlate them with genetic heritability estimates taken from imaging studies of family members (Winkler et al., 2010), then we find that there was a significant relationship to those parcels having significant (t score) percentile area (but not thickness) advantages favoring males across our 138 subjects [Area: Pearson r=-0.33, p<0.05; Thickness: r=0.07, n.s.]. Thus, a genetic explanation for the pattern of small regional area asymmetries is indicated here [for the OASIS data the heritability/area difference Pearson correlation was r=-0.52].\n\nOverall, we found few significant regional sex differences in cortical thickness, consistent with some recent reports (Fjell et al., 2009; Im et al., 2006; Im et al., 2008) but not others (Im et al., 2010; Luders et al., 2006; Sowell et al., 2007). The larger differences reported by others may reflect uncontrolled factors, e.g., age, handedness, or education, that were controlled in the current study. This underscores the importance of using appropriate regression functions for ICV correction (Im et al., 2008). It is also possible, however, that the two sexes in our relatively well-educated group may be more homogeneous than in a broader community sample of young adults (Luders & Toga, 2010) or in other non-western populations.\n\nImportantly, the well-established sex difference of 8–10% in cortical volume (Peters et al., 1998; Ruigrok et al., 2014) appears to be almost completely attributable to differences in surface area, as one might expect if sex differences follow the scaling relationships between brain area and cortical thickness seen in mammalian brains (Toro et al., 2008). In this sense our sex difference results resemble those of researchers (Brun et al., 2009; Im et al., 2008; Leonard et al., 2008) who found that individual differences in overall brain size explain far more variance in brain anatomy than do residual differences due to sex.\n\nWhen considering subcortical sex differences, a recent study using similar FreeSurfer methodology (Li et al., 2014) found, as we did, that all female subcortical structures were smaller on average than corresponding male ones. When normalized linearly by total brain volume, Li et al. found that the hippocampus of females was relatively larger than that of males. However, simple normalization using total brain volume or ICV would overcompensate for relative hippocampal volumes, especially as the hippocampus has the smallest ICV exponent (Table 2). We found no significant normalized volume sex differences in the hippocampus.\n\nUsing optimized exponents for ICV, we found significant differences in the volumes of the amygdala and the cerebellum between males and females (Table 3), consistent with most previous studies (Bramen et al., 2011; Kim et al., 2012; Neufang et al., 2009; Peper et al., 2009). However, one issue with the amygdala sex difference is that the fractional ICV scaling adjustment was the least well-fitting of any subcortical structure when estimated from the OASIS data, perhaps stemming from a difference in scaling relations between the two sexes in our dataset (see Supplemental Table S1). Nevertheless, we obtained a significant sex difference in the amygdala when we analyzed the subcortical volumes of the OASIS-130 dataset (Supplementary Table S4) and also when we analyzed the 43 ICV-matched pairs (male-female) subset of the primary dataset (F1,42=12.9, p=0.0009, M>F 7.3%) using no brain-size covariate. Finally, we note that there have been measurements (Lentini et al., 2013; Neufang et al., 2009) showing that testosterone levels in adolescents and adults track the size of the amygdala (and the hippocampus, but not the basal ganglia), suggesting an important role of sex steroids in the development of the amygdala.\n\n\nConclusions\n\nAfter appropriately correcting for the large, intracranial volume-related overall differences in the cortical surface area and subcortical structure size between male and female subjects, cortical and subcortical sex differences were reduced in magnitude and generally showed small effect sizes. Males had larger amygdala after ICV volume correction, and there were subtle differences in some cortical regions as well, with relative omnibus area differences favoring males and thickness differences favoring females. The regional distribution of cortical morphometric sex differences also indicated being related to genetic and developmental processes of brain growth.\n\nOur emphasis on the use of mammalian brain scaling in looking at sex differences echoes the suggestion of Im et al. (2008) and extends it to subcortical structures. This avoids the overcorrections of area, volume and especially thickness values that have, we believe, in the past produced results improperly assigning larger relative regional areas, volumes and thicknesses to female subjects. Also, we also repeat Im et al.’s (2008) call to avoid using VBM for investigating group differences when groups have sizeable mean ICV differences because a larger mammalian brain is not an affine-scaled smaller mammalian brain: the WM/GM ratio is larger in the former and cortical volume differences are not evenly distributed geometrically; i.e., volume increases are overwhelmingly increases in area resulting in more folding. Lastly, we endorse the empirically-derived recommendations of Barnes et al. (2010) to use ICV as a covariate when comparing cortical area measures but not when comparing thicknesses.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw Data, 10.5256/f1000research.6210.d44689 (Herron et al., 2015).\n\nNeither our IRB-approved MRI imaging protocol nor our informed consent document signed by our subjects permit us to make the original T1 MRI images publically available. However, we do provide all extracted, anonymized subject data used in the above statistical analyses as well as for the corresponding OASIS data subset that we used.\n\nThe androgen/estrogen receptor density level data referred to in the Discussion section from (Goldstein et al., 2001) was taken from Table 2. Only cortical values were used.\n\nThe heritability (h2) estimates referred to in the Discussion section were taken from Table 3 in (Winkler et al., 2010).\n\n\nConsent\n\nAll subjects provided informed written consent following procedures approved by the Institutional Review Board of the Northern California Health Care System of the Department of Veterans Affairs.", "appendix": "Author contributions\n\n\n\nDLW, XK, and TJH designed the study. XK collected the imaging data. TJH and XK developed the statistical analyses. XK wrote the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by a VA Research and Development Grants CX000583 and CX001000 to DLW. The content is solely the responsibility of the authors and does not necessarily represent the official views of Department of Veterans Affairs or the U.S. Government.\n\nI confirm that the 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 Bill Yund and the Radiology Service at the Martinez VA Clinic for help in collecting the imaging data. We also thank Marc Ettlinger for helpful comments.\n\n\nSupplementary material\n\nNo significant sex differences found except with the Current-138 dataset’s amygdala (p<0.05). However we note that in subcortical volumes the female exponents are always bigger except in the OASIS cerebellum (12 of 13 (1 tie), p<0.01 sign test, post-hoc).\n\nMean ± Standard Deviation. The limbic lobe is the only one showing a difference in estimated exponents between the two datasets, and the insula is the only lobe showing a seemingly reliable different (lower) exponent than the rest of the lobes in both hemispheres.\n\nThe multifactorial regression’s dependent variable was structure mean volume across hemispheres; with age and sex as factors as well as ICV – to the power estimated in Table 1 (right column) - as an independent variable to see how much overall brain size reduce sex differences. An ANOVA over sex (rightmost column) was used to identify unadjusted factors contributing to volume values. * p<0.01, p<0.001, * p<0.0001, Bonferroni corrected. Significant results in boldface.\n\n43 ICV-matched male-female pairs of subjects from the current-138 group were selected. ICV differences between the 43 matched pairs averaged 0.31%. Area sex and hemisphere differences are not adjusted for ICV and are analyzed using repeated measures ANOVA for sex as well as hemisphere. Significance is at p<0.01 Bonferroni corrected for all 34 × 3 × 3 comparisons. The asymmetric parcels are similar to those found in all 138 subjects (see Kang et al., 2015) and provide contrast to the complete lack of localizable, significant parcel-level sex differences (all p>0.07, corrected) or sex interactions.\n\n\nReferences\n\nAbe O, Takao H, Gonoi W, et al.: Voxel-based analysis of the diffusion tensor. Neuroradiology. 2010; 52(8): 699–710. PubMed Abstract \| Publisher Full Text\n\nAcer N, Cankaya MN, Isci O, et al.: Estimation of cerebral surface area using vertical sectioning and magnetic resonance imaging: a stereological study. Brain Res. 2010; 1310: 29–36. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8272", "date": "16 Apr 2015", "name": "Lutz Jäncke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general this is an interesting and important study again showing that brain size (or ICV) is quite important for morphometric analyses and especially when comparing the sexes. In this context I would like to draw the attention of the authors to a recent paper paper of our group (Jäncke et al., 2015) in which actually the same has been done (with a few exceptions) as in this study. However, in that paper the authors have used a much larger sample (n=856) and identified small or even non-existing sex influences on several brain compartments when ICV and/or FBV has been used as control variable. This finding and the finding report by the authors is in line with several studies of our group (Jäncke et al., 1997; Lüders et al., 2002) but also with studies from other groups (Leonard et al., 2008, Lemaître et al., 2005). Thus, sex differences are small or non-existing when one use brain size corrections. However, what is unknown so far (in my opinion) is whether there are indeed some sex differences, which are still there but which we do not \"see\" using the more or less cars methods we are all using. Thus, we have to be carefully in making to firm conclusion in terms of using potential anatomical sex differences as the basis of possible sex-gender-differences in behavior. Thus, the authors should add some comments on that, too.", "responses": [] }, { "id": "8267", "date": "05 May 2015", "name": "Gina Rippon", "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\nMethodologically, this study is sound although it should be noted that, with respect to intracranial volume (ICV) estimations, there was apparently no correction for height and weight (a flaw in the Ruigrok et al, 2014 review which these authors themselves acknowledge). Matching of participants for levels of education is also a welcome addition to standard practice.My substantive comments are confined to observations on the disconnect between the findings as reported in the paper itself and those inferred in the abstract (and indeed in the title of the paper). The paper is entitled “Sex differences in cortical and subcortical human brain anatomy” and the abstract includes the sentence; “Significant sex differences were observed”. Yet if we look at what was actually found, at the very least the abstract should report “very few significant sex differences were observed” and, by rights, the paper should be entitled: “On the failure to find sex differences….” Or “Few sex differences in cortical and subcortical human brain anatomy”A brief summary of the results of the search for sex differences follows:Looking at “whole hemisphere sex differences in cortical thickness, area and folding”, it is reported that there were no sex differences in thickness and folding, and a correlation with area was reduced to a trend when there was a correction for ICV (pg. 6). Looking at sex differences in cranial structures there were no differences in thickness and folding (pg. 6) There were sex differences in frontal lobe area (M>F), but no area differences at the parcel level in any lobe (pg. 6) and no significant sex by hemisphere interactions in any parcel in any lobe in thickness, area or BED (pg. 7) Of the 7 subcortical structures, only the amygdala showed significant sex differences ( M>F).The authors acknowledge in their discussion “Sex differences are generally small in the current study (pg.7) “ and that “effect sizes were small” (pg8) and even “our sex difference results resemble those of researchers who found that individual difference in overall brain size explain far more variance in brain anatomy that do residual difference due to sex”. (pg. 8).On balance, the findings of this paper are that there are very few, if any, substantive, sex differences and, when appropriate corrections are made, these may well disappear.I would draw the authors’ (and indeed any readers’) attention to the coverage of this type of issue raised in a recent paper from our group (Rippon et al., 2014). My concerns are that, given the title and the claims in the Abstract, this paper will be taken as an addition to the body of evidence proving that there are reliably identifiable differences between the brains of females and males. I think the authors should be invited to recast their paper to acknowledge the difficulties they have encountered in demonstrating any “Sex Differences in cortical and subcortical human brain anatomy”.", "responses": [] } ]	1	https://f1000research.com/articles/4-88
https://f1000research.com/articles/4-51/v1	24 Feb 15	{ "type": "Research Article", "title": "Adherence to Artemisinin Combination Therapy for the treatment of uncomplicated malaria in the Democratic Republic of the Congo", "authors": [ "M. Ruby Siddiqui", "Andrew Willis", "Karla Bil", "Jatinder Singh", "Eric Mukomena Sompwe", "Cono Ariti", "Andrew Willis", "Karla Bil", "Jatinder Singh", "Eric Mukomena Sompwe", "Cono Ariti" ], "abstract": "Between 2011 and 2013 the number of recorded malaria cases had more than doubled, and between 2009 and 2013 had increased almost 4-fold in MSF-OCA (Médecins sans Frontières – Operational Centre Amsterdam) programmes in the Democratic Republic of the Congo (DRC). The reasons for this rise are unclear. Incorrect intake of Artemisinin Combination Therapy (ACT) could result in failure to treat the infection and potential recurrence. An adherence study was carried out to assess whether patients were completing the full course of ACT.One hundred and eight malaria patients in Shamwana, Katanga province, DRC were visited in their households the day after ACT was supposed to be completed. They were asked a series of questions about ACT administration and the blister pack was observed (if available).Sixty seven (62.0%) patients were considered probably adherent. This did not take into account the patients that vomited or spat their pills or took them at the incorrect time of day, in which case adherence dropped to 46 (42.6%). The most common reason that patients gave for incomplete/incorrect intake was that they were vomiting or felt unwell (10 patients (24.4%), although the reasons were not recorded for 22 (53.7%) patients). This indicates that there may be poor understanding of the importance of completing the treatment or that the side effects of ACT were significant enough to over-ride the pharmacy instructions.Adherence to ACT was poor in this setting. Health education messages emphasising the need to complete ACT even if patients vomit doses, feel unwell or their health conditions improve should be promoted.", "keywords": [ "Adherence", "artemisinin", "compliance", "treatment failure", "artesunate-amodiaquine", "side effects", "malaria", "Plasmodium falciparum" ], "content": "Introduction\n\nDespite a wealth of natural resources, the Democratic Republic of the Congo (DRC) has one of the lowest per capita incomes in the world1 and in 2014 ranked 186th (out of 187 countries) in the United Nations Development Programme’s human development index2.\n\nMédecins Sans Frontières-Operational Centre Amsterdam (MSF-OCA) has been working in the provinces of North Kivu and South Kivu since the early 1990s and in Katanga since 2003. MSF-OCA operates health programmes in Mweso and Walikale in North Kivu; Baraka and Kimbi Lulimba in South Kivu; and Shamwana, Katanga (DRC).\n\nBetween 2011 and 2013, the number of recorded malaria cases more than doubled and between 2009 and 2013, increased almost 4-fold in MSF-OCA programmes in DRC (Figure 1a). The number of projects that MSF-OCA supported between 2009 and 2013 has however varied during that time. Hence, restricting the analysis to the fixed programmes only (in Baraka (South Kivu), Shamwana (Katanga) and Mweso (North Kivu)), the general trend still appears to be a marked increase in malaria cases (Figure 1b).\n\nIn Katanga the general trend is less clear for malaria cases. However this is likely due to the variable numbers of projects supported between 2009 and 2013 (Figure 2a). Focussing on the fixed project in Shamwana, there has been a marked increase in malaria cases (Figure 2b).\n\nIn 2013 MSF clinic staff in Shamwana began to identify patients returning to the clinic with complicated severe malaria after having already been prescribed the anti-malarial treatment. It was not known if these were treatment failures or re-infections.\n\nThis was an alarming increase in malaria incidence which cannot be explained by displacement of non-immune populations from non-endemic regions (mountains) to endemic malaria regions as the populations have been relatively stable since 2008.\n\nThe World Health Organisation (WHO)-recommended strategies for malaria control fall into two major areas: prevention and case management3. These strategies work against both the transmission of the parasite from mosquito vector to humans (and from humans to mosquitoes) and the development of illness and severe disease in humans3. The former is achieved in Shamwana through the distribution of long-lasting insecticide-treated bednet (LLIN) in a targeted manner (pregnant women and under 5 year old malaria patients) and the latter through Artemisinin Combination Therapy (ACT) of confirmed malaria cases (the national protocol of DRC is Artesunate and Amodiaquine (ASAQ) administered in a fixed dose combination (FDC) as a first-line ACT). These strategies do not seem to be having an impact on malaria incidence in MSF settings in DRC.\n\nThe current study explored whether this increase in cases could be explained by patients failing to complete the full three day course and adhering to the time schedule and dosage prescribed for ASAQ. Poor adherence increases drug pressure and thus the risk of developing resistance. Moreover, incorrect or incomplete intake could result in failure to treat the infection and potential recurrence.\n\n\nMaterials and methods\n\nThe study was conducted in Shamwana, Katanga, DRC. Katanga has an average temperature of 24°C and two seasons, rainy between November and March and dry from April to October. MSF-OCA has been working in the province of Katanga since 2003. MSF-OCA supports Shamwana hospital to provide free secondary health care and supports more than four health centres. The MSF catchment population included Shamwana town and the nearby village of Nsangwa.\n\nFor previous ACT adherence assessments, adherence was always approximately 60%4–9. The sample size was therefore based on the conservative estimated adherence of 50% (in order to obtain the maximum sample size). With a precision of 10% and an α-error of 5%, n=97 patients were required. Assuming a proportion of patients lost to follow-up or withdrawn of 20%, the total sample size needed for the home visits was thus n=117 patients. Similarly, a sample size of 117 was calculated for the exit interviews.\n\nTo prevent bias due to a change in prescribing and educating behaviour by health staff, the health staff were informed that a study would start but the exact purpose of the study (measuring patient adherence) was at no time revealed to them. The explanation was that this was a mapping exercise of where patients attending the health centre were mainly coming from.\n\n\nStudy procedure and questionnaires\n\nAt the health centre level, all patients were interviewed using a short “centre”-questionnaire. This included the patient’s name, age, sex, the number and type of prescriptions, and the patient's address in sufficient detail.\n\nStudy patients for the “home”-questionnaire were selected through systematic sampling of eligible patients (aged more than 1 year with a positive malaria rapid diagnostic test (RDT) result that had been prescribed ASAQ) from the “centre”-questionnaires. Study patients were visited at home on Day 3 (the health centre visit was on Day 0) and interviewed using the “home” questionnaire to assess patient adherence. Socio-demographic questions about the household were followed by a systematic account of how the pills were taken. A correct treatment schedule instrument was used to guide the questions on adherence. Lastly, some questions about knowledge of malaria cause and prevention were asked. Any sick patients were referred to the health centre.\n\nAfter all ‘home’-questionnaires were completed, exit interviews were carried out at the health centre using the “exit”-questionnaire to provide further information on patient/caretaker understanding of ACT and pharmacy dispensing practices.\n\n\nAdherence definitions\n\nA dose was considered correctly taken if according to the patient or caretaker’s verbal account the prescribed amount of pills was taken and it was neither spat out nor vomited.\n\nThere are no standard definitions for adherence so the following were used (Table 1).\n\nCertain incomplete intake or certain non-adherence: Any patient that showed blister packaging still containing any ASAQ pills was certain to have taken an incomplete treatment. There was absolute certainty that the treatment schedule was not completed.\n\nProbable incomplete intake or probable non-adherence: According to the patient/caretaker’s account, all pills were not taken so this patient was probably non-adherent. The blister packaging presented was empty or could not be shown.\n\nProbable incorrect intake or probable non-adherence: If according to the patient/caretaker’s account ASAQ was not taken following the prescribed time schedule or dosage, it was assumed the patient took the complete treatment but in an incorrect way. The blister packaging presented was empty or could not be shown.\n\nProbable correct intake or probable adherence: Only those patients who stated they had taken the complete treatment schedule, following the treatment protocol exactly were considered “probably adherent” (this was not ‘certain’ as each dose had not been observed). The blister packaging presented was empty or could not be shown.\n\n\nData management and analysis\n\nData for each subject were entered into EpiData 3.1 software10. Data were summarised by number and percentages for categorical variables. Continuous variables were summarised using means, medians and ranges. Adherence status was calculated and expressed as a percentage. Confidence intervals were calculated using binomial and multinomial formulae. To calculate the associations between risk factors and adherence status logistic regressions was used and the results expressed as odds ratios and 95% confidence intervals. Data analysis was conducted using Stata 13.011.\n\n\nResults\n\nIn total, 274 people completed the ‘centre’ questionnaires. Among those, 150 (54.7%) were diagnosed with malaria and given a prescription for ACT. Of these malaria patients 17 were aged <1 year so were excluded. Onehundred and seventeen (42.7%) of the 133 (48.5%) patients that satisfied the inclusion criteria were selected for home visits as these were the maximum that could be achieved during daily visits. However, only 108 (39.4%) of these eligible patients completed home questionnaires. The remaining 9 (3.3%) patients were considered lost to follow-up (Table 2). A total of 117 exit questionnaires were completed for patients who had received an ACT prescription in one day (Table 2). Nobody refused to participate in the study.\n\nReasons for loss to follow up included; temporarily out of the village (6), wrong address (1), age was <1 year (1), household already visited. No patients were lost to follow-up due to admission to hospital.\n\nThe mean age of the patients was 10.5 years and ranged from 1 to 57 years (median 5 years, Q25–Q75: 2–12.5, Table 3). Sixty (55.6%) patients were aged ≤5 years. The sex ratio was 0.7 (male/female 45/61). The age of the 28 patients that responded to the questionnaire ranged from 9 to 57 years (though parents/caretakers were present to give informed consent for the eight respondents that were aged <18 years). The age of the patients for whom a parent/caretaker responded ranged from 1 to 17 years. The most common caretakers were parents (74, 92.5%).\n\nEducation levels of the patients were low with no patients completing secondary education or higher (Table 3). The education levels of the patient/caretakers that attended the clinic (i.e. those likely administering the ACT treatment to children) were not assessed.\n\nThe mean household size was 8.6 members and ranged between 1 and 26 (median: 8, Q25–Q75: 5–10.5). The mean number of children aged <5 years per household were 2.6 and ranged between 0 and 13 (median: 2, Q25–Q75: 2–3) (Table 4). The main profession of heads of households was subsistence farming alone (46, 42.6%), and this occupation was named in combination with others for an additional 40 households (86, 79.6%).\n\nIncludes blacksmith (1), coalman (1), dressmaker (1), mason (1), sells fish oils (1), sells wood (1). †Includes health worker (2), student (2), no work (2), workman (2), trader (2), daily worker + other (1), daily worker + teacher (1), trader + other (1), farmer for trading + workman (1). ‡Includes guard (2), no work + other (1), daily worker + health worker (1), trader (1), farmer for trading + daily worker (1), farmer for trading + trader (1).\n\nA total of six (5.6%) patients had one or more pills left at the time of the home visit on Day 3 when all pills should have been taken (Table 5). They were defined as certainly non-adherent. Thirtyfive (32.4%) patients were defined as probably non-adherent after describing an incomplete intake (including eight patients that took ASAQ pills on Day 3) and 67 (62.0%) patients were described as probably adherent after describing a complete and correct intake (Table 5). Merging the two non-adherent groups gave 41 (38.0%) non-adherent patients.\n\nHowever it should be noted that in order to analyse these data, assumptions had to be made for missing data, primarily an assumption had to be made for 25 patients on Day 0 that ASAQ had been taken only once that day (at the clinic) and had been taken at the same time as the dose on Day 1 and Day 2 (which actually matched each other in all cases).\n\nThis adherence was calculated based on the number of pills taken, the number of times pills were taken per day and the number of days completed. However the timing of the daily dose and whether the medication was retained (not vomited or spat out) are also important. Both could result in a reduction of anti-malarial activity during the treatment phase and potential treatment failure. Using this stricter definition of adherence, 21 patients that had been defined as probably adherent became probably non-adherent (data not shown). Twelve (57.1%) of these patients did not take doses at the correct time of day, eight (38.1%) patients vomited or spat their pills and one (4.8%) patient vomited or spat their pills and also did not take doses at the correct time of day.\n\nReasons for 1) pills remaining or 2) a description of incorrect intake or 3) a description of correct intake were noted for each patient. However, 22 (62.9%) of the 35 probably non-adherent patients had missing data for reasons for incorrect intake and 19 of the 67 (28.4%) probably adherent patients had missing data for reasons for correct intake.\n\nThis analysis only considered the individuals classified into the appropriate categories (non-strict adherence). The main reason for pills remaining (incomplete treatment intake) were forgetting to give/take the treatment and patient didn’t feel better/treatment wasn’t working + felt unwell (2, 33.3% respectively) (Table 6). Unfortunately the design of the questionnaire did not allow analysis of whether a replacement dose was taken.\n\nThe most common reasons for incorrect intake were that the patient forgot to take/caretaker forgot to give the pills, claimed the wrong instructions were given at the clinic or the patient was vomiting (3, 8.5% respectively) (Table 6).\n\nTaken together the most common reason patients gave for incomplete and/or incorrect intake was that they were vomiting or felt unwell (10 patients (24.4%)).\n\nThe most common reasons for correct ACT intake was that the correct instructions were given at the clinic (32, 47.8%) and the patient/caretaker had been given the same medicine before and knew how to take it + Correct instructions were given at the clinic(13, 19.4%) (Table 6).\n\nUnivariate analyses of potential risk factors for non-adherence were explored (using the non-strict adherence classification). These included sex, weight classes (instead of age groups), education level of the patient, knowledge that mosquito bites can cause malaria, knowledge that LLINs prevent malaria and presence of observed LLINs in the household. Significant association with adherence was observed only for sex (χ2 = 8.1, P= 0.017, Table 7). Women were more likely to adhere to ACT treatment. Sex remained significantly associated when considering strict adherence (χ2 = 8.9, P= 0.031, Table 8) and when merging certain and probable non-adherence into a single ‘non-adherence’ category (χ2 = 8.1, P= 0.004 for non-strict adherence only, Table 9, Table 10).\n\nThese risk factors were analysed in a logistic regression where the sex of the patient only was significantly associated with adherence (Table 11).\n\n\nExit-questionnaires\n\nPatients or caretakers/parents in the exit population had broadly the same socio-demographic and clinical characteristics as those who were interviewed at home for the adherence study (i.e. sex of patient, caretaker relation to patient, education level of the patient).\n\nThe weight categories showed significantly higher infants and adults in the exit group (χ2=9.34, P=0.026 for age group and χ2=9.16, P=0.05 for weight class).\n\nIn the exit-questionnaires group, a high number of the malaria patients or their respective caretakers (114, 97.4%) knew the name of the disease or could name the correct signs/symptoms of malaria (Table 12). Similarly a high number (101, 86.3%) were able to identify the ACT pills among all the given pills and to indicate to the interviewer that they were an anti-malarial. Similarly, a high proportion of patients/caretakers (116, 99.2%) correctly said they would take the next dose the following day (though only six, 5.1% mentioned the time), correctly said they would take the next dose the day after (116 (99.2%), though only five, 4.3% mentioned the time), claimed that they would take all ACT pills and would therefore not have a balance at the end of the ACT course (117, 100.0%), claimed they would continue taking ACT treatment the following day even if their condition improved (115, 98.3%) and stated they would return to the clinic if their condition did not improve after 3 days (114, 97.4%) (Table 12). This indicated a high level of understanding of ACT administration. However, only 87 (74.4%) patients/caretakers could repeat the number of times per day pills should be taken and 76 (65.0%) could repeat the number of pills to take (Table 12).\n\nHowever, the Outpatient Department (OPD)/pharmacy performance was not consistent. Despite 107 (91.5%) of the patients/caretakers being asked if they had understood the instructions given to them, only 41 (35.0%) had been asked to repeat these instructions and only 90 (76.9%) were given additional information, mainly advised to return to the clinic to get a dose if they vomited or spat up a dose (51, 43.6%) and informed that ACT can cause fatigue but they must continue the treatment (16, 13.7%). Also a high but not optimal number of patients (101, 86.3%) had taken a dose of ACT at the clinic under supervision.\n\n\nDiscussion\n\nThis study found that six (5.6%) patients were certainly non-adherent; therefore approximately one out of 18 patients did not complete the course of ACT treatment within the timeframe of 3 days. From the verbal account of the patients or caretakers, another 35 (32.4%) patients took the treatment in a different way to how it was prescribed (probable non-adherence). Therefore, only 67 (62.0%) patients could be considered probably adherent.\n\nThis did not take into account the patients that vomited or spat their pills or took the pills at the incorrect time of day. In that case the adherence dropped to 46 (42.6%). The effect of a decrease in anti-malarial activity that would follow vomiting/spitting or failure to take a timely dose is not clear but it is thought that this risks a period of monotherapy to the partner drug in the ACT combination. Advice about timing of ACT dosages is not clear but it is thought that a 3-hour margin either side of the expected timing is acceptable12.\n\nThe most common reason patients mentioned for incomplete (67%) and incorrect (20%) intake was that they were vomiting or felt unwell. This appears to indicate that there was a poor understanding of the seriousness of the disease and the importance of finishing the treatment or that the side effects of ASAQ treatment were significant enough to over-ride the pharmacy instructions, unlike previous MSF studies using Coartem®8.\n\nMore than a third of the study population claimed not to know the cause of malaria nor how to prevent malaria during home visits, but this was not significantly associated with non-adherence to ACT. Only sex of the patient was associated with adherence (males were significantly less likely to adhere to ASAQ). As the side effects were unlikely to affect males more than females, this may demonstrate an indifference to malaria in the male population, a common disease seen regularly in Shamwana. Similarly in Ethiopia, a study of factors associated with non-use of bed nets found that malaria was not perceived by the population to be a problem despite high prevalence of the disease13.\n\nWhen assessing the main reason for correct ACT intake, 45 (66.2%) patients claimed they were given the correct instructions at the OPD/pharmacy. When assessing the exit-questionnaires the quality of ACT prescriptions and the related instructions given at the OPD/pharmacy were less clear: 86.3% of the patients in the exit-questionnaires group were able to identify ACT as the anti-malarial treatment given to them amongst other concomitant treatments and 99.2% could correctly repeat the number of days that ACT should be taken. However, only 74.4% could repeat the number of times per day pills should be taken and 65.0% could repeat the number of pills to take.\n\nUnfortunately the education level of the patients only was assessed, and not that of the caretakers. Therefore, any link between education level and adherence to antimalarials, which have been shown previously to be associated, could not be deduced5,7. Knowledge that mosquito bites cause malaria did not show a statistically significant association with ACT adherence unlike findings of an ACT adherence study in Bo, Sierra Leone8. No association was seen between adherence and age despite some observations in MSF projects that adherence is poor amongst adolescents.\n\nThree ACT adherence studies carried out in MSF malaria projects had similar findings; certain non-adherence was 22.9% and probable non-adherence 28.8% in a rural area of Sierra Leone, 18% and 29% in a remote area in South Sudan and 21% and 39% respectively in a refugee settlement in Zambia respectively6–8. These study settings had similar characteristics to Shamwana, such as a remote rural study area, low education level, treatment intake over 3 days, and treatment already implemented for a certain amount of time. Furthermore, our results are in line with a study carried out in two districts in Western Kenya that found a certain non-adherence of 31.7% and probable non-adherence of 4.2%14 and another study in rural Sri Lanka using post-treatment interviews where 26% of patients self-reported defaulting. The main reasons given for not taking the entire regimen were side effects and disappearance of symptoms15.\n\nHowever, studies in Uganda and Ghana showed certain non-adherence to be as low as 7% and probable non-adherence to be 3%7,16. In Tanzania, non-adherence was as low as 25%17. These higher adherences might be explained by the fact that adherence assessments were carried out after the successful introduction of a new malaria treatment. The introduction of a new, efficient treatment is always accompanied by special training for health workers, which is linked with greater motivation, and giving a more detailed verbal explanation to patients.\n\nA recent systematic review found 37 studies that measured ACT adherence18. However all had varying definitions of adherence and used different study designs and methods to measure adherence. Standardised methodologies for both self-report and bioassay measurements would improve the evidence base on ACT adherence and effectiveness.\n\nTwo different methods were used to measure adherence in patients: the pill count (observation of the blister pack) and a systematic questionnaire. Both assessment methods have their limitations: the pill count is a more objective measurement but gives incomplete information; the patient’s account is subjective and not verifiable but provides more information. By classifying patients as either certainly or probably non-adherent, the study accounted for the limitations of both methodologies.\n\nThis study has shown that ACT adherence in this setting is inadequate and may have contributed to the increase in malaria cases (if recrudescence was a major cause). It should be noted however that there is no recommended target for adherence levels. Artemisinin is the best drug available to treat malaria and currently there are no real alternatives. Artemisinin resistance has likely arisen in Cambodia due to the use of artemisinin monotherapy19. Combination treatment with ACT when taken correctly reduces, but does not completely eliminate, the chance of resistant strains developing. Poor adherence could potentially expose greater numbers of parasites to the more slowly-eliminated partner drug in ACTs, increasing the risk of resistance to the partner drug20. Once resistance has developed to the partner drug the ACT treatment would effectively become artemisinin monotherapy and hence render artemisinin vulnerable to resistance.\n\nThe effectiveness of ACT relies on both the efficacy of the drug components and on correct compliance. Adherence to ACT should not be taken for granted. At the community level, health communication campaigns that improve malaria knowledge and help them understand the importance of prevention and correct treatment should be carried out. At the clinic level, first ACT dose should always be observed at the pharmacy/OPD clinic and complete and clear patient instructions, including the importance of completing a course of ACT treatment and what to do if a dose is vomited or spat, should be given. The need to complete three sequential days of malaria treatment even if patients feel unwell or improve should be emphasised at both the community and clinic level.\n\n\nConsent\n\nWritten informed consent was obtained from the patient/parent/caretaker for the home questionnaires.\n\nVerbal informed consent was obtained from the patient/parent/caretaker for the exit questionnaires.", "appendix": "Author contributions\n\n\n\nMRS and KB conceived and designed the study.\n\nAJW carried out the field component of the study.\n\nJS and EMS facilitated the field component of the study.\n\nCA and MRS carried out all data analysis and wrote the manuscript.\n\nAll authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nJules Lugoma, Serge Kisenga, Mwilambwe Kaulu Desire, Mwilambwe Ngoy Etienne, Kabwe Mwilambwe Pierre, Kabwe Ngongwe Pierre, Kaluba Ilunga Maurice completed the questionnaires.\n\nThe MSF team, including Gemma Garcia Gonzalez and Pedro Calibiera, in Shamwana provided transportation and supplies and provided context and background. The MoH staff supported setting up and facilitating the study.\n\n\nReferences\n\nReuters: Alertnet. 2012. Reference Source\n\nUNDP: U. N. D. P. Humanitarian Development Report: Sustaining Human Progress: Reducing Vulnerabilities and Building Resilience. 2014. Reference Source\n\nOrganization, W.H.: World Malaria Report 2013. 2013. Reference Source\n\nAnsah EK, Gyapong JO, Agyepong IA, et al.: Improving adherence to malaria treatment for children: the use of pre-packed chloroquine tablets vs. chloroquine syrup. Trop Med Int Health. 2001; 6(7): 496–504. PubMed Abstract \| Publisher Full Text\n\nDepoortere E, Guthmann JP, Sipilanyambe N, et al.: Adherence to the combination of sulphadoxine-pyrimethamine and artesunate in the Maheba refugee settlement, Zambia. Trop Med Int Health. 2004; 9(1): 62–67. PubMed Abstract \| Publisher Full Text\n\nDepoortere E, Salvador ET, Stivanello E, et al.: Adherence to a combination of artemether and lumefantrine (Coartem) in Kajo Keji, southern Sudan. Ann Trop Med Parasitol. 2004; 98(6): 635–637. PubMed Abstract \| Publisher Full Text\n\nFogg C, Bajunirwe F, Piola P, et al.: Adherence to a six-dose regimen of artemether-lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Uganda. Am J Trop Med Hyg. 2004; 71(5): 525–530. PubMed Abstract\n\nGerstl S, Dunkley S, Mukhtar A, et al.: Successful introduction of artesunate combination therapy is not enough to fight malaria: results from an adherence study in Sierra Leone. Trans R Soc Trop Med Hyg. 2010; 104(5): 328–35. PubMed Abstract \| Publisher Full Text\n\nSiddiqui MR: Adherence to Artemisinin-based Combination Therapy (ACT) in the MSF catchment area of Boguila, Central African Republic (CAR). (Medecins Sans Frontieres, 2010).\n\nThe Epidata Association: EpiData 3.1. Reference Source\n\nStataCorp: Stata 13.0. Reference Source\n\nMarit de Wit: Personal communication. Reference Source\n\nBaume CA, Reithinger R, Woldehanna S: Factors associated with use and non-use of mosquito nets owned in Oromia and Amhara Regional States, Ethiopia. Malar J. 2009; 8: 264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawford H, Zurovac D, O'Reilly L, et al.: Adherence to prescribed artemisinin-based combination therapy in Garissa and Bunyala districts, Kenya. Malar J. 2011; 10: 281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReilley B, Abeyasinghe R, Pakianathar MV: Barriers to prompt and effective treatment of malaria in northern Sri Lanka. Trop Med Int Health. 2002; 7(9): 744–749. PubMed Abstract \| Publisher Full Text\n\nAsante KP, Owusu R, Dosoo D, et al.: Adherence to Artesunate-Amodiaquine Therapy for Uncomplicated Malaria in Rural Ghana: A Randomised Trial of Supervised versus Unsupervised Drug Administration. J Trop Med. 2009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKachur SP, Khatib RA, Kaizer E, et al.: Adherence to antimalarial combination therapy with sulfadoxine-pyrimethamine and artesunate in rural Tanzania. Am J Trop Med Hyg. 2004; 71(6): 715–722. PubMed Abstract\n\nBanek K, Lalani M, Staedke SG, et al.: Adherence to artemisinin-based combination therapy for the treatment of malaria: a systematic review of the evidence. Malar J. 2014; 13: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDondorp AM, Nosten F, Yi P, et al.: Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009; 361(5): 455–467. PubMed Abstract \| Publisher Full Text\n\nNosten F, White NJ: Artemisinin-based combination treatment of falciparum malaria. Am J Trop Med Hyg. 2007; 77(6 Suppl): 181–192. PubMed Abstract\n\nUnited Nations Cartographic section & Section, U. N. C. Map of Democratic Republic of the Congo (DRC). Reference Source" }	[ { "id": "7807", "date": "02 Mar 2015", "name": "Anne Moore", "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\nThis is an interesting paper from MSF epidemiologists that aims to determine some of the reasons for significant increases in malaria cases in DRC. It is imperative to prevent the emergence of anti-malarial drug resistance and patient adherence and compliance is critical to this issue. The data presented here provides evidence of what is happening in the field and touches on patients’ attitudes and knowledge in this area. The authors focus on patient compliance and statistically examine socio-demographic and other factors for non-adherence to the prescribed time schedule and dosage. A key finding is the low rate of adherence, which has significant impact on drug-based treatment of malaria. Non-adherence due to vomiting or feeling unwell in a proportion of individuals is an important finding for the drug formulation community; how can this be overcome by changing the formulation or possibly the route, in the future? Timing was also highlighted as an obstacle to correct drug administration; again this is an obstacle that should be tackled both by drug developers, practitioners in the field and those interested in health literacy. The authors should consider minor revisions to their paper to discuss these issues in more depth. For example, based on their data, what are the next critical steps that should be focused on; from both a practice, and more long-term, drug design perspective? How can we succeed in patient education relating to the importance of adherence and designing and implementing a system that results in better patient adherence? Given that the authors found that gender is a significant factor; can the authors suggest how this finding should be used to develop strategies to improve patient compliance? Finally, the authors discuss the problems of trying to compare across studies due to different study designs and methods. From a methodology perspective, therefore, it would be useful if the authors included the full questionnaires used in this study as an appendix, with the objective of replication of the study in other areas.", "responses": [] } ]	1	https://f1000research.com/articles/4-51
https://f1000research.com/articles/3-223/v1	17 Sep 14	{ "type": "Software Tool Article", "title": "An object oriented implementation of the Yeadon human inertia model", "authors": [ "Christopher Dembia", "Jason K. Moore", "Mont Hubbard", "Jason K. Moore", "Mont Hubbard" ], "abstract": "We present an open source software implementation of a popular mathematical method developed by M.R. Yeadon for calculating the body and segment inertia parameters of a human body. The software is written in a high level open source language and provides three interfaces for manipulating the data and the model: a Python API, a command-line user interface, and a graphical user interface. Thus the software can fit into various data processing pipelines and requires only simple geometrical measures as input.", "keywords": [ "For dynamic analyses", "it is typical to treat the human body as a collection of linked rigid bodies. For accurate simulation and analysis", "the inertial properties (mass", "center of mass location", "and moments of inertia) of each of the body segments must be estimated. Human inertial properties have been measured and estimated in a number of ways. Bjornstrup1 gives a detailed overview of mostly invasive methods up to 1995. Many other methods exist", "including cadaver measurements2–4", "photogrammetry5", "ray scanning techniques6", "7", "water displacement8", "rotating platforms9", "and geometrical estimation of the body segments10." ], "content": "Introduction\n\nFor dynamic analyses, it is typical to treat the human body as a collection of linked rigid bodies. For accurate simulation and analysis, the inertial properties (mass, center of mass location, and moments of inertia) of each of the body segments must be estimated. Human inertial properties have been measured and estimated in a number of ways. Bjornstrup1 gives a detailed overview of mostly invasive methods up to 1995. Many other methods exist, including cadaver measurements2–4, photogrammetry5, ray scanning techniques6,7, water displacement8, rotating platforms9, and geometrical estimation of the body segments10.\n\nYeadon’s mathematical method10 is attractive because it requires only a set of simple geometric measurements from a human and provides reasonably accurate estimates of an individual’s body segment parameters using simple, straightforward computations. Yeadon himself developed a Fortran program called ISEG in his doctoral work11, to efficiently compute the inertial parameters. The original source code is available in his dissertation but is not adaptable for inclusion in modern software packages, is not copyrighted under a liberal reuse license (i.e., Creative Commons Attribution-NonCommercial-NoDerivs 2.5), and is not very user friendly.\n\nBecause we often make use of Yeadon’s model in our research, we developed a modern object oriented program under a permissive license that allows for incorporation into other software packages and includes a graphical user interface for ease of use.\n\n\nYeadon’s model\n\nIn 1990 Yeadon published a four-paper series based on his dissertation work concerning simulating human aerial movement, particularly twisting somersaults. His first paper10 describes a method for obtaining joint angles from film data, and accordingly develops a scheme to define the orientation of the whole body and the relative orientation of its parts. The second paper12 describes in detail the geometry of the human model. The description of the model configuration is contained in the third paper13, which also details the analytical calculation of the angular momentum. The last paper in the series14 compares a film recording of the trajectory of an aerial flight to a computer simulation using the model developed in the first three papers.\n\nYeadon provides a lucid explanation of the human inertia model in the series described above. In this section, we merely seek to summarize his work. Note that we are not concerned with the angular momentum of the model; rather we are solely interested in the model’s inertial properties.\n\nThe model is defined in terms of segments, levels, and solids. These three elements of the model are all shown in Figure 1.\n\nThe human is defined in terms of segments, levels, and solids. Segments are distinguished by alternating colors, and are denoted as P, A1, etc. Levels are denoted as “L<s>#”, where <s> denotes a segment of the body (e.g. j for the left leg) and # denotes the index of the level in that segment. The solid that is proximal to level L<s># is denoted as “<s>#”. The model is personalized via 95 measurements of 4 types: lengths L along the longitudinal axis of the segments, perimeters p about the segments, mediolateral widths w, and anteroposterior depths d. Black dots denote joint centers10.\n\nSegments The human is assumed to be composed of eleven rigid segments. Each of the four limbs has two segments, and the remainder (head and torso) consist of three more. Each of these segments is a rigid body, and has at least one rotational degree of freedom with respect to the adjacent segment to which it is attached. The segments are labeled C, A1, etc.\n\nLevels Each segment is defined by a series of parallel transverse cross sections, referred to as levels, both in Yeadon’s work and in ours. The model contains a total of 45 levels, labeled Ls0, La0, etc. Each level has the shape of a stadium (see Figure 2). A stadium can be defined by any two of the following five attributes: its perimeter p, radius r, thickness t, width w, and depth d. However, there are a few situations in which the stadium degenerates into a circle. The choice of which two attributes are used to define a given stadium depends on its location in the body. For example, it is difficult to measure a perimeter around the shoulders (Ls4), so a depth is used instead.\n\nSolids The inertial properties of each segment are computed by viewing each segment as a solid lofted through all the levels in the segment. This defines N − 1 solids in a segment with N levels. The solids are labeled s0, a0, etc. All solids in a segment share the same longitudinal axis. The shape of a given solid is defined by its two bounding stadia and the longitudinal distance between them (the solid’s height). These are termed stadium solids. The only exception is s7, the solid above the ear, which is a semi-ellipsoid. The model contains a total of 40 solids. Note that in this formulation we begin numbering the solids from 0, while Yeadon numbers the solids from 1. This is simply to match Python’s 0-based indexing.\n\nThe levels that define the segments are all in the shape of a stadium, with one exception. A stadium is defined by any two of its attributes: perimeter p, radius r, thickness t, width w, and depth d.\n\nA key feature of this inertia model is that it can be personalized to a given individual (it is subject specific). The model is personalized via 95 anthropometric measurements. These serve to define each stadium, and to specify the distances between the stadia (the heights of the stadium solids).\n\nIn addition, much of the model’s utility comes from the ability to specify the configuration of the human in which the inertial properties are desired. The configuration is specified using 21 joint angles between the various segments; these are described in Figure 3.\n\nThe configuration of the human is defined using 21 joint angles, represented by green vectors. Green crosses and circles represent vectors into and out of the page, respectively. The direction of rotation for a joint angle is given by the right-hand rule about its vector. Labels beside the vectors are the names of the configuration variables in the code. The black dot on each segment denote its joint center. The origin of the fixed coordinate frame is at the bottom center of the pelvis segment. The local coordinate frame of each segment is specified by the pair of perpendicular black vectors in or next to the segments. Despite the locations of these black vectors, the origin of a segment’s local coordinate system is always at its joint center13.\n\nThus, Yeadon’s model is defined via segments, levels, solids, and configuration angles. One can personalize the model to an individual using measurements, and obtain inertial parameters for this individual in any desired configuration. The only additional data needed are the densities of the solids. We use Dempster’s segmental densities2 by default, as does Yeadon. But the user has the option to reassign these to alternative values if desired. The model is then completed with analytical expressions for a stadium solid’s and semiellipsoid’s center of mass location and moments of inertia, using the parallel axis theorem to combine the inertial properties of multiple stadium solids. Yeadon provides these explicit formulae in12. In the next section, we describe in more detail the measurements required for the model and the way the configuration is defined.\n\n\nImplementation of Yeadon’s model\n\nThe previous section makes no departures from Yeadon’s work. However, we will now make some changes that are important for computational implementation and that serve to generalize his work. These are summarized at the end of this section.\n\nThe experimentalist provides all of the measurements, and optionally the configuration angles, in two human readable YAML formatted text files. The details of these inputs follow.\n\nMeasurements are of four types: lengths (L), perimeters (p), widths (w), and depths (d) and are always tied to a level. For example, the symbol La1p denotes the perimeter at the La1 level.\n\nAlthough we require the heights of the individual stadium solids, the experimentalist does not measure these independently. Instead, measurements are made of the longitudinal distance across multiple stadium solids. For example, the Ls2L measurement is not the distance between the Ls1 and Ls2 levels. Instead, Ls2L is measured as a distance from Ls0. To learn the levels from which one measures the various lengths, see Table 1.\n\nThe length measurements are not simply the heights of the stadium solids. They are defined relative to a certain preceding level in their segment.\n\nThere are a few exceptions to the general measurement scheme we have described thus far. While most of the lengths are measured directly, some are determined by other lengths. For example, we set La1L to be half of La2L, and so the experimentalist does not measure La1L directly. This means, however, that La1p must be measured halfway down segment A1. This scenario arises in each limb.\n\nAll stadia are oriented mediolaterally except the heels (levels Lj6 and Lk6) which are oriented anteroposteriorily. Note from Figure 1 that one measures a depth at these levels instead of a width. This depth is in fact the width of a stadium that is rotated through 90 degrees. Other necessary information about exceptions in the measurement scheme is contained in the notes of Figure 1.\n\nSince the densities for the model are provided, we can readily estimate the human’s total mass. However, if the experimentalist measures the mass of the subject, that mass can be used to proportionally scale all densities in the model so that the model’s total mass matches the subject’s measured mass.\n\nIn this section we describe, relying heavily on Figure 3, how the configuration of the model is implemented. The joint center of each segment is located with a black dot: it is always located at the center of the base stadium of the segment. The black arrows on each segment indicate its local coordinate frame, whose origin is always at the joint center of the segment. For each segment, the local z-axis is the longitudinal axis of the segment. Each of the green arrows represents a degree of freedom, and indicates the direction and sign of the corresponding joint angle via the right-hand rule. Configuration variables are labeled with the names of the two segments at the joint, and a physiological description of the joint angle. Thus, K1K2flexion is the right knee flexion angle, and PK1abduction is the right hip abduction angle (positive for abduction, negative for adduction), etc. The exceptions to this naming convention are somersalt, tilt, and twist, which specify the orientation of the P segment with respect to the fixed coordinate frame.\n\nMost joints enable more than one degree of freedom, but only four have all three rotational degrees of freedom. Since rotations are not commutative, we must specify the order of rotations at multi-degree of freedom joints. Each child segment is rotated relative to its parent segment using Euler X-Y-Z angles in a body fixed fashion. Any joints with fewer than three angles follow this same order, e.g. X-Y.\n\nThe default configuration is that in which all configuration variables have a value of zero. In the default configuration, the local coordinate basis vectors of all segments align with the global fixed coordinate basis vectors. This means that for each segment, in the default configuration, the local x-axis lies in a coronal plane and the local y-axis is directed posteriorly. Furthermore, it is assumed that in this configuration, the palms of the hands face anteriorly. We now provide the locations of joint centers in our implementation of Yeadon’s model; this information is not in his original papers. The location of the joint centers of segments A1 and B1 are at the most distal points of level Ls4 on the respective sides of the body. Joint center locations for segments J1 and K1, respectively denoted as pJ and pK, can be expressed in the local coordinate frame of the pelvis P:\n\n\n\n\n\nwhere iˆ is a unit vector along the x-axis of the pelvis, tLs0 is the thickness of the stadium at level Ls0 and rLs0 is its radius. This choice is informed by calculations present in the ISEG code published in11. Joint center locations in all other segments are at the center of the last stadium in the preceding segment.\n\nThere are a few ways in which our implementation of the human inertia model differs from that presented in10,12–14. Some of these differences arise from the fact that his work was tailored for aerial movement, more specifically for twisting somersaults. We expect, however, that our implementation of the model can be used in a more general set of investigations.\n\nSymmetry of limbs Yeadon averages the measurements for the left and right limbs so that the model is symmetric. We provide the user with the option of imposing this symmetry, but the averaging is not performed by default.\n\nAcromion stadia One can see that there are actually two different cross sections at the acromion level Ls5: we use the wider one for solid s4 in the chest and the thinner one (actually, a circle) for solid s5 in the head. The perimeter measurement at Ls5 is used for the bottom of s5. In our implementation, the stadium at the top of s4 is determined internally by the Ls4 stadium by:\n\n\n\n\n\nwhere r and t are the radius and thickness for the top stadium of s4, respectively, and rLs4 and wLs4 are the radius and width of the stadium at level Ls4, respectively. This issue is not addressed in10,12–14, and our implementation disagrees with the ISEG code found in11 (see page 358 line 251). The justification for our choice is to agree with a more recent version of Yeadon’s code, provided to us in a personal communication.\n\nHip joint center stadia in the thigh The experimentalist makes no measurements at the Lj0 or Lk0 stadia, though these stadia must be defined to define solids j0 and k0. In our implementation, these stadia are circles with the same radius r:\n\n\n\nwhere rLs0 and wLs0 are the radius and width of the Ls0 stadium, respectively. As with the acromion stadia mentioned above, the justification is that this has been implemented in the more recent version of the code shared with us.\n\nRelationships between configuration variables Yeadon enforced relationships between certain configuration variables, such as symmetric movement of the legs with respect to the pelvis. We neither assume nor impose any relationships between the 21 configuration variables; all are independent. As a result our model allows the body to assume any geometrically feasible configuration with physiological bounds.\n\nInconsistent measurements The ratio of a stadium’s perimeter to its width must be greater than 2 and less than π. If the measurements do not satisfy these constraints, then the stadium is assumed to be a circle. This scenario is not discussed by Yeadon.\n\nDegenerate stadia In the case where a stadium has zero thickness (a circle), the stadium is degenerate and some equations have a zero in the denominator. In this scenario, Yeadon still employs the formulae for stadium solids but sets the thickness to be very small12. Instead, we manipulate the equations so that the approximation is not necessary.\n\nJoint center of chest-head segment We locate the joint center between the torso T and the chest-head C at the center of level Ls3. This is in accordance with Figure 1 of13. However, this is a departure from11, in which the joint center of the chest-head is placed at the midpoint of the shoulder joint centers.\n\n\nSoftware design\n\nWe implemented the inertia model in the Python language as a package named yeadon. Python was chosen due to its ease of use, wide adoption, its stable infrastructure for distributing open source software, and its strong scientific community (SciPy).\n\nThe input to yeadon consists of (1) geometric measurements of a subject, and (2) joint configuration angles. Using these two inputs, the inertial properties of the subject in this configuration can be calculated.\n\nThe yeadon package contains 5 modules: human, segment, solid, ui, and gui. The human module contains the public interface of the package, and the segment and solid modules are used internally to construct objects available in the human module. The user interacts either directly with the human module, or via the ui or gui modules, both of which are clients to the human module. The human module contains only the Human class, the segment module contains only the Segment class, and the solid module contains the Stadium, Solid, StadiumSolid, and Semiellipsoid classes. The package relies heavily on composition. The Human constructor constructs all Stadium’s, Solid’s, and Segment’s, and ties together these objects appropriately. A visual description of the class hierarchy is shown in the UML diagram in Figure 4. The GUI is built using Mayavi15 which is both a high level Python interface to the Visualization Tool Kit16 and a lightweight application framework. We utilized Mayavi’s ability to rapidly create cross platform graphical interfaces to expose the underlying yeadon classes through interactive widgets. The graphical user interface shown in Figure 5 allows the user to load measurement data files, adjust configuration variables interactively, view the human body’s mass center location, visualize its inertia ellipsoid, and view the resulting whole-body inertial properties.\n\nThe Human class constructs all Segment’s, Solid’s, and Stadium’s, and assembles them appropriately. The classes StadiumSolid and Semiellipsoid inherit from Solid. This diagram does not reveal the entire public interface of the classes shown. The user interacts with the software through the attributes or methods of the Human class, or via the ui or gui modules.\n\nA screenshot of the Mayavi GUI application (QT backend) running on Ubuntu 14.04 is shown. The user can supply the path to measurement YAML files to obtain inertial properties for a specific subject. The graphics window on the left shows a 3D view of the model. The viewing angle, camera perspective, and other details can be manipulated with a mouse or from the toolbar above the window. Tabs on the right provide sliders and text inputs for all 21 configuration variables and allow the user to interactively set the configuration. The “Reset Configuration” button sets all the configuration variables to zero. The mass center and inertia ellipsoid for the entire body can be toggled with the checkboxes. Finally, the whole-body mass, center of mass coordinates, and inertia tensor are displayed in the bottom right and are updated interactively as the configuration is altered.\n\nThe primary outputs of the software include:\n\nWhole-body inertial properties of the model in any given configuration with respect to any point and reference frame.\n\nSegment-fixed inertial properties for any single solid or any combination of solids.\n\nVisual depiction of the model in a given configuration. These can be exported to bitmap files.\n\nThe software provides inertial properties (mass, center of mass location, and moments of inertia) for the whole human, for an individual segment, for an individual solid, or for any combination of segments and solids. In the first case of the whole human, center of mass locations and moments of inertia are expressed in the global coordinate frame. This frame has its origin at the bottom center of solid s0, and is aligned with the local frame of segment P when somersalt, tilt, and twist are zero. In the cases of individual segments or solids, inertial properties are expressed in either the local frame of the particular segment or in the fixed frame. Inertial properties for any other combination are expressed in the fixed frame.\n\n\nUsage\n\nWe begin our description of how one uses yeadon with an example of an ice skater performing a spin. As is commonly taught in high school physics, an ice skater can change his angular velocity by altering their moment of inertia due to conservation of angular momentum. By what factor can an ice skater increase his angular velocity by bringing in his arms? The commands in Listing 1 perform this calculation.\n\nThe subject represented by the measurements in male1.txt can increase his angular velocity (about the vertical axis) by a factor of 2.9 by bringing the arms in toward the body from an extended position. Figure 6 shows a rendering of a human in a more complicated and asymmetrical ice skating spin pose to demonstrate that the model is capable of complex configurations. We can also obtain the mass and center of mass location of the whole human with the code presented in Listing 2.\n\nAn ice skater can increase the axial moment of inertia by extending their limbs away from the spin axis. The right image shows both the center of mass sphere and the inertia ellipsoid for this pose. Note that the center of mass is actually outside of the body for the gravitational force resultant to be directed through the ground contact point.\n\nWith a measurement of the subject’s actual mass, we can scale all the segmental densities so that h.mass is the same as our experimentally measured mass; see Listing 3.\n\nIt is also possible to calculate the combined inertia properties of various segments and/or solids. For example, we can obtain the mass, center of mass location, and inertia tensor of the entire right arm via the code in Listing 4.\n\nAll of the methods have rich docstrings accessible via the Python help() function. For example the previous method’s docstring shows the three returned values in Listing 5.\n\n\n\nListing 1. Python interpreter session showing how one could compute the spin moment of inertia of an ice skater in two configurations.\n\n\n\nListing 2. Python interpreter session demonstrating accessing the attributes for mass and center of mass.\n\n\n\nListing 3. Python interpreter session which demonstrates segment density scaling.\n\n\n\nListing 4. Python interpreter sessions which demonstrates collecting inertial properties of multiple segments.\n\n\n\nListing 5. An example docstring for a method in the Human class.\n\n\nAdvanced example\n\nThis software was originally developed as part of an effort to easily compute the inertial properties of a human rider seated on a bicycle. A common way to model the bicycle/rider dynamics is to assume that the rider is rigid and fixed to the bicycle rear frame17. Our studies18 included a variety of bicycles and riders, for which various combinations of the inertial properties of the bicycle rear frame and rider were required.\n\nAs an advanced example, we will configure the model using the yeadon software such that the human is seated on the bicycle, feet at the bottom bracket axis, and hands on the handlebars with arms hanging down. The inertia of the human will be computed first with respect to its center of mass and then combined with that of the bicycle rear frame using the parallel axis theorem to give the total inertia of the human rigidly affixed to the bicycle rear frame in the bicycle’s reference frame.\n\nWe use the definitions and parameters of the benchmark bicycle model17 defined using the standard SAE vehicle coordinate system (which is different from Yeadon’s coordinate system). In addition to the geometrical parameters in the benchmark bicycle, the bicycle rear frame and handlebar location are defined by several geometric and inertial parameters. Furthermore, a single measurement of the rider’s forward lean angle relative to the bicycle frame was made.\n\nAn interactive IPython19 notebook in the supplementary materials provides a detailed walk through this advanced example. This example is also included in the Yeadon 1.2.0 software source files and a rendered version is viewable with NBViewer. The following briefly summarizes the steps involved in the computation and further illustrates use of the yeadon software.\n\n1. Geometric and inertial properties of the bicycle were estimated with an independent method18. Inertial properties of the rear frame of the bicycle were expressed in the SAE coordinate system described above.\n\n2. We solve for a configuration of the human that enforces the human rider to be seated properly on the bicycle, for any bicycle. The SymPy Mechanics package20 is used for these computations.\n\n3. The Human.inertia_transformed method is used to express the human’s inertia in the bicycle’s reference frame.\n\n4. The combined mass and center of mass location of the human and the rear frame of the bicycle are computed and the parallel axis theorem is employed to express the moments of inertia of each body about the combined center of mass, where they are then summed to get the combined moments of inertia.\n\nThe example shows how to set a complex configuration of the Yeadon model and extract the geometric and inertial properties expressed in arbitrary reference frames and relative to arbitrary points as well as how to visualize the configuration.\n\n\nConclusion\n\nWe have presented an open source software package that implements a widely used inertial model of a human. This package is available in public repositories under a permissive copyright license. The software provides an API for use as a library, and also has both a command-line user interface and a graphical user interface for interactive high level use as a standalone application. The structural design of the software is presented as an introduction to the source code which is available in a public repository that is open for contributions and modifications. Finally, we have described both simple and advanced use cases for the API, one in the text of the paper and one in the supplementary IPython notebook.\n\n\nSoftware availability\n\nhttp://pypi.python.org/pypi/yeadon/\n\nhttp://www.github.com/chrisdembia/yeadon\n\nhttps://github.com/F1000Research/yeadon/releases/tag/V1.2\n\nhttp://dx.doi.org/10.5281/zenodo.1157921\n\nyeadon is licensed under the 3 clause BSD license which permits both non-commercial and commercial use.", "appendix": "Author contributions\n\n\n\nJ.K.M and M.H. conceived of the software tool. C.D. was the primary developer of the software and primary author of the documentation and the paper. J.K.M contributed to the development of the software and authorship of the documentation and the paper. M.H. advised the project and contributed to the writing of the paper. C.D and J.K.M prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nThe authors have no financial, personal, or professional competing interests that could be construed to unduly influence the content of this article.\n\n\nGrant information\n\nThis material is partially based upon work supported by the National Science Foundation under Grant No. 0928339. The recipients of this grant are Mont Hubbard and Ronald Hess. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.\n\n\nAcknowledgements\n\nWe thank M.R. Yeadon for sharing his source code and measurement methodologies. His generosity has greatly improved the quality of our software.\n\n\nReferences\n\nBjørnstrup J: Estimation of Human Body Segment Parameters Historical Background. Technical report, 1995. Reference Source\n\nDempster WT: Space requirements of the seated operator. Technical report, Wright-Patterson Air Force Base, Dayton, OH. 1955. Reference Source\n\nClauser CE, McConville JT, Young JW: Weight, volume and center of mass of segments of the human body. Technical Report AMRL TR 69–70, Wright-Patterson Air Force Base, Ohio. 1969. Reference Source\n\nChandler RF, Clauser CE, McConville JT, et al.: Investigation of inertial properties of the human body. Technical Report AMRL TR 74–137, Wright-Patterson Air Force Base, Ohio, 1975. Reference Source\n\nJensen RK: Estimation of the biomechanical properties of three body types using a photogrammetric method. J Biomech. 1978; 11(8–9): 349–358. PubMed Abstract \| Publisher Full Text\n\nZatsiorsky V, Seluyanov V: The mass and inertia characteristics of the main segments of the human body. In H Matsui and K Kobayashi, editors, Biomechanics VIII-B, Illinois, Human Kinetic, 1983; 1152–1159. Reference Source\n\nZatsiorsky V, Seluyanov V, Chugunova L: In vivo body segment inertial parameters determination using a gamma-scanner method. In N Berme and A Cappozzo, editors, Biomechanics of Human Movement: Applications in Rehabilitation, Sports and Ergonomics, Ohio, Bertec. 1990; 186–202. Reference Source\n\nPark SJ, Kim CB, Park SC: Anthropometric and biomechanical characteristics on body segments of Koreans. Appl Human Sci. 1999; 18(3): 91–99. PubMed Abstract \| Publisher Full Text\n\nGriffiths IW, Watkins J, Sharpe D: Measuring the moment of inertia of the human body by a rotating platform method. Am J Phys. 2005; 73(1): 85–92. Publisher Full Text\n\nYeadon MR: The simulation of aerial movement--I. The determination of orientation angles from film data. J Biomech. 1990; 23(1): 59–66. PubMed Abstract \| Publisher Full Text\n\nYeadon MR: The mechanics of twisting somersaults. Doctoral, Loughborough University of Technology, Thesis, 1984. Reference Source\n\nYeadon MR: The simulation of aerial movement--II. A mathematical inertia model of the human body. J Biomech. 1990; 23(1): 67–74. PubMed Abstract \| Publisher Full Text\n\nYeadon MR: The simulation of aerial movement--III. The determination of the angular momentum of the human body. J Biomech. 1990; 23(1): 75–83. PubMed Abstract \| Publisher Full Text\n\nYeadon MR, Atha J, Hales FD: The simulation of aerial movement--IV. A computer simulation model. J Biomech. 1990; 23(1): 85–9. PubMed Abstract \| Publisher Full Text\n\nRamachandran P, Varoquaux G: Mayavi: 3D Visualization of Scientific Data. Comput Sci Eng. 2011; 13(2): 40–51. Publisher Full Text\n\nSchroeder W, Martin K, Lorensen B: Visualization Toolkit: An Object-Oriented Approach to 3D Graphics, 4th Edition. 2006. Reference Source\n\nMeijaard JP, Papadopoulos JM, Ruina A, et al.: Linearized dynamics equations for the balance and steer of a bicycle: a benchmark and review. Proc R Soc A: Math Phys Eng Sci. 2007; 463(2084): 1955–1982. Publisher Full Text\n\nMoore JK: Human Control of a Bicycle. Doctor of Philosophy, University of California, Davis, Davis, CA, 2012. Reference Source\n\nPérez F, Granger BE: IPython: A system for interactive scientific computing. Comput Sci Eng. 2007; 9(3): 21–29. Publisher Full Text\n\nGede G, Peterson DL, Nanjangud AS, et al.: Constrained multibody dynamics with Python: From symbolic equation generation to publication. In Volume 7B: 9th International Conference on Multibody Systems, Nonlinear Dynamics, and Control, Portland, Oregon, USA, 2013; DETC201313470. Publisher Full Text\n\nDembia C, Moore JK: yeadon-1.2.0. 2014. Data Source" }	[ { "id": "6185", "date": "30 Oct 2014", "name": "Alison L. Sheets", "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\nThe aim of this manuscript is to describe a software tool that was developed to calculate the inertia properties of the human body and segments of the body using the method developed by M. R. Yeadon. Yeadon's method represents the body as a series of solid shapes, and is frequently used in biomechanical analyses. The software described in this paper makes the method developed by Yeadon faster and easier to implement. The manuscript is very well written, includes numerous examples that highlight the software flexibility and capabilities, and the host site for the software is well documented. Additionally, the detailed description of the inertia model clarifies a few ambiguities from Yeadon’s original text, and modifications were made to enable the model to answer more general questions. The figures are detailed and descriptive.My only minor suggestion is for the authors to add definitions for terms in Figure 4 that are not self-descriptive. For example, it is not clear what rel_center_of_mass and rel_inertia are defined relative to.", "responses": [ { "c_id": "1062", "date": "05 Nov 2014", "name": "Jason Moore", "role": "Author Response", "response": "Thank you for the review and the approval. As far as the method/attribute names are concerned in the UML diagram (Fig 4), we specifically didn't include great detail, trying to focus on the object hierarchy and the the most important methods/attributes of the objects. All of the methods and attributes are explained in the online software documentation, for example:http://yeadon.readthedocs.org/en/latest/segment.htmlWe can add some of those details in the paper if you think it will clear up things, or maybe link to the online documentation. I will discuss with the co-authors." }, { "c_id": "1283", "date": "08 Apr 2015", "name": "Jason Moore", "role": "Reader Comment", "response": "Dear Alison L. Sheets,Thank you for your time and expertise in this review and we are happy that you have approved with minor revisions. You had one specific concern in your review:\"My only minor suggestion is for the authors to add definitions for terms in Figure 4 that are not self-descriptive. For example, it is not clear what rel_center_of_mass and rel_inertia are defined relative to.\"In version 2, we have updated the text to clarify what these methods names mean and how we define the word \"relative\", see:https://github.com/chrisdembia/python-yeadon-paper/pull/61/filesAlso note that the docstrings for these methods explain that these produce the inertial properties relative to the segment's local coordinate system. For example see:http://yeadon.readthedocs.org/en/latest/segment.html#yeadon.segment.Segment.rel_inertiaSincerely,Chris Dembia, Jason K. Moore, and Mont Hubbard" } ] }, { "id": "6948", "date": "02 Feb 2015", "name": "Arnaud Barré", "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\nGeneral commentsThe aim of this manuscript is to present a software tool used to: 1. facilitate the computation of the body segment inertial parameters proposed by M.R. Yeadon; 2. visualize in three dimensions the posture of an avatar; 3. request/compute some body segment inertial parameters related to the posture set. The description of the Yeadon's model is very well detailed as well as all the modifications realized. The illustrations and examples presented shows also the possibilities of the software.However, it would be interesting to have a better comparison of the different methods proposed to compute body segment inertial parameters using direct and indirect methods. For example, the authors mentions the facility of Yeadon's method compared to other studies, but the number of measurements is large compared to others studies not cited, like de Leva (1996) or Dumas et al. (2007). These indirect methods have also error as explained in Rossi et al. (2013). What is the advantage of Yeadon's method compared to others? A discussion on the limitations of the model proposed (joint center accuracy, joint range, mass estimation, density scaling, etc.) would be also of interest. The authors modified some parts of the original model, why did they not add joint centers for ankles and wrists? This would give a better general behavior to the avatar.Specific commentsI would suggest to the authors to clarify the points listed below:Figure 1: The authors should give the abbreviations of the body parts. It is not obvious why all the levels for the torso does not use the P,T,C letters, but 's' instead. I understand this is the original nomenclature used by M.R. Yeadon, but this should be detailed. Moreover, as explained later in the manuscript, some segments have two levels at the same location (for example, shoulder and neck levels are abbreviated by Ls5), they would be distinguished for clarity. Thus, this would help to better understand the departures proposed (like in Acromion stadia).Figure 1: In the legend \"segments, label, solids\", the segment A2 is defined between a2 and s6. I assume it is a6 and not s6.\"Measurements\": fifth paragraph: The authors should mention the potential accuracy issue related to the scaling of the densities (homogeneous scaling over the body, possibly increasing the error with the body segment inertial parameters). This could be in the discussion.\"Configuration\" paragraph 1: The authors wrote \"somersalt\" instead of \"somersault\". (Also in page 7).Formulas 1 and 2: These formulas give only the x coordinates of the hip center joint. What about the y and z coordinates?Formulas 3, 4, 5: The authors should use a suffix to the computed parameters to clarify the associated level. \"Relationships between configuration variables\": The authors wrote \"21 configuration variables;\". Please modify to \"21 joint angle configuration variables to clarify\".\"Degenerate Stadia\": What are the manipulations? Please detail.\"Software design\": Please add a reference for \"Python\" as this is the only one tool that does not have one (compared to Mayavi or VTK). The authors should also indicate that the library is implemented with Python 2.7. Their code could not be compatible with Python 3.Figure 5: How did the authors fix the joint angular range? This seems to be empirical but it should be at least mentioned in the manuscript.", "responses": [ { "c_id": "1212", "date": "10 Feb 2015", "name": "Jason Moore", "role": "Reader Comment", "response": "Thank you for the thorough and helpful review. We appreciate all of your suggestions and plan to address them in the second version. If you are interested, we have all of your comments in our Github repository (https://github.com/chrisdembia/python-yeadon-paper/issues) and will be discussing and dealing with them there." }, { "c_id": "1284", "date": "08 Apr 2015", "name": "Jason Moore", "role": "Reader Comment", "response": "Dear Arnaud Barré,Thank you very much for lending your time and expertise for this review. We are happy that you have approved the article and appreciate your thorough review. We have submitted a second revision to address your comments and suggestions. The responses for each of your comments follow:\"However, it would be interesting to have a better comparison of the different methods proposed to compute body segment inertial parameters using direct and indirect methods. For example, the authors mentions the facility of Yeadon's method compared to other studies, but the number of measurements is large compared to others studies not cited, like de Leva (1996) or Dumas et al. (2007). These indirect methods have also error as explained in Rossi et al. (2013). What is the advantage of Yeadon's method compared to others? A discussion on the limitations of the model proposed (joint center accuracy, joint range, mass estimation, density scaling, etc.) would be also of interest.\"We also think that a paper showing the disadvantages and advantages of these various models would be very beneficial but we believe that this undertaking is out of the scope of this paper. We are leaving these discussions to other literature and would like to keep the scope of this paper about the specific software implementation of one method that has been described, judged, and commented on elsewhere. If the reviewer would like to work on this idea with us for a future publication we could certainly discuss possibilities.\"The authors modified some parts of the original model, why did they not add joint centers for ankles and wrists? This would give a better general behavior to the avatar.\"We aimed to create a faithful representation of Yeadon's model and thus did not include extra joints. But the object oriented nature of our software design would allow relatively easy sub-classing to implement other models with more joints.Another reason we did not worry much about the ankle and wrist joints was due to our intended application: whole body inertia of a bicycle rider. The inertial difference in a whole body model with and without ankle and wrist joints is very small, in our case negligible. It is only important when one is worried about those specific segments of the body. Yeadon's model is likely not suitable for models that focus on the hand or foot. It was not necessarily designed to predict the body segment parameters on a individual basis but to predict whole body inertia, and this is how we use the model.The text has been updated to reflect this, see:https://github.com/chrisdembia/python-yeadon-paper/pull/63\"Figure 1: The authors should give the abbreviations of the body parts. It is not obvious why all the levels for the torso does not use the P,T,C letters, but 's' instead. I understand this is the original nomenclature used by M.R. Yeadon, but this should be detailed. Moreover, as explained later in the manuscript, some segments have two levels at the same location (for example, shoulder and neck levels are abbreviated by Ls5), they would be distinguished for clarity. Thus, this would help to better understand the departures proposed (like in Acromion stadia).\"We clarified more on the P, T, C letters here:https://github.com/chrisdembia/python-yeadon-paper/pull/64But we felt that we adequately addressed the single level that shares two different sized stadia and that further improvement with, for example, more subscripts would be overkill.\"Figure 1: In the legend \"segments, label, solids\", the segment A2 is defined between a2 and s6. I assume it is a6 and not s6.\"Keen eye!, fixed here:https://github.com/chrisdembia/python-yeadon-paper/pull/52\"\"Measurements\": fifth paragraph: The authors should mention the potential accuracy issue related to the scaling of the densities (homogeneous scaling over the body, possibly increasing the error with the body segment inertial parameters). This could be in the discussion.\"We agree that there are potential accuracy issues with scaling the densities by mass, but we also believe that this is a logical thing to do and that it more than likely reduces error in the inertial estimates. We've added a sentence to warn the user that there can be pitfalls:https://github.com/chrisdembia/python-yeadon-paper/pull/59\"\"Configuration\" paragraph 1: The authors wrote \"somersalt\" instead of \"somersault\". (Also in page 7).\"Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/51\"Formulas 1 and 2: These formulas give only the x coordinates of the hip center joint. What about the y and z coordinates?\"We've clarified how the origin of the model is defined and thus the y and z coordinates are zero by definition.Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/58\"Formulas 3, 4, 5: The authors should use a suffix to the computed parameters to clarify the associated level.\"Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/57\"\"Relationships between configuration variables\": The authors wrote \"21 configuration variables;\". Please modify to \"21 joint angle configuration variables to clarify\".\"Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/55\"\"Degenerate Stadia\": What are the manipulations? Please detail.\"Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/66\"\"Software design\": Please add a reference for \"Python\" as this is the only one tool that does not have one (compared to Mayavi or VTK). The authors should also indicate that the library is implemented with Python 2.7. Their code could not be compatible with Python 3.\"Thanks, it is good to note the Python 3 incompatibility. We are unfortunately tied to the Mayavi/VTK tool chain which does not yet support Python 3.Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/54\"Figure 5: How did the authors fix the joint angular range? This seems to be empirical but it should be at least mentioned in the manuscript.\"Fixed here: https://github.com/chrisdembia/python-yeadon-paper/pull/62Sincerely,Chris Dembia, Jason K. Moore, and Mont Hubbard" } ] } ]	1	https://f1000research.com/articles/3-223
https://f1000research.com/articles/4-87/v1	07 Apr 15	{ "type": "Research Article", "title": "Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin", "authors": [ "Penny Beckerson", "Dimitri Svistunenko", "Brandon Reeder", "Penny Beckerson", "Dimitri Svistunenko" ], "abstract": "The reaction of hydrogen peroxide with ferric human cytoglobin and a number of distal histidine variants were studied. The peroxidase activity of the monomeric wildtype protein with an internal disulfide bond, likely to be the form of the protein in vivo, exhibits a high peroxidase-like activity above that of other globins such as myoglobin. Furthermore, the peroxidatic activity of wildtype cytoglobin shows increased resistance to radical-based degradation compared to myoglobin. The ferryl form of wildtype cytoglobin is unstable, but is able to readily oxidize substrates such as guaiacol. In contrast distal histidine mutants of cytoglobin (H81Y and H81V) show very low peroxidase activity but enhanced radical-induced degradation. Therefore, the weakly bound distal histidine appears to modulate ferryl stability and limit haem degradation. These data are consistent with a role of a peroxidase activity of cytoglobin in cell stress response mechanisms.", "keywords": [ "Cytoglobin", "distal histidine", "peroxide", "ferryl", "hexacoordinate", "peroxidase", "monomer" ], "content": "Introduction\n\nCytoglobin (Cygb) is a haem protein ubiquitously expressed in vertebrate cells1,2. Cygb has a coordinating distal histidine in the deoxygenated ferrous form, giving a hexacoordinate haem iron similar to neuroglobin (Ngb)1,3,4. Several possible physiological functions of Cygb have been proposed including nitric oxide (NO) dioxygenase activity, involvement in collagen synthesis and oxidative stress response5–8. The mechanisms by which Cygb affects cellular responses to oxidative stress are unknown, however it has been suggested that protection of cells may occur by Cygb functioning as a scavenger of reactive oxidative species (ROS) or as a peroxidase9,10. In vivo studies have shown that Cygb expression is up-regulated by hydrogen peroxide suggesting a role in oxidative stress protection6. This is supported by Cygb knockdown studies showing sensitisation of cells to oxidative stress, with over-expression providing protection5. The specific role of Cygb in these stress responses is unclear.\n\nThe reaction of Cygb with peroxides has not been extensively studied. However, the reactions of pentacoordinate globins, such as myoglobin (Mb) and haemoglobin (Hb), with hydrogen peroxide have been well characterised11,12. The reaction of ferrous or ferric Mb and Hb with peroxides leads to the formation of the ferryl species ([Fe4+=O2-]2+). Protein based radicals are also formed in the reaction of ferric Mb or Hb with peroxides. Unlike classical peroxidase enzymes, the radical in these systems is inherently unstable leading to oxidative modifications to the haem and protein. In pentacoordinate globins the distal histidine (e.g. H64 in Mb) is important in providing a hydrogen bond to the partially deprotonated bound peroxide (Fe3+-OOH-)13. This facilitates the scission of the di-oxygen bond to form the ferryl iron and porphyrin cation radical (Compound I), followed by a rapid migration and partial decay of the radical to form compound II14–16. Proteins that lack the distal residue (e.g. H64Q in Mb) lead to a transient stabilisation of the peroxide-bound intermediate (Compound 0) over the millisecond timescale13,17. In Ngb the hexacoordinate configuration of the haem iron prevents the formation of a ferryl species18.\n\nThis study examines the mechanism of the interaction of Cygb with peroxide and the role of the distal histidine in such reactions. Our data show that the monomeric wildtype (WT) Cygb, when reacting with peroxides, is characterised by a high peroxidase activity (in comparison to other globins) and that the ferryl formed (compound II) is more unstable than seen in Mb or Hb. The distal histidine is particularly important for this peroxidase activity since the mutants lacking this residue show a low substrate oxidation and high rates of haem degradation. This study supports a role of Cygb peroxidase activity in stress responses in vivo.\n\n\nMaterials and methods\n\nThe human Cygb gene was purchased from OriGene (via Cambridge Bioscience, UK), transferred from the cloning vector into expression vector pET28a (Merck Millipore, UK), expressed and purified as previously described19,20. Cygb pET28a plasmid was mutated using site-directed mutagenesis based on a modified Agilent Quikchange II method to give the H81V and H81Y mutation. Initial denaturation was 95°C for 2 min followed by 20 cycles of 95°C for 50 s, 55°C for 60 s and 68°C for 13 min. Primers were purchased from Eurofins, UK. The mutated Cygb were also expressed and purified using the same protocol as for the wildtype (WT) protein19. The monomeric, dimeric and polymeric Cygb were separated using G75 Superdex column (GE Healthcare) equilibrated with 0.1 M sodium phosphate buffer (Fischer, UK) pH 7.4. Fractions were collected corresponding to absorbance peaks at 280 nm. Monomeric Cygb variants were used for all subsequent experiments.\n\nA Varian Cary 5E UV-vis spectrophotometer was used to measure UV-visible spectra. Extinction coefficients of the Cygb distal histidine mutants were calculated as previously described using HPLC20.\n\nFar UV CD spectra of monomeric WT, H81V and H81Y Cygb (5 μM) in 20 mM sodium phosphate buffer (pH 7.4) were measured using an Applied Photophysics Chirascan CD spectrophotometer between wavelengths 180–300 nm in a 1 ml quartz cuvette at 20°C. Scan speed was 60 nm min-1. Three spectra were taken and averaged and corrected for buffer baseline.\n\nMonomeric WT, H81V and H81Y Cygb (5 μM) in 0.1 M sodium phosphate buffer pH 7.4 were reacted with hydrogen peroxide (5 mM) in the presence of 8.94 mM guaiacol (0.1% v/v, Sigma, UK). The peroxidase activity of Cygb was monitored by the formation of tetraguaiacol (470 nm) due to the oxidation of guaiacol by the ferryl iron (equation 1):\n\n\n\nThe reaction was followed using an Agilent 8453 diode array spectrophotometer.\n\nFerric monomeric WT, H81V and H81Y Cygb (50 μM), in 20 mM phosphate buffer (pH 7.4), were reacted with a range of hydrogen peroxide (Sigma, UK) concentrations (0–5 mM) and incubated at room temperature for 18 hours. The samples were analysed by both non-reducing (4% stacking, 12% resolving) and reducing SDS-PAGE (4% stacking and 12% resolving) gel electrophoresis.\n\nOxidative damage to the haem in these samples was measured by reverse phase HPLC using an Agilent HP1100 HPLC fitted with a diode array spectrophotometer. The column used was a Zorbax StableBond 300SB C3 250 mM × 4.6 mM fitted with a 12 mm × 4.6 mm guard column. Solvent A was 0.1% trifluoroacetic acid (TFA) (Fisher, UK) and solvent B was acetonitrile (VWR, UK) containing 0.1% TFA. The gradient started at 35% solvent B for 10 min, increasing to 37% for 5 min, increasing to 40% for 1 minute with a final increment to 43% for 10 min. The flow rate was 1 ml min-1 and the temperature was 25°C21. The haem concentration was calculated from the area under the peak at ~14 min.\n\nThe kinetics of the formation of ferryl Cygb was measured using an Applied Photophysics SX20 stopped-flow spectrometer fitted with a diode array unit. Ferric monomeric WT, H81V and H81Y Cygb (5 µM after mixing, in 50 mM sodium phosphate buffer pH 7.4) were rapidly mixed with a range of hydrogen peroxide concentrations (0–10 mM final). Absorbance changes following mixing were recorded using the proSX software and kinetics fitted globally using the Applied Photophysics ProKinetist software.\n\nMonomeric WT, H81V and H81Y Cygb (80 μM) in 20 mM sodium phosphate buffer (pH 7.4) were reacted with hydrogen peroxide, either at 1:1 at (80 μM) or 1:10 (800 μM) protein:hydrogen peroxide ratio in Wilmad SQ EPR tubes (Wilmad Glass). The reaction was halted by flash-freezing in dry ice cooled methanol at various time points (0, 20, 45, 90, 180 s). The samples were stored in liquid nitrogen (77 K) prior to measurement. Electron paramagnetic resonance (EPR) spectra were taken using a Bruker EMX EPR spectrophotometer (X-band) equipped with a spherical high-quality resonator SP9703 and an Oxford Instruments liquid helium system. The modulation frequency was 100 kHz. Accurate g values were obtained using the built-in microwave frequency counter and a 2,2-diphenyl-1-picrylhydrazyl powder standard, the g value for which is g = 2.0027 ± 0.000222,23.\n\n\nResults\n\nThe expression of WT Cygb displays mixed monomer and dimer conformations as previously described20. However, the expressed H81V and H81Y mutants were predominantly monomeric as shown in the protein elution profiles from gel filtration (Figure 1A). Herein only monomeric protein was used in this study. The circular dichroism (CD) spectra of Cygb distal histidine mutants are shown in Figure 1B. All proteins display the typical double minima at 209 and 222 nm characteristic of protein structures that is predominantly α-helical, essentially identical to the WT protein as previously reported24. Therefore the mutations did not significantly alter the secondary structure and the recombinant protein has folded correctly.\n\n(A) Size exclusion chromatography (G-75 Superdex) of recombinant WT cytoglobin showing two major peaks, (i) at 56 ml corresponding to the 43 kDa dimer and (ii) at 67 ml corresponding to the 21 kDa monomer respectively. A small peak at ~45 ml corresponding to a minor fraction of polymerised Cygb. The mutant Cygb show the same peaks, however the peak corresponding the monomer (i) is the most prominent. (B) CD spectra of Cygb distal histidine mutants displaying characteristic double minima at 209 and 222 nm identifying α-helical content.\n\nThe optical spectra of Cygb are shown in Figure 2A in the ferric, deoxyferrous and ferrous-CO bound forms. These spectra are typical of those previously shown for the WT protein19,24. The ferrous-CO spectrum of H81V and H81Y Cygb is essentially identical to that of the WT protein with a Soret of 423 nm and α and β peaks of 570 and 542 nm respectively19,24. Mutation of the distal histidine results in significant changes of the optical properties of the protein when in the absence of exogenous ligands (Figure 2B and Figure 2C respectively). In the deoxyferrous state both of the distal histidine mutants exhibit only a single αβ band with a low intensity shoulder at higher wavelength in comparison to the WT which has two distinct peaks. The α and β bands of the WT protein is characteristic of a hexacoordinated haem iron, similar to that observed in Ngb or cytochrome c3,25. The two mutant proteins deoxyferrous optical characteristics are more typical of a pentacoordinate haem iron ligation as seen in Mb and Hb11. However, the broad Soret band of deoxyferrous H81V Cygb is wider than observed with the H81Y protein and Mb or Hb, suggesting that H81V has a subpopulation of hexacoordinated species. In the ferric oxidation state the H81Y mutant displays three peaks in the visible region (Figure 2C) and exhibits a green appearance due to the 600 nm band compared to the orange-brown appearance of the other ferric proteins11. Other haem proteins with a distal tyrosine also display these characteristic absorbance peaks such as the homodimeric haemoglobin from Mycobacterium tuberculosis (HbN)26 or in myoglobin with a distal tyrosine mutation27.\n\n(A) The optical spectra of WT monomeric Cygb as previously described20. (B) The optical spectra of H81V Cygb. The ferric (solid) has a Soret of 415 nm and low intensity α and β peaks at 550 and 588 nm respectively. The reduction of the protein by dithionite (dashed) led to a bathochromic shift with a broadening of the Soret accompanied by appearance of a single broad αβ peak. The binding of CO (thin solid) led to a hypsochromic shift of the Soret with an increase in intensity. This is accompanied with the appearance of the α and β peaks to 580 and 550 nm respectively. (C) The optical spectra of H81Y Cygb. The ferric (solid) has a Soret of 411 nm and three peaks in the visible region at 500, 543 and 605 nm. The reduction of the protein by dithionite (dashed) led to a bathochromic shift of the Soret accompanied by an appearance of a single αβ band with a shoulder. The binding of CO (thin solid) led to a hypsochromic shift of the Soret with an increase in intensity. This is accompanied with the appearance of the α and β peaks at 570 and 542 nm respectively.\n\nThe EPR spectrum of monomeric ferric WT Cygb is shown in Figure 3 and is consistent with that previously reported20, comprising of a high spin (HS) signal at g = 5.95, a low-spin (LS) signal at g = 3.15 and a small component at g = 4.3 representative of iron ferric ion in rhombic coordination mainly originating from damaged haem28. Both of the distal histidine mutants exhibit different EPR spectra to the WT monomer. WT Cygb is partially HS showing a weak distal histidine coordination in the ferric form of the protein as previously described19,20. In the H81V mutant the HS signal is much greater compared to the WT protein, indicating the H81V is primarily pentacoordinate in the ferric state. This is also the case for the H81Y variant. Although the HS signal intensity for the H81Y appears lower, there is a notable contribution from the rhombic HS ferric haem signal with gx = 6.68 and gy = 5.39 which makes the whole EPR signal wider than the axial EPR line at g┴ = 5.95. This accounts for a higher spin concentration at a similar intensity of the EPR signal.\n\nEPR spectra of ferric cytoglobin (80 µM): wild type monomer with internal disulfide bond (upper spectrum), histidine 81(E7) to valine mutation (middle spectrum) and histidine 81 to tyrosine mutation (lower spectrum). The wild type protein is predominantly in a HisF8–Fe(III)–HisE7 co-ordination (g3 = 3.20, g2 = 2.05, g1 is off scale) as previously reported19. The H81V protein is mainly high spin with a HisF8–Fe(III)–H2O configuration with a minor (but sharper) LS component (g3 = 2.76, g2 = 2.29, g1 is off scale). The H81Y protein is again predominantly HS ferric haem rhombic signal with g = 6.68 and g = 5.39.\n\nUnlike WT Cygb, the mutants do not exhibit the broad LS ferric haem form spectrum with g = 3.15, which accounts for a significant fraction of protein in the WT. There is a different LS ferric haem EPR signal seen in both mutants, with g = 2.76 and g = 2.29. The concentration of this haem form is 5.2 times higher in the H81V mutant as compared to the H81Y mutant. The g-factors of this LS ferric haem form are close to some bis-His LS forms observed in other proteins29,30. We speculate that a different residue can be the cause of this LS ferric haem state, likely resulting from a nitrogen ligand that can be positioned at the 6th haem coordination site. The best candidate for such a residue is the nearby arginine (R84). One can speculate why a LS ferric haem EPR signal is stronger in the valine mutant compared with the tyrosine mutant – most likely because tyrosine is larger allowing lower R84 occupancy.\n\nThe reaction of WT Cygb with hydrogen peroxide does not form a ferryl species that can be monitored by standard optical spectroscopy, unlike other haem proteins such as Mb and Hb17,31. Therefore the peroxidatic activity was monitored using the guaiacol peroxidase activity assay which uses guaiacol as a substrate for ferryl Cygb, generating a tetrameric oxidation product of guaiacol that can be monitored at 470 nm (Figure 4A)32. The formation of the tetrameric oxidation product of guaiacol following the reaction of H81V and H81Y with hydrogen peroxide is negligible in comparison to the WT protein (figure 4D). This initially suggests that there is limited ferryl formation at the peroxide concentration studied (5 mM). However, the bleaching of the haem moiety, which is particularly rapid with the H81V protein, signifies that the guaiacol is not reacting with the ferryl haem iron hence radical damage from redox cycling is prominent. Therefore an alternative method was used to attempt to measure the formation of ferryl haem iron directly using stopped-flow spectroscopy.\n\n(A) Spectra of the oxidation of guaiacol (8.9 mM) from the reaction of WT Cygb (5 µM) and H2O2 (5 mM), showing the rapid formation of tetrameric guaiacol. (B and C) Conditions are for those described in (A) except with H81V (B) and H81Y (C) variants of Cygb. There is little guaiacol oxidation observed but extensive haem bleaching within the time course of the experiment. (D) Time course of guaiacol oxidation showing a high peroxidase activity of WT Cygb.\n\nThe reaction of monomeric Cygb with high concentrations of hydrogen peroxide (1 to 10 mM) displayed monophasic time course (Figure 5A). The spectrum of the ferryl species has a Soret peak of 418 nm and α and β peaks of 562 and 542 nm respectively (Figure 5B), consistent with the spectrum of ferryl Mb31. The rate of formation of the ferryl species was hydrogen peroxide concentration dependent with a second order rate constant of 312.3 ± 16.3 M-1s-1 (Figure 5C). This value is very similar to previously reported values of the second order rate constant of myoglobin reacting with hydrogen peroxide (170 M-1s-133 and 267 M-1s-116). There were no changes in amplitude in response to hydrogen peroxide concentration.\n\n(A) Time course of monomeric WT Cygb (5 µM) reacting with H2O2 (5 mM). The changes in absorbance at 420 nm over time were fitted to a single exponential. (B) UV-visible changes observed during the reaction of monomeric Cygb (5 µM) with 5 mM H2O2, identifying the ferryl species of Cygb with a Soret peak of 418 nm and α and β peaks of 542 and 562 nm respectively. (C) The hydrogen peroxide concentration dependence on ferryl formation observed rate constants of monomeric WT Cygb (5 µM) giving a second order rate constant of 312.3 ± 16.3 M-1s-1.\n\nThe reaction of the distal histidine mutants with hydrogen peroxide showed no measurable formation of ferryl haem iron, with the main optical changes indicative of haem degradation from radical damage (Figure 6A–C). There is a transient increase in absorbance at ~600–700 nm for H81V, which may be suggestive of an oxidative modified intermediate species.\n\n(A) UV-visible changes of H81V (5 µM) due to the reaction with 100 mM H2O2 over the time course of 1 s. (B) UV-visible changes of H81Y (5 µM) due to the reaction with 100 mM H2O2 over the time course of 1 s. (C) Time course of H81V and H81Y (5 µM) reacting with H2O2 (40 mM). The changes in absorbance at 420 nm over time were fitted linearly.\n\nTo monitor radical damage to the haem of Cygb, the protein (50 µM) was reacted with hydrogen peroxide (0–5 mM) at 25°C and the oxidative damage to the protein assessed after reaction was complete by HPLC analysis and gel electrophoresis. The HPLC results (Figure 7) show that WT Cygb is more resistant to haem damage compared to Mb, with 2500 µM peroxide required to degrade or modify 50% of the haem compared to 500 µM for Mb. However, the distal histidine mutants were more susceptible to peroxide induced haem damage than the WT protein with H81Y showing 50% haem damage at 500 µM peroxide and 60 µM for H81V (~1:1 peroxide: protein ratio). Interestingly Mb and H81Y show identical resistance to peroxide induced haem damage at all peroxide concentrations studied.\n\n(A) HPLC analysis of cytoglobin (50 µM) following reaction with H2O2 (0–5 mM) showing unmodified haem concentration. (B) Time course of radical concentration formed in the reaction of cytoglobin (80 µM) and H2O2 (at either 1:1 or 1:10 molar excess), as measured by the EPR spectroscopy. (C) Example EPR spectra (t=90 s) exhibiting a primarily doublet radical line shape of WT cytoglobin compared to the singlet line shape of H81Y and H81V.\n\nThe non-reducing PAGE analysis of monomeric Cygb before hydrogen peroxide treatment showed two bands that correspond to the monomeric protein, one with an intramolecular disulfide bond and the other with a reduced disulfide bond (Figure 8A) as described previously20. These bands lose intensity upon reaction with low concentrations of hydrogen peroxide (50–100 μM) accompanied with the appearance of bands representative of dimeric and higher order conformers. At hydrogen peroxide concentrations above 1 mM the protein forms aggregates as shown by minimal gel migration.\n\nCygb (50 μM) was reacted for 18 hours with 0, 50, 100, 250, 500, 1000, 2500, 5000 μM H2O2 (lanes 1–8 respectively). SDS-PAGE analysis of WT, H81V and H81Y Cygb are shown in A, B and C respectively and non-reducing PAGE analysis of WT, H81V and H81Y Cygb are shown in D, E and F respectively. The monomer and dimer conformations are shown as (M) and (D) respectively.\n\nBoth of the distal histidine mutants exhibit bands similar to that of the WT monomeric protein showing the presence and absence of an intramolecular disulfide bond (Figure 8B and Figure 8C). The mutants appear to be more resistant to peroxide induced protein damage than the WT with significant concentrations of the monomeric forms at even 5 mM peroxide (100× excess). There is dimerization of the mutant proteins at hydrogen peroxide ratios as low as 1:1 (protein:peroxide) similar to the WT protein. These dimers are still apparent at high peroxide concentrations in the mutant protein unlike the WT Cygb.\n\nMonitoring the same reaction products using reducing gels (SDS-PAGE, Figure 8D–F) showed a well-defined band at ~21 kDa indicative of monomeric Cygb. In comparison with the non-denaturing gels the monomer and dimer bands lose intensity with increasing peroxide concentrations, which is more evident in the WT protein relative to the mutants. As the denaturing gel eliminates disulfide bonds the peroxide-induced formation of dimers and higher oligomers are likely to arise from covalent di-tyrosine links resulting from the termination of surface-exposed tyrosine radicals on two or more protein chains as have been reported for Mb and Hb34,35.\n\nEPR spectra of the radicals formed from the reaction of Cygb with H2O2 are shown in Figure 7C. All proteins studied react with hydrogen peroxide to form protein-based radicals. However, the shape of the radical signal in WT differs significantly from the two mutants. While the WT radical exhibits a doublet-like EPR signal, the two mutants show a singlet EPR signal. An EPR signal that lacks a hyperfine structure is always difficult to interpret. However, with respect to the 15–18 G wide singlet EPR spectrum of the mutants, it should be noted that similar EPR signals of protein-bound free radicals have been reported in a number of occasions and were recently interpreted as superposition of several non-specific protein radicals formed as a result of disperse radical propagation during the radical character decay process36. The WT monomer Cygb, on the other hand, shows a doublet-like EPR line shape pointing to a possibility that the radical originates more likely from a single site on the protein, rather than many different sites. The formation rate and stability of the protein radical for WT and H81Y proteins are essentially identical for both 1:1 and 1:10 protein:peroxide ratios and are stable over the period of 180 seconds examined. The formation rate of the radical in the H81V protein is marginally larger but the radical itself is less stable.\n\n\nDiscussion\n\nThis study shows Cygb as having a high peroxidase-like activity in comparison to other pentacoordinate globins such as Mb. This is in contrast to previous reports that Cygb has a no appreciable peroxidase activity37. This discrepancy could be explained by the different conformations of Cygb, with the current study using the monomeric protein with an internal disulfide bond between C38 and C83. Previous studies have not identified the conformation of the protein under investigation. We have previously shown that this disulfide bond is important for the biochemical properties of the protein with differences reported for ligand binding and redox chemistry20. The nature of the Cygb conformation in vivo is unknown, however, at micromolar concentrations the protein is monomeric38, which is consistent with the cellular concentrations.\n\nPrevious studies with Cygb have shown that mutation of the distal histidine affects ligand binding24. The reaction of WT Cygb with peroxide showed the formation of the transient ferryl species (Figure 5) and ferric-ferryl redox cycling (Figure 4). This is in contrast to the H81V and H81Y mutants that appear not to exhibit ferryl or any significant guaiacol oxidation (<5% compared to WT). The lack of a distal histidine in Mb leads to a transient formation of peroxide-bound species (Compound 0)17, which is not observed with our mutants of Cygb. However, the rapid degradation of haem as reported in the mutants by HPLC, stopped flow and EPR spectroscopy supports a conjecture that the proteins react fast with peroxide, but the ferryl formed in the reactions is extremely unstable as it is not observed in the millisecond timescale. This suggests that the lack of guaiacol oxidation in the H81V and H81Y proteins is due to the inability of the guaiacol to donate an electron to the ferryl before it decays. The instability of the ferryl could account for the extensive haem and protein damage and is in agreement with extensive protein radical formation on multiple free radical sites in the mutants as compared to the WT, resulting in unresolved singlet EPR signal (Figure 7 and Figure 8).\n\nThe introduction of a tyrosine at the distal site increases resistance to peroxide-induced haem degradation as compared to valine, but degradation of the H81Y is still higher than that of the WT. This is likely due to the access to the haem being sterically hindered more by the larger cyclic amino acid. The polarity of the distal residue has also been suggested to affect the reaction with H2O2 - by providing a hydrogen bond to the ligand at the distal site17. Both mutants show a partial low spin form (Figure 3). This is not wholly unexpected for the H81Y mutant as the tyrosine could coordinate to the haem iron. However, the LS signal for this species is identical to that observed with H81V (but with different intensity), suggesting that another residue is coordinating to the iron. The EPR signal is similar to that observed in distal histidine mutants of Ngb39. The identity of this residue is unknown, however; a nearby arginine (R84) is a position to potentially coordinate to the haem iron.\n\nIn summary, our study identifies the requirement of the distal histidine for the formation of a ferryl species in monomeric Cygb and prevention of rapid haem degradation by free radical activity. This is similar to what has been reported for distal histidine mutations (H64Q/H64V) in Mb17. Monomeric Cygb with the internal disulfide bound has a weaker coordination of the distal histidine and is therefore more pentacoordinate-like. This is consistent with the EPR data (Figure 3) that show partial penta-coordination which can also be increased through lipid binding19,20. This pentacoordinate-like nature of the Cygb allows for the reaction with peroxide, which is not observed with fully hexacoordinate Ngb. Although the ferryl Cygb is more unstable compared to this haem oxidation state in other pentacoordinate globins, the distal histidine introduces a resistance to free radical damage above that of other globins that show peroxidatic activity. These findings are consistent with a potential peroxidatic-like role of monomeric Cygb in vivo.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for peroxidatic activity of monomeric cytoglobin, 10.5256/f1000research.5971.d4506640", "appendix": "Author contributions\n\n\n\nPenny Beckerson and Brandon Reeder planned the experiments. Penny Beckerson performed most of the experiments assisted by Dimitri Svistunenko with the EPR data (Figure 3 and Figure 7C). Brandon Reeder designed and supervised the project. Penny Beckerson, Dimitri Svistunenko and Brandon Reeder analysed the data and wrote the paper. All authors contributed to the revision of the paper and proofread before submission.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Prof. Michael T. Wilson for useful discussions.\n\n\nReferences\n\nTrent JT 3rd, Hargrove MS: A ubiquitously expressed human hexacoordinate hemoglobin. J Biol Chem. 2002; 277(22): 19538–45. PubMed Abstract \| Publisher Full Text\n\nBurmester T, Ebner B, Weich B, et al.: Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Mol Biol Evol. 2002; 19(4): 416–21. PubMed Abstract \| Publisher Full Text\n\nDewilde S, Kiger L, Burmester T, et al.: Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family. J Biol Chem. 2001; 276(42): 38949–55. PubMed Abstract \| Publisher Full Text\n\nSawai H, Makino M, Mizutani Y, et al.: Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin. Biochemistry. 2005; 44(40): 13257–65. PubMed Abstract \| Publisher Full Text\n\nFang J, Ma I, Allalunis-Turner J: Knockdown of cytoglobin expression sensitizes human glioma cells to radiation and oxidative stress. Radiat Res. 2011; 176(2): 198–207. PubMed Abstract \| Publisher Full Text\n\nLi D, Chen X, Li WJ, et al.: Cytoglobin up-regulated by hydrogen peroxide plays a protective role in oxidative stress. Neurochem Res. 2007; 32(8): 1375–80. PubMed Abstract \| Publisher Full Text\n\nGardner AM, Cook MR, Gardner PR: Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes. J Biol Chem. 2010; 285(31): 23850–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMimura I, Nangaku M, Nishi H, et al.: Cytoglobin, a novel globin, plays an antifibrotic role in the kidney. Am J Physiol Renal Physiol. 2010; 299(5): F1120–F33. PubMed Abstract \| Publisher Full Text\n\nKawada N, Kristensen DB, Asahina K, et al.: Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells. J Biol Chem. 2001; 276(27): 25318–23. PubMed Abstract \| Publisher Full Text\n\nHodges NJ, Innocent N, Dhanda S, et al.: Cellular protection from oxidative DNA damage by over-expression of the novel globin cytoglobin in vitro. Mutagenesis. 2008; 23(4): 293–8. PubMed Abstract \| Publisher Full Text\n\nAntonini E, Brunori M: Hemoglobin and Myoglobin in their Reactions with Ligands. Neuberger A TE, editor. Amsterdam: North-Holland Publishing Company. 1971. Reference Source\n\nGeorge P, Irvine DH: Reaction of methmyoglobin with hydrogen peroxide. Nature. 1951; 168(4265): 164–5. PubMed Abstract \| Publisher Full Text\n\nSvistunenko DA, Reeder BJ, Wankasi MM, et al.: Reaction of Aplysia limacina metmyoglobin with hydrogen peroxide. Dalton Trans. 2007; (8): 840–50. PubMed Abstract \| Publisher Full Text\n\nMatsui T, Ozaki Si, Watanabe Y: On the formation and reactivity of compound I of the His-64 myoglobin mutants. J Biol Chem. 1997; 272(52): 32735–8. PubMed Abstract \| Publisher Full Text\n\nEgawa T, Shimada H, Ishimura Y: Formation of compound I in the reaction of native myoglobins with hydrogen peroxide. J Biol Chem. 2000; 275(45): 34858–66. PubMed Abstract \| Publisher Full Text\n\nKhan KK, Mondal MS, Padhy L, et al.: The role of distal histidine in peroxidase activity of myoglobin--transient-kinetics study of the reaction of H2O2 with wild-type and distal-histidine-mutanted recombinant human myoglobin. Eur J Biochem. 1998; 257(3): 547–55. PubMed Abstract \| Publisher Full Text\n\nBrittain T, Baker AR, Butler CS, et al.: Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket. Biochem J. 1997; 326(pt 1): 109–15. PubMed Abstract \| Free Full Text\n\nHerold S, Fago A, Weber RE: Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role against oxidative stress. J Biol Chem. 2004; 279(22): 22841–7. PubMed Abstract \| Publisher Full Text\n\nReeder BJ, Svistunenko DA, Wilson MT: Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress. Biochem J. 2011; 434(3): 482–92. PubMed Abstract \| Publisher Full Text\n\nBeckerson P, Wilson MT, Svistunenko DA, et al.: Cytoglobin ligand binding regulated by changing haem-coordination in response to intramolecular disulfide bond formation and lipid interaction. Biochem J. 2015; 465(1): 127–37. PubMed Abstract \| Publisher Full Text\n\nReeder BJ, Svistunenko DA, Sharpe MA, et al.: Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin. Biochemistry. 2002; 41(1): 367–75. PubMed Abstract \| Publisher Full Text\n\nWeil J, Bolton J, Wertz J: Electron Paramagnetic Resonance. Wiley, New York. 1994.\n\nReeder BJ, Grey M, Silaghi-Dumitrescu RL, et al.: Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes. J Biol Chem. 2008; 283(45): 30780–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSawai H, Kawada N, Yoshizato K, et al.: Characterization of the heme environmental structure of cytoglobin, a fourth globin in humans. Biochemistry. 2003; 42(17): 5133–42. PubMed Abstract \| Publisher Full Text\n\nShechter E, Saludjian P: Conformation of ferricytochrome c. IV. Relationship between optical absorption and protein conformation. Biopolymers. 1967; 5(8): 788–90. PubMed Abstract \| Publisher Full Text\n\nCouture M, Yeh SR, Wittenberg BA, et al.: A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1999; 96(20): 11223–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHargrove MS, Singleton EW, Quillin ML, et al.: His64(E7)-->Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. J Biol Chem. 1994; 269(6): 4207–14. PubMed Abstract\n\nde Groot JJ, Veldink GA, Vliegenthart JF, et al.: Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1. Biochim Biophys Acta. 1975; 377(1): 71–9. PubMed Abstract \| Publisher Full Text\n\nWang J, Boldt NJ, Ondrias MR: Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH. Biochemistry. 1992; 31(3): 867–78. PubMed Abstract \| Publisher Full Text\n\nPeisach J, Blumberg WE, Ogawa S, et al.: The effects of protein conformation on the heme symmetry in high spin ferric heme proteins as studied by electron paramagnetic resonance. J Biol Chem. 1971; 246(10): 3342–55. PubMed Abstract\n\nKing NK, Winfield ME: The mechanism of metmyoglobin oxidation. J Biol Chem. 1963; 238(4): 1520–8. PubMed Abstract\n\nMaehly A, Chance B: The assay of catalases and peroxidases. Methods Biochem Anal. 1954; 1: 357–424. PubMed Abstract \| Publisher Full Text\n\nCarlsen CU, Skovgaard IM, Skibsted LH: Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates. J Agric Food Chem. 2003; 51(19): 5815–23. PubMed Abstract \| Publisher Full Text\n\nLardinois OM, de Montellano PR: Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide. J Biol Chem. 2003; 278(38): 36214–26. PubMed Abstract \| Publisher Full Text\n\nLardinois OM, Ortiz de Montellano PR: Autoreduction of ferryl myoglobin: discrimination among the three tyrosine and two tryptophan residues as electron donors. Biochemistry. 2004; 43(15): 4601–10. PubMed Abstract \| Publisher Full Text\n\nRajagopal BS, Edzuma AN, Hough MA, et al.: The hydrogen-peroxide-induced radical behaviour in human cytochrome c-phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant. Biochem J. 2013; 456(3): 441–52. PubMed Abstract \| Publisher Full Text\n\nTrandafir F, Hoogewijs D, Altieri F, et al.: Neuroglobin and cytoglobin as potential enzyme or substrate. Gene. 2007; 398(1–2): 103–13. PubMed Abstract \| Publisher Full Text\n\nLechauve C, Chauvierre C, Dewilde S, et al.: Cytoglobin conformations and disulfide bond formation. FEBS J. 2010; 277(12): 2696–704. PubMed Abstract \| Publisher Full Text\n\nUno T, Ryu D, Tsutsumi H, et al.: Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps. J Biol Chem. 2004; 279(7): 5886–93. PubMed Abstract \| Publisher Full Text\n\nBeckerson P, Svistunenko D, Reeder B: Dataset 1 in: Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin. F1000Research. 2015. Data Source" }	[ { "id": "8224", "date": "28 Apr 2015", "name": "Mark Hargrove", "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\nThese authors have been publishing quality data with Cygb over the past few years, and the work presented here is a continuation of the same line of investigation. The experiments conducted are fairly straight forward, and the immediate results are clear (with the possible exception of Figure 7, as discussed below), but the conclusions drawn from them are not as strong. The goal of the work was to test the hypothesis that Cygb might serve as a peroxidase in vivo. Since no one yet knows the actual function of Cygb in vivo, the present investigation must compare Cygb to something else. Two point mutants of Cygb and myoglobin are used for comparison. Absorbance and EPR spectroscopy, along with reactions with H2O2, and a guaiacol-based peroxidase assay provide the bulk of the data to test this hypothesis. Interesting/positive points about this article: Cygb and Mb react differently in the guaiacol assay (Cygb actually doesn’t react much at all). However, when an assay specific for formation of ferryl heme is used, both proteins react similarly (with respect to their bimolecular rate constants for the spectral change following mixing with H2O2). This means that Cygb can be oxidized by H2O2 just like Mb, but does not then oxidize a substrate such as guaiacol, as does Mb. Remaining questions/criticisms of the article: It is evident that interesting things happen when Cygb is mixed with H2O2, and that different things happen to the two point mutants. It is not at all clear what is learned from the EPR spectra. The conclusion is that the introduced Tyr might occlude R84 coordination, but this is very speculative and has little to do with the point of this article. Figure 7 is not an overwhelming endorsement of the stability of Cygb against hydrogen peroxide. The H2O2 dependence of Cygb modification is very strange, at first responding like Mb (for the first four points of the curve), then resisting the approach to 50% reduction of unmodified heme until the reported value of 2500 µM. I think it is a bit misleading to judge these curves by the concentration of H2O2 needed to damage 50% of the heme, when the does-response is so complex. For example, if the parameter used to measure Mb versus Cygb were “concentration of H2O2 needed to damage 40% of the heme”, Mb and Cygb would be the same. Why in the discussion is it said that Cygb has a higher peroxidase activity than Mb? As measured by guaiacol, it does not, and as measured by the formation of the ferryl species, it is the same. Furthermore, the final conclusion for how these data test the primary hypothesis is that they are “consistent with a potential peroxidatic-like role of monomeric Cygb in vivo”. While these results are interesting, I am not sure how they could fail in support of this hypothesis, short of Cygb not reacting at all with H2O2. Based on the data reported here, these results also support a role for Mb having a peroxidatic-like activity in vivo.", "responses": [] }, { "id": "8551", "date": "01 May 2015", "name": "Rakesh Pravinchandra Patel", "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\nCytoglobin in regulating redox reactions with H2O2. The study design and methods used are appropriate to address the proposed questions. The data nicely show that distal His residues of this protein play key roles in maintaining heme stability during reactions with H2O2.Details of how many replicates and statistical analyses used to discern differences should be provided. Is the heme loss observed after reactions with H2O2 and His mutants due to heme release and/ or heme modification that in turn would preclude extraction and measurement by HPLC. The authors should provide some thoughts on how they think reactions with H2O2 (in the His mutants) is causing the heme to be released or degraded? Are the effects of the distal His exclusive for reactions with H2O2? Could the authors speculate a little more on whether their results are more generalizable to other biologically relevant peroxides (LOOH, ONOO(H) etc.).", "responses": [] } ]	1	https://f1000research.com/articles/4-87
https://f1000research.com/articles/4-85/v1	01 Apr 15	{ "type": "Case Report", "title": "Case Report: Nitrofurantoin-induced interstitial lung disease", "authors": [ "Suhail Basunaid", "Helena Pilate", "Melanie Schoutteten", "Rooy Sprooten", "Helena Pilate", "Melanie Schoutteten", "Rooy Sprooten" ], "abstract": "Nitrofurantoin is widely used for urinary tract infection (UTI) prophylaxis. Long-term use is known to be able to cause serious adverse effects including pulmonary and hepatic toxicity. The prevalence of nitrofurantoin-induced pulmonary injury is on the increase again as the drug regains popularity as a urinary antiseptic.We describe a previously healthy 83-year-old woman who presented to our emergency department in early 2012 with progressive dyspnoea since two weeks. This was not preceded by cough. She had no fever, wheezing, chest pain, or sputum production. She was a 50 pack per year ex-smoker. She had no previous exposure to tuberculosis or industrial chemicals. However, she suffered from recurrent symptomatic UTIs and was on a long-term prescription of nitrofurantoin for prophylaxis.Respiratory examination revealed dullness on percussion at both lung bases and widespread fine inspiratory crackles throughout both lungs. Arterial blood gas analysis showed hypoxia and complete compensation of respiratory acidosis.Initial treatment with co-amoxiclavulanic acid was initiated. CT scanning of the chest showed widespread ground-glass appearance in both lungs with organising pneumonia. A diagnosis of nitrofurantoin-induced interstitial lung disease (NIILD) was suspected. Nitrofurantoin was subsequently stopped and prednisone treatment at 30 mg OD was initiated. Follow-up chest X-ray showed marked improvement.", "keywords": [ "Nitrofurantoin", "urinary tract infection", "pulmonary injury nitrofurantoin-induced interstitial lung disease", "NIILD" ], "content": "Case report\n\nA previously healthy 83-year-old Caucasian woman presented with progressive dyspnoea. She was a 50 pack per year ex-smoker. She suffered from recurrent symptomatic urinary tract infections (UTIs) and was prescribed long-term nitrofurantoin 50 mg daily for prophylaxis by her GP.\n\nOn examination she appeared dyspnoeic. She was afebrile and normotensive with respiratory rate of 31 per minute and oxygen saturation was 91% while receiving supplementary oxygen at a flow of 5 liter per minute. Respiratory examination revealed fine inspiratory crackles throughout both lungs. Arterial blood gas showed hypoxia with PaO2 5.4 kPa (without oxygen suppletion), PaO2 8.3 kPa (with 5 liter/min oxygen suppletion) and complete compensation of respiratory acidosis with pH 7.37, and PaCO2 6.9 kPa on 5 liter oxygen suppletion with base excess 2.9 mmol/l [Figure 1]. Laboratory findings showed increased leucocytes and C-reactive protein level [Figure 2]. Auto-immune laboratory findings including the antinuclear antibody and rheumatoid factor tests were negative. Chest X-ray revealed diffuse bilateral interstitial infiltrates [Figure 3]. CT scanning of the chest showed widespread ground-glass appearance with organizing pneumonia [Figure 4]. Initial treatment with co-amoxiclavulanic acid was started at a dose of 1.2 gram 4 times daily.\n\n\nCourse\n\nNitrofurantoin was subsequently stopped and prednisolone treatment at 30 mg OD was initiated. She had a short hospital course of 12 days and was finally discharged without long term oxygen treatment. Follow up chest X-ray before discharge and after withdrawal of nitrofurantoin (Figure 5) showed marked improvement compared to the X-ray upon admission.\n\nThe patient was seen after two months during outpatient control; her symptoms had improved dramatically and a follow-up chest X-ray showed further normalization.\n\n\nDiscussion\n\nThe differential diagnosis of nitrofurantoin induced interstitial lung disease (NIILD) includes pulmonary edema, cryptogenic organizing pneumonia and idiopathic interstitial pneumonias1. The auto-immune markers tested, antinuclear antibody and rheumatoid factor, were negative indicating a reaction to nitrofurantoin rather than an underlying systemic pathology.\n\nPrompt resolution following the discontinuation of nitrofurantoin further supports the diagnosis2,3. The diagnosis is based on the history of nitrofurantoin use and the absence of another explanation for the patient’s symptoms and radiographic abnormalities.\n\nDiscontinuation of nitrofurantoin results in the regression of symptoms and radiographic abnormalities3. Systemic corticosteroids are occasionally administered and it remains unclear how much corticosteroids contribute to improvement beyond drug cessation alone4.\n\n\nConclusion\n\nLong-term use of nitrofurantoin as prophylaxis for UTIs can cause serious pulmonary side effects. Our patient received antibiotics and corticosteroids because of diagnostic uncertainty. This may represent frequent clinical practice, however there are no specific symptoms to separate NIILD from other interstitial lung diseases1.\n\n\nInformed consent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the next of kin.", "appendix": "Author contributions\n\n\n\nSuhail Basunaid: data collection, drafting and revising the manuscript for intellectual content. Pilate Helena: clinician assessing and looking after the patient. Schoutteten Melanie: clinician assessing and looking after the patient. Sprooten Rooy: the chief clinician looking after the ward.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nAt the completion of this case study, I am very thankful for Dr. Rohde Gernot’s help, and without his support this case study would have never come into its present form.\n\n\nReferences\n\nCamus P, Fanton A, Bonniaud P, et al.: Interstitial lung disease induced by drugs and radiation. Respiration. 2004; 71(4): 301–26. PubMed Abstract \| Publisher Full Text\n\nMendez JL, Nadrous HF, Hartman TE, et al.: Chronic nitrofurantoin-induced lung disease. Mayo Clin Proc. 2005; 80(10): 1298–302. PubMed Abstract \| Publisher Full Text\n\nPeall AF, Hodges A: Concomitant pulmonary and hepatic toxicity secondary to nitrofurantoin: a case report. J Med Case Rep. 2007; 1: 59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheehan RE, Wells AU, Milne DG, et al.: Nitrofurantoin-induced lung disease: two cases demonstrating resolution of apparently irreversible CT abnormalities. J Comput Assist Tomogr. 2000; 24(2): 259–61. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8203", "date": "23 Apr 2015", "name": "Demosthenes Bouros", "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 describe a case of Nitrofurantoin-induced interstitial lung disease. However a lot of important data are missing from this review.The authors do not report the duration of nitrofurantoin therapy. This is of utmost importance as there are 3 types of reactions regarding nitrofurantoin related lung toxicity: acute, sabacute and chronic. Determining the type of reaction is critical as this is associated with the clinical features of the patient, the radiographic findings and the time needed for their resolution after nitrofurantoin withdrawal. The authors report: “CT scanning of the chest showed widespread ground-glass appearance in both lungs with organising pneumonia”.First, in the CT image provided (figure 4) is of poor quality and the prominent pattern is not that of ground glass opacities (GGO). Second, organizing pneumonia is a histology pattern. No radiology pattern is conclusive of organizing pneumonia. It is important not to confuse histology with radiology patterns. There are two CXR presented in the article, on admission and at discharge (figure 3 and 5). The second CXR shows marked (almost complete improvement). Given the fact that the patient received co-amoxiclav (1.2gr x 4 iv), a likely scenario is that of lower respiratory tract infection. The patient presented with hypercarbia. In the chronic form of nitrofurantoin related lung toxicity there is usually underlying fibrosis. This leads to a rapid shallow pattern of breathing causing hypocarbia and not hypercarbia. The history of smoking (50 pack years) in this context favors the possibility of lower respiratory tract infection. No data are given regarding pulmonary function tests.Based on the above, a diagnosis of nitrofurantoin related lung toxicity cannot be robustly proven. This cases refers to a well known drug-induced ILD in patients taking nitrofurantoin. The first sentence into discussion is erroneous. ---COP is included in the idiopathic interstitial pneumonias! It should be termed as non cryptogenic if the cause is known...", "responses": [ { "c_id": "1360", "date": "22 May 2015", "name": "Suhail Basunaid", "role": "Author Response", "response": "Thank you very much for the feedback regarding this case report.Our patient had nitrofurantoin therapy several times for a recurrent UTI. The last one was longer than normal. I am not sure if this was mentioned in my abstract. I could revise it and include this. For sure it was not a chronic use due the self-discontinuation by the patient every time her UTI was cured. The aim of this article was to raise awareness regarding lung toxicity due to some medications like nitrofurantoin. I believe that the term organizing pneumonia could be also mentioned by radiologists in describing X-rays or CT-scans. I am familiar that this term is a pathologic finding but this was the description of our radiologist, together with associated GGO during the acute presentation. However this latter is usually not specific and could be seen in many differential diagnoses like superinfection, pulmonary edema and many others. I will try to find a better quality CT scan in my next revise, however I am sure that the case here has nothing to do with an organizing pneumonia due to the quick improvement and evidence of recurrent scenario after tapering of the steroids. Unfortunately, I am not sure of your intended meaning here. In most cases as I said there is a superinfection and in most situations adding a broad spectrum antibiotic is a pragmatic issue. Or is the practice in your country different from here in the Netherlands? As I said the case was not a chronic scenario, otherwise the patient would not have improved so quickly. Again the prognosis was very good and did not lead to fibrosis. I was not familiar with term hypercarbia, thank you for the added information. Due to the quick response in the case and the fact that the patient was discharged within 1 week there was no pulmonary function test performed. This was done later during the follow up and was surprisingly good despite the fact that our patient is an ex-smoker. Unfortunately, again, I am not sure of your intended meaning here. Are you trying to add some information, because I do not believe that this is a COP case, - the term drug induced ILD is a general term and might refer to the use of nitrofurantoin." } ] }, { "id": "10196", "date": "03 Sep 2015", "name": "Klaus Dalhoff", "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\nA case of drug-induced ILD after Nitrofurantoin Treatment due to recurrent UTIs is presented. Although there are some questions left (DD pulmonary infection, documentation on the CT scan shown) the report seems plausible. This is an important topic since NF use is increasing due to changes in international recommendations regarding antimicrobial treatment of UTIs (restriction of other antibiotic classes such as Co-trimoxazole and quinolones). I would however, recommend in comparable cases to perform a BAL for excluding infection and confirming the aetiology by a BAL cytology compatible with drug induced ILD (typically mixed pattern with lymphocytosis, eosinophilia and some PMN).", "responses": [] } ]	1	https://f1000research.com/articles/4-85
https://f1000research.com/articles/4-84/v1	01 Apr 15	{ "type": "Case Report", "title": "Case Report: Primary squamous cell carcinoma of the bladder secondary to chronic renal fungal ball and recurrent polymicrobial urinary tract infections.", "authors": [ "Andrew Keller", "Benjamin Shepherd", "Arief Mulyadi", "Ahmad Ali", "Benjamin Shepherd", "Arief Mulyadi", "Ahmad Ali" ], "abstract": "IntroductionSquamous cell carcinoma (SCC) of the bladder is a rare malignancy in Western countries accounting for only 5% of all primary bladder cancers. Chronic irritation is the predominant risk factor, with chronic infections, bladder stones and long term catheterisation common precursors. The highest incidence of SCC occurs in patients with spinal cord injuries who rely on indwelling or self-catheterisation for bladder drainage. We report a case of primary SCC of the bladder secondary to a fungal ball located in the renal pelvis.Case reportA 72 year-old lady was referred to our unit for further investigation of recurrent polymicrobial urinary tract infections associated with intermittent flank pain and complicated by sepsis. Investigations into the cause for her recurrent urinary tract infections identified a mass in her left renal pelvis. Pyeloscopy demonstrated no tumour, but a fungal ball. Attempts to clear the fungal ball via pyeloscopy resulted in recurrent intensive care unit (ICU) admission for urosepsis. Several months after her last pyeloscopy she returned with haematuria. Cystoscopy at this time revealed a large bladder mass. Biopsy revealed primary SCC of the bladder invading muscle. At cystectomy the mass had invaded pubic bone and was unresectable and a palliative ileal conduit was formed. The patient passed away less than 4 months following diagnosis.ConclusionWe report what we believe to be the first case of primary SCC of the bladder secondary to a renal pelvis fungal ball. Despite frequent surveillance of her urinary tract the tumour developed rapidly and was unresectable at diagnosis.", "keywords": [ "Urinary bladder cancer", "Squamous cell carcinoma", "Non-urothelial bladder cancer", "Fungal ball" ], "content": "Introduction\n\nSquamous cell carcinoma (SCC) of the bladder is a rare malignancy in Western countries, accounting for only about 5% of all primary bladder cancers1. Chronic irritation is the predominant risk factor, with recurrent infections, bladder stones and long term catheterisation common precursors, and the highest incidence occurring in patients with spinal cord injuries who rely on indwelling or self-catherisation for bladder drainage2,3. In the Middle East and Northern Africa, where schistosomiasis is endemic, the incidence is much higher, with SCC being the predominant subtype of bladder cancer4. We report a case of primary SCC of the bladder secondary to a fungal ball located in the renal pelvis.\n\n\nCase report\n\nA 72 year-old Caucasian lady was referred to our unit for further investigation of recurrent polymicrobial urinary tract infections (UTI) complicated by sepsis and associated with intermittent left flank pain. An ultrasound performed by the treating team revealed a poorly defined, poorly vascularised mass in the lower pole calyx of her left kidney associated with moderate ipsilateral hydronephrosis.\n\nThe patient had extensive medical co-morbidities including chronic renal impairment, ischaemic heart disease with unstable angina, aortic stenosis and mitral regurgitation in addition to a previous cerebrovascular event (CVA) 8 years prior with residual left sided weakness and blindness, and a known but unclipped middle cerebral artery aneurysm.\n\nWe arranged further investigations including a mercaptoacetyltriglycine (MAG 3) renogram, computerised tomography (CT) of her renal tracts and urine cytology. The MAG3 renogram demonstrated obstructed drainage of urine from the left renal pelvis. CT again demonstrated left-sided hydro-nephrosis, with a poorly defined mass in the lower pole calyx, with no obvious contrast enhancement. Urine cytology was negative for malignancy with only non-specific inflammatory changes present.\n\nCystoscopy, retrograde pyelogram (RGP) and insertion of ureteric stent were arranged to further investigate her renal mass and hydronephrosis. Cystoscopic examination of the bladder was unremarkable. RGP demonstrated a “moth-eaten” left lower pole filling defect (Figure 1). Washings from the renal pelvis were taken via ureteric catheter. A ureteric stent was placed and she was discharged on the same day.\n\nThe day following her procedure she presented to the emergency department with urosepsis. Her urine and blood failed to culture a causative organism and she was subsequently discharged on oral trimethoprim 300mg daily for seven days after clinical improvement following intravenous piperacillin/tazobactam, 4.1g three times daily.\n\nUrine cytology from operative renal pelvis washings were again inconclusive, with non-specific inflammatory changes only, and flexible pyeloscopy was arranged two weeks later in order to better assess her renal mass. The bladder was again unremarkable on cystoscopic examination. Flexible pyeloscopy demonstrated a poorly defined mass in the lower calyx. Visualisation of the mass was poor due to debris in the renal pelvis and contact bleeding. Washings and tissue biopsies were retrieved and her ureteric stent was replaced. The patient was discharged the following day on oral norfloxacin 400mg twice daily for seven days.\n\nBiopsies and washings following pyeloscopy demonstrated no evidence of malignancy, but fungal elements were identified on microscopy. Re-look pyeloscopy was arranged in two weeks to exclude urothelial malignancy as visualisation of the mass at the previous pyeloscopy had been poor.\n\nAt repeat flexible pyeloscopy we again saw no macroscopic evidence of a renal pelvis tumour. However, a white plaque was seen extending over the lower pole calyces suggestive of a fungal ball. After subtotal removal of the fungal plaque with an endoscopic basket, a temporary ureteric catheter was placed. Post-operatively, the patient became haemodynamically unstable and unresponsive to fluid resuscitation and was transferred to the ICU. Treatment with teicoplanin (800mg twice daily loading dose, following by 400mg once daily dosing) and piperacillin/tazobactam (4.1g three times daily) was initiated due to her history of polymicrobial UTI. Intravenous caspofungin (70mg once daily loading dose, 50mg once daily maintenance) was used in addition to cover for possible fungaemia. Vasopressor support necessitated ICU stay for three days prior to a further seven days of intravenous piperacillin/tazobactam on the surgical ward. She was discharged on oral fluconazole 200mg once daily for two weeks.\n\nAs a result of urosepsis repeatedly complicating any urological procedure, the patient remaining currently asymptomatic and her multiple medical comorbidities, we decided to manage her fungal ball conservatively and no further procedures were planned at this stage.\n\nAfter several uneventful months, the patient represented with a further episode of urosepsis again unresponsive to fluid resuscitation. A further extended stay in ICU on vasopressor support was required. CT of the urinary tract at this time demonstrated recurrent mild left hydronephrosis and reappearance of debris in the lower pole calyces. A ureteric stent was placed in case upper tract obstruction had precipitated her sepsis. In view of her continued septic episodes and recurrence of her fungal ball, we decided in conjunction with the patient to prepare for an elective simple nephrectomy. Infectious diseases prescribed oral voriconazole 200mg twice daily to be continued prophylactically on discharge until her planned nephrectomy which was tentatively scheduled in 1 months’ time.\n\nPrior to her planned nephrectomy she was re-admitted for a further episode of urosepsis, this time complicated by the new symptom of macroscopic haematuria with passage of multiple clots. After failed conservative management of her haematuria with bladder irrigations, flexible cystoscopy was performed to identify the source of ongoing bleeding. A large ulcerated erythematous mass was found, adjacent to her left ureteric orifice (Figure 2 and Figure 3). CT confirmed an enhancing bladder mass 5cm in diameter, arising from the left wall of her bladder (Figure 4). Resection of bladder tumour was performed and confirmed a pure squamous cell carcinoma of the bladder with invasion into muscularis propria (Figure 5, Figure 6, Figure 7 and Figure 8).\n\nCystoscopic examination revealed a large irregular ulcerated bladder mass with adherent blood clot.\n\nSquamous carcinoma in situ is seen overlying a focus of SCC which has invaded into the lamina propria.\n\nSCC is here seen invading into the muscularis propria.\n\nInvasive carcinoma with squamous differentiation with inclusion of a keratin pearl.\n\nSuperficial squamous metaplasia without atypia is seen overlying SCC which has invaded the lamina propria.\n\nThe patient was booked for a radical cystectomy and formation of an ileal conduit. At operation the tumour was found to have invaded pubic symphysis and was deemed unresectable. The planned cystectomy was aborted and instead a palliative urinary diversion was created. The patient was reviewed by medical and radiation oncology in regards to her suitability for adjuvant chemo-radiotherapy but the consensus was that she would not benefit from further aggressive treatment.\n\nOne month post-operatively she developed recurrent bleeds from her bladder with difficulty and much discomfort expelling the clots. She received two fractions of palliative radiotherapy to her bladder.\n\nPrior to receiving further planned doses she was again admitted to hospital with another urinary tract infection and further bladder haemorrhage. Whilst in hospital she continued to deteriorate despite antibiotics and after discussion with the patient and her family, comfort cares were initiated. She passed away just over three months following her urinary diversion and less than four months following her diagnosis of squamous cell carcinoma.\n\n\nDiscussion\n\nSCC of the bladder is an uncommon primary malignancy of the bladder. Whilst chronic irritation, and chronic urinary tract infection are well known predisposing factors in Western populations, there is to date, no reported cases of bladder SCC secondary to fungal balls2,3.\n\nSCC of the urinary tract typically presents at an advanced stage. In a large series of 1422 non-bilharzial SCC cases 85% were muscle invasive at diagnosis5. Additionally 56% of all bladder SCC were graded as American Joint Committee on Cancer (AJCC) stage T3 or T4 at diagnosis5.\n\nSCC of the urinary tract has a significantly higher mortality than primary urothelial carcinoma, even after adjustment for higher tumour stage for SCC at diagnosis5. Scosyrev et al. found that for T4 tumours, even with cystectomy two year survival rates for SCC were low at 28%, where-as for stage matched urothelial carcinoma survival was significantly improved at 42%. Two year survival rates for T4 SCC without cystectomy is poor at only 5%5.\n\n\nConclusion\n\nWe present, we believe, the first documented case of primary SCC of the bladder secondary to an upper tract fungal ball. Despite the patient being under close urological surveillance for her fungal ball at the time of tumour development, the cancer demonstrated trans-mural invasion at initial diagnosis and was surgically unresectable. The disease was rapidly progressive and the patient passed away fewer than four months following diagnosis.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient’s next of kin.", "appendix": "Author contributions\n\n\n\nAK writing and literature review. BS provided histological images and contributed to article structure. AM helped in writing of the article and provided clinical images. AA concept of article and proofing of article. All authors have read and approved the final proof of this manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSerretta V, Pomara G, Piazza F, et al.: Pure squamous cell carcinoma of the bladder in western countries. Report on 19 consecutive cases. Eur Urol. 2000; 37(1): 85–9. PubMed Abstract \| Publisher Full Text\n\nLocke JR, Hill DE, Walzer Y: Incidence of squamous cell carcinoma in patients with long-term catheter drainage. J Urol. 1985; 133(6): 1034–5. PubMed Abstract\n\nAbol-Enein H: Infection: is it a cause of bladder cancer? Scand J Urol Nephrol Suppl. 2008; 42(218): 79–84. PubMed Abstract \| Publisher Full Text\n\nGhoneim MA, el-Mekresh MM, el-Baz MA, et al.: Radical cystectomy for carcinoma of the bladder: critical evaluation of the results in 1,026 cases. J Urol. 1997; 158(2): 393–9. PubMed Abstract \| Publisher Full Text\n\nScosyrev E, Yao J, Messing E: Urothelial carcinoma versus squamous cell carcinoma of bladder: is survival different with stage adjustment? Urology. 2009; 73(4): 822–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8953", "date": "16 Jul 2015", "name": "M. Hammad Ather", "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\nThis is an interesting case as it shows a rather dramatic development and progression of squamous cell cancer of the bladder.I have few observations on this submission.In retrospect were there any observation on the part of bladder which subsequently showed any changes like hyperemia? The CT showed no evidence of T4 disease, however the per operative findings were quite different. What was the time frame between the CT and surgery?", "responses": [ { "c_id": "1489", "date": "03 Aug 2015", "name": "Andrew Keller", "role": "Author Response", "response": "Thanks for your comments Dr. Ather. 1. Retrospectively there were some erythematous patches over the area prior to the initial resection but the erythema was not limited to only that area. They were assumed to be a reaction to the chronic cystitis this lady experienced secondary to her fungal ball. 2. The time frame between the CT which demonstrated the mass, and the attempted cystectomy was just over five weeks. Cystectomy was attempted just under four weeks after initial tissue confirmation of SCC via TURBT. Due to the patients extensive medical history some of the delay was due to the necessary anaesthetic work-up in order to establish her fitness for surgery." } ] }, { "id": "10705", "date": "07 Oct 2015", "name": "Richard J Ablin", "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 interesting Case Report, particularly relative from the aspect of implications of fungi, their mycotoxins and a possible role in malignancy, e.g., See: Kaufmann et al. Oncology News, 9, 164-66, 2014, and references therein. Given the implications, it is surprising the authors did not do more of a thorough infectious disease workup, whereby the patient’s compromised physiological status may have made her vulnerable to kidney and bladder infections. It is reasonable that whereby, their use of catheters, the fungus may have arisen from a contaminated catheter. The latter begs the question as to why the authors’ did not identify the fungus?", "responses": [ { "c_id": "1654", "date": "13 Oct 2015", "name": "Andrew Keller", "role": "Author Response", "response": "Thanks Dr. Ablin for your review and comments. It is indeed a glaring omission that I did not include the cultured fungus in the report. The cultures all grew candida albicans, which was broadly sensitive to all tested anti-fungal agents at low minimum inhibitory concentrations.Whilst it is acknowledged that urinary tract instrumentation precipitated many of her septic episodes, her presenting complaint of recurrent poly-microbial urinary tract infections and renal pelvis mass (which on investigation was revealed to be a fungal ball) occurred in the absence of previous urinary tract instrumentation.The location of her tumour was exactly where the vesical loop of the ureteric stent would have been situated. This does make me suspect that the ureteric stent, via chronic inflammation from both stent colonization and mechanical irritation, was contributory to her carcinogenesis." } ] } ]	1	https://f1000research.com/articles/4-84
https://f1000research.com/articles/4-63/v1	11 Mar 15	{ "type": "Case Report", "title": "Case Report: Bone fragment in the third ventricle of a 22 year-old woman", "authors": [ "Sunil Munakomi", "Balaji Srinivas", "Iype Cherian", "Balaji Srinivas", "Iype Cherian" ], "abstract": "Here we present a very rare case of a woman with a bone fragment in the third ventricle of the brain following compound-depressed skull fractures due to a road traffic accident.There are only few case reports of bullets and textiloma being removed from the third ventricle. Following operative removal of the fragment, the patient was started on cortisol, mineralocorticoid and thyroid hormone replacement. However, the patient eventually died of the severe traumatic hypothalamic insult.", "keywords": [ "bone fragment", "brain surgery", "compound-depressed fracture" ], "content": "Case report\n\nA 22 year-old female, with no significant past medical and surgical illnesses, was brought to the casualty room with a Glasgow coma scale of 6/15 following a collision between two bikes three hours earlier. Local examination revealed two compound depressed skull fractures in the frontal and the parietal region with egress of brain matter. Following primary resuscitation, computed tomography (CT) of the head confirmed the local findings along with the presence of one bone fragment in the third ventricle (Figure 1). The patient was taken for debridement of the wound and craniotomy with retrieval of the bone fragment (Figure 2, Figure 3) following hematoma tracking. Intraventricular drain was placed and neurosurgical intensive care was provided. Repeated CT scans showed hypodensities around the third ventricle (Figure 4). On the second post-operative day, the patient was started on ionotropic support because of the refractory hypotension, and was also replaced with hydrocortisone, fludrocortisone and thyroid hormones. Wound dressing and the ventricular drain care was continued. Cerebrospinal fluid (CSF) culture from the drain resulted sterile. The patient died on the 8th post-operative day because of the traumatic severe hypothalamic insult.\n\n\nDiscussion\n\nAs brain abscesses may result from driven bone fragments and other retained foreign bodies in the brain, the removal of readily accessible foreign bodies has received much attention3–6. Migration of foreign bodies can occur because of gravitational force. Other routes of migration can be subdural, parenchymal, transventricular or along streamlining along the white matter track7. The removal of foreign bodies is mostly done via craniotomy8,9, but other methods such as burr hole, stereotaxy10 and sometimes by ventriculoscopy11 have also been described.\n\nThe goals of modern treatments include removal of the foreign body under a controlled environment in the neurosurgical operation setting. Surgical principles include removal of bone fragments, intracerebral hematoma, control of hemorrhages and prevention of further loss of neural tissue. Patients should receive a broad spectrum intravenous antibiotic therapy along with tetanus prophylaxis. Monitoring and control of elevated intracranial pressure with maintenance of cerebral perfusion pressure plays a significant role in the patient’s survival and outcome. The follow-up of such patients is essential, considering known complications like cerebrospinal fluid fistula in the early post-operative period and brain abscesses and seizures which may occur years after injury. Outcome after a penetrating head injury is directly related to the Glasgow coma scale at the time of presentation, which is the reflection of the extent of brain tissue damage caused directly by the primary impact. Intensive post-operative monitoring of intracranial pressure, cardio-respiratory function and metabolic status are required for optimizing the outcome of victims of penetrating craniocerebral injuries12. Penetrating head injuries have a higher mortality and morbidity than blunt trauma even in a civilian set up13. Even after timely removal of the penetrating objects and intensive medical management, the outcome may remain poor.\n\n\nConsent\n\nInformed written consent for publication of images and clinical details was obtained from the patient’s husband.", "appendix": "Author contributions\n\n\n\nSunil Munakomi wrote and submitted the manuscript. Balaji Srinivas, Binod Bhattarai and Iype Cherian formatted and reviewed the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGreenwood J Jr: Removal of foreign body (bullet) from the third ventricle. J Neurosurg. 1950; 7(2): 169–72. PubMed Abstract \| Publisher Full Text\n\nLavrnic S, Stosic-Opincal T, Gavrilovic S, et al.: Intraventricular textiloma with granuloma formation following third ventricle colloid cyst resection - a case report. Cent Eur Neurosurg. 2009; 70(2): 86–8. PubMed Abstract \| Publisher Full Text\n\nCarey ME, Young H, Mathis JL, et al.: A bacteriological study of craniocerebral missile wounds from Vietnam. J Neurosurg. 1971; 34(2 Pt 1): 145–154. PubMed Abstract \| Publisher Full Text\n\nHagan RE: Early complications following penetrating wounds of the brain. J Neurosurg. 1971; 34(2 Pt 1): 132–141. PubMed Abstract \| Publisher Full Text\n\nHammon WM: Analysis of 2187 consecutive penetrating wounds of the brain from Vietnam. J Neurosurg. 1971; 34(2 Pt 1): 127–131. PubMed Abstract \| Publisher Full Text\n\nHammon WM: Retained intracranial bone fragments: analysis of 42 patients. J Neurosurg. 1971; 34(2 Pt 1): 142–144. PubMed Abstract \| Publisher Full Text\n\nRengarchy SS, Carey M, Templer J: The Sinking Bullet. J Neurosurg. 1992; 30(2): 291–294. PubMed Abstract \| Publisher Full Text\n\nCampbell WP, Howard WO, Weary WB: Gunshot wounds of the brain. Report of two unusual complications: bifrontal pneumocephalus and a loose bullet in the lateral ventricle. Arch Surg. 1942; 44(5): 789–798. Publisher Full Text\n\nSternbergh WC Jr, Watts C, Clark K, et al.: Bullet within the fourth ventricle. Case report. J Neurosurg. 1971; 34(6): 805–807. PubMed Abstract \| Publisher Full Text\n\nSujita K, Doi T, Sato O, et al.: Successful removal of intracranial air-gun bullet with stereotaxic apparatus. J Neurosurg. 1969; 30(2): 177–181. PubMed Abstract \| Publisher Full Text\n\nDandy W: The brain. In: Lewis Practice of Surgery. Hagerstown, Md.: Wf Prior Co Inc. 1932; 12. : Chap 1, 279–280.\n\nRosenberg WS, Harsh GR: Penetrating wounds of the head. In Williams, Rengachary, editors. Neurosurgery. New York: McGraw-Hill; 1996; 2813–9.\n\nBlack KL, Hanks RA, Wood DL, et al.: Blunt versus penetrating violent traumatic brain injury: frequency and factors associated with secondary conditions and complications. J Head Trauma Rehabil. 2002; 17(6): 489–96. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7934", "date": "24 Mar 2015", "name": "Andrey Belkin", "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\nIt seems obvious that surgery even on symptomatic intracerebral foreign bodies is determined by the anatomical availability and physiological fact. In this case, initially, it seems that the risk of surgery was higher than the risk of complications due to the presence of a foreign body and, therefore, the intervention was not justified. In order to give a more detailed review we would need to have a tomogram data on the patient’s condition. Based on the currently available outline of the situation, this patient would require more conservative treatment (aggressive antibiotic therapy in the presence of the slightest signs of SIRS or antibiotic prophylaxis from the first hours). Only if the patient on admission had a detailed diencephalic syndrome (and judging by the description they did not) and clinical signs of meningitis, would we try endoscopic removal of the foreign body (if it is of small size, somewhere up to 1 cm). Thus one alternative conservative intervention is to attempt endoscopic evacuation. If endoscopic evacuation is not possible (large size foreign bodies, technical inaccessibility through the ventricle), another possible surgical action that I would have done (but only in the case of ventriculitis) is external ventricular drainage. No more.", "responses": [] } ]	1	https://f1000research.com/articles/4-63
https://f1000research.com/articles/4-82/v1	31 Mar 15	{ "type": "Research Article", "title": "Tweets from the forest: using Twitter to increase student engagement in an undergraduate field biology course", "authors": [ "Lauren Soluk", "Christopher M. Buddle", "Christopher M. Buddle" ], "abstract": "Twitter is a cold medium that allows users to deliver content-rich but small packets of information to other users, and provides an opportunity for active and collaborative communication. In an education setting, this social media tool has potential to increase active learning opportunities, and increase student engagement with course content. The effects of Twitter on learning dynamics was tested in a field biology course offered by a large Canadian University: 29 students agreed to take part in the Twitter project and quantitative and qualitative data were collected, including survey data from 18 students. Students published 200% more public Tweets than what was required, and interacted frequently with the instructor and teaching assistant, their peers, and users external to the course. Almost 80% of students stated that Twitter increased opportunities for among-group communication, and 94% of students felt this kind of collaborative communication was beneficial to their learning. Although students did not think they would use Twitter after the course was over, 77% of the students still felt it was a good learning tool, and 67% of students felt Twitter had a positive impact on how they engaged with course content. These results suggest social media tools such as Twitter can help achieve active and collaborative learning in higher education.", "keywords": [ "Twitter", "active learning", "medium theory", "communication" ], "content": "Introduction\n\nSocial media tools have infiltrated teaching practices across many educational settings and a body of research shows that the tools can increase engagement (Junco et al., 2010; Luttrell, 2012; McCool, 2011). However, understanding how social media may directly influence learning dynamics remains understudied, and more detailed empirical studies are required. This is especially important as more social media tools are developed and gain popularity, research needs to be redirected to investigate the impacts of specific media across the full spectrum of teaching and learning.\n\nTwitter, a prevalent social media tool, has become a major player in education. While some skepticism remains by some teaching faculties, there are many who recognize the perceived benefits of using Twitter in the classroom, including the following:\n\nRestricting posted content to 140 characters forces student to clarify thoughts into concise information bullets. Bergtrom (as cited in Tinti-Kane, 2013) shares that integrating Twitter is not used for the sole purpose of social connecting but “the 140-character strength forces student to gather their thoughts and state clearly a hypothesis or a conclusion” (p. 1).\n\nShort posts encourage discussion. While Twitter is asynchronous by nature, the short posts can happen in live time and can facilitate a synchronous-like conversation. These conversations can happen both in and out of class between students, teaching faculty and others interested in the topic, increasing communication (Hedge, 2012). Hedge (2012) explains that posting tweets is similar to passing notes between students, which results in a more cohesive community.\n\nTwitter has the ability to authenticate course material. When information is posted to the online Twitter forum, outside members have the ability to interact with the posted content. This interaction can validate student work (Hawks, 2012; Hedge, 2012). Students are then accountable to the public and not just the professor.\n\nTwitter has the ability to archive digitally all course content if students use a course hashtag (#). Using the hashtag, students have on-demand access to relevant course material (Hawks, 2012; Hedge, 2012).\n\nDarling et al. (2013) explain that Twitter can help establish connections to like-minded researchers, subsequently helping students to evolve ideas into actual scientific output. From this perspective, students can transition from student to researcher. This provides validation for the student’s work.\n\nThese insights provide a foundation to understand the possible positive effects of Twitter in an educational setting. However, there must be a clear recognition that the tool itself will influence the messages sent by students, and received by others. This relates to McLuhan’s (1964) thinking about how the medium through which content is delivered can impact the way the message and content is received and understood by the receiver, or, “the medium is the message” (McLuhan, 1994, p. 7). The stimulation of different senses through the medium dictates the way in which the message and content is received, making the medium and the message inseparable. McLuhan’s (1964) medium theory also suggests that a medium has the ability to change between hot and cold.\n\nHot media deliver large packets of content in high definition that fill the recipient with information (McLuhan, 1964). Since hot media offer a high quantity of content, the recipient requires low levels of participation and interaction with the content to construct understanding. The learning acquisition metaphor (AM) is a learning theory that helps to explain and support McLuhan’s understanding of hot media. The acquisition metaphor explains that learning can occur by direct content transmission from teacher to student. This type of direct content transmission reduces the levels of student participation (Sfard, 1998). Active learning principles suggest that for students to make meaning of the content, students must actively engage with the content in different ways. Since hot media deliver larger packets of information, students do not need to actively construct meaning of the content; all information and meaning is provided through the medium. An example of this is a traditional post-secondary lecture (McLuhan, 1964), which may be associated with lower levels of interaction and engagement.\n\nCold media, in contrast, send small amounts of information thereby requiring recipients to “fill in the blanks” in order to make meaning of the content (McLuhan, 1964, p. 22). This requires a high level of active participation and interaction with the content. Active learning pedagogy suggests that when learners have autonomy and ownership over content through active participation, material and content will be better understood (Jonassen & Land, 2012). Sfard (1998) explains that this type of learning can be characterized using a participation metaphor (PM). The PM emphasizes a shift in the role of the learner. The learner in the PM is actively engaged with the content and other students to construct their own knowledge structures (Sfard, 1998). This is considered to be effective pedagogical practice.\n\nThis change between hot and cold media is dependent on the context in which the recipient engages with the material, suggesting the personal motivations of a student, for example, can impact their participation level, thereby influencing the medium. We posit that Twitter is a cold medium, since Twitter restricts content to a small amount of content and it follows that the recipients (i.e., students) are required to engage actively with the content through high levels of participation to make meaning of it. The medium, Twitter, would be directly responsible for shaping the message.\n\nWe sought to directly quantify the effects of Twitter on learning dynamics in an undergraduate field biology classroom, and to place our findings in the context of how the medium shapes the message, and on the medium type. Our first objective was to test the effects of the medium, Twitter, on student engagement, and our second objective was to demonstrate how Twitter affected communication patterns and interactions within the course parameters.\n\n\nMethods\n\nA field biology course at a large Canadian post-secondary institution was the selected research site as part of the course was designed around student-led, group-based research projects about natural history (Ernst et al., 2014). For about a one-month period of the course, and over several intense four-hour laboratory periods, groups of student worked together on a research project about a species occurring within a forest setting. As part of these projects, students were required to write about their research projects using various tools, including Twitter. To study the effects of Twitter on undergraduate learning dynamics, it was essential that the course had a defined and well-articulated learning outcome that could be associated with Twitter. In the field biology course, the fourth learning outcome was concerned with science communication: “(iv) Communicate about Natural History & Ecology to general and specialized audiences (including the instructor), through a range of media (e.g., social media, written works, oral presentations),” thus ensuring that Twitter integration into the course design was both purposeful and meaningful. To achieve this, students were provided an outreach strategy, using Twitter, worth 15%, in the course syllabus. The outreach strategy stated:\n\nOutreach (15%) – during November, groups will be publishing blog posts about their selected study species and their research project. The post is to include an overview of the biology and natural history of the study species, outline of research project, personal anecdotes related to the projects, links to other resources and scientific literature, and photos or other media. This is worth 5% as each group member shall receive the same grade on the blog post. Throughout the term, groups will also use Twitter as a means to disseminate information about their study species, and as a means to network and collaborate about natural history within and beyond the course boundaries. Early in the term, the groups will make an Twitter account for their project, and will be required to tweet at least 4 times within a 24 hour time period of each field laboratory (i.e., including those outside of the research project laboratories). Tweets will be ‘relevant’ to [course]. At least 10 tweets are to be released within 48 hours of each group’s blog post being published. Grading will be based on quality of tweets, evidence of communication within the course, and with people beyond the boundaries of the course. This is worth 10% and each group member shall receive the same grade.\n\nTo maximize the potential for student success, students were given a “How-to” Twitter guide, the assignment details and a document containing reasons for using Twitter in the course.\n\nWithin the Twitter assignment details, it was emphasized that Twitter was engaging, collaborative, and provided networking capabilities. It was explained that Twitter enables tracking and archiving of content, has academic and teaching value, and provides validation (i.e. it gives students an audience other than the professor and teaching assistant). There were explicit instructions and expectations, and student were given the following grading rubric as related to Twitter:\n\n1. Quantity and format: groups met the minimum requirements for tweets, hashtags used appropriately.\n\n2. Etiquette and Professionalism: tweets and interactions on Twitter were mature, professional and written with appropriate tone.\n\n3. Engagement: evidence of collaboration, networking and engagement, both within the course and beyond; use of replies, direct tweets, and interactions.\n\n4. Quality: content of tweets of high quality, relevant to study species, research projects and the overall course content.\n\n5. Links and Photos: appropriate use of linked content in tweets, including photos (e.g., study).\n\nThe purpose of our project was to specifically assess how the use of Twitter in the field biology course affected learning dynamics, and as such, the tweeting process, and tweets themselves, became our quantitative data. Data about the students’ tweets, collected using various methods including, observations, tweet analysis, and individual student interviews, were conducted by the first author. First, pending consent, students were asked to provide a copy of their Twitter profile statistics, which were obtained through Twitter. Each groups’ tweets were analyzed in two distinct ways: content (i.e., the body of the tweet) and the tweet interactivity levels (i.e., number of tweets, tweets to and from different users) in order to provide understanding of how students shaped content to fit within the Twitter restrictions and interaction levels. All participants were asked to complete a survey to gain insight into their thoughts around Twitter use; 18 students (out of the 29 who consented to the survey, out of the 43 registered in the class) participated in the survey. The complete survey can be found in Appendix A. Field observations were also conducted throughout during the laboratory periods which provided insights into the different levels of Twitter users in each group. Subsequently, two members from each group were asked to participate in interviews (one high level Twitter user and one low level Twitter user). Combined, a total of five users participated in the interviews (three active users, two low-level users). The qualitative interview results were used to supplement the quantitative data from the tweets, and the quantitative data from the survey results.\n\n\nResults and discussion\n\nBased purely on the number of tweets sent by our four study groups, students clearly used Twitter, sending over 200% more tweets than minimally required (Figure 1). Of these, less than 1% of the tweets were text only, meaning that almost all tweets were content-rich, including a link, photograph, other user, or hashtag. Student groups were able to gather followers, and connect directly with other users (Figure 1), and they used Twitter for various means. Example tweets illustrate how students used Twitter to promote their own writing about their project (Figure 2), ask questions of other groups (Figure 3), or ask questions of experts from outside of the classroom, to troubleshoot in the field (Figure 4).\n\nData from surveying 18 of 29 consenting students (see Appendix A for full survey details and Appendix B for raw data from survey).\n\nIn this Tweet, the student are promoting a link to their own published blog post.\n\nIn this Tweet, one group of students is asking a question to another student group, about something they observed during a field laboratory.\n\nIn this example, a Twitter user, external to the course and from a different country, interacted directly with the students, in response to their question.\n\nActive learning pedagogy states that when students are actively involved in the learning process, it can result in enhanced memory recall and learning (Cherney, 2008; Craik & Lockhart, 1972). Two important features of active learning are manipulation of content by students through discussion and hands-on activity, and second, student collaboration and cooperation (American Psychological Association, 1997).\n\nThe general results (Figure 1) illustrate how Twitter promoted active learning: the medium allowed students to actively engage with other learners, collaborate in a hands-on manner, cooperate among each other and with other users, and actively engage with course content. Although the majority of the interactions were between students and other members of their direct learning community (Figure 1), it is notable that 17% of interactions were with users external to the class: without Twitter these interactions would not have occurred.\n\n\nMedium is the message\n\nGiven that Twitter is only a medium, in order to engage with it, students had to choose which content to communicate through the channel. The only constraints that students encountered were the content instructions (i.e., content had to be related to course material) and the 140-character restriction. Together, the limited restrictions gave students control of how to use Twitter (e.g. network building) and control over the content of tweets. Given that the students could control content, we categorized tweets post hoc, and were able to separate the 521 tweets into the following distinct functions, in descending order of frequency: outreach (i.e. tweets to interact with users, 33%), informal communication (i.e. general purpose tweets or status updates, 30.7%), promotion (i.e. trying to promote a topic, idea, and/or link, 15.9%, e.g., Figure 2), information (i.e. tweets with informational and/or educational content, 14.5% of tweets), and question (i.e. users asking for help and/or guidance, 5.7%, e.g., Figure 4). The content and text structure of each tweet changed as Twitter was used for different purposes suggesting that the medium has various uses in a classroom context.\n\nThe 140-character restriction provided a limitation on tweet content. The students did not all view the 140-character favourably and found it frustrating. For example, Andrew (All names were given pseudonyms to protect student identity) explained,\n\nYeah, and many times I felt like well, come on I want this word in but how can I, you know, cause I didn’t want to shorten words cause it was still for the class. So I was trying to find a way to rephrase, and you’d rephrase that and because you rephrased it the second sentence would make sense. So you’d have to, it was much harder grammatically than like, that I expected. (Andrew)\n\nDespite this, the students indicated the restriction had an impact on their thinking: the limitation helped students clarify thoughts, as they had to consciously think about how to structure the text and content of the tweet. This idea is supported by Emily, as she stated,\n\nIt’s hard to say because like since it’s limited it also gets people to write like meaningful things, like straight to the point instead of just dragging on sentences and stuff. (Emily)\n\nThis restructuring of tweet content is a characteristic of active learning as the students actively manipulated the content to fit within the character restriction of a tweet.\n\nGiven that 83% of the students had never used Twitter, the newness of the tool shaped the impact of the medium as the students had to learn how to use it. Despite this, the students evaluated Twitter and its associated benefits and drawbacks. There were few negative judgments of Twitter. For example, even a low-level Twitter user was able to see the potential benefits of using Twitter:\n\nSo it’s interesting because despite you saying that you don’t use Twitter you still have a very positive view of it? (Lauren)\n\nYeah I know really because I kind of like, I don’t know how to say... because I kind of think about Facebook and I know how it’s like a great, because I just don’t know Twitter enough. But maybe if I knew Twitter how I know Facebook and if I used Twitter how I, how I used Facebook I know it would really like I would really like it. And I would really like students to go on there and to share things, and to, but it’s just the social media [the professor] chose I’m not really comfortable with. But I know it’s a great, a great idea to use social media to learn because, well Facebook I would have been really more comfortable but it’s okay. (Kristen)\n\nEven though many students found that Twitter was a useful tool in the classroom, a large number of participants indicated that they would not continue to use it after the class finished: 67% of respondents said they would no longer continue to use Twitter outside of the classroom, where 28% said they would (one student said “maybe”) (Appendix B). These results were unexpected as we assumed that if students have a favourable reaction to Twitter, they would continue to use it in additional settings. Simply put, they may just prefer other tools for their personal use. Since Twitter was limited to the classroom context, picking the tool, as an instructor, is extremely important as it has a direct impact on student engagement and potential successful interactions with course content. The medium must serve a direct purpose, which is outlined by a learning outcome. Moreover, students’ recognition of the value of such tools is critical as it provides them with understanding of how specific tools might be potentially useful in their future endeavours.\n\n\nInteractivity and communication\n\nSince Twitter was used in a group setting, it was anticipated that the medium would have an effect on group dynamics, intra-class dynamics, and communication with outsiders. Given that the Twitter responsibilities were associated with a particular group, not individuals, the integration of Twitter had strong implications for relations within groups, as the students had to identify individual Twitter roles. This design was purposeful as it was a strong belief of the second author (based on previous experience) that not all students would gravitate towards the medium; the survey results and field observations supported this belief.\n\nThe interviews and field notes suggested that there was one group member who was often responsible for the majority of tweets and other group members’ involvement ranged from non-participation to active involvement with Twitter, suggesting the emergence of a Twitter hierarchy, yet despite the varying levels of Twitter use, 78% of participants still felt that they contributed to creating tweets. Some interviewees indicated that they participated through idea sharing and research:\n\nTwitter was not my, I didn’t fully use Twitter. I posted one picture one time and some, some little comments, but most of the time I was like, I found some, some articles on the Internet. And I was like I tell them on Facebook I sent them, and [Marie] was like okay I’m gonna post this. And name was more like the, the poster and I, and I kind of did a little research. (Kristen)\n\nMost of the tweets I participate in is when we were doing the data collection in the-, so it was like on the moment I would say, “Oh, you should tweet that, it’s fun,” or something like at. (Carla)\n\nDespite the different levels of Twitter use, the excerpts also suggest that the students still worked together to form their tweets. The data provided supports the claims by the second author as there were different levels of Twitter users. It is reasonable to suggest that the group format directly contributed to the successful integration of Twitter as it provided those who favoured Twitter the opportunity to use the medium and those who were less inclined to use the medium and alternative way of contributing to the work without penalization.\n\nIn the laboratory setting, the research groups were dispersed across a large forested reserve. Without communication devices, groups would have had limited contact with each other and integrating Twitter into the course design provided students with a tool to connect with peer groups and outsiders. The survey data demonstrates that Twitter provided student groups with a unique opportunity to interact with each other and the instructor in an online setting (Figure 1), and students generally believe that this across-group communication is beneficial to their own learning (Figure 1). Qualitative data supported this as the majority of students indicated that across-group dialogue was enhanced by Twitter integration and that increased peer dialogue was beneficial for student learning. For example, one student mentioned that outsider participation resulted in engagement and provided a more interactive student experience.\n\nBecause we really get to see other like in a simple classroom like wouldn’t really know the other team’s research. But on Twitter it’s public and everyone can see it, and like everybody that’s not in the class also can add their own little thing to do it. I don’t know it’s more engaging and interactive. (Kristen)\n\nTwitter provided the students with an opportunity to interact and communicate with anonymous outsiders. To use Twitter effectively, students had to build publicly accessible networks and to do so required active participation from students. The students had to actively search and follow like-minded users, as it was their responsibility to develop networks for communication. Quantitative data from the individual group Twitter interactivity profiles demonstrated that students had numerous unique connections with outside users and they often had repeat connections with these users (Figure 1). The students indicated that the interactions and communication with outsiders as well as other groups provided them with the opportunity to interact with individuals to discuss topics associated with their research goals and course content.\n\nWell yeah I can say [it’s helpful] because some, some people when they were asking us questions we, it gave us another way of thinking about stuff. And, and sometimes they gave us also papers also to prove their point, so it did help me, well us in a team just to have more papers also to look at to answer our questions, so yeah. (Marie)\n\nCombined, 56% of interactions were with the instructor or teaching assistant, 27% of interactions were with peers groups, and 17% of interactions were with external users (Figure 1). Through collaboration, students can work together to discuss and manipulate content thereby creating, and participating in, communities of practice (Wenger, 2000). Such communities help develop and maintain competencies that contribute to learning and identity, all of which align with the characteristics of active learning pedagogy (Craik & Lockhart, 1972).\n\n\nContributions to engagement\n\nThe various findings, including tweet content and creation and interactivity and communication suggest that student levels of engagement were affected by using Twitter. Engagement indicators are based on a variety of criteria, including academic challenge (i.e., higher order thinking, learning strategies, reflective and integrative learning, and quantitative reasoning), learning with peers (i.e., collaborative learning, discussions with diverse others), experiences with faculty (student-faculty interactions and effective teaching practices), and campus environment (quality of interactions and supportive environment) (National Survey of Student Engagement, 2014, p. 1). Using Twitter in the classroom addressed three of the engagement indicators: students were required to actively collect data and reflect and synthesize this content to share out using Twitter (academic challenge), although not required, students engaged in active peer discussion through the medium, as well as collaborated with outside anonymous users, and finally, there was a high degree of interaction between the instructor/teaching assistants and the students (experiences with faculty). Students, when surveyed, felt that Twitter impacted how they engaged with course content (Figure 1), which subsequently contributed to generally positive outlooks of Twitter as a learning tool (Figure 1). Since we suggest that Twitter is a cold medium, its usefulness is finally determined by the students’ willingness to engage with it. Students must actively engage with the cold medium of Twitter and the content to get the full benefit as the tool itself cannot alter teaching and learning but only encourage active participation.\n\n\nImplications for Teaching and Learning\n\nOur results have implications for many educators. Twitter is determined by user interactions with the platform and in our work, the results were positive because the majority of students felt that they contributed, directly and indirectly, to the Twitter project. This resulted in increased communication, collaboration, and critical thinking, all of which was mostly related to course content. This demonstrates that students were actively involved in the learning process. A contributing factor to the successful integration of Twitter was its group form given that it allowed for differing levels of individual contribution based on comfort levels without impacting academic performance.\n\nThe results showed that students had different levels of interactions with the medium. This meant that students who enjoyed using the medium could do so, and the students who were not drawn to the medium could have lower participation. Essentially, the group regulation, a by-product of classification, resulted in agreed upon Twitter roles that were comfortable for group members. As a functioning and regulated group they could effectively use Twitter and achieve the intended learning goals.\n\nIf Twitter were integrated into course design as an individual-based project, we speculate that the results would not have been as positive since not all students are drawn to Twitter. This is evident by the differing levels of student interaction with Twitter. Also, Bernstein (1996) suggests that uncontrolled communication can threaten identity and security. Given that is it essential for students to develop networks to facilitate dialogue and collaboration with outsiders, Twitter profiles should remain public. If Twitter profiles were individualized and public, students might not have been likely to approach Twitter since uncontrolled communication can be considered a threat to identity and security. To counter this, if students had private profiles, networks would have been more difficult to establish, thereby reducing the probability and ease of dialogue and communication with outsiders. These factors suggest that to effectively integrate Twitter into course design, Twitter may best be approached as a group task, and a group-based Twitter handle and alias ensure privacy of individual students.\n\nIt is also important to consider the interactivity levels of users for an additional purpose: given that not all students prefer to use the medium, the students must be provided with additional activities to engage with course material. Educators must employ various strategies to engage students; Twitter is only one tool that can facilitate engagement and collaboration.\n\nA key concern for educators is about how to integrate Twitter successfully into pedagogical practice. Educators must establish boundaries to guide student use of Twitter. In this study, students were required to create tweets related to course content. This restriction forced the students to use the medium primarily for educational purposes, thereby facilitating increased engagement directly with course content. In addition, there was a clear grading schema to assess and evaluation of student’s use of Twitter: this provided an important expectation and framework for the use of social media. Given the various interactivity levels with the medium, we would speculate that without a grade attachment, Twitter uptake would not be as successful. Also, given some students’ unfamiliarity with Twitter, providing documents or lessons on Twitter provides students with the tools to be effective Twitter users.\n\nTo effectively build and establish networks, Twitter profiles must remain public. This allows for an easy, bi-directional relationship between followers and users. The benefit of network building is increased access to new and alternative learning materials through tweets. Also, with a larger Twitter network, students will have the opportunity to communicate and collaborate with various users. However, this also means that the instructors of teaching assistants should also be familiar with the tools, and leverage their own networks as required.\n\n\nConclusion\n\nThe purpose of the study was to investigate the effects of Twitter on undergraduate learning dynamics. To do so, we explored the functionality and purpose of tweets, Twitter’s effect on communication, and the impact of Twitter on engagement. The data analysis revealed that using Twitter:\n\nrequired students to learn about Twitter and evaluate its usefulness in a classroom setting,\n\nfacilitated engagement with course content,\n\nfacilitated and enhanced intra-group dialogue,\n\npromoted and enhanced communication and collaboration with group outsiders, and,\n\nfacilitated active learning with students being responsible for actively manipulating and restricting content to form, and response to, tweets.\n\nTwitter can be effectively used in an undergraduate classroom and can be effectively integrated into a course design. As a result of Twitter use, students actively participated in their learning, which resulted in collaboration, increased communication, and facilitated student engagement. The message of the medium is that active and collaborative learning can be achieved, it matters, and it should be recognized and supported.\n\n\nData availability\n\nFigshare: Raw data from student survey on the use of Twitter to increase engagement in biology courses. Doi: http://dx.doi.org/10.6084/m9.figshare.1360179 (Soluk & Buddle, 2015).\n\nTwitter data were analyzed in the following manner: first, participant groups provide a copy of their Twitter user profile; these profiles included every Twitter interaction that occurred during the semester, including interactions with users, tweet content, and date and time stamps. This information was into a larger document and analyzed tweet functions. Tracking included: overall profile statistics, including the number of followers, tweets sent from mobile and stationary devices, and the number of picture and video tweets, number of user interactions with the professor, teaching assistant, peer groups, and anonymous users. After this analysis was complete, this information was set aside and incorporated with other data at a later date.\n\nSecond, individual interview transcripts were analyzed. In this process, each interview and coded the data based on ‘what was happening’; the categories were descriptive of actions and events; each category had a rule of inclusion.\n\nFinally, the survey results were integrated into the thematic analysis to provide a level of dimensionality to the findings. While interviews lend insight into the students’ consciousness, the survey data provided quantitative information from all selected study participants. It helped to provide an overview of student feelings. These results provided a general understanding of student perspective.\n\nAll data was integrated under the themes established from the student interviews, thus resulting in the final product. Raw data from the survey is accessible through this article.", "appendix": "Author contributions\n\n\n\nLauren Soluk was responsible for project design, development and execution, data collection, analysis and writing.\n\nChristopher Buddle assisted with project development and execution, overall framework for the field course, and helped with writing and interpretation.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors would like to sincerely thank Ralf St. Clair for supervising the lead author, and providing guidance in the project development. Long-time Teaching Assistant Crystal Ernst is thanked for helping with this project, and providing timely input. Teaching and Learning Services at McGill, especially Adam Finkelstein and Laura Winer, also helped to facilitate this work, and provided opportunity for the authors to discuss, debate and lean about using social media tools in higher education. McGill’s Research Ethics Board for their approval of the work. McGill’s Department of Natural Resource Sciences (specifically Prof. James Fyles, chair) continually supports teaching innovation, and allows courses to be incubators for new approaches to teaching: thank you.\n\n\nSupplementary material\n\nLink to Appendix A\n\nLink to Appendix B\n\n\nReferences\n\nAmerican Psychological Association. Learner-centered psychological principles: A framework for school reform and Redesign. 1997. Reference Source\n\nBernstein B: Pedagogy, symbolic control and identity. London: Taylor & Francis. 1996. Reference Source\n\nCherney ID: The effects of active learning on students’ memories for course content. Active Learning in Higher Education. 2008; 9(2): 152–171. Publisher Full Text\n\nCraik FIM, Lockhart RS: Levels of processing: A framework for memory research. J Verb Learn Verb Beh. 1972; 11(6): 671–684. Publisher Full Text\n\nDarling ES, Shiffman D, Côté IM, et al.: The role of Twitter in the life cycle of a scientific publication. Ideas Ecol Evol. 2013; 6: 1–31. Reference Source\n\nErnst CM, Buddle CM, Soluk L: The value of introducing natural history field research into undergraduate curricula: A case study. Bioscience Education. 2014. Publisher Full Text\n\nHawks J: Best practices and tips for Twitter in the higher-ed classroom. 2012. Reference Source\n\nHedge S: Teaching with Twitter. Insider Higher Ed. 2012. Reference Source\n\nJonassen D, Land S: Theoretical foundations of learning environments. New York & Oxon: Routledge. 2012. Reference Source\n\nJunco R, Heibergert G, Loken E: The effect of Twitter on college student engagement and grades. J Comput Assist Lear. 2011; 27(2): 119–132. Publisher Full Text\n\nLuttrell R: Social networking sites in the public relations classroom: A mixed methods analysis of undergraduate learning outcomes using WordPress, Facebook, and Twitter. (Ph.D., California Institute of Integral Studies). ProQuest Dissertations and Theses. 2012. Reference Source\n\nMcCool LB: The pedagogical use of Twitter in the university classroom. Iowa State University. ProQuest Dissertations and Theses. 2011; 66. Reference Source\n\nMcLuhan M: Understanding media: the extensions of man. Cambridge, Mass.: MIT Press. 1964. Reference Source\n\nNational survey of student engagement. From benchmarks to engagement indicators and high-impact practices. 2014. Reference Source\n\nSeaman J, Tinti-Kane H: Social media for teaching and learning. Babson Survey Research Group. 2013. Reference Source\n\nSfard A: One two metaphors for learning and the dangers of choosing just one. Educational Researcher. 1998; 27(2): 4–13. Publisher Full Text\n\nSoluk LC, Buddle CM: Raw data from student survey on the use of Twitter to increase engagement in biology courses. Figshare. 2015. Data Source\n\nTinti-Kane H: Overcoming hurdles to social media in education. Educause Review. 2013. Reference Source\n\nWenger E: Communities of practice and social learning systems. Organization. 2000; 7(2): 225–246. Publisher Full Text" }	[ { "id": "8181", "date": "11 May 2015", "name": "Joshua Drew", "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\nWhile many people on twitter sing its praises for use in class, I think there is a real lack of worked examples of how exactly one would use the medium. Moreover, cases that have demonstrated impacts are precious few. This piece addresses both of these shortcomings with a convincing and even handed approach to how one could use technology to augment an undergraduate field based class. They show that it works well for a given period of time, but that like all tools, there are appropriate and inappropriate uses for twitter. Having used the tool in several of my cases, I can wholeheartedly agree with the lack of engagement post-class - the medium simply isn't for everyone. However I think the authors here clearly show how using Twitter can help a class expand beyond the traditional boundaries and help students learn to engage in informal science discourse.", "responses": [] }, { "id": "8893", "date": "07 Jul 2015", "name": "Gavan P. L. Watson", "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\nAs the uses of social media in post-secondary instruction continue to proliferate across across disciplines, engaging in research that describe classroom approaches and measures outcomes is a valuable addition to this area of scholarship. I'm happy to approve of this paper for: 1) its contributions to the scholarship of practice related to using Twitter in the classroom and 2) its methodology to measure the tool's impact. Innovative, is the pedagogical design of a group-level use of the tool. The strengths of the research begin with the strong link and alignment between the course-level learning outcomes and the use of Twitter. This finding, and the implications of the importance of choosing e-Learning tools aligned with student learning, are important contributions beyond the field of teaching and learning with social media. Broadly, I would encourage the authors to make explicit links to the teaching and learning literature in the opening of the paper to strengthen the underpinning pedagogical arguments. For example, there are also more recent (and compelling) references than McLuhan in the literature to support the claim that large-class lectures are more passive. They write that active student engagement is \"…considered to be effective pedagogical practice\"—again, a link to research that substantiates this claim would improve the argument being laid out.While it may be implicit, I assume that students' use of Twitter on smartphones was integral to the tool's positive impact on engagement across the forested reserve. In this case, it is not just Twitter that had an impact, rather it was the combination of the platform (Twitter) and the internet-connected mobile device. This is a broad consideration for future research: how does the mobile, ubiquitous nature of smartphones, used in conjunction with a tool such as Twitter, impact student engagement and outcomes?", "responses": [] } ]	1	https://f1000research.com/articles/4-82
https://f1000research.com/articles/4-78/v1	25 Mar 15	{ "type": "Case Report", "title": "Case Report: Primary dural based diffuse large B-Cell lymphoma in a 14 year-old boy", "authors": [ "Sunil Munakomi", "Binod Bhattarai", "Balaji Srinivas", "Iype Cherian", "Binod Bhattarai", "Balaji Srinivas", "Iype Cherian" ], "abstract": "Primary dural lymphoma is a subentity of primary leptomeningeal lymphoma which represents 0.1% of all non-Hodgkin’s lymphomas. Only five cases have been reported so far. We report a very rare case of primary dural-based lymphoma in a 14 year-old boy presenting with mass effect. The patient was managed with excision of the lesion and removal of the involved bone. Post-operatively, the patient showed good recovery. He was then referred to the oncology unit for further chemo- and radiation therapy. A high index of suspicion should therefore be kept in order to diagnose the condition in a timely fashion and then plan for appropriate management since diffuse large cell lymphoma has a relatively benign clinical prognosis.", "keywords": [ "Surgical Resection", "Primary dural diffuse large B-cell lymphoma", "Paediatric cancer", "Herniation syndrome", "Intracranial lymphoma" ], "content": "Introduction\n\nPrimary dural diffuse large B-cell lymphoma (DLBCL) is an extremely rare entity with only five cases reported so far1. The symptoms are nonspecific. The main differential diagnosis of the condition remains meningioma2. Currently there is no standard treatment due to a paucity of cases3. A high index of suspicion should be kept in order to diagnose the condition in a timely fashion and then plan for appropriate management since diffuse large cell lymphoma has a relatively benign clinical prognosis4. Here we report a case of a primary dural based DLBCL in a 14 year-old boy presenting with herniation syndrome, who improved after surgical excision and is currently on chemotherapy.\n\n\nCase report\n\nA 14 year-old Tharu boy, from Siraha (a remote village in Nepal) presented to our emergency department with a sudden onset altered sensorium which lasted for 1 day. The patient had a history of intermittent headaches and vomiting over the last 3 months. The patient’s parents also noticed significant weight loss and the presence of scalp swelling for the last 2 months. There was no remarkable family history. Previous treatment history revealed that the patient had been taken to India 1 month back, where fine needle aspiration cytology (FNAC) of the scalp lesion in the parietal region had revealed Non-Hodgkin’s lymphoma. The patient party was told the prognosis and advised for chemo- and radiation therapy but this was refused because of their poor financial status and so the family returned back to Nepal.\n\nOn initial examination at our ER room, the patient attained a Glasgow Coma Scale (GCS) of E2M4V2 with anisocoria on the left side. There were two scalp swellings on the left parietal and the frontal regions (Figure 1) which were soft and fluctuant. Serology performed was negative for human immuno-deficiency virus (HIV) and hepatitis B and C. Computed tomography (CT) scan of the head was performed, revealing a dural-based hyperintense lesion on the frontal and parietal region with subfalcine herniation (Figure 2 and Figure 3) and honeycomb appearance of the involved bone (Figure 4). Ultrasonography of the abdomen revealed no significant lymph nodes.\n\nBecause the child was already herniating, he was started on intravenous dexamethasone (4mg over 8 hours) and a single 100ml dose of 25% mannitol was given. Parents were counseled and written consent was taken for operative management. Surgery revealed a dural-based lesion (Figure 5) that was moderately vascular, soft and friable in consistency with involved bone showing a moth-eaten appearance (Figure 6). Both extra- and intra-dural extension (Figure 7 and Figure 8) of the lesion was seen. Scalp lesions, involved bone, and the dural and intradural component were all excised and sent for histopathological (HPE) study. A post-operative scan showed gross excision of the lesions and absence of mass effect (Figure 9). The HPE revealed an immunoblastic variant of diffuse large cell lymphoma (Figure 10).\n\nPostoperatively, the patient improved to GCS 15. The steroids were slowly tapered off as the mass effect and edema were absent on repeat CT image and also prolonged usage would hamper healing of scalp surgical wound. The patient was thoroughly counseled and then referred for free chemo- and radiation therapy in a government oncology hospital.\n\nThe patient’s GCS at 2 weeks follow-up was 4. Patient has been started on chemotherapy and is also advised for regular follow-ups.\n\n\nDiscussion\n\nPrimary dural lymphoma, first described by Oberling5, is an exceedingly rare disease entity. Only five cases of primary dural diffuse large B-cell lymphoma have been described so far with a median age at diagnosis of around 50 years1. Trauma, inflammation and viral infection have been postulated as probable causes6. The symptoms of the disease are variable and non-specific. The radiological findings are indistinguishable from other dural-based lesions such as meningiomas and hemangiopericytomas2. Since the prognosis of intracranial DLBCL is favourable4, it is important to make a correct and timely diagnosis. Rapid progression of the symptoms, lytic lesions on the bone and restricted diffusion in magnetic resonance imaging (MRI) may provide additional clues to the diagnosis. In cases where there are no obvious neurological symptoms, it may be advisable to take a needle biopsy of the scalp tumor as described by Ochiai et al.7. There has been no consensus on the correct treatment protocol in the management of dural large-cell lymphoma due to a paucity of cases3. Previous cases have been treated with tumor resection followed by cyclophosphamide, hydroxy-doxorubicin, oncovin (vincristine) and prednisone (CHOP) with or without rituximab- or methotrexate-based chemo regimes. Additional radiation was also tried in some cases1. This case is the youngest age where the entity has been observed and showed good recovery despite initial presentation with herniation syndrome. Therefore, we suggest that maintaining a high index of suspicion and timely intervention is the key to better outcome in the patients.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and images was obtained from the father of the patient.", "appendix": "Author contributions\n\n\n\nDr. Sunil prepared and formatted the paper, Dr. Binod and Dr. Balaji revised the paper and Dr. Cherian approved the final draft. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to acknowledge the Department of Pathology of the College of Medical Sciences for providing us with the histopathological image in preparation of the manuscript.\n\n\nReferences\n\nBrito AB, Reis F, de Souza CA, et al.: Intracranial primary dural diffuse large B-cell lymphoma successfully treated with chemotherapy. Int J Clin Exp Med. 2014; 7(2): 456–60. PubMed Abstract \| Free Full Text\n\nJohnson MD, Powell SZ, Boyer PJ, et al.: Dural lesions mimicking meningiomas. Hum Pathol. 2002; 33(12): 1211–1226. PubMed Abstract \| Publisher Full Text\n\nIwamoto FM, Abrey LE: Primary dural lymphomas: a review. Neurosurg Focus. 2006; 21(5): E5. PubMed Abstract \| Publisher Full Text\n\nYamada SM, Ikawa N, Toyonaga S, et al.: Primary malignant B-cell-type dural lymphoma: Case report. Surg Neurol. 2006; 66(5): 539–543. PubMed Abstract \| Publisher Full Text\n\nOberling C: Les reticulosarcomeset les reticuloendotheliosarcomes de la moelleosseuse (sarcomas d'Ewing). Bull Assoc Fr Etude Cancer. 1928; 17: 259–296.\n\nTanimura A, Adachi Y, Tanda M, et al.: Primary peripheral B cell lymphoma, Burkitt-like, of the cranial vault. Acta Haematol. 2005; 113(4): 258–261. PubMed Abstract \| Publisher Full Text\n\nOchiai H, Kawano H, Miyaoka R, et al.: Primary diffuse large B-cell lymphomas of the temporoparietal dura mater and scalp without intervening skull bone invasion. Neurol Med Chir (Tokyo). 2010; 50(7): 595–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "8460", "date": "15 May 2015", "name": "Guo-Yi Gao", "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\nPrimary dural lymphoma is the disease to most neurosurgeons and this case report provided details of a young patients' clinical treatment including diagnosis, surgery, and following chemotherapy and radiotherapy choices. It is worth indexing to improve the management of dural lymphoma.", "responses": [] }, { "id": "9101", "date": "24 Jun 2015", "name": "Amit Thapa", "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\nThe authors Munakomi et al. have written a nice case report which highlights a rare disease. Diffuse large cell, mixed and immunoblastic lymphomas of B cell origin should be considered together as aggressive lymphomas1 which unlike MALT associated primary dural lymphomas are not indolent. However certain facts need to be discussed.Even though rare, this case is not the sixth reported in literature. Other extra axial locations noted are orbit2, Meckel cave3,4, parasellar5,6, zygoma2,7, cerebellopontine angle8. In 2010, 93 cases with dural based lymphomas with 10 having extra CNS (systemic involvement) had been reported9. Tumor immunophenotyping using flow cytometric analysis (CD5, CD10, CD19, and CD20) and by histochemical evaluation of the lymphoid cells is mandatory. These exhibit strong immunoreactivity both to leukocyte common antigen and to B-cell marker (L26). No staining should be noted for chromogranin, synaptophysin, or the epithelial markers AE1 and AE31. Even though financial restriction is of concern in our country, contrast enhanced CT chest and abdomen should be the protocol to see for lymph node involvement rather than insensitive USG study. This may be a typo error, however to correct the same: Mannitol comes in 20% and not in 25% in Nepal. Image (figure 9) show a limited craniotomy causing brain herniation from the site of craniectomy and has been taken at a level lower than lesion previously shown in Figure 2. To allow comparison, it is suggested to provide CT images of same level or plane. Differential diagnosis should include meningiomas, metastases,extradural abscesses with osteomyelitis or extradural hematomas. Clues for diagnosis include a rapid progression of symptoms with permeative growth pattern with large soft tissue component in galeal and extradural compartment, restricted diffusion on MRI, and the presence of bone lytic lesions on CT, limited periosteal reaction and an angiographically avascular lesion except for blush or vascular encasement1,10. The treatment of choice for primary CNS lymphoma is chemotherapy (Adriamycin containing CHOP protocol) followed by radiation of the involved field and surgical excision is limited to conditions where mass effect is life threatening.Recognising this entity is important because an early diagnosis and rapid initiation of treatment, in certain cases with sole chemotherapy, is associated with a high response rates and favourable outcome10.", "responses": [] } ]	1	https://f1000research.com/articles/4-78
https://f1000research.com/articles/4-77/v1	24 Mar 15	{ "type": "Opinion Article", "title": "A new hypothesis on HIV cure", "authors": [ "Florian Hladik" ], "abstract": "In this opinion article, I provide the rationale for my hypothesis that nucleoside reverse transcriptase inhibitors (NRTIs) may prevent human immunodeficiency virus (HIV) cure by promoting the survival of cells with integrated provirus. If correct, we may be closer to a cure than we realize.", "keywords": [ "AIDS", "HIV Latency", "NRTIs", "Tenofovir", "Antiretroviral drugs", "Mucosa", "Berlin patient", "Integrase Inhibitors" ], "content": "Introduction\n\nCurrent antiretroviral treatment (ART) is extremely effective in controlling replication of human immunodeficiency virus (HIV) and in many patients suppresses the number of virions measurable in peripheral blood, i.e., the HIV viral load, to undetectable levels. Nevertheless, whenever ART is stopped, HIV levels rebound and the disease returns. This lack of eradication is attributed to a stable latent reservoir of HIV-1 in resting CD4+ T lymphocytes and perhaps other susceptible cell types such as macrophages1. These cells harbor HIV in the form of replication-competent proviruses that are integrated into the host genome. During effective ART this reservoir decays so slowly that it would theoretically require treatment for 60 years or longer to eliminate it2.\n\nFor this reason, HIV-related research efforts are increasingly being devoted to understanding the nature of this latent virus reservoir and how to eradicate it. Two aspects of the latent virus reservoir have emerged as crucial in maintaining infection. First, HIV is not transcribed or translated from latently infected cells, allowing them to escape detection from the immune system. Second, cells with integrated provirus persist and even expand despite continuous ART3–5. To circumvent viral persistence, “kick and kill” strategies have been proposed that attempt to reactivate HIV with latency-reversing agents and then destroy these cells with the help of targeted active or passive immunization strategies. Unfortunately, reactivating cells from infected individuals ex vivo has thus far not shown promising results6,7 and the hurdle for effective immune control of cells reactivated from latency may be high8,9.\n\nWhy cells carrying HIV proviruses continue to expand during ART without expressing viral proteins in the process remains an unresolved paradox. To solve this conundrum tremendous effort is going into characterizing the latent virus reservoir and understanding ongoing immune activation during ART. It is hoped that a synthesis of findings in both areas may provide important clues about navigating available and newly arising treatment options toward a cure.\n\n\nMucosal effects of tenofovir\n\nA third area that has been little considered is the effect of ART drugs, both on viral latency and immune activation. Modern antiretroviral combination therapy provides tremendous clinical benefits for HIV-infected patients, dramatically improving quality of life and prolonging life expectancy. Thus, the possibility that a component of ART could paradoxically decrease the chance of a cure being effective had never crossed my mind when we initiated a systems biology evaluation of an ART drug topically applied to the rectal mucosa in a phase I clinical safety trial. This trial, MTN-007 (www.clinicaltrials.gov/ct2/show/NCT01232803), tested the safety and tolerability of a gel containing 1% tenofovir, a phosphonated nucleoside reverse transcriptase inhibitor (NRTI) in development for potential use to prevent rectal HIV transmission10. A gel containing 2% nonoxynol-9 (N-9) (a temporary mucosal toxin) was included as a positive control arm, and hydroxyethyl cellulose gel and no gel served as negative controls. Our original hypothesis was that the effects of 1% tenofovir gel on the mucosal transcriptome would be negligible whereas N-9 would activate inflammatory genes. However, upon unblinding of the microarray data, we were surprised to find that tenofovir caused many more genes to change than N-9, more often suppressing than enhancing gene expression11.\n\nTenofovir caused three particular changes that bear potential relevance to the HIV cure agenda. First, it strongly inhibited the transcription of a large number of nuclear transcription factors; second, it inhibited the anti-inflammatory function of mucosal epithelial cells; and third, it stimulated signatures of increased cell proliferation and viability. Results obtained from rectal biopsies were replicated in primary vaginal epithelial cells, which also proliferated significantly faster in tenofovir’s presence. In addition to the breadth of transcriptional changes, individual effects caused by tenofovir could be large. For example, both in vivo and in vitro, the drug blocked transcription and protein production of interleukin 10 (IL-10) in the range of 90%11.\n\n\nAn emerging hypothesis\n\nFrom these data grew my first suspicion that tenofovir, and perhaps more generally NRTIs, could have unappreciated effects on HIV latency, and may in fact prevent HIV cure by promoting the survival of cells with integrated provirus (Figure 1). Before developing this concept below, I want to caution that many of the statements are preliminary and/or hypothetical, intended to serve as a stimulus to the field for further investigation and verification.\n\nBased on the pronounced inhibitory activity of tenofovir on the transcription of many genes, I hypothesize that it also inhibits transcription of provirus integrated into such genes. Host gene transcriptional activity has been shown to be an important determinant of integrated HIV transcription12. This ‘integrated virus transcription inhibitor’ (IVTI) effect of tenofovir and other NRTIs could explain the transcriptional silence of integrated provirus during ART, since nearly all patients receive an ART regimen containing not just one but two NRTI drugs. Tenofovir’s IVTI activity is supported by the preliminary finding that genes reported in two recent studies to be preferential sites of HIV integration after periods of ART appear to overlap with genes inhibited in our studies by tenofovir3,4. A preliminary analysis of the lists of genes highlighted in the two papers and those found to be strongly inhibited by tenofovir in the rectum showed considerable overlap, including CREBBP, IL6ST, KIF1B, FBXW7, DDX6, IKZF3, ZNF652, DST, CLIC5, GRB2, CEPT1, TAOK1 and PAK2. No overlap was found with genes inhibited by N-9. If true, this overlap would imply that over time NRTIs select for cells in which latent HIV survives because of the drugs’ inhibitory effects on transcription of genes hosting integrated provirus.\n\nThe IVTI function of NRTIs could be complemented in favoring latency by their inhibitory effect on the immune system’s anti-inflammatory circuits. In our study, tenofovir was not directly inflammatory, but its strong inhibition of IL-10, as well as of pathways downstream of the immune homeostatic factor TGF-β, indicated that once inflammation is triggered by an outside event, which could be HIV infection itself, it could be prolonged or perpetuated in the presence of tenofovir. I call this the ‘anti-anti-inflammatory’ action of tenofovir.\n\nIn our cohort of individuals at low risk for inflammation and HIV infection, we did not detect overt inflammation, although tenofovir did significantly increase the density of CD3+ and CD7+ lymphocytes in the rectal mucosa. The participants in CAPRISA 004 (www.clinicaltrials.gov/ct2/show/NCT00441298), an efficacy trial that demonstrated an overall 39% protective effect of vaginal 1% tenofovir gel13, were at much higher risk for inflammation and HIV infection. This may have uncovered an interesting paradoxical effect of tenofovir: unpublished data in a subset of CAPRISA 004 participants suggest that in the presence of inflammation the risk of HIV infection increased markedly more in the tenofovir than the placebo arm (personal communication). Further analyses by CAPRISA 004 investigators are ongoing. If confirmed, I would hypothetically attribute this effect to tenofovir’s anti-anti-inflammatory action.\n\nThus, the anti-anti-inflammatory effect of NRTIs could explain why the massive immune activation caused by primary HIV infection never completely reverses despite effective ART. Interestingly, a similar persistence of immune activation is observed in HSV infection treated with acyclovir, a nucleoside analogue related to NRTIs, which also inhibits DNA synthesis by terminating the growing strand. That HSV-induced local immune activation does not resolve well with acyclovir treatment has been identified as a possible reason why the HSV-associated increase in HIV susceptibility does not reverse when women with genital HSV infection receive acyclovir14–16. Perhaps acyclovir has some of the same anti-anti-inflammatory properties as tenofovir.\n\nResidual immune activation perpetuated by NRTIs could drive the ongoing expansion of cells harboring integrated provirus, and their IVTI function could simultaneously limit transcription of these proviruses. Indeed, our analysis so far indicates that the genes generally turned on by cell activation and the HIV-hosting genes inhibited by tenofovir are different, potentially explaining this apparent paradox. Additionally, the direct cell proliferation- and viability-enhancing effects of tenofovir could contribute to the persistence and expansion of latently infected cells.\n\n\nART and anatomic sites of HIV latency\n\nIn theory, the HIV latency-inducing effects of NRTIs would likely be strongest where drug concentrations are highest in vivo. Studies on tenofovir’s biodistribution after oral administration show that it highly enriches in gut tissues17–19, which is believed to harbor a major portion of the latent HIV reservoir20. Estimates also indicate that the rectal concentrations of tenofovir diphosphate, the active intracellular metabolite, are comparable after seven days of oral tenofovir dosing or a single dose of intrarectal 1% tenofovir gel (personal communication, Dr. Craig Hendrix, Johns Hopkins University). Thus, it is likely that some of the effects we observed in the rectal mucosa after topical application also occur after oral dosing, in particular with years of administration and in combination with a second NRTI.\n\nOf note, after oral dosing, NRTI drug concentrations may be even higher in the small intestine than in the colon and rectum, because in the upper gastrointestinal tract locally dissolving drug likely adds to drug distributing from the blood stream. If NRTIs do indeed promote latency, then high drug concentrations would make the small intestine favorable for HIV latency, consistent with the observation that within the gut the duodenum and ileum were preferential sites of residual HIV DNA and unspliced RNA in ART-suppressed patients20,21. In fact, if NRTIs did not enhance latency, it would be difficult to explain why residual HIV is found precisely where antiretroviral drug concentrations are highest.\n\n\nTwo special cases of cure without ART\n\nCircumstantial evidence suggests that pharmacological ART is not required to cure HIV/simian immunodeficiency virus (SIV) infection. The only adult patient ever cured of HIV infection, the “Berlin patient” Timothy Brown, received a stem cell transplant from a donor homozygous for a 32-bp deletion in the CCR5 allele, which provides resistance against HIV-1 infection22. He took suppressive ART until the point of his first stem cell transplant, at which point he stopped all ART and never resumed it. Of course, he received a powerful alternative to pharmacological ART in the form of two CCR5-deficient stem cell transplants, carried out about one year apart. However, he did not achieve complete chimerism for some time after transplantation, because CCR5-expressing macrophages were still present in rectal biopsies 5.5 months following the stem cell transplants22,23, and thus potential HIV target cells were not completely eliminated at that point. This could have provided a hold for residual HIV. Perhaps removing the hypothetical latency-favoring activity of the NRTI drugs could have contributed to his cure.\n\nIn contrast, two HIV-1-infected patients in Boston who also received stem cell transplants continued ART in the peri- and post-transplantation period, and were not cured24. Notably, though, these two patients did not receive CCR5-negative stem cells, which provided a less favorable scenario than in the Berlin patient’s case.\n\nThe only animals ever cured from a highly pathogenic SIV infection were rhesus macaques who had been vaccinated before SIV challenge with SIV-protein-expressing rhesus cytomegalovirus vectors25. Although the vaccinated rhesus macaques all showed signs of ongoing systemic infection for weeks or months after challenge, protected monkeys lost all indications of SIV infection over time, consistent with immune-mediated clearance of an established lentivirus infection. None of these animals ever received ART.\n\nWhile it was suggested that establishment of a latent SIV reservoir might have been prevented by the persistently high frequencies of vaccine-induced SIV-specific CD8+ T lymphocytes, early on many of these animals showed clear signs of productive infection, which requires viral integration. Thus, a latent reservoir was likely established. However, in the absence of the latency-prolonging effects of NRTIs the decay rate of provirus-containing cells could hypothetically have been accelerated, due to faster natural cell death, less cell expansion, and higher expression of viral proteins, allowing immune recognition by the SIV-specific cytolytic T cells. No viral blips were detected in any animals beyond 70 weeks, perhaps offering a clue as to the time frame required to eradicate a latent reservoir in the absence of NRTIs. However, the pool of latently infected cells was likely small in these animals, and eradication of a larger reservoir may take longer.\n\n\nConclusion and outlook\n\nIn summary, given that (1) NRTIs may prevent immune detection of latently infected cells by inhibiting transcription of integrated virus, (2) NRTIs may increase persistence of cells with integrated virus by perpetuating inflammation and enhancing cell proliferation, (3) the only monkeys ever cured of SIV infection never received ART, and (4) the only adult patient ever cured of HIV infection discontinued ART before initiating another powerful antiviral therapy, I hypothesize that effectively suppressing HIV with a strategy that does not contain an NRTI component has curative potential.\n\nOnly a few years ago, finding a similarly suppressive alternative to an NRTI-containing ART regimen would have posed a dilemma26. Today, powerful second-generation integrase inhibitors and non-NRTI drugs (NNRTIs) are entering early human clinical trials27–29. Active vaccination, passively infused neutralizing antibodies and vector-expressed CD4/CCR5 co-mimetics show promise as therapeutic immune interventions25,30–34, and even more complex strategies such as HIV receptor deletion and specific destruction of integrated viral DNA sequences are progressing35. We are thus moving into a phase where effective NRTI-sparing strategies are becoming reality and could offer hope for a cure.\n\nOne phase IIb trial (www.clinicaltrials.gov/ct2/show/NCT02120352) is registered to switch HIV-1-infected patients who are initially suppressed with an NRTI-containing regimen to an NRTI-free combination of GSK744 LA, a long-acting injectable formulation of the novel integrase inhibitor GSK126574427, and TMC278 LA, a long-acting injectable formulation of the novel NNRTI TMC278 (ripilvirine)29. Though not designed to test a cure, this regimen may in fact have curative potential. The study sponsors should consider adjusting their design for that purpose.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI thank M. Juliana McElrath, Fred Hutchinson Cancer Research Center, for helpful discussions, and Sean Hughes, University of Washington, for editing the manuscript. A prior version of this opinion article was posted July 21st 2014 on the preprint server bioRχiv (www.biorxiv.org/content/early/2014/07/21/007294).\n\n\nReferences\n\nSiliciano RF: Opening Fronts in HIV Vaccine Development: Targeting reservoirs to clear and cure. Nat Med. 2014; 20(5): 480–1. PubMed Abstract \| Publisher Full Text\n\nFinzi D, Blankson J, Siliciano JD, et al.: Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med. 1999; 5(5): 512–7. PubMed Abstract \| Publisher Full Text\n\nWagner TA, McLaughlin S, Garg K, et al.: HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection. Science. 2014; 345(6196): 570–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaldarelli F, Wu X, Su L, et al.: HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science. 2014; 345(6193): 179–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKearney MF, Spindler J, Shao W, et al.: Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy. PLoS Pathog. 2014; 10(3): e1004010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBullen CK, Laird GM, Durand CM, et al.: New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nat Med. 2014; 20(4): 425–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCillo AR, Sobolewski MD, Bosch RJ, et al.: Quantification of HIV-1 latency reversal in resting CD4+ T cells from patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A. 2014; 111(19): 7078–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukazawa Y, Lum R, Okoye AA, et al.: B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers. Nat Med. 2015; 21(2): 132–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeng K, Pertea M, Rongvaux A, et al.: Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature. 2015; 517(7534): 381–5. PubMed Abstract \| Publisher Full Text\n\nMcGowan I, Hoesley C, Cranston RD, et al.: A phase 1 randomized, double blind, placebo controlled rectal safety and acceptability study of tenofovir 1% gel (MTN-007). PLoS One. 2013; 8(4): e60147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHladik F, Burgener A, Ballweber L, et al.: Mucosal effects of tenofovir 1% gel. Elife. 2015; 4: e04525. PubMed Abstract \| Publisher Full Text\n\nHan Y, Lin YB, An W, et al.: Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough. Cell Host Microbe. 2008; 4(2): 134–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdool Karim Q, Abdool Karim SS, Frohlich JA, et al.: Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science. 2010; 329(5996): 1168–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCelum C, Wald A, Hughes J, et al.: Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial. Lancet. 2008; 371(9630): 2109–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu J, Hladik F, Woodward A, et al.: Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med. 2009; 15(8): 886–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYi TJ, Shannon B, Chieza L, et al.: Valacyclovir therapy does not reverse herpes-associated alterations in cervical immunology: a randomized, placebo-controlled crossover trial. J Infect Dis. 2014; 210(5): 708–12. PubMed Abstract \| Publisher Full Text\n\nPatterson KB, Prince HA, Kraft E, et al.: Penetration of tenofovir and emtricitabine in mucosal tissues: implications for prevention of HIV-1 transmission. Sci Transl Med. 2011; 3(112): 112re4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHendrix CW, Andrade A, Kashuba AD, et al.: Tenofovir-emtricitabine directly observed dosing: 100% adherence concentrations (HPTN 066). 21st Conference on Retroviruses and Opportunistic Infections. Boston. 2014; 104. Reference Source\n\nLouissaint NA, Cao YJ, Skipper PL, et al.: Single dose pharmacokinetics of oral tenofovir in plasma, peripheral blood mononuclear cells, colonic tissue, and vaginal tissue. AIDS Res Hum Retroviruses. 2013; 29(11): 1443–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChun TW, Nickle DC, Justement JS, et al.: Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. J Infect Dis. 2008; 197(5): 714–20. PubMed Abstract \| Publisher Full Text\n\nYukl SA, Shergill AK, McQuaid K, et al.: Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010; 24(16): 2451–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHutter G, Nowak D, Mossner M, et al.: Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009; 360(7): 692–8. PubMed Abstract \| Publisher Full Text\n\nAllers K, Hutter G, Hofmann J, et al.: Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood. 2011; 117(10): 2791–9. PubMed Abstract \| Publisher Full Text\n\nHenrich TJ, Hu Z, Li JZ, et al.: Long-term reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation. J Infect Dis. 2013; 207(11): 1694–702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansen SG, Piatak M, Ventura AB, et al.: Immune clearance of highly pathogenic SIV infection. Nature. 2013; 502(7469): 100–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAchhra AC, Boyd MA: Antiretroviral regimens sparing agents from the nucleoside(tide) reverse transcriptase inhibitor class: a review of the recent literature. AIDS Res Ther. 2013; 10(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrews CD, Spreen WR, Mohri H, et al.: Long-acting integrase inhibitor protects macaques from intrarectal simian/human immunodeficiency virus. Science. 2014; 343(6175): 1151–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrews CD, Yueh YL, Spreen WR, et al.: A long-acting integrase inhibitor protects female macaques from repeated high-dose intravaginal SHIV challenge. Sci Transl Med. 2015; 7(270): 270ra4. PubMed Abstract \| Publisher Full Text\n\nAzijn H, Tirry I, Vingerhoets J, et al.: TMC278, a next-generation nonnucleoside reverse transcriptase inhibitor (NNRTI), active against wild-type and NNRTI-resistant HIV-1. Antimicrob Agents Chemother. 2010; 54(2): 718–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarouch DH, Whitney JB, Moldt B, et al.: Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature. 2013; 503(7475): 224–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPicker LJ, Deeks SG: HIV: Antibodies advance the search for a cure. Nature. 2013; 503(7475): 207–8. PubMed Abstract \| Publisher Full Text\n\nKlein F, Nogueira L, Nishimura Y, et al.: Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants. J Exp Med. 2014; 211(12): 2361–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShingai M, Nishimura Y, Klein F, et al.: Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature. 2013; 503(7475): 277–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardner MR, Kattenhorn LM, Kondur HR, et al.: AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature. 2015; 519(7541): 87–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeterson CW, Younan P, Jerome KR, et al.: Combinatorial anti-HIV gene therapy: using a multipronged approach to reach beyond HAART. Gene Ther. 2013; 20(7): 695–702. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "8673", "date": "22 Jun 2015", "name": "Eric Hunter", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the absence of a highly effective preventative vaccine to protect against HIV infection, and with a growing burden of HIV-1 infected individuals currently on or soon to be eligible for antiretroviral treatment, there is a major research effort underway to seek approaches that might yield a functional cure for the disease. As Florian Hladik points out in this Opinion Article, a major challenge and obstacle to the goal of developing an HIV Cure is the fact that, despite years of suppressive antiretroviral therapy, a stable latent virus reservoir persists that can seed the rapid rebound in HIV-1 replication. An event that is consistently observed whenever treatment of chronically infected individuals is interrupted.In this Opinion Article, Hladik draws on recent research from his laboratory, in which a systems biology approach was taken to understand the impact of a topically applied nucleoside reverse transcriptase inhibitor (NRTI), tenofovir, on gene expression in the rectal mucosa. The results of this study showed that topical tenofovir inhibited the transcription of a large number of nuclear transcription factors, inhibited anti-inflammatory functions of mucosal epithelial cells, in particular IL-10 expression, and stimulated signatures of increased cell proliferation and enhance viability. Similar results were observed in primary vaginal epithelial cells in culture (author reference 11).Based on this research, Hladik hypothesizes that tenofovir might paradoxically enhance formation and maintenance of the latent reservoir by inhibiting the transcription of proviruses integrated into the suppressed genes, and that such transcriptional silencing could explain the lack of HIV expression in such cells. Support for this theory comes from the fact that genes shown recently to be preferential sites for HIV-1 integration (refs) overlap with those inhibited by tenofovir. Moreover, Hladik proposes that by inhibiting anti-inflammatory functions, tenofovir and other NRTIs could perpetuate residual immune activation and drive ongoing expansion of cells harboring an integrated provirus. An effect that could be exacerbated by the cell proliferation and enhancement of viability also observed following administration of the drug. Because NRTIs are generally delivered orally, the author argues that the above effects would be expected to be strongest where drug concentrations are highest, consistent with observations that residual viral DNA is preferentially found in the duodenum and ileum.Given the strong base to this interesting hypothesis, it is unfortunate that the author uses somewhat anecdotal examples of situations, where non-NRTI approaches have resulted in apparent “cure” of the latent reservoir, to support his theory. While the mechanism of latent virus clearance has not been defined, in the first of these, the Berlin patient Timothy Brown, did stop ART, but as pointed out by the author, underwent two CCR5-deficient stem-cell transplants that could through a combination of resistant cells and graft versus host reaction have cleared residual latent cells. The second example of apparent cure from ongoing early infection is that observed with cytomegalovirus SIV vaccine vectors. While approximately 50% of infected vaccinated animals do appear to clear SIV infection, the mechanistic basis for this is unknown, and it would seem to be a stretch to argue that this is simply because the animals were not ART treated.The hypothesis put forward in this Opinion Article – that drugs highly effective in suppressing viral replication might actually play a role in sustaining, in a latent state, the very virus that it so effectively inhibits – seems at first counter-intuitive. Nevertheless, the gene expression data from the phase I clinical trial of tenofovir as a rectal microbicide, do provide a plausible underpinning for the hypothesis and it is one that should be tested experimentally. Indeed, given the diversity of treatment regimens that are ongoing for patients, it seems likely that existing clinical samples may well be available to allow such a study to be performed. Moreover, as noted by the author, clinical trials that have or propose to switch patients from an NRTI-based ART regimen to one lacking NRTIs would provide clinical samples with which to test the hypothesis.Although not discussed by the authors in the eLife manuscript, if the effect of tenofovir on gene expression is reversible – something that could be tested in vitro – then one might anticipate that individuals switching from a NRTI-based regimen to a combination of NNRTI, protease inhibitor, or integrase inhibitor, might be expected to exhibit a more rapid decline in the latent reservoir compared to those remaining on an NRTI regimen. Moreover, it should be possible using RT-SHIVs to test whether non-NRTI suppressive antiretroviral regimens result in a reduced latent virus reservoir compared to those on a tenofovir-based regimen.A minor point – in the Abstract the author states “(NRTIs) may prevent human immunodeficiency virus (HIV) cure”. This should be softened to “may reduce the likelihood of a cure for human immunodeficiency virus”, since one cannot predict the efficacy of future “cure” approaches.Finally, while the hypothesis is novel and if proven may provide clues to limiting the latent viral reservoir in patients on ART, it is important to note that tenofovir and a second NRTI, Emtricitabine (FTC) form the base of the 3 drug regimen that is the low-cost mainstay of initial HIV-1 treatment across the continent of Africa and other developing countries. While the long-term goal of a cure for HIV, along with an effective preventative vaccine, will likely be key to controlling the epidemic, it is equally important not to undermine confidence in these highly effective components of current therapy.", "responses": [] }, { "id": "8703", "date": "24 Aug 2015", "name": "Leonid Margolis", "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\nThis work by Hladik addresses one of the most important problems of HIV research: the persistence of a “latent reservoir” in spite of the virtually complete suppression of viral replication. Indeed, despite dramatic progress in the “functional” cure of HIV infection, we have failed to eradicate virus from the infected organism. The author suggests that the nucleoside reverse transcriptase inhibitors (NRTIs), in particular tenofovir, have a side effect: they suppress a natural anti-inflammatory activity, thus, probably indirectly, facilitating inflammation. Also, ART does not prevent cell proliferation and in some cases even facilitates it, thus increasing the number of cells with the integrated provirus. This proliferation may contribute essentially to the establishment and increase of the HIV reservoir.The hypothesis suggested in this paper was not tested directly, but the author presents significant arguments in favor of it. His arguments are based on his and others’ research on the effect of tenofovir on tissue explants. One of the strongest effects of tenofovir on human tissues is a blockade of transcription and protein production of IL-10. Moreover, analysis of gene activation in rectal tissue of patients under ART led the author to imply that because of the drugs’ inhibitory effects on transcription of genes hosting integrated provirus, NRTIs select over time for cells in which latent HIV survives.Finally the author discusses the case of a Berlin patient as well as the curing of non-human primates (rhesus macaques) who have been vaccinated before SIV challenge with SIV protein-expressing rhesus cytomegalovirus vectors. In none of these unique examples of cures was ART used.In summary, the author suggests that ART suppresses anti-inflammatory responses, indirectly promoting inflammation. This may be true not only for tenofovir and HIV but also for acyclovir and HSV, as these drugs, despite suppressing HSV facilitate HIV in the treated individuals.This is an original and interesting hypothesis that may explain some aspects of HIV infection that are not understood yet. By the way, it may explain why HIV-1 patients under ART never return to normal even if their virus, which has been suppressed for years, is undetectable, while the immune activation persists, probably leading to various AIDS-unrelated diseases and to premature aging. The author may further develop his hypothesis to cover the area of age-related diseases in functionally cured patients under ARTOn the other hand, it is important for the author to emphasize more strongly the success of modern ART as a universal treatment. Even if the author’s hypothesis be proved, the effects he described would be at most the side-effects of the successful treatment, although very important ones. Moreover, in my opinion, the establishment and persistence of the HIV reservoir hardly can be explained by only one factor.Finally, the association of the cure with the lack of ART application in just three cases is of course anecdotal, and this fact should be emphasized more strongly.All these critical remarks are rather of an editorial nature, and Hladik’s original hypothesis certainly deserves to be indexed and to be tested in targeted basic and epidemiological research.", "responses": [] } ]	1	https://f1000research.com/articles/4-77
https://f1000research.com/articles/4-74/v1	20 Mar 15	{ "type": "Research Article", "title": "Digital collaborative learning: identifying what students value", "authors": [ "Claire Hemingway", "Catrina Adams", "Molly Stuhlsatz", "Catrina Adams", "Molly Stuhlsatz" ], "abstract": "Digital technologies are changing the learning landscape and connecting classrooms to learning environments beyond the school walls. Online collaborations among students, teachers, and scientists are new opportunities for authentic science experiences. Here we present findings generated on PlantingScience (www.plantingscience.org), an online community where scientists from more than 14 scientific societies have mentored over 14,000 secondary school students as they design and think through their own team investigations on plant biology. The core intervention is online discourse between student teams and scientist mentors to enhance classroom-based plant investigations. We asked: (1) what attitudes about engaging in authentic science do students reveal, and (2) how do student attitudes relate to design principles of the program? Lexical analysis of open-ended survey questions revealed that students most highly value working with plants and scientists. By examining student responses to this cognitive apprenticeship model, we provide new perspectives on the importance of the personal relationships students form with scientists and plants when working as members of a research community. These perspectives have implications for plant science instruction and e-mentoring programs.", "keywords": [ "Attitudes", "Plant Science", "E-mentoring", "Authentic Science", "Student-Teacher-Scientist Partnership" ], "content": "Introduction\n\nA revolution in digital learning is underway. The number of students taking online courses in the United States has skyrocketed to 7.1 million in higher education institutions (Allen & Seaman, 2014) and almost 750,000 in public primary and secondary schools (Evergreen Research Group, 2014). Digital technologies offer new mechanisms to support reform-based approaches and increase student engagement. Transforming traditional college and pre-college classrooms into active-learning environments where students interact with peers and instructors to collectively construct and apply knowledge can positively impact student attitudes towards science (Armbruster et al., 2009; Gibson & Chase, 2002; Taraban et al., 2007; Ward et al., 2014). A significant challenge to the tremendous potential for the digital learning revolution is transferring authentic science investigations to digital learning environments. There is a particular need to investigate students’ attitudes about technology-enhanced science investigations in precollege settings, as this is a critical time when interest in science can set the direction of future career goals (Maltese & Tai, 2011).\n\nHow students and teachers experience plant science is a focus of concern; alarming trends in U.S. formal education show that plants are under-represented in teaching materials, and poorly understood. A decline in botanical literacy is part of the continuing U.S. crisis in science literacy, although some underlying causes are unique to botany. The best-selling U.S. high school biology texts feature primarily animals (Uno, 1994). Teachers also place a focus on animals; when choosing material to teach biological concepts, teachers reported preferring to use animal examples over plant examples (Flannery, 1999; Link-Perez & Schussler, 2013). Pre-service teachers (Krantz & Barrow, 2006) and young learners (Barman et al., 2006) hold many of the same misconceptions (or alternate conceptions) about plants. High school biology teachers from across the U.S. report being least confident about plant biology when surveyed about five fundamental topics, and just 46% of those with 6 years or less teaching experience report having ever had a botany course (Horizon Research Inc., 2002; Horizon Research Inc., 2013). The problem is not restricted to the U.S. Research on the uptake of plant sciences in the United Kingdom shows that the majority of UK students entering university biology courses have little interest in or knowledge of plants (Stagg et al., 2009).\n\nCompounding these documented issues is the human tendency to overlook plants, known as ‘plant blindness’ (Wandersee & Schussler, 1999), which has both cultural and physiological underpinnings (Balas & Momsen, 2014). Educators, students, and the public who generally don’t notice plants in the environment are not likely to see that plants are of utmost importance to the food, fuel, fibers and pharmacology of everyday life, as well as the functioning of our global ecosystem. A future workforce prepared with an understanding of plant science and cross-cutting concepts applied in innovative solutions will be needed to meet societal challenges, such as coping with climate change, feeding an increasing population, and generating sustainable energy sources (National Research Council, 2009). Engaging scientists as mentors has the potential to inspire interest and to link classroom learning to real-world authentic science. For plant science, meaningful and early exposure may be critical:\n\n“The presence of a plant mentor earlier in one’s life (someone who helped the mentee observe, plant, grow, and tend living plants) is a key predictor of that person’s awareness, appreciation, and understanding of plants throughout the lifespan.” (Wandersee & Clary, 2006).\n\nGiven the need to enhance teaching and learning about plants in formal U.S. education and the promise of student-scientist partnerships (Houseal et al., 2014; Summers & Hrabowski, 2006), we have been engaged in an approach to foster student learning of scientific practices and plant biology through interactions with scientist mentors. The PlantingScience program was intentionally developed as a blended approach to student-centered experiential learning taking place in the classroom, supplemented by communication and collaboration with peers and experts online. The online platform (www.plantingscience.org) not only eliminates geographic limitations, its design features make student thinking visible, enabling students, teachers and mentors to monitor thinking and learning and provide feedback. An impetus for this study was to take a systems approach to examining the inputs and outputs of PlantingScience. A previous study examined the techniques mentors used in online discourse with student teams (Adams & Hemingway, 2014). Here we examine the attitudes (affective responses) of students participating in collaborative plant investigations. We present qualitative data on what students value about a digital learning environment in which science practices and content are integrated and science experts and novices collaborate, as it occurs in authentic science research. We ask what major themes emerge from the student responses and how the exploratory analyses relate to design features of the program.\n\n\nMethods\n\nStudents ages 11 to 18 are the focus of this study. The students mentored by volunteer scientists enter the program through their teachers, who typically are seeking inquiry learning opportunities for their students. Participating classrooms (60% high school, 40% middle school) come from a variety of demographics; rural, urban, public and private schools. Classroom teachers choose one of the eight available investigation themes and decide whether the 3–12 week long projects would be limited to controlled experimentation or include observational studies; their past experience with inquiry often determines how guided or open student projects will be. The research questions and plants used by student teams vary widely; however, all investigations intend for students to collaboratively develop a research question on a core idea in plant biology, plan and carry out an investigation to answer the question, analyze the data, and make sense of the findings.\n\nEach student team is assigned a unique project page where they are encouraged to post information about their research project, as well as engage in asynchronous dialog with the mentor matched to their team. The program’s 988 registered mentors, from undergraduate students to professor emeriti, belong to more than 14 scientific societies that partner in the program. Student pre- and post-tests are not mandatory, and they are administered through the online platform, which is a customization of the open-source content management system Zikula. The Institutional Review Board of Texas A&M University granted approval for collection of these data, and we obtained permission from schools, students, and parents where appropriate, for publication. Over 4000 team projects with associated dialog, archived since 2005 are available at www.plantingscience.org.\n\nFor this study we analyzed students’ open-ended responses to the post-test survey question, “What did you like most about this experience?” Following six online mentored inquiry sessions between 2010 and 2012, only 2.7% of the students who initiated the online survey did not complete the open-ended question. A total of 2,617 responses from middle school (n = 947) and high school (n = 1,670) students were analyzed. The students completing surveys over this period were in classrooms of 20 middle school and 42 high school teachers. These classrooms encompassed a range of private and public schools, including two international classrooms. As expected given that “Wonder of Seeds” is the most frequently used module, more than half (54.6%) of the students who completed the surveys had conducted germination and/or seedling growth studies.\n\n\n\nWe downloaded the student responses from the archives in the online platform, removed errant duplications, and then imported an aggregated file into IBM® SPSS® Text Analytics for Surveys version 4 (IBM copyright 2010). As responses to the open-ended survey question typically ranged from one to several sentences, the computational linguistics text mining tool simplified the creation of broad sets of categories across responses. From the initial automated categorization of results, we discussed refinement to the categories through several iterations. In particular, we identified where automated codes were not applied appropriately, automated categories were conceptually related, and custom terms needed. For example, student comments about observations, collection of data, measurement, and analysis of data were manually grouped in an overarching category on the practice and processes of science. Similarly, text referring to seeds and germination were grouped, and the students’ various descriptions of doing experiments, labs, or projects were defined as synonyms. This iterative, exploratory process results in a robust view of the elements of most interest to the students as well as allowing one to investigate connections between areas of interest.\n\n\nResults\n\nTo put the student survey data in perspective, we first present statistics on content of the project pages as a way to quantify the student experience and to provide some context of what the experience involved during the period investigated here (Table 1). Most student projects included information on the team’s research question, prediction, experimental design and conclusion. Projects variably included supplemental documentation about their team research. The number of asynchronous posts between student teams and mentors ranged widely. There are many factors that account for the wide range in student post numbers, from teachers’ directions whether all students should post or appoint a team spokesperson, to the number of days computers are available, to teachers’ grading structures, to individual student motivation levels.\n\nAcross all survey results, students mentioned most frequently four themes as favored elements: plants (26.9%), scientist-mentor (20.3%), growing (15.7%), and experiments (13.8%). While some students responded to the open-ended question by noting only one thing that they liked most, other comments mentioned multiple elements in the same sentence(s). Relationships between themes mentioned by students illustrate a complex network of the four major nodes and how they are interconnected and also cross-linked to other favored elements (Figure 1). We next narrowed the selection for a view of the networks associated with each of the four major nodes in turn.\n\nThe size of the node represents the number of total respondents liking that element, and the width of the line between nodes is weighted by the number of shared responses. Items with fewer than 20 respondents are filtered out to simplify the lexical web. The size of the nodes corresponds to the number of responses in a given category, while the thickness of the lines represents the number of links between categories.\n\nWhat many students explicitly liked about plants was growing them, although the comments about liking plants formed a relatively dense web of connections to other items (Figure 2). Looking closely at the language that students use about plants, we saw two subtle, distinct descriptions about interactions with their study organisms. Students expressed a sense of enjoyment as a result of their interaction with plants, which was often connected to closely observing their plants: “I enjoyed seeing my plants grow and display their traits.” Less commonly but importantly students expressed a personal relationship, often a personal responsibility, for tending to their plants. (1) “Working with and caring for the plants was my favorite part.” (2) “The caring and effort you had to put into it. It was kind of like babysitting a child you could say. Because just like a child you had to watch and care for it.” Students mentioned liking the plants together with a wide array of other aspects of the PlantingScience experience such as the procedural aspects of manipulating variables and the social aspects of working with friends, classmates, and scientists. Taken together student comments about liking “plants” and “growing” account for the majority of favored program elements.\n\nThe size of the node represents the number of total respondents liking that element, and the width of the line between nodes is weighted by the number of shared responses. Items with fewer than 10 respondents are filtered out to simplify the lexical web.\n\nThe network of comments that students made about liking most their mentor shows strong connections to many aspects of doing and learning science as part of a science community (Figure 3). When commenting about liking their mentor, students also noted the uniqueness and the global viewpoint of the online experience. For example, comments included references to experiments, answering a question, communication, advice, team, help, and advantages. Three student quotes that illustrate several of these connections in context are: (1) “It was great working with a scientist who took the time to give us meaningful feedback. It really helped me learn and experiment.” (2) “The ability to interact with real life scientists was interesting and unprecedented in my life. Our group bonded with our mentor and it provided an awesome experience overall, as well as the chance to create and enact our own experimental design.” (3) “I liked having the advantage to speak with scientists from other areas around the world and looking at other experiments being done by other students. This to me helped give my group and I more ideas for our experiment and it kind of showed us how we could improve ours and make it a little more detailed with less problems.”\n\nThe size of the node represents the number of total respondents liking that element, and the width of the line between nodes is weighted by the number of shared responses. Items with fewer than 20 respondents are filtered out to simplify the lexical web.\n\nAlthough students less commonly cited liking most the experience of engaging in scientific practices and experiments, comments on this fourth major theme were also connected to the mentor, the collaborative team research, and the liberation that student-led inquiry offers (Figure 4). Phrases such as “we got to” or “I was able to” or “had the freedom to” were common signs that students valued the ownership of their research project and ideas. Students appreciated getting to choose variables, particular techniques, particular species of plants as subjects, and the research question. Students also valued the combination of independence and collaboration of the environment in terms of working with and learning from others. Two student quotes capture both of these themes: (1) “It was interesting to see what kinds of experiments different people came up with, and how they went about testing their hypothesis. I also like coming up with our idea in itself,” (2) “I liked how personal it was with our mentor, I also really enjoyed the freedom we had on deciding what we wanted to do and how we wanted to do it. We just picked our project then we tested everything ourselves and planned out everything and presented it just like real scientists would.”\n\nThe size of the node represents the number of total respondents liking that element, and the width of the line between nodes is weighted by the number of shared responses. Items with fewer than 10 respondents are filtered out to simplify the lexical web.\n\n\nDiscussion\n\nThe aggregated responses provide a picture of the major features that students appreciate most from the blended learning experience of conducting team-led plant investigations in their classrooms and collaborating with domain experts and peers online. Students highly value communicating with their mentors; it both creates a personal connection and provides students with a contextualized experience, working as part of a scientific community. Student comments about scientific practices demonstrated the importance they place on ownership of their own learning and the value of integrating authentic practices like data collection and interpretation into the experience. This study also indicates that students highly value, perhaps most of all, the interactions they have with plants as their study organisms.\n\nAs an examination of student attitudes toward program design features, this analysis suggests that key objectives are being met and it hints to some challenges for digital learning environments. Many students responded favorably to the design features that promote experiential and collaborative learning with plants, connections to scientists, and digital opportunities for feedback and reflection. A previous study documented that participating scientists use an array of mentoring techniques including socializing students into science, modeling scientific thinking, and combining content and practices naturally (Adams & Hemingway, 2014). Here we see that participating students respond by expressing appreciation for the personal relationships they form with scientists, plants, and peers while working as members of a research community. As digital connection to domain experts was a key program intervention, the stronger student response to plants than mentors warrants discussion and further exploration. Students interacted daily with plants and intermittently with their mentors during the course of the team investigations. Challenges of asynchronous discourse with mentors that likely play a role include communication delays, computer access and school schedules and the classroom being a “black box” to some scientists. The frequency of exposure may also influence the high frequency with which students cited liking plants. We would argue the students’ strong positive attitude towards plants as their study subjects reflects an authentic trajectory for developing scientists. Mentors may open the door to the scientific enterprise, and once through it is the discovery process that captures students’ intellectual curiosity. Mentors, advisors, sponsors—more senior experts by any name and at any stage of the career path—are facilitators or cultivators, not generators, of individual wonder and talent.\n\nPrior studies have shown that students studied in several countries find plants less interesting than animals, uninteresting, or downright boring (Fancovicova & Prokop, 2010; Kinchin, 1999; Randler et al., 2012). While our study was not designed to test students’ relative interest in plants versus animals, our findings of students’ strongly positive attitude about working with plants in this learning setting indicates that how students are exposed to plants matters a great deal. This view is not new (Uno, 2009), and it is reinforced by other studies. For example, learning experiences that emphasized observations in the local environment enhanced interest in both plants and animals among Swiss precollege students (Lindemann-Mathhies, 2005). Similarly, improvements in student attitudes towards plants accompany shifts in curricular approach to student-centered, active learning in an undergraduate botany course (Goldberg & Ingram 2011; Ward et al., 2014). The appreciation and caring relationships that students expressed for plants in this study invoke a sense of biophilia, which Wilson (1984) describes as the human urge to affiliate with other forms of life. To counter ‘plant blindness’ students need opportunities to experience the lives of plants: “this experience taught me that plants are more than what they just appear to be, they are creatures that develop in ways that are so different than mammals, humans, etc. they are a very beautiful type of species and they are very amazing to learn about.”\n\nStudents’ attitudes, learning, achievement, and career path are linked in close and complex ways (Osborne et al., 2003). Analyses of attitudes about science from open-ended text analysis are less common than analyses based on Likert scale responses and pose unique challenges compared to measurement of student learning (Lovelace & Brickman, 2013). While there is a clear need for assessments that produce evidence of student content knowledge and proficiency in science practices (National Research Council, 2014), the affective domain is a powerful and under-utilized body of evidence in the development of student learning (Trujillo & Tanner, 2014). In this analysis we began to tease out some of the indicators that students self-report as being positive to their learning experience. We see these as potentially important to the development of future instructional materials and activities and as an indicator of the effectiveness of the PlantingScience program.\n\nThese findings have implications for other projects that use technology to support more authentic science practices in science classrooms. Just doing an investigation is not sufficient for deep learning (Bell et al., 2003) and digital tools for collaboration and communication used effectively can enhance student motivation and understanding (Mistler-Jackson & Songer, 2000). Digital learning environments generally accumulate large amounts of data rapidly. Without being prohibitively time consuming, the text-analysis approach allowed us to reveal broad patterns in a large set of open-ended data. Students participating in PlantingScience appear to give primacy to the personal science experience with the digital collaboration serving as an enhancer of the experiential learning. Placing students in an environment where they are asked to behave and think like scientists as they conduct investigations, while at the same time providing a mentor who can model scientific thinking, is a powerful combination for students to experience change in their worldview about science, scientists, and plants.\n\n\nData availability\n\nDataset 1. Student Responses. http://dx.doi.org/10.5256/f1000research.6223.d44181 (Hemingway et al., 2015).", "appendix": "Author contributions\n\n\n\nAll authors contributed to the study design and writing of the paper. MS conducted the IBM Text Analysis. All authors have agreed to the content of the final draft of the manuscript.\n\n\nCompeting interests\n\n\n\nCatrina Adams currently leads the PlantingScience program; Claire Hemingway led the program until 2013.\n\n\nGrant information\n\nFunding from the National Science Foundation (NSF DRL 0733280) supported in part the program activities of PlantingScience from 2007 to 2012. Contents of this publication are solely the responsibility of the authors and do not necessarily represent official views of the NSF. No grants were involved in supporting the study presented here.\n\nI confirm that the 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 PlantingScience participants, steering committee, and collaborators, in particular Jane Larson and Carol Stuessy, who served as external evaluator and co-principle investigator, respectively, during the timeframe of this study.\n\n\nReferences\n\nAdams CT, Hemingway CA: What Does Online Mentorship of Secondary Science Students Look Like? BioScience. 2014; 64(11): 1042–1051. Publisher Full Text\n\nAllen IE, Seaman J: Grade Change: Tracking Online Education in the United States. Babson Survey Research Group. 2014. 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Publisher Full Text\n\nWard JR, Clarke HD, Horton JL: Effects of a Research-Infused Botanical Curriculum on Undergraduates’ Content Knowledge, STEM Competencies, and Attitudes toward Plant Sciences. CBE Life Sci Educ. 2014; 13(3): 387–396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson EO: Biophilia . Harvard University Press Cambridge. 1984. Reference Source" }	[ { "id": "8133", "date": "07 Apr 2015", "name": "Melanie Link-Perez", "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\nThis article by Hemingway and co-authors presents the results of a lexical analysis of an open-ended survey question presented to secondary school students participating in the PlantingScience (www.plantingscience.org) program. The survey question probed what students most-liked about participation in the inquiry-based classroom research experience supported by online discourse with a scientist mentor.The article is a well-written and, in my opinion, important contribution to the literature about science education in general and botanical education in particular. The major themes that emerged from the research are that students value their personal relationships both to their scientist mentor and their study organisms, and that the ability to exert some control over choice of study organism, research question, or methodology encouraged students to take ownership in their learning experience. The program’s intervention of connecting student researchers with plant scientist mentors via online discourse seems particularly important given the lack of botanical training of many primary and secondary teachers and the significance of a plant mentor for stimulating lifelong interest and awareness of plants. This is an important article for increasing visibility of the PlantingScience program, but the findings have broader implications for botanical education; in particular, it provides a strong example of the value of directly exposing students to the lives of plants as an effective means of replacing plant blindness with plant appreciation and interest. Some general comments:Abstract is concise and clear; provides adequate context for study and presents the major conclusions.Discussion related to Figure 4 was illuminating and suggests ways that educators can encourage students’ interest in inquiry-type projects of any discipline.The combination of the lexical webs and example student comments was an effective illustration of the major themes identified by the analysis.Excellent selection of student quotes.Methods used for analysis of survey data are sound and the conclusions are justified by the data.The paper generally is clearly written but could be improved by making some of the text more explicit. For example, consider this sentence: “For plant science, meaningful and early exposure may be critical…” As a reader, I am unclear about what the authors are implicitly stating here, and it would be better for them to be explicit about their meaning. When I read, “For plant science” in that sentence, I am left feeling that something is missing, that this thought is incomplete (What about plant science? For plant science to do what? For plant science to be considered important, valid, of interest? Critical for what?). The writing will be stronger when the connections are explicitly and completely stated. Please elaborate on “cross-cutting concepts” in the reference to the National Research Council (2009) (“cross-cutting concepts applied in innovative solutions”), since this meaning is unclear. I refrained from reading with an eye toward grammatical issues, but I would like to make one suggested revision: “The online platform (www.plantingscience.org) not only eliminates geographic limitations but also makes student thinking visible through its design features, enabling students, teachers and mentors to monitor thinking and learning and to provide feedback.” Thank you for the opportunity to review this excellent paper.", "responses": [] }, { "id": "8776", "date": "01 Jun 2015", "name": "Graham Scott", "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\nThis paper makes an important contribution to the literature about engagement with botany in formal schooling. It presents a project that harnesses the enthusiasm of practicing scientists and the power of the internet to enable children (and their teachers) to learn about plants and practical science in an enabling environment. The paper presents an interesting lexical analysis of the comments of a very large sample of participating children and draws out conclusions that will be of value to those individuals who care about the botanical (and wider scientific literacy) of the next generation. I found the discussion around figure 2 and the interaction between the affective domain and cognitive domains in the context of growth particularly interesting. It complements the findings of others who have suggested that pedagogies which enhance this interaction are particularly effective and should be a focus of those of us trying to re-connect children and young people with plants and animals in the natural world. I do have some very minor suggestions for alterations to the current version of the manuscript that the authors might like to consider:Lexical analysis is not particularly common and I think therefore that it will be unfamiliar to the much of the readership of this paper. For this reason I suggest that the authors might provide a little more detail in the description of figure 1. For example, four key nodes are highlighted in the text (plants; scientist mentor; growing and experiments) but from the figure (at the resolution provided) a reader might see/infer five (plants; scientist mentor; experiment or lab project; personal pleasure in growth or plant; grow or growth; and, group or team). Perhaps a little more text to expand the methods section to explain how the four that are used were chosen (what cut off criterion was applied for example) and how these relate to the figures would be helpful. I feel that the sentence “The network of comments that students made about liking most their mentor shows strong connections …” is a little difficult to follow and suggest that it might be helpfully re-worded to improve the clarity of the message.", "responses": [] } ]	1	https://f1000research.com/articles/4-74
https://f1000research.com/articles/4-4/v1	08 Jan 15	{ "type": "Research Note", "title": "Observations of membrane fusion in a liposome dispersion: the missing fusion intermediate?", "authors": [ "Marianna Foldvari" ], "abstract": "Early intermediate structures of liposome-liposome fusion events were captured by freeze-fracture electron microscopic (EM) technique. The images show the morphology of the fusion interface at several different stages of the fusion event. One of the intermediates was captured at a serendipitous stage of two vesicles’ membranes (both leaflets) merging and their contents starting to intermix clearly showing the fusion interface with a previously unseen fusion rim. From the morphological information a hypothetical sequence of the fusion event and corresponding lipid structural arrangements are described.", "keywords": [ "membrane fusion", "liposome", "fusion mechanism", "freeze-fracture", "fusion intermediate" ], "content": "Introduction\n\nPhospholipid vesicles (liposomes) are suitable models for studying biological membrane behaviour. Liposomes can be made from a single lipid or lipid mixtures that can provide opportunities to investigate membrane-related events under different conditions and in the presence of additives. The fusion process has been investigated in various artificial membrane systems using electron microscopic, small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and fluorescence-based kinetic techniques1–5. Despite the efforts, complete understanding of the molecular structural and kinetic details of the fusion event is still lacking.\n\nThe first fusion mechanism from early studies proposed ‘lipidic particles’ (intermediates between lamellar and HII hexagonal phases of the phospholipids or inverted micelles) as the possible intermediate in the fusion process of model lipid vesicles6–8, since these particles seemed to be present at attachment sites of lipid vesicles and could be visualized by freeze-fracture EM. These intermediates could also be detected in fusion processes of biological membranes (exocytosis, myoblast fusion, protoplast fusion). For example, Satir et al.9 observed small particles arranged in rosettes at the site where fusion of the mucocysts in Tetrahymena pyriformis was initiated. Other authors questioned the existence of lipidic particles as dynamic fusion intermediates10,11 because these particles could not be observed every time at fusion interfaces, and suggested that ‘lipidic particles’ develop subsequent to the fusion process. However, it was suspected that some type of a non-bilayer structure formed in the fusion event.\n\nAnother mechanism proposed was the ‘stalk mechanism’12–14, which involved the formation of a trilaminar structure between the closely apposed bilayers such that the outer monolayers bend to the side to allow joining of the inner monolayers (trans-monolayer contact (TMC)15), which form a stalk at the attachment site of the two membranes12,15–17.\n\nThe theoretical sequence of events in model lipid membrane fusion can be summarized as follows: 1) close apposition of the two bilayers (<1 nm); 2) local dehydration of phosphorus head groups; 3) destabilization of bilayers; and either 4a) formation of inverted micelle intermediates (IMI) at the attachment site; or 4b) formation of stalk and TMC); and 5) completion of fusion, i.e. the leakless mixing of contents of two vesicles.\n\nMost of these stages of membrane fusion were described and indirectly measured or modeled, but direct visual evidence is still lacking. Siegel18 and Cullis et al.19, and more recently Lentz et al.20,21 speculated that the reason why only some of the actual intermediate structures were detected or visualized is the short lifetime (1 msec or less18) of any given fusion intermediate, making the capture very challenging even with rapid freezing, 31P-NMR or SAXS techniques.\n\nIn this study, we have observed some fusion intermediate structures in a liposome system in the presence of glycerol by freeze-fracture EM. One of these intermediates, the ‘fusion rim intermediate’ may provide new structural/morphological information on membrane fusion events.\n\n\nMethods\n\nLiposomes (small unilamellar vesicles, SUVs) were prepared with soybean lipids (Centrolex P; Central Soya, Fort Wayne, IN). The liposomes were prepared by high shear dispersion using Microfluidizer M110 (Microfluidics Inc. Newton, MA). The liposomes were freeze-fractured without glycerol or after preincubation in 30% v/v glycerol for 30 minutes at room temperature. A drop of the liposome suspension was placed on a gold specimen stub and rapidly frozen in liquid nitrogen cooled Freon 22 (-158°C). All samples were fractured at -105°C in a Balzers 360 freeze-fracture unit. The fracture surfaces were shadowed at 45° angle with a thin layer of platinum-carbon followed by vertical deposition of a carbon layer for replica support. The replicas were floated onto the surface of distilled water and subsequently cleaned with sodium hypochlorite (5% chlorine) and 60% sulfuric acid. After the final washing in distilled water the replicas were picked up on 200 mesh copper grids and examined in a Phillips 200 EM and photographed on Kodak fine grain positive film.\n\n\nResults\n\nThe fusion of liposomes was induced by glycerol and several fusion intermediates were captured in the replicas (Figure 1). Without glycerol, there was no liposome fusion (Figure 1A). The relatively low concentration of glycerol provided slow dehydration at the phospholipid head group regions which made it possible to observe vesicles still in the fusion process. The micrographs captured vesicles (assumed to be) at various stages of the membrane fusion event (Figure 1A–C). Figure 1B (large arrow) depicts the initial contact between vesicles – their external monolayers fused but no communication between the two aqueous compartments started. Figure 1D shows a previously unseen moment of liposome-liposome fusion. During the freeze-fracturing procedure one of the liposomes was fractured on the outside surface (it shows the E face), while the other shows the cytoplasmic (P) face (the interior surface of liposome). The leakless intermixing of the aqueous contents of the liposomes had started. At the perimeter of the fusion interface small, 10–12 nm diameter, particles can be distinguished, which probably correspond to the inverted micellar intermediates, so called, lipidic particles. This fusion rim intermediate structure is depicted in the insert of Figure 2. The presence of these lipidic particles could be suspected from another micrograph (Figure 1C, large arrow), which may represent a preceding intermediate state of fusion.\n\nVarious stages of liposome-liposome fusion event were captured by rapid freezing. A) Liposomes were freeze-fractured without the addition of glycerol. B–D) Liposomes were preincubated with 30% v/v for 30 minutes before freezing. B) The first stage of liposome-liposome fusion: the joining of the outer monolayers of the liposomes (large arrow) is apparent without communication between their internal spaces. C) Early liposome-liposome fusion intermediate (large arrow) fractured at the exterior surface of the membrane. The rim between the two vesicles (small arrow) is indicative of non-bilayer intermediate structures. D) Late liposome-liposome fusion intermediate (large arrow). One of the liposomes was fractured on the external surface, the other on the internal surface with its internal space is being visible and exposing the fusion interface. The presence of small (10–12 nm) particles can be distinguished at the fusion rim. E) Schematic representation of the identified fusion intermediates. Numbers on micrographs correspond to the numbers of the diagrams. Freeze-fracture nomenclature27: ES – external surface; PS – cytoplasmic surface (in this case interior surface of liposome); PF and * - fractured face of the lipid monolayer adjacent to the interior space of the vesicle; arrows in left corners of micrographs indicate the shadowing direction. Bars 250 nm.\n\nOn the basis of this morphological evidence we constructed a schematic set of drawings to represent a modified model for phospholipid vesicle fusion (Figure 2). When the bilayers of two separate vesicles are in close apposition (Figure 2 I), an initial fusion product will form. The outer leaflets of the bilayers of the two vesicles fuse, while their inner leaflets form one common bilayer at the attachment site (Figure 2 IIa). This stage is followed by the formation of inverted micelles (lipidic particles) around the attachment site (Figure 2 IIb). The organization of the next fusion intermediate (Figure 2 IIc and Figure 1D) involves the formation of a fusion orifice. At the perimeter of this orifice, the fusion rim can be seen (Figure 2, insert), which contains inverted micelles all around. The development of an intermediate like this appears feasible from both energetic and morphological viewpoints, if we take into consideration that the excess phospholipid molecules cleared from the attachment site at this particular stage should be accommodated somewhere, until incorporated into the expanded bilayer of the single larger liposome.\n\nI. Close apposition of two bilayers and formation of an aggregation site when bilayers touch each other. II. Merging of two bilayers. a) formation of the initial fusion product: the outer leaflets of the bilayers of both vesicles join (fuse), while their inner bilayer leaflets form one common bilayer at the attachment site. b) phospholipid molecules from the outer leaflet of the vesicle bilayers, ie. From the attachment site are pushed sideways and form inverted micelles; at the attachment site transient bilayer form composed of the inner bilayer leaflets of the vesicles. c) formation of the fusion orifice: the phospholipid molecules from the attachment site are used for the formation of the outer monolayer of the inverted micelles. Inverted micelles are situated all around the fusion orifice. Mixing of the contents of the vesicles has started. Boxed insert shows a horizontal section of the fusion area viewed from above. III. Expansion of the bilayer to form a single larger liposome.\n\n\nDiscussion and conclusions\n\nGlycerol-induced fusion seen in this study may bear similarities to polyethylene glycol (PEG)-induced fusion20,22 with dehydration at the liposome attachment site contributing to close contact between the bilayers. It is recognized that fusion is a very dynamic event and certain stages of the fusion are easier to demonstrate than others. Due to the low frequency of vesicle collisions and short lifetime of the actual fusion event and the fact that fusion of vesicles in an aqueous medium is not a synchronous event, visualization of intermediary fusion features on all liposomes in a sample is difficult.\n\nMost of the morphological freeze-fracture studies in the literature show liposomes just before fusion or at the stage already well undergoing fusion. It would be important to clarify events at the stage, where bilayers of the two vesicles are merging and communication between their aqueous spaces begins. The freeze-fracture results in this work supplement those previously reported in the literature and potentially add a new visual image of an intermediate structure to the model of membrane fusion. The initial fusion product (Figure 2 I) must be very similar to the one proposed by Kozlov and Markin12 on the basis of theoretical considerations. The molecular arrangement of lipids in the ‘fusion rim intermediate’ (Figure 1D and Figure 2 IIc) could provide an alternative interpretation of the IMI8,23 or could be considered the next step after the previously described stalk and TMC intermediate15,17 (this latter may correspond to the image on Figure 1B), and may also be similar to membrane hemifusion events involving proteins21,24–26.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declares that no grants were involved for the funding of this work.\n\n\nAcknowledgements\n\nThis paper is dedicated to the memory of Dr Michael Mezei, my mentor and friend. I specifically thank Dr Michael Mezei for providing the liposome dispersion and inspiring the study. I am also grateful to Dr Gary T. Faulkner for guidance with the freeze-fracturing and useful discussions of the micrographs.\n\n\nReferences\n\nMarsden HR, Tomatsu I, Kros A: Model systems for membrane fusion. Chem Soc Rev. 2011; 40(3): 1572–85. PubMed Abstract \| Publisher Full Text\n\nStark B, Pabst G, Prassl R: Long-term stability of sterically stabilized liposomes by freezing and freeze-drying: Effects of cryoprotectants on structure. Eur J Pharm Sci. 2010; 41(3–4): 546–55. PubMed Abstract \| Publisher Full Text\n\nQian S, Wang W, Yang L, et al.: Structure of transmembrane pore induced by Bax-derived peptide: evidence for lipidic pores. Proc Natl Acad Sci U S A. 2008; 105(45): 17379–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilschut J, Düzgüneş N, Fraley R, et al.: Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents. Biochemistry. 1980; 19(26): 6011–21. PubMed Abstract \| Publisher Full Text\n\nLee J, Lentz BR: Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry. 1997; 36(21): 6251–9. PubMed Abstract \| Publisher Full Text\n\nCullis PR, Hope MJ, Tilcock CP: Lipid polymorphism and the roles of lipids in membranes. Chem Phys Lipids. 1986; 40(2–4): 127–44. PubMed Abstract \| Publisher Full Text\n\nVerkleij AJ, van Echteld CJ, Gerritsen WJ, et al.: The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions. Biochim Biophys Acta. 1980; 600(3): 620–4. PubMed Abstract \| Publisher Full Text\n\nCullis PR, Hope MJ: Effects of fusogenic agent on membrane structure of erythrocyte ghosts and the mechanism of membrane fusion. Nature. 1978; 271(5646): 672–4. PubMed Abstract \| Publisher Full Text\n\nSatir B, Schooley C, Satir P: Membrane fusion in a model system. Mucocyst secretion in Tetrahymena. J Cell Biol. 1973; 56(1): 153–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBearer EL, Düzgünes N, Friend DS, et al.: Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion. Biochim Biophys Acta. 1982; 693(1): 93–8. PubMed Abstract \| Publisher Full Text\n\nWilschut J, Hoekstra D: Membrane fusion: lipid vesicles as a model system. Chem Phys Lipids. 1986; 40(2–4): 145–66. PubMed Abstract \| Publisher Full Text\n\nKozlov MM, Markin VS: On the theory of membrane fusion. The adhesion-condensation mechanism. Gen Physiol Biophys. 1984; 3(5): 379–402. PubMed Abstract\n\nMarkin VS, Albanesi JP: Membrane fusion: stalk model revisited. Biophys J. 2002; 82(2): 693–712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarkin VS, Kozlov MM, Borovjagin VL: On the theory of membrane fusion. The stalk mechanism. Gen Physiol Biophys. 1984; 3(5): 361–77. PubMed Abstract\n\nSiegel DP: The modified stalk mechanism of lamellar/inverted phase transitions and its implications for membrane fusion. Biophys J. 1999; 76(1 Pt 1): 291–313. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChernomordik LV, Kozlov MM: Membrane hemifusion: crossing a chasm in two leaps. Cell. 2005; 123(3): 375–82. PubMed Abstract \| Publisher Full Text\n\nKozlovsky Y, Chernomordik LV, Kozlov MM: Lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm. Biophys J. 2002; 83(5): 2634–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiegel DP: Inverted micellar structures in bilayer membranes. Formation rates and half-lives. Biophys J. 1984; 45(2): 399–420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerkleij AJ, Leunissen-Bijvelt J, de Kruijff B, et al.: Non-bilayer structures in membrane fusion. Ciba Found Symp. 1984; 103: 45–59. PubMed Abstract\n\nLentz BR: PEG as a tool to gain insight into membrane fusion. Eur Biophys J. 2007; 36(4–5): 315–26. PubMed Abstract \| Publisher Full Text\n\nLentz BR, Siegel DP, Malinin V: Filling potholes on the path to fusion pores. Biophys J. 2002; 82(2): 555–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee J, Lentz BR: Outer leaflet-packing defects promote poly(ethylene glycol)-mediated fusion of large unilamellar vesicles. Biochemistry. 1997; 36(2): 421–31. PubMed Abstract \| Publisher Full Text\n\nHafez IM, Cullis PR: Roles of lipid polymorphism in intracellular delivery. Adv Drug Deliv Rev. 2001; 47(2–3): 139–48. PubMed Abstract \| Publisher Full Text\n\nLangosch D, Crane JM, Brosig B, et al.: Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity. J Mol Biol. 2001; 311(4): 709–21. PubMed Abstract \| Publisher Full Text\n\nHernandez JM, Kreutzberger AJ, Kiessling V, et al.: Variable cooperativity in SNARE-mediated membrane fusion. Proc Natl Acad Sci U S A. 2014; 111(33): 12037–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai HH, Chang CM, Lee JB: Multi-step formation of a hemifusion diaphragm for vesicle fusion revealed by all-atom molecular dynamics simulations. Biochim Biophys Acta. 2014; 1838(6): 1529–35. PubMed Abstract \| Publisher Full Text\n\nBranton D, Bullivant B, Gilula NB, et al.: Freeze-etching nomenclature. Science. 1975; 190(4209): 54–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7365", "date": "22 Jan 2015", "name": "James McNew", "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 intriguing observation of potential liposomal fusion intermediates. While the method of inducing protein-free lipid mixing and fusion is unconventional, the structures observed by freeze fracture are unique. I think it would be important to confirm these intermediates under conditions that have been studied by other means. The wealth of information on PEG-mediated liposome fusion could be brought to bear if similar conditions were used. Additionally, it would be beneficial to examine the lipid requirements of such intermediates. While the lifetime of these structures are likely short making their identification challenging, altered lipid compositions such as at the inclusion of hexagonal phase ii forming lipids like phosphatidylethanolamine may increase the propensity of observing the types of intermediated like the \"fusion rim\" or \"fusion orifice\" depicted in this work. Overall, the proposed intermediate are interesting, but require further experimental evidence to support their role as bona fide fusion intermediates.", "responses": [ { "c_id": "1269", "date": "18 Mar 2015", "name": "Marianna Foldvari", "role": "Author Response", "response": "The reviewer’s comments are appreciated and will stimulate further work regarding the better understanding of fusion mechanism. It is agreed that the proposed fusion intermediate observed in this liposome system only represents one possible lipid organization at the fusion interface and further studies with the addition of other lipids, proteins or fusogenic agents could provide more clarity of the fusion event." } ] }, { "id": "7364", "date": "22 Jan 2015", "name": "M Joseph Costello", "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\nConventional freeze fracture experiments were performed on small unilamellar vesicles prepared from soybean lipids using a microfluidizer. Glycerol was used to induce fusion, which was random producing a few pairs of vesicles in the process of fusion. The images of intermediate states are interpreted to support the presence of a ring of lipidic particles at the critical point of bilayer-bilayer fusion. This approach is promising and the images are interesting; however, there are major concerns with the controls and with the interpretation of the images. Specific comments: Figure 1A. The image does not display a freeze fracture experiment. There is no fracture plane through the lipid vesicles. If such a fracture plane existed in this region, then vesicles with both convex and concave surfaces would be visible (as in subsequent images). Only convex surfaces are visible. In addition the convex surface of the larger structures have a revealing pattern of density: the four larger vesicles have a dark rim and light center. This is due to the carbon layer that builds up on the curved surface to be thick near the edges but thin enough in the center to be nearly transparent. It appears that a layer of sample, perhaps adjacent to a fractured region, was shadowed to reveal some vesicles but not the expected vesicle size distribution or fracture pattern of a control. Figure 1B. The vesicles exposed in this image are consistent with a normal fracture pattern where both convex and concave vesicles are visible. It should be noted that the fracture step through the monolayer of lipid is present on concave vesicles and, as the authors point out, on the large vesicle where a flap has been fractured away. The difficulty with this image is that the background ice is not smooth as would be expected for samples prepared in 30% glycerol. The authors should explain why the background here is different from those in subsequent freeze fracture images. One possibility is that some etching occurred. Figure 1C. The main emphasis in this image is the mound or bump near the fusion interface of the vesicle pair labeled 2. The interpretation is that the bump represents a fracture through a ring of lipidic particles. The problem is that a single bump is not consistent with a ring of particles and the hydrophobic plane of fracture around a lipid particle may not appear as a bump or a bulge (as a whole particle might). Fractures from several different viewpoints and proceeding around the fusion site are needed to properly interpret this pattern in terms of fusion intermediate structures. A single image showing a slight protrusion is not convincing even if the interpretation turns out to be correct. Figure 1D. This shows an interesting image of a fusion contact site (labeled 3). The interpretation of this pattern as a fracture through a ring of lipid particles is difficult to justify based on the irregular size of the particles in the ring. More such images are needed showing particles of consistent size and distribution. In addition the image shows some other features that should be explained, such as the large number of oblong (or rod-like) vesicles, up to about five, and the large number of small particles in the background ice (also present in 1C). It is not clear whether these particles are from contaminant proteins in the solution or from artifacts deposited during the fracturing or shadowing steps. Figures 1E and 2. The diagrams and molecular interpretations emphasize the role of lipidic particles in the fusion process. These particles have been implicated in bilayer and membrane fusion for decades without clear resolution about their presence in native fusion events within cells. The use of glycerol to induce fusion implies that glycerol does not significantly interact with the lipid bilayers or induce lipidic particles. At least one report suggests that glycerol (unlike polyethylene glycol) can intercalate within the lipid head groups to the extent of inducing interdigitation of lipid chains in the gel phase (BBA 731:97-108, 1983). Less interaction is seen for fluid phase lipids as used here but glycerol is not a passive inducer of fusion. Furthermore, Bearer et al. (ref. 10) suggest that glycerol induces lipidic particles and that such particles are not typically found in the fusion process in their model system. The critical point is that the glycerol-induced fusion of soy lipids may not represent natural lipid fusion and thus is not likely to expose the long sought lipid pore intermediate in natural fusion events.", "responses": [ { "c_id": "1268", "date": "18 Mar 2015", "name": "Marianna Foldvari", "role": "Author Response", "response": "The author thanks the reviewer for the comments and alternative ideas. The freeze-fracture/etching technique used in this experiment utilized a Balzers 360 instrument which has certain limitations related to manual operation and timing of the sample mounting, temperature adjustment, fracturing and shadowing steps, providing some inconsistencies within sample areas. Nevertheless, the vesicles and fusion events can be observed and some new useful information may be gleaned from this liposome system.Previous synchrotron x-ray scattering studies examined fusion events in various lipid membrane compositions in aligned multilayer lipid deposit samples [Yang and Huang (2002) Science 297:1877-1879; Yang and Huang (2003) Biophys J. 84:1808-1817; Wang et al. (2006) Biophys. J. 91:736-743 and Qian et al (2008) PNAS 105:17379-83.] In these studies the electron density distribution was used to map the merging of apposed lipid bilayers and the presence of transmembrane pores and the stalk structure. However, the scarcity of visual information on lipid vesicle interactions captured at different time points lead us to present these images as possible additional information.In the paper by Bearer et al. (1982) the authors have examined PE:PS and PC: CL vesicles by quick freezing in the presence of Ca2+ or glycerol and Ca2+. It was stated that no lipidic particles were observed after the addition of Ca2+ and very few vesicles showed lipidic-particle-like structures after preincubation with glycerol followed by Ca2+ addition and this was only after long incubations of 1-2 h, although fusion-related images were frequently observed. We think that the findings in this study do not exclude the possibility of existence of various fusion intermediates just because they were not captured in the samples viewed. The authors actually suggest that the absence of lipidic particles or other intermediate structures may not mean that some dynamic process is taking place and that fusion intermediates could be unstable and convert to different polymorphic forms.As the authors state: “This conclusion [ie. “lipidic particles (as defined by their morphology in freeze-fracture electron microscopy) are not involved as an intermediate in the stages of fusion”], however, does not exclude the possibility of a transitory intermediate at the site of membrane fusion which involves a lipid conformation distinguishable from the unmodified stable bilayer configuration. This 'elusive' intermediate, which is not visualized at present in any morphological studies, could be an inverted micellar or some other non-bilayer structure or a small domain of a more condensed or crystalline lipid bilayer. For lack of concrete evidence at this point, this 'intermediate' could be characterized simply as a local perturbation of the lipid bilayer structure, which allows mixing of lipid molecules between the two closely apposed membranes.” While it is agreed that one image is not sufficient to base final conclusions, this note (or ‘case report’) attempts to report an interesting morphological observation that others in the field may find motivating.We have revised the paper to clarify that the morphological observation and the schematic interpretation are from a single examination and limited to one lipid system and fusogenic agent combination that will require other studies and confirmation in the future." } ] }, { "id": "7233", "date": "30 Jan 2015", "name": "Jesse C. Hay", "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 submission uses freeze-fracture electron microscopy to observe fusion intermediates in glycerol-induced fusion of unilamellar liposomes. Though fusion in this system is asynchronous, several images are presented that presumably correspond to different sequential intermediates in membrane fusion. One particularly suggestive image is interpreted to indicate that the hemi-fusion to full-fusion transition proceeds through an intermediate containing a circular array of lipidic particles just beneath the junction between the fused outer leaflets. The lipid particle model is proposed to potentially reflect the mechanism of membrane fusion induced by biological fusion protein machinery. The lipid particle model is not a new idea, but has been controversial and lacked direct support. The paper is interesting and makes a unique contribution to understanding the lipidic intermediates in fusion. However, it was disappointing that only a single image of the key intermediate is shown and therefore the characteristics of the intermediate cannot be confirmed. If more examples were analyzed, it should be possible to estimate quantitative parameters such as the approximate number of lipid molecules in the particles, how uniform they are and how often they occur per unit of membrane, etc., that might allow future energetic understanding of the proposed process. Likewise I found it somewhat disappointing that only glycerol was used to induce fusion. If the proposed intermediate is generalizable to biological membrane fusion, it should be apparent using another fusogen.", "responses": [ { "c_id": "1267", "date": "18 Mar 2015", "name": "Marianna Foldvari", "role": "Author Response", "response": "Thank you for the valid comments on the limited number of events in the experiment. In this case we have mostly observed fusion-related events at stages that are not revealing the moment of complete merging of the membranes. Following the field in the past two decades indicate that capturing a fusion event at the most revealing time point is indeed a challenge. Due to the rarity of such image, it is important to present this to the scientific community. As we indicate the serendipity of such finding, it is recognized that this paper can only be a small piece of information that stimulates further studies." } ] } ]	1	https://f1000research.com/articles/4-4
https://f1000research.com/articles/4-72/v1	18 Mar 15	{ "type": "Case Report", "title": "Case Report: A 54 base pair inactivating mutation of LHCGR in a 28-year old woman with poor ovarian response", "authors": [ "Ravi Krishna Cheemakurthi", "Gottumukkala Achyuta Rama Raju", "Thota Sivanaryana", "Kalagara Madan", "Kota Murali Krishna", "Godi Sudhakar", "Ravi Krishna Cheemakurthi", "Thota Sivanaryana", "Kalagara Madan", "Kota Murali Krishna", "Godi Sudhakar" ], "abstract": "The luteinizing hormone/choriogonadotropin (LH/CG) receptor plays an important role in male and female infertility. Many studies have demonstrated that mutations at specific sites in LHCGR gene may result in mild or complete loss of receptor function. Insertions in exon-1 of LHCGR gene were first studied in male Leydig cell hypoplasia and later extended to female reproductive disorders. Previous studies have shown that these insertions play an important role in intrauterine insemination (IUI) and in vitro fertilization (IVF) outcome. Here we report a 54bp insertion in a 28-year old woman with infertility, recurrent cyst formation and failed stimulated IUI cycles. As the patient showed a blunted response to the ovarian stimulation and human chorionic gonadotropin (hCG) stimulation test, follicle stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin (LHCGR) gene sequencing was performed. Gene sequence analysis revealed a 54bp homozygous insertion (GCTGCTGAAGCTGCTGCTGCTGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTGCA) in the exon-1 of LHCGR gene. This mutation might have caused a decrease in receptor function in the present infertile patient, thus resulting in poor ovarian response.", "keywords": [ "LHCGR", "insertions", "exon-1", "poor ovarian response" ], "content": "Introduction\n\nLuteinizing hormone (LH) is a key regulator of the female menstrual cycle1 and is produced by the anterior pituitary gland of gonadotroph cells. During ovulation, the LH surge is not only essential for the release of an egg from the follicle, but also regulates the formation of the corpus luteum. The corpus luteum in turn produces progesterone, which helps in the maintenance of pregnancy2,3. Therefore, LH along with its receptor complex has a cardinal role during reproductive processes4,5. The heterodimeric glycoproteins LH and chorionic gonadotrophin (CG) are mediated by luteinizing hormone/choriogonadotrophin receptor (LHCGR), which is a member of the G-Coupled receptor protein family. This receptor is expressed by the LHCGR gene located on chromosome 2p21 that consists of 11 or 12 exons and codes for around 600 amino acids. The LH receptor is located on Leydig cells in males, and theca, granulosa cells of the ovaries20. The receptor activation and inactivation are brought about by the conformation of amino acids in their localized regions6.\n\nWomen, whose LH receptor is down regulated with GnRH agonists or antagonists, may have decreased concentrations of luteinizing hormone and follicle stimulating hormone. This results in poor outcome due to low endogenous LH levels. Results from our earlier study demonstrated more number of oocytes and clinical pregnancies after use of recombinant LH(rLH) combined with recombinant follicle stimulating hormone (rFSH) in assisted reproductive therapy (ART)7. Although supplementation of LH is beneficial in a selected category of patients, a number of studies have also demonstrated that LHCGR mutations at specific amino acid regions result in mild or complete loss of receptor function8.\n\nLHCGR gene mutations were first studied in men with Leydig cell hypoplasia, later they were extended to female reproductive disorders11. Studies have shown that inactivating base pair insertions in exon-1 of LHCGR gene are associated with low oocyte yield and also negatively associated with intrauterine insemination (IUI) and in vitro fertilization (IVF) outcome in infertile population9–11. A recent investigation by Bentov et al., 2012 reported a 6bp insertion in exon-1 of LHCGR gene which was associated with recurrent IUI and IVF failures. Although many factors like age, low anti-mullerian hormone (AMH) etc. are indications for decreased oocyte yield, poor ovarian response and low ovarian reserves, recent publications have focused on FSHR and LHCGR mutations/polymorphisms. A study was initiated to delineate the possible role of LHCGR dysfunction in patients with poor ovarian response, low and moderate ovarian reserves11.\n\n\nMaterials and methods\n\nA total of 114 patients was screened for LH receptor gene polymorphisms/mutations with the following inclusion criteria: number of antral follicle count <8, normal testosterone levels, low antimullerian hormone (AMH) and poor response to previous infertility treatment. Out of 114 patients, 3 patients showed a 6bp homozygous insertion and 23 patients showed heterozygous 6bp insertion as described previously11,12. However, a 54bp insertion in exon-1 of LHCGR gene was detected in a patient with a clinical history of poor response to human menopausal gonadotrophin (hMG) stimulation and recurrent cyst formation.\n\nThe study was approved by the institutional ethics committee and informed consent was obtained from the participants.\n\nA 28-year old Indian woman with a marital life of 5 years suffering with infertility referred to the Krishna IVF clinic for evaluation and treatment. Physical examination revealed normal breast development, normal external and internal female genitalia with irregular menstrual cycles. The patient has a sibling with two children.\n\nAt day 2, basal ultrasound examination showed an anteverted uterus volume:48ml, a right side ovary volume of 14.43 ml with a growing follicle and a left side ovary volume of 6.32 ml. Antral follicle count was 6 combining both ovaries. The patient underwent diagnostic laparoscopy and hysteroscopy and the ovaries had a normal volume with thick capsules as shown (Figure 1). On patient’s request, gonadotrophin/IUI was planned three weeks after laparoscopy. Ultrasound examination detected a large functional cyst which was managed using oral contraceptive pill. In the first cycle, the patient was super ovulated with a dose of hMG of 150 units/day. No response was observed after 4 days and 6 days of stimulation. As the AMH (0.2 ng/dl) and the ovarian reserves were low and there was no other option available, in the next cycle patient was super ovulated with a hMG dose of 225 units/day for 10 days. On the 9th day of stimulation, one follicle with 18 to 20 mm diameter, 2 follicles with 1 to 9 mm and endometrial thickness of 10 mm were observed. A trigger dose of hCG 5000 IU was administered. Luteal support was provided with susten 200 mg vaginally. The patient had started bleeding p/v from 6 days after trigger dose of hCG. A day 2 scan in the subsequent cycle showed an ovarian cyst formation again.\n\nBecause of the discrepancy between AFC, AMH and the ovarian volume, and the poor response to IUI stimulated cycles and the recurrent cyst formation, hCG stimulation test and LHCGR gene sequencing were performed.\n\nThe hCG stimulation test was performed in a natural cycle. Baseline data of the patient hormonal profile were recorded. These showed serum FSH 10 mIU/ml, serum LH 8 mIU/ml, serum dehydroepiandrosterone (DHEA-S) 3.71 mcg/ml, serum testosterone 20 ng/dl, serum prolactin 14.1 ng/ml, indicating normal values, and serum AMH showed a value of <0.2 ng/ml. hCG increases the serum concentrations of androstendione and testosterone by acting through LHCGR. A dose of 10,000 units of hCG was administered subcutaneously. A blunted response to hCG was observed after 4 hours of administration as there was no significant change in the serum testosterone concentration (22 ng/dl) compared to baseline values. Based on the patient’s poor response to ovarian stimulation and hCG stimulation test, the patient was further referred to the molecular genetics department to investigate the possible LHCGR mutations/polymorphisms.\n\nGenomic DNA from peripheral blood of patient’s was isolated using a modified salting out method as described previously13. All the coding exons of the gene were amplified by polymerase chain reaction using primers designed using primer express software from Applied Biosystems, the amplified product was observed on 2% agarose gel electrophoresis and purified with Exo-Sap enzyme from Thermo Scientific21. The purified product was sequenced using big dye cycle sequencing kit on 3500 genetic analyzer and analyzed using Seq Scape (Life Technologies, USA).\n\nThe sequencing results revealed a 54bp insertion (GCTGCTGAAGCTGCTGCTGCTGCTGCA)2 in exon-1 as shown (Figure 2). This insertion sequence gave rise to glutamine, lysine and leucine rich regions. Insertion in exon-1 of LHCGR gene, according to previous authors11,12,14, will have a profound effect on the LHCGR function as reflected by the patient history and hCG test.\n\nA) A dark yellow portion showing the reference sequence of LHCGR exon 1 from NCBI database and the insertion sequence shown in pink. B) Wild type protein sequence and the inserted aminoacids shown in red.\n\n\nResults and discussion\n\nLH regulates a number of reproductive functions in males as well as in females. LH and CG hormones act through the LH/CG receptor which facilitates the activation of different cell groups in the ovaries. LH triggers follicular maturation, ovulation and formation of corpus luteum and increases progesterone secretion in luteal phase2,3,14. Different chronological, hormonal, functional biomarkers are used to assess the ovarian response during controlled ovarian stimulation (COS)15. Along with these biomarkers, the possible roles of LHCGR mutations associated with different female reproductive disorders like oligo amenorrhea, recurrent pregnancy loss, ovarian resistance, infertility and IVF outcome have been studied by previous authors10,11,16,17. A 6bp (CTGCAG, amino acids: LQ) exon-1 insertion was first described in LHCGR after nucleotide position 54 in Leydig cell hypoplasia9,18. Later, a homozygous 33bp and a 27bp insertions (CTGCTGAAGCTGCTGCTGCTGCTGCAG, amino acids: ) were described by two authors after nucleotide position 54 presenting Leydig cell hypoplasia in males explaining a possible role in signal transduction12,19.\n\nHere we present a homozygous mutation with a 54bp insertion (GCTGCTGAAGCTGCTGCTGCTGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTGCA) in the exon-1 of LHCGR of an infertile woman. Inactivating mutations of the hLHR are known to affect signaling pathways, decrease LH and its receptor binding. Improper folding of this receptor protein structure in the endoplasmic reticulum may result in decreased LHR expression which leads to reduced cell response14. One of the above stated mechanisms might have played a role in the poor ovarian response in the present case. The serum hCG stimulation test also showed a blunted response indicating reduced receptor function. The data from the present case are highly significant as the involvement of a possible genetic cause was delineated at an early stage of the infertility treatment. LHCGR mutations may lead to a reproductive catastrophe. Supplementation of LH in patients with LHCGR polymorphisms might offer a benefit for those who opt for IVF treatment. However the concept of LH supplementation may not yield a good response in such a type of homozygous LHCGR mutations. A blunted response might be observed at a very high doses with mature and immature oocytes. In such situations, in vitro maturation of oocytes, where in the dependence of hCG is less to cause the follicle maturation, can be an option for such patients. This study concludes that examination of LHCGR mutations/polymorphisms would be a useful investigation for classifying response, prognosis and management in infertile patients with low ovarian reserves.\n\n\nConsent\n\nInformed written consent to publish clinical data was obtained from the patient.", "appendix": "Author contributions\n\n\n\nRavi Krishna: carried out the research.\n\nThota Sivanarayana: played a role in standardization and provided necessary technical support.\n\nMadan: reviewed the literature and played an important role in designing the whole study.\n\nMurali Krishna: wrote the manuscript.\n\nSudhakar: helped us in developing the technique and writing the manuscript.\n\nRama Raju: designed the project and provided the whole clinical support.\n\nAll authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Kavitha Lakshmi and Dr. Kavitha for their help in coordinating the patient/s.\n\n\nReferences\n\nDekel N: Master Regulators of female fertility. N Engl J Med. 2009; 361(7): 718–9. PubMed Abstract \| \|Publisher Full Text \|\n\nFilicori M, Cognigni GE, Samara A, et al.: The use of LH activity to drive folliculogenesis: exploring uncharted territories in ovulation induction. Hum Reprod Update. 2002; 8(6): 543–57. PubMed Abstract \| \|Publisher Full Text \|\n\nKumar P, Sait SF: Luteinizing hormone and its dilemma in ovulation induction. J Hum Reprod Sci. 2011; 4(1): 2–7. PubMed Abstract \| \|Publisher Full Text \| \|Free Full Text \|\n\nMinegishi T, Nakamura K, Ibuki Y: Structure and regulation of LH/CG receptor. Endocr J. 1993; 40(3): 275–87. PubMed Abstract \|\n\nPierce JG, Parsons TF: Glycoprotein hormones: structure and function. Annu Rev Biochem. 1981; 50: 465–95. PubMed Abstract \| \|Publisher Full Text \|\n\nTroppmann B, Kleinau G, Krause G, et al.: Structural and functional plasticity of the luteinizing hormone/choriogonadotrophin receptor. Hum Reprod Update. 2013; 19(5): 583–602. PubMed Abstract \| \|Publisher Full Text \|\n\nRama Raju GA, Teng SC, Kavitha P, et al.: Combination of recombinant follicle stimulating hormone with human menopausal gonadotrophin or recombinant luteinizing hormone in a long gonadotrophin-releasing hormone agonist protocol: a retrospective study. Reproductive Medicine and Biology. 2012; 11: 129–33.\n\nLatronico AC, Seagaloff DL: Naturally occurring mutations of the luteinizing-hormone receptor: lessons learned about reproductive physiology and G protein-coupled receptors. Am J Hum Genet. 1999; 65(4): 949–958. PubMed Abstract \| \|Publisher Full Text \| \|Free Full Text\n\nWu SM, Hallermeier KM, Laue L, et al.: Inactivation of the luteinizing hormone/chorionic gonadotropin receptor by an insertional mutation in Leydig cell hypoplasia. Mol Endocrinol. 1998; 12(11): 1651–60. PubMed Abstract \| \|Publisher Full Text \|\n\nArnhold IJ, Lofrano-Porto A, Latronico AC: Inactivating mutations of luteinizing hormone beta-subunit or luteinizing hormone receptor cause oligo-amenorrhea and infertility in women. Horm Res. 2009; 71(2): 75–82. PubMed Abstract \| \|Publisher Full Text \|\n\nBentov Y, Kenigsberg S, Casper RF: A novel luteinizing hormone/chorionic gonadotropin receptor mutation associated with amenorrhea, low oocyte yield, and recurrent pregnancy loss. Fertil Steril. 2012; 97(5): 1165–68. PubMed Abstract \| \|Publisher Full Text \|\n\nRichter-Unruh A, Martens JW, Verhoef-Post M, et al.: Leydig cell hypoplasia: cases with new mutations, new polymorphisms and cases without mutations in the luteinizing hormone receptor gene. Clin Endocrinol (Oxf). 2002; 56(1): 103–112. PubMed Abstract \| \|Publisher Full Text \|\n\nNasiri H, Forouzandeh M, Rasaee MJ, et al.: Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent. J Clin Lab Anal. 2005; 19(6): 229–32. PubMed Abstract \| \|Publisher Full Text \|\n\nLatronico AC, Anasti J, Arnhold IJ, et al.: Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med. 1996; 334(8): 507–12. PubMed Abstract \| \|Publisher Full Text \|\n\nAlviggi C, Humaidan P, Ezcurra D: Hormonal, functional and genetic biomarkers in controlled ovarian stimulation: tools for matching patients and protocols. Reprod Biol Endocrinol. 2012; 10: 1–9. PubMed Abstract \| \|Publisher Full Text \| \|Free Full Text\n\nToledo SP, Brunner HG, Kraaij R, et al.: An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female. J Clin Endocrinol Metab. 1996; 81(11): 3850–54. PubMed Abstract \| \|Publisher Full Text \|\n\nStavrou SS, Zhu YS, Cai LQ, et al.: A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters. J Clin Endocrinol Metab. 1998; 83(6): 2091–98. PubMed Abstract \| \|Publisher Full Text \|\n\nWu SM, Jose M, Hallermeier K, et al.: Polymorphisms in the coding exons of the human luteinizing hormone receptor gene. Mutations in brief no. 124. Online. Human Mutat. 1998a; 11(4): 333–4. PubMed Abstract \| \|Publisher Full Text \|\n\nSinha SK, Bhangoo A, Ten S, et al.: Leydig Cell Hypoplasia due to Inactivating Luteinizing Hormone/Chorionic Gonadotropin receptor gene mutation presenting as a 46,XY DSD. Adv Exp Med Biol. 2011; 707: 147–148. PubMed Abstract \| \|Publisher Full Text \|\n\nMcFarland KC, Sprengel R, Phillips HS, et al.: Lutropin-choriogonadotropin receptor: an unusual member of the G protein-couples receptor family. Science. 1989; 245(4917): 494–9. PubMed Abstract \| \|Publisher Full Text \|\n\nWerle E, Schneider C, Renner M, et al.: Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res. 1994; 22(20): 4354–4355. PubMed Abstract \| \|Publisher Full Text \| \|Free Full Text \|" }	[ { "id": "8534", "date": "01 May 2015", "name": "Han Zhao", "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 article has identified a 54bp homozygous insertion in exon-1 of LHCGR gene which was related to poor ovarian response and concluded that screening of LHCGR mutations/polymorphisms would be useful for prognosis and management of patients with low ovarian reserves. However, there are several concerns that must be adequately addressed and require a detailed response or additional data. The authors performed sequencing of LHCGR gene in patients with history of poor ovarian response, but detailed criterion of poor ovarian response was not characterized. The article mentioned that the patient with the 54bp homozygous insertion in exon-1 of LHCGR gene has a sibling with two children. Whether the sibling has been sequenced of LHCGR gene or not? The result of the sibling could help understand the origin and the function of the homozygous insertion. As described in the article, the patient with LHCGR gene insertion had a history of poor ovarian response and recurrent cyst formation. Why not try transvaginal oocyte retrieval which might improve the outcome? A minor detail is that although the language is excellent for a paper from a non-English speaking country, there are still a lot of grammatical errors that would benefit from editing by a native speaker.", "responses": [] }, { "id": "9293", "date": "21 Jul 2015", "name": "Zi-Jiang Chen", "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\nCorrection from F1000Research – 22nd July 2015: This report was mistakenly published with the summary status of ‘Approved’ and has now been corrected to the referee’s intended status of ‘Approved with Reservations’. No changes have been made to the text of the report. We apologise for this error.This is an interesting report in which a 54bp homozygous insertion in exon-1 of LHCGR have been found that are associated with poor ovarian response. That should be helpful for understanding the effect of short sequence repeat in LHCGR on ovarian response. But there still are a number of concerns that need to be addressed.The sequence of 54bp insertion should be (CTGCTGAAGCTGCTGCTGCTGCTGCAG)2, but not (GCTGCTGAAGCTGCTGCTGCTGCTGCA)2, which did not change the amino acid sequence but consist with the previous reported insertions of 6bp (CTGCAG), 33bp and 27bp (CTGCTGAAGCTGCTGCTGCTGCTGCAG). Moreover, it would be interesting to discuss whythe 6bp to 54bp duplication in the same region adversely affect LHCGR response to hCG/LH. How about the LHCGR sequence of the patient’s parents and sibling? Whether the insertion is an autosomal recessive disorder or a kind of dynamic mutation? “Insertions in exon-1 of LHCGR gene were first studied in male Leydig cell hypoplasia.” In present study, the patient with 54bp insertion had ovaries with thick capsule. Is there some relationship? There are some typos and awkward phrasing in the manuscript. E.g. “Later, a homozygous 33bp and a 27bp insertions (CTGCTGAAGCTGCTGCTGCTGCTGCAG, amino acids:) …”. There should be the sequence of amino acids, right? So please review carefully for those.", "responses": [] } ]	1	https://f1000research.com/articles/4-72
https://f1000research.com/articles/3-282/v1	18 Nov 14	{ "type": "Review", "title": "Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis", "authors": [ "Natasha G. Caminsky", "Eliseos J. Mucaki", "Peter K. Rogan", "Natasha G. Caminsky", "Eliseos J. Mucaki" ], "abstract": "The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, a companion software program that computes changes in information content for any splice site substitution, linked to corresponding publications containing these mutations. The accuracy of IT-based analysis was assessed in the context of experimentally validated mutations. Because splice site information quantifies binding affinity, IT-based analyses can discern the differences between variants that account for the observed reduced (leaky) versus abolished mRNA splicing. We extend this principle by comparing predicted mutations in natural, cryptic, and regulatory splice sites with observed deleterious phenotypic and benign effects. Our analysis of 1727 variants revealed a number of general principles useful for ensuring portability of these analyses and accurate input and interpretation of mutations. We offer guidelines for optimal use of IT software for interpretation of mRNA splicing mutations.", "keywords": [ "mRNA", "splicing", "mutation", "genetic disease", "rare disease" ], "content": "Introduction\n\nPre-mRNA splicing is a necessary step in the production of a functional protein product. It consists of the recognition of intron/exon boundaries, and the subsequent excision of the introns. It is important to distinguish between alternate splicing isoforms and mutant splice forms. The former consists of using different combinations of splice sites for the same gene. It is estimated to occur in over 60% of human genes, some of which will have multiple alternate isoforms1,2. For example, NF1 is reported to produce 46 splice variants3. The cell regulates this naturally occurring process through the availability of tissue-specific splice factors. Alternative splicing is not generated by changes in the unspliced RNA sequence, whereas mutations that produce non-constitutive splice forms are the result of dysregulation of natural splice site recognition. Mutations can have various consequences to RNA processing, such as exon skipping, cryptic splicing, intron inclusion, leaky splicing, or less frequently, introduction of pseudo-exons into the processed mRNA. A broad range of molecular phenotypes are possible depending on the type and severity of the mutation, making it imperative to understand the consequences of splicing mutations. For the purposes of this review, we consider sequence changes in genes that affect transcript structure or abundance to be mutations, regardless of their allele frequencies. Although spliceosomal recognition and RNA binding factors are operative in mutation-derived and normal alternative mRNA splicing events, this review is focused on aberrant sequence changes that alter constitutive splicing, and often result in clinically abnormal phenotypes.\n\nThe process of U1/U2-based mRNA splicing involves the recognition of a number of key sequence components4,5, with exons defined by both intronic and exonic features4,6. The exonic and intronic sequences flanking the 5´ end of an intron is termed the donor site and the 3´ end, the acceptor site. In typical mRNA splicing, the natural donor and acceptor splice sites span intervals of 10 and 28 bases in length, respectively. It is a common misconception that these sequences (especially the dinucleotides immediately intronic to the exon) are invariant. Although highly conserved, these sequences vary at different splice junctions within a gene as well as between genes. The particular combination of nucleotides at each position within the same splice site determines its overall strength, which dictates the likelihood of recognition by the U1 and U2 spliceosomes.\n\nIn addition, binding sites for splicing regulatory elements have been shown to reside over a range of distances from the corresponding natural splice sites7; the impact of these sites appears to be related to their binding affinities to the cognate RNA binding proteins and to their distance from the proximate intron/exon boundary8. Recognition sites for these regulatory proteins can reside either within introns or exons. Those within exons are commonly referred to as exonic splice enhancers or silencers (ESE or ESS, respectively), whereas the corresponding designations for intronic elements are ISE or ISS. Sequence variants affecting these protein-binding sites (or mutations in the binding proteins themselves) have been documented as contributing to aberrant splicing and pathogenic phenotypes. We focus on variants occurring in cis with target genes, as opposed to those in the splicing complex (in trans), leading to abnormal splicing. The efficiency and specificity of splicing depends on the combination of natural splice site strengths and the binding of splicing regulatory proteins that orchestrate exon recognition9.\n\nMutations that affect pre-mRNA splicing account for at least 15% of disease-causing mutations10 with up to 50% of all mutations described in some genes11,12. Interpreting the effects that these variants have on splicing is not straightforward because natural and regulatory splice sites exhibit considerable sequence variation. Furthermore, performing in vitro experiments to verify the consequences of each variant is costly and time consuming, and may not be practical. In silico prediction methods have become essential resources for analyzing these variants. Software programs for splicing analysis use a wide variety of bioinformatic approaches. Several splice site prediction tools compare the predicted mutant sequence to a consensus sequence, based on a set of functional acceptor or donor splice sites13. A drawback of this approach is that low-frequency nucleotides present in functional splice sites are not represented, which can lead to misinterpretation and false-positive mutation predictions. One example of this was illustrated by Rogan and Schneider (1995), in which the variant, IVS12-6T>C in MSH2, described by Fishel et al. (1993) was predicted to be benign, despite being located 6 nt from the natural acceptor splice junction14,15. The consensus sequence fails to indicate that C and T at this position are nearly equally probable, which reclassified this transition as a polymorphism rather than a pathogenic variant. This conclusion is supported by evidence that ~10% of normal individuals without predisposition to non-polyposis colon cancer harbour this alternate allele16.\n\nOver the last 20 years, we and others have developed an information theory (IT)-based approach for prediction of splicing mutations, and their impact on mRNA structure and abundance. The effects of these mutations is founded on the formal relationship between IT and the second law of thermodynamics, in that the change in information ascribed to a sequence variant within a splice site is directly related to thermodynamic entropy and free energy of binding17,18. A weight matrix consisting of the Shannon information (product of the probability of each nucleotide and –log2 of its probability) at each position of the splice site is constructed. The individual information for a splice site (Ri, in bits) is defined as the dot product of this weight matrix and the unitary vector of a particular splice site sequence. The magnitude of the information content of a nucleotide within a given site is an indication of its level of conservation relative to a set of functional sites. This method retains all of the sequence variability inherent in each model of donor and acceptor splice sites. By contrast, each base in the consensus sequence has the maximum Ri value, which is actually rare in the human genome, and is generally not representative of the preponderance of natural splice sites. Prior to the introduction of IT-based approaches, consensus sequence-based methods were widely used13. Also, the use of neural networks, trained on sequences experimentally determined to be “bound” and “unbound”, was another early approach used to predict splice sites19. However, these unbound set of sequences are known to harbour some contaminating functional sites20,21, which can limit the sensitivity and specificity of these networks22.\n\nThere are instances when IT does not accurately predict the consequences of a splice variant. This can often be attributed to instances involving multiple sites or multiple regulatory factors, which are not components of current splicing models. In addition, splicing regulatory proteins can share overlapping and degenerate binding sites, and may exert conflicting effects (for example, serine-arginine [SR] vs. hnRNP proteins), making in silico prediction less reliable and accurate in these cases23. Finally, functional cryptic splicing motifs occurring deep within the introns can be challenging to identify, because they tend to be less well conserved than natural splice sites24,25.\n\nNevertheless, a number of authors have recommended IT methods for analysis of splice site variants (N = 29; Supplementary Table 1). In fact, this approach has been described as equivalent to using a general reference textbook as a diagnostic tool, which complemented by functional assays, may provide a complete molecular diagnosis26. Most of the applications of IT for splicing mutation analysis have involved predominantly rare diseases, as well as some low frequency variants associated with more common genetic conditions. This is because IT has been used to assess how well computed changes in binding affinity conform to levels of expression and/or patient phenotypes.\n\nMany IT studies have focused on sequence variants in individual disorders or genes. Our synopsis of the broader implications of this work sets the stage for this compilation of peer-reviewed variants with accompanying IT analyses. We cover all publications retrieved through PubMed and Google Scholar that cite the use of IT (N = 367; Supplementary Bibliography) before September 2014. These items include primary research articles, review articles, presentations, and theses. Of all references, 216 publications reported variants or other results or analyses pertinent to this review (Supplementary Table 2). In the remaining studies, analyses were either not performed, insufficient information was provided to reproduce the reported result, or authors described unrelated applications of IT-based analysis. We summarize the spectrum of variants analyzed to obtain a global perspective of splicing mutations resulting in genetic disease. We also highlight common errors that can occur in variant analysis and interpretation, and offer guidelines for optimal use of our software programs for interpretation of splicing mutations.\n\n\nInformation theory and splice site analysis\n\nIT was first introduced by Claude Shannon in 1948 and is now used in a variety of disciplines to express the average number of bits (i.e. the information content) needed to communicate symbols in a message27. Bits are the basic unit used in computing and can have one of two values (typically the answer to a yes/no, true/false, or +/- problem). In nucleic acid molecular biology, the symbols in the message comprise a group of related, aligned sequences, with the average number of bits in the set corresponding to the amount of information in the message. This is determined from the information content at each position in the sequence, summed over all positions28. The average information is depicted graphically by a sequence logo, which stacks the individual nucleotides at each position ranked by frequency, and where the height of the stack is the position-specific contribution to the average information29. If the set of sequences are functional binding sites recognized by the same factor, the individual information in each site (i.e. Ri value) is related to thermodynamic entropy, and thus, to the free energy of binding18.\n\nThe information content of a nucleic acid binding site is related to the affinity of its interaction with proteins and other macromolecular complexes, such as the case during mRNA splicing18. Information theory-based position weight matrices (PWM; Ri [b,l] - also referred to as a ribl - where b and l correspond to the nucleotide and position in the splice site) can be determined for set of known binding sites, in this case, for the purpose of calculating individual and average sequence information28. Figure 1 shows an example of sequence logos for the canonical acceptor (or 3´, recognized by the U2 spliceosome) and donor (or 5´, recognized by the U1 spliceosome) splice sites, computed from the majority of constitutive sites at annotated splice junctions in the human genome30. The information contained within the natural splice donor site is distributed between the last codon of each exon and the adjacent 6 nucleotides of intronic sequence, whereas the acceptor sites are almost entirely intronic, extending 26 nucleotides upstream from the exon boundary.\n\nA) The sequence logo for human acceptor and donor splice sites based on the positive (+) strand of the October 2000 (hg5) genome draft. The logo shows the distribution of information contents (Ri in bits) at each position over the region of 28 nucleotides for acceptor [-25, +2] and 10 nucleotides for donor [-3, +6] from the first nucleotide of the splice junction (position 0). Nucleotide height represents its frequency at that position. The horizontal bar atop each stack indicates the standard deviation at that position. This figure was modified from Rogan et al. (2003) to include splice sites in genes on both strands of the annotated human reference genome30. B) The distribution of deleterious single-nucleotide variants reported at the natural acceptor (left) and donor (right) splice sites. The variants used to populate this graph (Supplementary Table 8) were included only if they were reported to negatively affect splicing (N = 419 for acceptors, 599 for donors). The image was aligned to the sequence logo (A) to illustrate potential correlation of number of splicing variants at a position to the information content at that position.\n\nThe distributions of Ri values for these sets are approximately Gaussian, with a couple of important exceptions, namely the distribution has defined upper and lower bounds18. The upper limit corresponds to the consensus sequence, as it is not possible to have stronger binding than an exact match to this sequence. The theoretical lower limit corresponds to Ri = 0 bits. An Ri value less than zero implies that energy would be required (ΔG > 0 kcal/mol) for a stable binding complex to form, i.e. that the event would not occur spontaneously without an exogenous source of energy. The minimum strength site is zero bits, the equilibrium state (ΔG = 0). Assuming the contacts at each position in the same binding site form independently, this approach is accurate and quantitative. Altering a nucleotide with high information (implying high prevalence and conservation at that position) will have a greater impact on binding, than if a less-well conserved base were altered. The change in information due to a mutation in a site (ΔRi) is the difference between Ri,final and Ri,initial values, where Ri,final is the information of the sequence containing the variant, and Ri,initial the information of the reference (wild-type) sequence. The minimum fold change in binding affinity resulting from the mutation is an exponential function based on ΔRi, or ≥ 2ΔRi (Ref.18).\n\n\nSoftware resources\n\nInformation analysis was originally performed using the Delila sequence analysis system, which included a language to process nucleic acid sequences, and a library of sequence tools to retrieve and process various types of sequence data31,32. Tools to measure information content of nucleic acid sequences were subsequently added to Delila28. Initially, models of information content of bacteriophage T7 RNA polymerase binding sites and other bacterial control systems were studied, and mRNA splice sites were subsequently developed28,33. Later, tools to display binding sites as sequence logos of average information, and sequence walkers showing individual information were incorporated into Delila20,29. The Automated Splice Site Analysis (ASSA) server introduced in 2004, and its successor, Automated Splice Site and Exon Definition Analysis server (http://splice.uwo.ca; ASSEDA), have been freely available throughout the last decade, and have been used for IT-based calculations on nucleic acid sequences for the preceding 20 years34,35. Both ASSA and ASSEDA still use the Delila program suite to retrieve sequences, calculate information content, and create sequence walker representations of individual binding sites.\n\nTo simplify mutation analysis, we built a web interface for variant analysis using Delila software as the processing backbone34. Our aim was to standardize and facilitate IT-based mutation analysis by using Human Genome Variation Society (HGVS)-approved variant nomenclature (which has since become the worldwide standard), employing server-based retrieval/processing, and reporting results as concise predictions in both tabular and sequence walker display formats. Initially, ASSA results described mutations in relation to genome annotations from the first finished genome release (hg15)34. While many publications cited this version of ASSA for novel splicing mutation analysis, continued improvements have introduced more accurate reference sequences, annotations, and models (for both constitutive and regulatory splice sites) based on more comprehensive sets of binding sites. The ASSA server contained the original donor and acceptor information position weight matrices derived by manual curation of GenBank entries33, murine donor and acceptor weight matrices, a subset of splicing enhancer elements (SF2/ASF, SC35 and SRp40), and the lariat branch point recognition sequence33. ASSA reported the strengths of all potential sites predicted within the window selected by the user, highlighted those with the largest changes in Ri, and computed the minimum fold change in binding affinity for each mutation or polymorphism. Tabular results were colour-coded. Unaltered sites above and below the Ri,min (described in Minimum splice site information content and exceptions) were highlighted grey and white, respectively. Pre-existing sites abolished by the variant (where Ri,final < Ri,min) were marked in red, while leaky natural sites (Ri,final ≥ Ri,min) were highlighted in blue. Cryptic sites that were created, strengthened, or weakened were highlighted in pink, green and teal, respectively. The server parsed any mutation type described precisely by the HGVS notation, including substitutions, insertions, deletions, and combinations of these changes36. Recapitulating variants described in articles before these guidelines were widely adopted proved to be time-consuming and error-prone22. Multiple binding factors had to be analyzed simultaneously; however, results were reported independently. The analysis did not consider other factors relevant to splice site recognition, such as the resulting exon size, or potential formation of cryptically spliced exons.\n\nASSEDA, the successor software to ASSA, provides a new isoform-oriented type of mutation interpretation, updates the coordinate system to HG19 (GRCh37), adds current gene and single nucleotide polymorphism (SNP) annotations (dbSNP135), and provides additional ribls for other splicing regulatory sites (SRp55, TIA1, ELAVL1, hnRNP A1, hnRNP H, and PTB). All models, except those for SRp55 and hnRNP H, have been built using sequences from publicly available CLIP-seq data, and are based on a larger number of binding site sequences. They have been tested by comparing predictions to validated binding sites from published primary literature, and to any splice-altering variants found within them35. ASSEDA introduces in silico exon definition analysis by computing the total splicing information across an exon35. Total exon information (Ri,total) is the sum of the corresponding donor and acceptor Ri values, and corrected for the gap surprisal term, which is based on the length of the potential exon formed using those sites (from RefSeq)37. The gap surprisal function is based on the genome-wide distribution of constitutive exon lengths, also known as self-information. This term ensures that exons are computationally defined using donor and acceptor splice sites in close proximity37,38.\n\nExons of uncommon length lead to large negative gap surprisal terms, which reduces Ri,total. When applied to predicted exons that activate a cryptic splice site, comparison of Ri,total values can more accurately predict cryptic site use than the strength of this site alone. The gap surprisal term decreases the predicted Ri,total value of particularly long internal exons (eg. the 3.4 kb long exon 11 of BRCA1; Ri,total = 1.4 bits), which tends to compensate for this effect with strong splice sites and other sequence elements that enhance natural splice site recognition and suppress internal cryptic splice sites.\n\nThe exon definition paradigm extends to the assessment of the impact of mutations in ESE/ISS elements. ASSEDA calculates Ri,total by adding the Ri value of a regulatory splicing element to the contributions of constitutive splice sites, and applying a second gap surprisal term based on the frequency of distance from the splicing element to the nearest natural site. Currently, the effect of only a single splicing factor can be evaluated by the software, although the approach itself is generalizable to multiple regulatory binding sites. If a variant causes changes in the Ri values of multiple sites, such as the simultaneous creation of both splicing enhancer and repressor elements, there will be less confidence in ASSEDA’s predictions.\n\nTwo distinct sets of IT-based models for donors and acceptors are available on ASSEDA. The manually curated ribls were originally determined from 1799 donor and 1744 acceptor sites33. We subsequently derived a set of ribl matrices from genome-wide exon annotations30. These models were automatically curated using the criteria that enforced Ri > 0 for correctly annotated sites. The resultant models consisted of 108,079 acceptor and 111,772 donor splice sites, however these were not formally implemented on the ASSA server until 201130. These genome-wide models are used in the calculation of Ri,total values. The ΔRi values for a single nucleotide splicing variant are similar for both sets of models. Variants having opposite predicted effects between the respective donor or acceptor ribls have not been reported. In general, the genome-wide models report slightly lower information contents, however the frequencies of nucleotides at the 5´ end of the acceptor site differ significantly. This results in differences in the weights in the -4 to -20 nt region between the manually-curated and the genome-wide acceptor ribl matrix, which can significantly lower Ri values based on the genome-wide model. In the genome, thymine is more prevalent than cytosine at these positions and has a higher positive contribution to the overall Ri. This can account for up to a 1.97 bit difference between the models. Guanine nucleotides within this sequence window significantly lower the Ri values computed from the genome-wide acceptor ribl, as well. While these differences contribute only a 0.1–0.4 bit difference to the Ri per nucleotide, the cumulative effect of multiple differences within this window can lead to significant differences between the acceptor Ri values.\n\nHigh-throughput DNA sequencing is generating a deluge of novel variants in patients with genetic diseases, most of which currently have unknown significance (VUS). For example, 20% of the patients with Pelizaeus-Merzbacher disease possess VUS, among which are single or compound heterozygous, rare pathogenic mutations39. Many solutions have been proposed, however prediction of pathogenicity by bioinformatic analyses is often inaccurate40. The Shannon Human Splicing Mutation Pipeline software predicts mutations at genome scale to predict which variants may alter mRNA splicing and is based on the same principles and IT models used in ASSA and ASSEDA41. However, this software processes ~5 million substitutions and/or indels in 10–15 minutes. While initially only available for the CLC-Bio Genomics platform, this software is now offered as a web service (http://shannonpipeline.cytognomix.com). Variants are batched in standard variant call format (VCF). The pipeline reports any genic variant that affects a known natural site or a cryptic site where Ri,initial or Ri,final are ≥ 0 bits and ΔRi ≥ 1.0 bits, however more stringent criteria for selecting variants with significant information changes can be applied.\n\nIn Shirley et al. (2013), all variants from the complete genomes of three cancer cell lines (A431, U2OS, U251; N = 816,275) were analyzed41. Variants that were common (≥ 1%) were removed. Variants that weakened natural sites, or strengthened cryptic sites to levels comparable to or exceeding the strength to the nearest natural site, were flagged. Variants that strengthen a natural site could have an effect on the splicing profile of a gene (i.e. reduce the frequency of exon skipping for the corresponding exon), but are less likely to cause a deleterious phenotype. The overall fraction of mutations flagged, after filtering out distant cryptic sites and small ΔRi values, averaged 0.016%, illustrating how the software can be used for prioritizing variants. Some of the prioritized variants occurred in genes with known defective functional and biochemical pathways in these cancer cell types, i.e. cytokine signalling (in A431), DNA replication and cell cycle (in U2OS). Natural splice mutations were confirmed by expression data to a greater extent than either leaky or cryptic splice site variants.\n\nIn a complete cancer cell line genome, the number of cryptic sites with altered Ri values greatly exceeds the number of affected natural splice sites. Many of these are weak decoys, which can occur throughout genes. Using the principle that novel cryptic sites that are likely to be activated must compete with the natural splice site for spliceosomal recognition, the relevant cryptic sites are restricted to those with Ri values comparable to or greater than the corresponding strength of the adjacent natural site of the same polarity22. Additionally, the proximity of potential cryptic sites to the natural site should be considered in assessing whether an exon could be formed with the natural splice site of opposite polarity. Cryptic sites that are considerably weaker than the nearest natural site of the same type, or cryptic sites that would lead to unusually large exons, diminish the likelihood of cryptic site activation. Benaglio et al. (2014) used the Shannon Pipeline to screen 303 sequenced patients and flagged five variants that each strengthened or created a different cryptic site42. While comparable in strength to the natural site, these were all distant (>400 nucleotides away) and thus, less likely to be recognized. The authors also stated that the ΔRi values for three of these sites were discordant with results obtained with NNSplice, a neural network-based splicing prediction program. In fact, both the Shannon Pipeline and NNSplice demonstrated strengthening of these decoy cryptic splice sites.\n\nShirley et al. (2013) evaluated the predictions of the Shannon Pipeline by manually inspecting RNAseq data for each variant with significant information changes in each cell line41. However, manual review is unfeasible for many large datasets, especially from tumors, because of the large numbers of potential somatic mutations affecting splicing in each genome. Veridical, an in silico method for validation of DNA sequencing variants that alter mRNA splicing, has been developed to provide high throughput, statistically-robust unbiased evaluation based on RNAseq data43. The method has been implemented as software for analysis of potential splicing variants from large datasets and catalogues their effects. Veridical takes Shannon Pipeline output from predicted genomic variants with effects on splicing and performs a case-control analysis of corresponding expressed transcripts that cover the same genomic region, taken from normal tissues. Upon Yeo-Johnson transformation of the expressed read count distribution, parametric statistics are used to compare normal and abnormal mRNA species (exon skipping, intron inclusion, and cryptic site use). Veridical is designed to be used with large data sets, as the statistical analysis gains power with increasing numbers of control samples. A recent study of 442 breast cancer tumors from the Cancer Genome Atlas Project revealed 5,206 putative splicing mutations using the Shannon Pipeline. Veridical was then used to confirm exon skipping, leaky or cryptic splicing of 988 of these variants44.\n\n\nNatural sites\n\nThe early splice site recognition literature often oversimplified the composition of the U1/U2-type 5´ donor and 3´ acceptor sites by presenting only consensus sequences and truncating the positions in each site13,45,46. However, the conserved tracts extend well beyond the canonical GT and AG dinucleotides adjacent to intron/exon junctions. Furthermore, a small, albeit significant, proportion of natural donor sites (~800, or 0.7%) contain cytosine at position +2 in the genome. This is reflected by a corresponding small decrease in average information at this position (Figure 1). Sequences adjacent to these positions are more variable, but are nevertheless essential for the accurate recognition by the spliceosome. Specifically, the donor site is defined by the three terminal nucleotides of each exon and the first seven bases of the downstream intron. Conversely, acceptor sites are represented by the first two bases of the exon and the last 26 bases of the upstream intron. Because ASSA and ASSEDA use an integer-based coordinate system, there is a zero coordinate at the first intronic base of each splice site (Figure 1), which is not used in the conventional numbering system. The coordinate ranges for the donor and acceptor site positions are therefore [-3, +6] and [-25, +2], respectively. Individual information analysis computes the Ri values over these intervals for normal and variant-containing splice sites. As discussed below, information content present in intronic intervals justifies sequencing and analyses of sequences well beyond the locations of the splice junctions themselves.\n\nCertain variants within donor and acceptor sites are tolerated and may even have benign effects, while others have a deleterious impact on spliceosomal recognition. IT accounts for all of these possible outcomes. Unusual donor sites (i.e. with cytosine at position +2) are detected by information analysis, but could be falsely called deleterious by consensus sequence-based methods. Although the terminal position of exons contributes significantly to donor splice sites with a preference for G, a significant proportion of sites naturally possess A or U at this position, or less frequently, C.\n\nOf the published IT-based variant analyses, single nucleotide variants (SNVs) that were reported to affect a natural splice site (multi-nucleotide and insertion/deletion variants are listed separately in Supplementary Table 3) were compiled and reanalyzed. After reducing this set to only those variants occurring within the intervals covered by the splice site information weight matrices described above, 1152 SNVs were reported to affect the strengths of either natural donor or acceptor sites. A variant was considered deleterious if it was predicted to affect splicing (either leaky expression or exon skipping), or if it was experimentally shown to reduce or abolish splicing of the corresponding exon. In instances where prediction and validation did not concur, the latter were used to determine the effect of the variant. Variants predicted to have a neutral effect but demonstrated to be deleterious in the validation study were classified as damaging. In total, 1010 deleterious natural splice site variants were analyzed (Supplementary Table 4).\n\nSequence conservation has long been considered a surrogate measure of evolutionary constraint and, by inference, functional significance. The average information quantitates the relative conservation at each of the positions within a binding site. We compiled the mutation spectra for all mutations that significantly affected the strengths of donor and acceptor splice sites and compared these with the average information contents at each position. The panels in figure 1b respectively indicate, at each position of the natural acceptor and donor sites, the frequencies of variants deemed deleterious by information analysis. Interestingly, when the sequence logo is overlaid with the histogram of the corresponding mutation spectra, the relative frequencies of deleterious mutations and the average information are comparable. Indeed, these frequencies and the information contents across each type of site are strongly correlated (r=0.95 for acceptors and 0.89 for donors). Our interpretation is that the susceptibility to deleterious mutation at a position is related to its overall conservation within the splice site, which reflects the contribution of that ribonucleotide to the stability of the interaction with the corresponding spliceosome. Nevertheless, there is an unstated bias in ascertainment in these mutation spectra. Variants occurring at sites with low information and/or that are benign are underrepresented, as they are less likely to be associated with genetic disease, and were less likely to be reported. Also, the distribution is dependent on the region sequenced by the authors of the reviewed publications; in early work, the full sequence interval containing the entire splice site was sometimes not included or unavailable for analysis.\n\nAn interactive website was created to summarize this set of SNVs. This software application renders interpretations of variant effects in a more practical, useful way than the corresponding table of supplemental data (Supplemental Table 10). The “Splicing Mutation Calculator” (SMC; http://splicemc.cytognomix.com) is a web service that amalgamates all published results for the same type of substitution in a natural splice site, regardless of genic context. Variants that create cryptic splice sites were not included, because we consider these cases to be sequence-specific as opposed to positional. With this program, users have the option of exploring mutation data (at present, only SNVs can be analyzed) linked to the original literature citations. SMC processes and provides literature support for the variants that occur within the defined regions spanned by natural splice sites. The user first selects the type of site (donor or acceptor), position (based on ASSEDA’s integer-based system), wild-type or reference nucleotide, and the alternate substitution at that position (Figure 2a). The software tool outputs the ΔRi and the number of variants that have been reported and analyzed to date using IT (Figure 2b). SMC provisionally classifies the reported variants based on the degree to which these predicted effects are expected to decrease spliceosomal affinity, and consequently splicing. The following criteria are empirically based on affinity changes and a summary of published phenotypes associated with these changes: “Deleterious” (if the site is weakened by more than 7.0 bits), “Probably Deleterious” (if the site is weakened such that -4.0 bits ≥ ΔRi ≥ -7.0 bits), “Leaky” (the site is weakened such that -1.0 bits ≥ ΔRi ≥ -4.0 bits), or “Benign, probable polymorphism” (if the site is weakened by less than 1.0 bits). In this first release of SMC, we have omitted “benign” variants, which are likely polymorphisms; these will be catalogued and included in a later version. It is important to appreciate that the ΔRi is a constant for a specific nucleotide change at a specific position, though the absolute strength of the splice site depends on the sequence context of the mutation. This context varies between mutations, and Ri,initial is not the same for each case, which can result in different Ri,final values for different mutations.\n\nA) Example mutation input for SMC (T>A at the 3rd intronic position of natural acceptor). The type of splice site is selected by clicking on the corresponding sequence logo (acceptor [left] or donor [right]). The purple slider bar appearing below the logo is used to select the position of the mutation. The reference and mutant nucleotides are then designated, and the variant is submitted to the software (‘Submit your selection’). SMC outputs a table indicating the user input, the number of instances in the literature where this substitution has been analyzed using IT, and the computed ΔRi values (in bits) using both the old (1992; top) and new (2003; bottom) ribls. The cell color for ΔRi values indicates the predicted severity of the inputted variant according to defined thresholds22,168. B) Tabular output detailing each instance of the selected mutation from the source table. The user may view, in a separate window, extensive details of all variants referred to in SMC output (Supplementary Table 10).\n\nBesides published sources, the software also can predict effects of mutations by computing ΔRi values directly. Particular substitutions that have not been reported in Supplemental Table 10 can nonetheless be provisionally interpreted. The ΔRi value is computed and reported from the ribl. While SMC enables rapid exploration of results for validated and novel mutations, it is, however, not a replacement for ASSEDA or the Shannon Pipeline, since it does not consider the sequence context, which can also influence the interpretation of deleterious, leaky, or benign variants.\n\nThe minimum theoretical information content of a binding site, Ri,min, is zero bits18. Comparison of the Ri, values of a series of inactivated and minimally active splice sites revealed the minimum strength of functional splice sites (Ri,min) to be at least 2.4 bits for the original donor and acceptor models of Stephens and Schneider (1992) (based on 103 mutations with functional validation, including 57 natural and 46 cryptic site activating mutations)22. This value was redefined based on information models from a genome-wide set of donor and acceptor models (Figure 1a) to be 1.6 bits using the identical set of mutations30. It is likely that the differences between these values are not significant and are attributable to the increased precision of the ribl using the ~50-fold larger set of sites. Weakened natural sites, with significantly reduced Ri values that remain above these thresholds, are considered to be leaky (lower affinity binding), whereas those below this threshold are found to completely abolish natural splice site recognition, resulting in either exon skipping or activation of neighbouring cryptic splice sites. However, these outcomes are not mutually exclusive, since leaky splice site mutations may also result in exon skipping and/or activate neighboring cryptic sites. Natural splice sites below these thresholds are extremely rare, and their recognition is likely enhanced through the binding of specific RNA binding proteins that promote exon definition (eg. XPC exon 4 acceptor and MYBP3 exon 12 acceptor47,48).\n\nLeaky natural sites have Ri values exceeding the Ri,min threshold, which, in theory, retain some capacity to be recognized by the spliceosome. There were 84 variants predicted to cause leaky splicing, of which 19 were shown experimentally to lead to exon skipping without any detectable residual natural splicing (Supplementary Table 2: #32, 120, 128/380, 195, 276.5, 355, 360, 363, 364, 365, 379, 409, 477/496/934, 573, 842, 853, 883/1589, 886, and 918). Of those, seven are donor splice site mutations at position +5 (ΔRi ~ -3.5 bits; #128/380, 195, 355, 842, 853, 883/1589, 886), four alter the first exonic nucleotide of a donor site (ΔRi ~ -3.0 bits; #276.5, 360, 379, 409), and three are donor mutations at position +4 (ΔRi ~ -2.6 bits; #120, 365, 573). The Ri,final values of these 19 inactivated natural sites range from 2.7 to 8.8 bits, which suggests the possibility that the variant may also simultaneously affect other adjacent or overlapping sites that preclude recognition of the mutated natural site. Additionally, weakening of 11 of these variants activates a neighbouring cryptic splice site, in which no residual natural splicing was detected. However, changes in splice site preference due to small changes in binding affinity within exons are probably related to the processive nature of donor splice site selection49.\n\nLeaky splicing mutations are readily detected when the expressed transcript contains the causative variant or a neighbouring polymorphism. However, there are a number of practical limitations on the methods for experimental validation of leaky splicing mutations. RT-PCR alone would only be considered reliable for confirmation of homozygous mutations (and in one case, a compound heterozygote where two separate variants abolished natural splicing of the same exon), unless combined with a secondary quantitative methodology50. Similarly, it is difficult to assess leaky splicing of heterozygotes using RNAseq data, as reduced levels of wild-type splicing are challenging to determine without adequate read coverage and controls for comparison. However, leaky splicing can be assessed by comparing the frequency of the causative allele to the normal allele in the same cell line when the variant is present within the sequenced reads41. These are special cases however, as the variant itself must either be expressed within an exon or, if intronic, must lead to an activation of a cryptic site further into the corresponding intron.\n\nWe previously suggested that weaker splice sites are more susceptible to mutational inactivation relative to stronger sites22. In the present study, all experimentally verified variants affecting natural sites (where leaky and abolished splicing could be differentiated) were analyzed (N = 98). Variants predicted to abolish splicing (Ri,final < Ri,min and/or ΔRi < 7.0 bits) were filtered out, as large changes in binding affinity will essentially abolish splicing, despite remaining binding strength and regardless of initial Ri value. Supplementary Figure 1 illustrates the frequency of inactivation by these variants relative to initial Ri value. Variants occurring at weak splice sites (Ri,initial < 4 bits) abolish splicing in 5 of 6 cases (where ΔRi < 7 bits), but are not represented as they all weaken the site below Ri,min. The remaining variant slightly weakens a site where Ri,initial is -0.1 bits (where ΔRi = 0.5 bits), and its recognition may be supported by SR elements47. Moderate strength splice sites (5–11.0) bits are inactivated in 25–60% of cases, and mutations at strong splice sites (Ri,initial ≥ 12 bits) tend to be leaky (Supplementary Figure 1b).\n\nMutations that abolish natural sites (without cryptic splice site activation) are expected to result in a complete loss of normal splicing. However, of the 94 variants that reduced natural splice site strength below Ri,min, 11 were reported to have residual normal splicing activity (Supplementary Table 2: #185/750, 275, 881, 914, 1315, 1321, 1325, 1326, 1361, 1380, and 1407)22,41,51,52. Two of these occurred at the G of the +1 position of the donor site (Supplementary Table 2: #185/750 and 1326), which is essentially invariant in functional splice sites. This suggests potential problems in IT or experimental analysis of these mutations. Surprisingly, the majority of these variants occur at the +2 position of a donor splice site and are T>G mutations, which are predicted to abolish splicing activity41. However, the analysis of RNAseq data for these variants showed no splicing defects (Supplementary Table 7: #1315, 1321, 1325, 1361, 1380 and 1407). One explanation is that resultant aberrantly spliced transcripts were subjected to nonsense-mediated decay (NMD) and degraded. Another possibility is that the coverage of these splice junctions is insufficient to distinguish expression of a single allele from that same allele plus the leaky splice junction. The remaining variants differ in the position within the splice site and decrease natural site strengths to between 0.9 to 2.2 bits22,51.\n\nTheoretically, a site lacking the canonical G at +1 (donor) or -1 (acceptor) position of a natural site may exceed Ri,min. Ozaltin et al. (2011) and Di Leo et al. (2009) each assessed mutations at positions +1 or -1, which weaken natural splice sites to Ri > Ri,min, and note that these sites are predicted to be leaky53,54. However, this is not the sole criterion for interpreting splice site mutations using IT-based methods. The overall change in binding affinity must also be considered, as both mutated sites were predicted to have only 0.4–0.5% of the binding affinity of the corresponding natural splice sites53,54.\n\nAlthough branch-point site (BPS) recognition occurs independently and post-exon definition, mutations in this sequence have also been described, due to its proximity to the natural acceptor site. Following the recognition of and binding to the 5´ss (upstream donor site) by the U1 snRNP, the U2 is recruited to the 3´ss (downstream acceptor) and recognizes the BPS, resulting in the formation of the pre-spliceosome55. Association of U2 with the BPS is essential, as it is the first energy-requiring step, allowing for the tri-snRNP complex of U4/U6 × U5 to be recruited to the BPS, which produces a catalytically active spliceosome56. The BPS typically contains a conserved adenosine and a downstream polypyrimidine tract. It is located within 40 nt of the natural 3´ss, however there are reported cases where it can be up to 400 nt away.\n\nRecognition of the BPS is thus a crucial step in proper splicing, and sequence variants can disrupt this event, impede lariat formation, and intron excision. The complete list of BPS variants analyzed using the ASSA and ASSEDA server can be found in Supplementary Table 5. The variants range in distance from 0–76 nt from the natural acceptor junction, and either weaken, abolish or strengthen the BPS. When validation assays were performed, the prediction by the server was correct in 9/11 cases. We deemed the two other cases to be partially discordant (NM_004628:c.413-24A>G and NM_005902:IVS8-55A>G). ASSEDA predicted these variants to abolish the BPS, but leaky and normal splicing was observed, respectively. The predictions are partially concordant with experimental findings because ASSEDA also predicted the existence of nearby alternative BPS, which if used, could account for the observed phenotype.\n\nAlthough IT-based prediction of a variant effects on BPS has been accurate, the number of validated sites used to compute the ribl is substantially smaller (N = 20), and it is not as reliable as those used to determine Ri values of natural acceptor and donor sites. Furthermore, these motifs are short and relatively frequent in unspliced mRNA. One possible explanation for the rarity of BPS mutations is that compensatory, alternative BPS sequences can be recognized and used. Furthermore, the weak constraint on the precision of the distance between the BPS and the 3´ (acceptor) splice site (Figure 3) further enables activation of these alternative sites. These factors increase the chance that a variant will be falsely predicted to affect a BPS. For example, variants within donor splice site sequences are routinely predicted to alter strength of false BPS. This error is easily avoidable if the potential recognition sequence is filtered for the genomic context of the variant, as well as its proximity to acceptor splice sites.\n\nSequence logo for information model for the branch-point site, created using 20 annotated branch-point sequences.\n\n\nActivation of cryptic splicing\n\nIt has been estimated that 1.6% of disease causing missense mutations can affect splicing and recent predictions suggest that approximately 7% of exonic variants in the general population may disrupt splicing, which includes cryptic splicing57,58. The genome is replete with pseudo (or decoy) splice sites with varying degrees of similarity to natural sites that are not recognized in constitutive splicing59. However, mutations that alter the strengths of either these decoys or the natural splice site of the same polarity may shift the balance of isoforms towards non-constitutive splice isoforms that predominate over or eliminate normal mRNAs (Figure 4). Mutations can create a cryptic splicing event by creating or strengthening a site in either intronic or exonic regions (Figure 4, Type 1), weaken the natural site while simultaneously altering an overlapping decoy site (Figure 4, Type 2), or exclusively weaken the natural site, leading to the activation of a pre-existing decoy site (Figure 4, Type 3). Although the contributions of cryptic splicing to genetic disease have long been recognized, IT analysis correctly predicts most, but not all, cases (Figure 4). The challenges in identifying potential cryptic sites or determining activation are attributable to our incomplete understanding of the requirements for activation60–62, which include exon length, processivity of donor site recognition, and involvement of splicing regulatory factors. A database of aberrant 3´ and 5´ splice sites has been compiled62.\n\nA prototypical internal exon (in purple) with flanking exons (in blue); introns are represented by black solid, and dashed lines (top). The three types of cryptic splice site activation are then illustrated. Type 1 cryptic splice site activation (left) is caused by the activation (green arrow) of a cryptic site by strengthening a pre-existing site, or by creating a novel splice site (blue). Type 2 (middle) results from the simultaneous weakening or abolition (red arrow) of the natural splice site while strengthening or creating (green arrow) a cryptic site. Type 3 (right) involves the activation of a pre-existing cryptic site due to the weakening or abolition of the natural splice site (indicated by orange triangle). The number of cases that have been reported in the literature that have been analyzed by IT for each type is indicated, with the percent accuracy in parentheses. The bottom row represents the resulting mRNA structure due to the activated cryptic splice site.\n\nThe frequency of validated cryptic splice acceptors (A) and donors (B) occurring at positions relative to the natural splice site. Positions are given using ASSEDA coordinates. Lower panel expands the cryptic site distribution of the region circumscribing the natural splice site.\n\nAnother bioinformatic method for cryptic site recognition relies on a training set composed of cryptic sites that are known to be used63. There are a number of drawbacks to this approach: the training set is itself not representative of all cryptic sites; and sites that are altered but unused cannot be discriminated from those that are activated (since the latter group also depends on the strength of the corresponding natural splice site). IT-based methods rank cryptic and cognate natural site strength in a way that predicts whether the site will be activated, as well as the abundance of each pair of splice isoforms. Furthermore, the structures of the prospective isoforms are presented by ASSEDA with relative quantitation of each, both prior to and post-mutation.\n\nDuring our review, we noted 203 variants with experimental support for cryptic splicing (Supplementary Tables 6–8). Of these, 38 variants resulted in Type 1 cryptic splicing. From those, site activation (existence of the site and strength ≥ 2.4 bits22) was correctly predicted by ASSEDA in 34 cases (89.5%). We identified 56 variants resulting in Type 2 splicing, 38 of which (67.7%) were accurately predicted, while the remaining 119 variants resulted in Type 3 cryptic splicing and 99 (90.8%) of the alternate splice sites matched predictions.\n\nPrediction of Type 3 cryptic splicing was more accurate than Types 1 or 2. The criteria for concordance with experimental data were that ASSEDA predicted both the cryptic site and that the variant weakened the natural site. However, the strength of a site is not the sole determinant of whether or not a site is activated. Unlike natural sites, novel cryptic sites are not under selection to maintain binding to the spliceosome, and their genomic context is less constrained than natural splice sites. The presence of cooperative splicing enhancer or repressor elements adjacent to cryptic sites, which could influence cryptic splice site activation, is not yet predictable. Additionally, many of the reported activated cryptic sites have been confirmed using non-quantitative approaches, and these may not constitute the predominant splice forms relative to constitutive exons with stronger natural sites. Finally, certain isoforms may not be detected; as aberrant transcripts are often subject to degradation and the tools used to evaluate functional splicing consequences do not always have sufficient resolution to distinguish small differences in isoform structure. All of these factors can affect the concordance of predicted cryptic site activation with experimental validation.\n\nWe also separated each sub-group of cryptic splice variants by location (intronic vs. exonic) and computed the average difference in strength between pairs of natural (post-mutation) and the activated cryptic sites. For intronic Type 1 variants, activated cryptic sites were 0.86 ± 5.28 bits stronger than the corresponding natural site (N = 12). There were eight Type 1 variants (4 at acceptors and 4 at donors) that were missed, because the Ri,final value of the natural site exceeded the strength of the corresponding cryptic site by ≥ 1.0 bits (variants with ΔRi < 1.0 bits are not reliably detected experimentally). We hypothesize that these cases could be explained by concomitant changes in surrounding regulatory binding site sequences. Exonic Type 1 variants were often slightly weaker than their cognate natural sites (-1.1 ± 3.8 bits; N = 26). Nearly all of these involved ectopic donor site activation (12 of 13), consistent with a processive mechanism for donor site recognition, which searches downstream from the acceptor splice site to the first donor site of sufficient strength to form an exon35. The opposite pattern was observed with intronic Type 2 cases, in which 20 of 21 exceptions occurred at acceptor sites. On average, the activated cryptic site exceeded the strength of the cognate natural site (1.3 ± 4.6 bits; N = 57). Activated, exonic Type 2 acceptor cryptic sites tended to be weaker than their natural site counterparts (-2.2 ± 3.3 bits; N = 4). This result may be attributable a low sample size, with 2 of these mutations exhibiting natural sites that were stronger (≥ 1.0 bits) than the corresponding cryptic site (1 donor and 1 acceptor). Finally, Type 3 activated intronic cryptic sites exhibited the greatest difference between the strengths of cryptic sites and cognate natural splice sites (6.3 ± 4.9 bits; N = 104). This category contained the fewest number of exceptional cryptic sites, with Ri values less than those of natural sites (5 acceptors and 3 donors). This is consistent with the idea that the intronic cryptic sites are generally not under selection for adjacent functional regulatory binding sites, and, in order to be activated, are required to be substantially stronger than the natural site. Although Ri,final values were stronger (2.1 ± 1.9 bits; N = 20) than the natural site, exonic Type 3 cryptic splice sites did not show as great a difference in strength with a single exceptional case (of an acceptor). Despite these exceptions, activated cryptic splice sites are generally stronger than the corresponding natural splice sites22.\n\n\nCombinatorial effects\n\nWhile functional natural splice sites and an intact BPS are integral for accurate and efficient splicing, other genetic elements have been shown to make essential contributions to exon definition64. Introns will often contain more than one potential splice site recognition sequence, but nevertheless, the correct natural site is consistently selected59. Differences among the strengths of potential sites, as determined by IT analysis, are a major, but not the sole, determinant of splice site utilization. The implication is that additional sequences within the gene are necessary to ensure specificity and precision of exon recognition. Studies of facultatively expressed alternative exon structures have revealed cis-acting sequence elements that function to enhance or repress exon recognition. These sequences cooperate with factors that recognize natural splice sites, whose sequences and relative strengths can vary considerably. Depending on their context, these elements have been referred to as exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs) or intronic splicing silencers (ISSs). In general, these elements serve as binding sites for trans-acting elements, which will either promote or impede the spliceosomal recognition of a splice site. The majority of enhancer elements will act through the recruitment of SR proteins and associate components of the U1 and U2 spliceosomes65,66. Silencers are often of the hnRNP class, which act through a diversity of mechanisms including steric hindrance, the formation of dysfunctional complexes, or blocking processiveness67–69. To add to the complexity of splicing regulation, it has recently been shown that SR protein function is dependent on context, i.e. whether the corresponding binding site is intronic or exonic70,71.\n\nTo improve accuracy of exon definition, the strengths of regulatory elements (i.e. their Ri values) have been incorporated into splicing mutation prediction. The significance of regulatory elements in disease has been demonstrated in many cases. For example, in the NF1 gene, ESE disruption is the primary cause of exon skipping72. Many other genes, including APC, SMN, BEST1, PDHA173–76 have been proven to harbour variants that disrupt ESEs and have a confirmed impact on mRNA splicing.\n\nAdding to the complexity, the recognition sequences for these RNA binding factors, while well defined, tend to be short, and can vary to the degree that the same sequence may contain overlapping elements of binding sites for multiple factors. However, this does not necessarily imply that such a sequence is bound with similar affinity by each factor or that it contributes to exon definition. At the same time, these sequences tend to be evolutionarily conserved and may be required for proper splicing77,78.\n\nASSEDA optionally incorporates PWMs for regulatory binding sites for mutation analysis (Table 1) in addition to the default donor and acceptor sites. The program selects the most proximate predicted ESE/ISS to the natural splice site when calculating Ri,total. The molecular phenotype, which dictates the splice isoforms that are predicted and their relative abundance, accounts for both the potential effect on the natural site and the most relevant splicing regulatory site. For these regulatory binding sites, a second gap surprisal term specific to the ESE/ISS of interest is applied to the Ri,total calculation35. The gap surprisal functions for SF2/ASF and SC35 have been previously described35, where the most common distance of the ESE/ISS is within 10nt of the natural site. The gap surprisal penalty gradually increases with distance from the natural site. Gap surprisal distributions for ELAVL1, TIA1 and SRp55 show a similar pattern, while hnRNPA1 and PTB binding sites are strongly clustered around splice junctions. It should be feasible to include the contributions of multiple splicing regulatory binding sites of the same or different RNA binding proteins in determining Ri,total; however this capability had not yet been implemented. Currently, if multiple sites of the same type are altered, the strongest (before or after mutation) is chosen by ASSEDA software.\n\nReported dominant effect is bolded. E – Enhancer; S – Silencer; N - Neutral.\n\niEnhancer activity by hnRNP A1 occurs at the junction84. iiPTB does not directly enhance splicing, but can do so indirectly by preventing the binding of splicing repressors91.\n\nAlthough the disruption of splicing regulatory sequences can cause aberrant splicing, the interpretation of variants affecting these sites is not as straightforward. Due to their degenerate nature, short sequence, and a lack of understanding of the context of their use, altered regulatory sites should be functionally validated before being deemed pathogenic7. Using variants from a number of different studies, ASSEDA accurately predicted experimentally determined changes in binding at a splicing regulatory site 75% of the time (N = 12)35. However, there were instances where regulatory sequences had been analyzed by IT, and considered to contribute to disease, but the results were not reproducible. For example, Kölsch et al. (2009) described SNPs associated with Alzheimer’s Disease, one of which strengthened and created SRp40 and SRp55 sites, respectively, but were reported by authors to be abolished94. This study did not report any evidence to support the significance of these predictions.\n\nFunctional validation of the effects of these mutations could contribute to understanding the roles of these factors in regulating constitutive splicing. Similarly, there is still little understanding on how multiple regulatory binding sites within the same region function as a unit. Using a pull-down assay, Olsen et al. (2014) demonstrated how different variants affect the binding of multiple regulatory proteins. One mutation was predicted to create and strengthen multiple hnRNPA1 sites and slightly strengthen an SF2/ASF (SRSF1) site. The pull-down studies showed up-regulation of hnRNPA1 binding and a decrease in SF2/ASF binding. However, SF2/ASF binding increased when a mutation disrupting hnRNPA1 affinity was introduced, suggesting that the strong hnRNPA1 sites outcompete the weaker SF2/ASF site.\n\nIn some instances, alterations in regulatory splice site recognition sequence and natural splice strength occurred concomitantly, with both predicted to have similar effects on splicing. Alteration of a regulatory sequence can sometimes provide a plausible explanation for discordant in silico prediction and experimental validation. As an example, Smaoui et al. (2004) analyzed a donor site mutation (NM_001040667:c.1327+4A>G) in HSF4 in a family with congenital cataracts50. This variant was predicted to cause leaky splicing (Ri,final = 5.4 bits; ΔRi = -2.6 bits; 67.5% residual binding), however RT-PCR showed complete exon skipping. Our further analysis showed that it is predicted to also create an overlapping hnRNPA1 site (Ri,final = 4.2 bits; ΔRi = 17.1 bits). Another case involved a mutation in the XPC gene (NM_004628:c.2033+2T>G) that created a novel intronic cryptic site 4 nt downstream of a natural donor site95. However, a weaker site 68 nt downstream from the natural site was activated. A possible explanation could be that activation of the cryptic site is influenced by a neighbouring hnRNPA1 site that is itself strengthened (Ri,final = 5.2 bits; ΔRi = 2.2 bits) and an SRp55 site that is significantly weakened (Ri,final = 1.9 bits; ΔRi = -4.0 bits).\n\nThe effects of changes in regulatory binding site strengths may ascribe potential functions to previous VUS. For example, Maruszak et al. (2009) present a PIN1 variant associated with late-onset Alzheimer’s Disease (NM_006221:c.58+64C>T)96. Based on IT, it is expected to abolish an intronic SC35 site, which could have either an enhancing or silencing effect (Table 1). A 2.82-fold decrease in transcript levels was demonstrated, which is concordant with previous findings reporting decreased PIN1 levels in the brains of Alzheimer’s Disease patients. Another study described an exonic missense variant within the ETFDH gene in a patient with multiple acyl-CoA dehydrogenase deficiency (NM_004453:c.158A>G) that showed evidence of exon skipping. The variant was predicted to be “benign” or “tolerated” when evaluated with PolyPhen and SIFT23. ASSEDA, on the other hand, predicted the creation of an hnRNPA1 site (Ri,final = 5.9 bits; ΔRi = 17.1 bits), a slightly strengthened hnRNPH site (Ri,final = 4.0 bits; ΔRi = 0.2 bits), the abolition of an SRp40 site (Ri,final = -3.3 bits; ΔRi = -6.3 bits) and two novel, weak SF2/ASF sites (Ri,final = -4.6 bits; ΔRi = 0.8 bits and Ri,final = -2.4 bits; ΔRi = 0.4 bits)23. The natural donor site was unaltered by the mutation. As indicated earlier, the mutation was confirmed experimentally to increase hnRNPH and hnRNPA1 and decrease SRp40 and SF2/ASF binding.\n\n\nValidation of results\n\nA number of early mutation studies did not perform expression analysis and relied solely on the ASSEDA or ASSA server to interpret potential mutations. This is not recommended, as there are limitations to any in silico predictive method, which impacts accuracy and precision of the prediction. Assuming that the impact of the mutation on expression can be detected, experimental validation of IT-based mutation analysis can reveal its limitations. We describe the various validation methods that were employed in the articles where expression data were available. Below, advantages and disadvantages of these approaches are explored, as well as how lower sensitivity validation can result in misinterpretation. Finally, we determine the accuracy of IT-based prediction, and point out some instructive, discordant cases.\n\nThe two most widely used methods for validating mutant mRNA splicing isoforms have been RT-PCR analysis of patient mRNA, and transfection of minigene constructs expressing the mutated exon into cell lines, followed by RT-PCR. These assays were, in some cases, accompanied by other techniques such as direct sequencing of cDNA, Western blotting, luciferase expression assays or immunostaining. A number of studies used quantitative RT-PCR or real-time PCR to estimate isoform abundance. RNA or cDNA sequencing and exon expression microarrays were also used in several studies to support in silico predictions. Certain functional assays that we reviewed were unique to a single study, including: allelic instability, exon trapping, immunoprecipitation of splicing factors, and flow cytometry23,97–99. Other indirect methods of justifying the association between a splice site variant and disease included fundoscopy, loss of heterozygosity, blood protein levels, and segregation with disease100–103. Because a variant may result in aberrant splicing but might not be accompanied by a detectable phenotypic change, we excluded the results of indirect assays of phenotype. Indirect measures of phenotype can support disease association, but do not inform about accuracy of splicing prediction.\n\nEndpoint RT-PCR and minigene assays probe the specific variant in question, but do not reveal relative abundance of each isoform, whereas qPCR does. Neither method resolves mRNA sequence at the nucleotide level, which can fail to confirm predicted splicing mutations, especially in instances where a small number of nucleotides are retained at the constitutive splice junction104. The resultant frameshifted mRNAs can cause premature truncation of the transcript (PTC), instability, and NMD, leaving no evidence of the mutated isoform (unless the cells had been treated with an NMD inhibitor). A disadvantage is that in cases where the protein is not degraded, but still impaired or dysfunctional, the result will be incorrectly categorized as benign. For example, Wessagowit et al. (2005) used sequencing of a COL7A1 variant (NM_000094:c.341G>T) to demonstrate a 87 nt deletion in the cDNA105. The authors also performed immunostaining of the corresponding protein with a monoclonal antibody, which showed no difference between wild-type and mutant samples because the epitope was not disrupted by the deletion. Had the authors only performed the binding assay, the variant would have likely been disregarded. NMD can be a predominant cause of false-negative results when validating splice variants. When aberrant splicing causes a frameshift and PTC, translation of truncated proteins is prevented, which otherwise can have dominant negative effects or exhibit gain-of-function106. However, if these transcripts are degraded and only the normal allele is detectable (in the case of a heterozygote or leaky splicing), then the splicing prediction will not be supported. Interestingly, Khan et al. (2004) were able to show that NMD had occurred by comparing levels of total message (qPCR) between wild-type and mutant samples47. Experimental methods have been developed to stabilize transcripts with premature termination of translation, thus circumventing NMD. The use of emetine, which inhibits translation and stabilizes RNA transcripts, can increase the relative amount of aberrant transcript observed107,108. However this approach can induce a stress response within the cell and further transcription must be halted using actinomycin D. This combination was used by Bloethner et al. (2008) in an approach called Gene Identification by NMD Inhibition109. Similarly, the use of puromycin and cycloheximide were shown to inhibit NMD and restore predicted aberrant splice forms97,110. Furthermore, certain mutations proximate to the penultimate exon evade NMD111,112.\n\nA number of assays have been developed to confirm direct effects of variants on splice site recognition, however fewer methods are available to measure effects of mutations at binding sites of splicing regulatory proteins113. The most reliable approach is to associate a change in splicing with a change in regulatory protein binding. A combination of electrophoretic mobility shift assay and RT-PCR were used to confirm that a predicted change in an SF2/ASF binding site caused exon skipping in the CFTR gene114. Others performed RNA affinity purification in combination with Western blotting23.\n\nAnother approach tests multiple variants at the same position through minigene assays. Anczuków et al. (2008) observed that two variants at the same position in BRCA1 (c.3600G>T and c.3600G>C) predicted different effects on regulatory sequences, as well as different observed effects on splicing115. The G>T variant predicted abolition of a SRp40 site and weakening of an SF2/ASF site by both ASSA and ESEfinder, and showed a significant reduction in the relative amount of normal transcript. The G>C variant, which did not elicit a change in splicing, was not predicted by ASSA to have a significant effect on either site (although ESEfinder predicted weakening of the SRp40 site below its default threshold). The difference in splicing efficiency could be due to the loss of binding by one or both of these regulatory proteins. This assay associates predicted changes to regulatory protein binding site strength to changes in splicing. A direct binding assay would lend key support for such predictions.\n\n\nAccuracy of IT-based prediction\n\nWe previously evaluated the accuracy of IT-based prediction using a set of validated splicing mutations (85.2%; N = 61)25. Other studies have also evaluated the accuracy of ASSA/ASSEDA while evaluating differences between multiple predictive programs and have shown varying levels of concordance (68.8%, N = 16; 90.1%, N = 22; 100%, N = 24)51,104,116. With a comprehensive list of all published variants analyzed using IT-based methods (Supplementary Bibliography), we perform a meta-analysis of all of these variants to minimize bias in interpretation and impact of ascertainment of specific phenotypes from individual studies. The list of variants is more extensive than any previous study examining accuracy of IT-based methods. The variants are not restricted to a single or even group of diseases, but rather cover over 150 different conditions (see Supplementary Table 2).\n\nIn total, 905 variants were reported in 122 different publications to have been validated for their effects on splicing (1,727 total variants analyzed from 216 papers – Supplementary Table 9). In all cases, the authors performed information analysis; however, the validation experiments were sometimes contained in the original reports and in other cases, later studies. In a minority of mutations, the validation results were either uninformative (N = 36) or the methods did not directly imply an effect on splicing (N = 2); these mutations were therefore excluded in determining the accuracy of predictions (shaded in grey in Supplementary Table 9).\n\nMore specifically, in order for experimental results and predictions to be considered concordant, one or more of the following criteria had to be met:\n\na. A variant predicted to abolish a splice site did abolish splicing, with no residual splicing observed. Exceptions to this were assays in which both the mutant and wild-type alleles were expressed in the same cell line or patient sample, and could not be discriminated from one another (i.e. RT-PCR);\n\nb. A variant predicted to be leaky exhibited residual normal splicing, with the exception of cases where a much stronger cryptic splice site was activated;\n\nc. A variant that strengthened the natural site and showed normal or increased levels of the wild-type isoform, consistent with it having a benign phenotype and/or polymorphic;\n\nd. A variant predicted to activate a pre-existing splice site, while also reducing the natural splice site strength, was demonstrated experimentally to result in cryptic splicing, regardless of whether it was predicted it to be the predominant isoform;\n\ne. A variant predicted to affect a splicing regulatory protein-binding site was consistent with validation experiments explicitly assessing binding affinity and associated splicing alterations.\n\nCumulatively, 87.9% of variants documented by expression studies (762 of 867) that satisfied these criteria were accurately predicted by ASSEDA. A minority of papers reported variants to be “partially concordant” (3.1%; 27/867), meaning that while the cryptic site observed was predicted, it was not the most likely splice isoform relative to other expressed cryptic exons. Because this method of scoring met our criteria (see point d above), we included these in our determination.\n\nLimitations of both the predictive model and the validation data/methods were the primary reasons for discordance. Where information analysis predicted a neutral change or no effect, but validation showed aberrant splicing, we hypothesize that there are either unrecognized splicing regulatory protein binding sites that are weakened or abolished, or that there are underlying mechanisms that are not currently addressed by current information models23,35,50–52,54,96,98,99,114,117–127. The validation methods used can also contribute to discordant results. We note that 41 discordant results originated from one of our own studies41. This study used RNAseq data to validate predictions, a genome-wide approach that should be used with caution when inferring changes resulting from potential splicing mutations. Until this study was published, IT-based mutation analysis was based on single or candidate disease gene studies. RNAseq reveals all changes in transcript levels for all genes, which although potentially relevant to splicing, may not necessarily contribute to the phenotype in question. This leads to the possibility, especially in cancer phenotypes, of bystander effects (global splicing dysregulation, natural alternative splicing) that are not directly attributable to the predicted mutations. Furthermore, because the sequence reads at splice junctions are short and often limited in number, a relevant splicing aberration may result from a given variant, but it was not detectable. Finally, the predictions of IT can pick up variants that should alter splicing for example, of rare recessive alleles, that that may not have any disease relevance.\n\n\nMisinterpretation of variant effects\n\nWhile preparing this review, several variants misinterpreted with IT-based tools were noted. These variants have been re-analyzed to disseminate the correct findings and to avoid making similar errors in the analysis of newly discovered variants. Supplementary Table 2 contains these results. The most common problems result from unfounded emphases on altered or pre-existing cryptic sites that are determined to be significantly weaker relative to the cognate natural site109,128–132, and from selectively reporting a single change in the Ri value when, in fact, multiple significant changes can be detected48,128,133–136. An example of the first type of error is exemplified by a variant in CGI-58 (ABDH5), where the natural splice site is 9.1 bits (or ≥ 549-fold) stronger than the reported cryptic site129. Henneman et al. (2008) selectively reported the effect of a mutation that weakens a natural donor splice site in APOA5, however only a change in the information content of an SC35 binding site was indicated.\n\nOther common problems include incorrect declaration of small ΔRi values as significant changes109,137,138, use of incorrect Ri,min values139,140, and the computation of predicted binding strength changes on a linear scale141 rather than the correct exponential function (i.e. ≤ 2ΔRi)18. Smaoui et al. (2004) described an 8.0 bit donor site as weak, which is actually equivalent in strength to Rsequence, the average strength33. Allikmets et al. (1998) and Ozaltin et al. (2011) both described an inactivating mutation as leaky, because the weakened site remained above the Ri,min. However, the variant mutation produces a site with < 0.7% of its original binding affinity, which would substantially reduce exon recognition and lead to exon skipping116. Also, cryptic sites created in the promoter regions of genes should not be considered to be splicing mutations142. Variants that are predicted to create a cryptic site upstream or overlapping a natural site of the opposite polarity (i.e. cryptic donor upstream of a natural acceptor) have been reported131,132,143, which would be inconsistent with established splicing mechanisms35. A rare exception that could render such a site active is to the creation of a cryptic exon that occurs in conjunction with a proximate, correctly oriented, pre-existing cryptic splice site of opposite polarity22,30. Insufficient numbers of examples of mutations creating cryptic exons have been reported to date for ASSEDA to accurately predict these exons by default.\n\nSeveral results were generated by incorrect entry of mutations in to ASSA/ASSEDA. For example, altered cryptic splice sites have been confused with natural sites48,137,144,145. Additionally, ‘residual binding strength’ displayed has been misinterpreted as a percent decrease144,146. Strong, pre-existing cryptic sites outside of the default sequence analysis window (54 nt circumscribing the mutation) have also been missed because the window was not expanded to include these sites147. Although the predicted isoform structure generated by ASSEDA will, by default, display skipping for mutated natural sites with ΔRi ≥ -7.0 bits (or ≥ 128-fold)116, smaller decreases in natural site strength of an internal exon can sometimes partially induce exon skipping. This value is adjustable, and it may be advisable to explore different thresholds depending on the particular susceptibility of a splice junction to exon skipping. Sharma et al. (2014) used the default threshold from ASSEDA to interpret CFTR mutations c.2988G>A (9.6 to 6.6 bits, natural donor site of exon 18) and c.2657+5G>A (9.1 to 5.6 bits, natural donor site of exon 16), but exon skipping was documented. IT analysis was not discordant for these variants, which significantly weaken the corresponding splice sites by ≥8- and 11-fold, respectively, and has been shown in other genes to lead to exon skipping, leaky splicing, or both of these outcomes. Aissat et al. (2013) tabulated variants that were predicted to affect strengths of ESE binding sites, but did not comprehensively report all findings even though predictions by ASSA and ESEfinder were concordant (eg. CFTR: c.1694A>G). Alternate mutation entry methods, which do not use contextual gene name annotations, such as entry by rsID, report predicted binding changes on both strands. A report indicating abolition of SRp40 binding sites on the antisense strand was confused with binding sites for CYP46A1, which is transcribed from the sense strand148.\n\nOther problems include inadvertent mislabelling of splice site type or location149–151, interchange of the terms information content and change in information (Ri and ΔRi)122, and unclear variant interpretation (i.e. “run on into the intron”)152. Moriwaki et al. (2009) claim ASSA did not predict a mutated natural donor site, but in fact, the site was present in our reanalysis153. Published Ri values from Rogan et al. (1998) and von Kodolitsch et al. (1999) are in some instances different from current values due to updates of the reference genome sequence. Nevertheless, the overall predicted effect did not change, but initial and final Ri values were inconsistent. Interpretations of certain mutations could not be reproduced in some instances103,145,154–156. Finally, we noted that ASSEDA can sometimes improperly parse indels entered using c. or IVS notation. Such errors have led to published false results67,116,157,158.\n\n\nInterpretation of published variants in studies that use information analysis\n\nThe severity of phenotype due to splicing mutations can be related to their effects on mRNA splicing, after careful consideration of the overall impact on mRNA levels and protein coding159. Significant information changes (where ΔRi ≥ 7.0 bits or where Ri ≤ 2.4 bits) of splicing variants in hemophilia patients (F8C and F9) were shown to correspond to the severe clinical phenotypes of the disease (reduced protein activity, increased clotting time, bleeding frequency)127. The overall effect on the coding potential of the mutated transcript should be considered, as skipping events that maintain the reading frame commonly lead to milder phenotypes160–162. Nevertheless, two variants that abolish splice site recognition in PTPRO in Idiopathic Nephritic Syndrome reported by Ozaltin et al. (2011) had similar phenotypes even though one retained the reading frame and the other caused a frameshift. The exon deleted by the in-frame skipping event is highly conserved53. Exon skipping events that cause frameshifts close to the carboxy-terminus may lead to mild phenotypes, as they avoid NMD112–163. Dominant negative mutations with either Ri > Ri,min or with modest decreases in ΔRi, may be less likely to cause severe phenotypes, as a residual amount of the natural isoform continues to be expressed103,117,141,164–168. The impact of cryptic site-activating variants on phenotype can be similarly assessed. Activated cryptic sites which shift the reading frame have been shown to be more severe clinically relative to those which maintain the same reading frame as the native gene105,162,169,170.\n\nIT-based tools exhibit high specificity for analysis of splicing neutral variants in hereditary breast/ovarian cancer and other disorders116. These predictions can reduce the requirement for experimental validation of low-priority candidate mutations with minimal changes in information content14,22. IT analysis has been used in numerous studies to infer neutral effects of variants14,34,97,109,116,119,128,129,151,156,157,171–185. Similarly, variants that strengthen natural splice sites186–188 are also likely to be neutral, though these variants can increase retention of exons that are otherwise frequently alternatively spliced189,190. However, binding site variants with minimal splicing information changes may still alter mRNA processing by disrupting mRNA secondary structure191.\n\n\nPolymorphisms and splicing\n\nEarly studies suggested that common polymorphic sequence variations at splice sites corresponded to small ΔRi values, consistent with these changes having little impact on mRNA abundance22. More recently, it has been appreciated that certain rare SNPs have significant genetic loads, can actively alter mRNA splicing profiles, and lead to non-obvious splicing phenotypes58,189. Nevertheless, it is not uncommon for reports to solely analyze novel variants and ignore known SNPs136,156,158,192, or limit results only to those that occur in the vicinity of natural splice sites184. We find that 56.4% of common SNPs (with population frequencies ≥ 1% in Supplementary Table 2) within natural sites significantly alter their strength (12.8% abolish and 28.2% cause leaky splicing, 15.4% modestly strengthen sites [ΔRi < 2.6 bits]), and 43.6% have insignificant ΔRi values, as expected (N = 39). The mean Ri,final and ΔRi values, for these natural sites are 7.9 ± 4.0 bits and -1.4 ± 3.0 bits, respectively, which suggests the effects of these polymorphisms on splicing are nil to limited. However, polymorphisms can significantly modulate splicing, as some common SNPs are predicted to abolish natural splicing (Supplementary Table 2: #1291, 1296, 1431, 1435, and 1436). These include rs10190751 in CFLAR, which modulates the production of two short isoforms, and is associated with an increased risk of lymphoma189,193, rs3892097, which alters exon inclusion in CYP2D630 and leads to a non-functional protein and altered drug metabolism194, and rs1805377 in XRCC434, which has been associated with oral cancer susceptibility195 and increased risk of gliomas196. There is also experimental support for common SNPs that have been predicted to affect splicing22,98,107,110,114,118,149,164,189,197. For example, experimental evidence for increased exon inclusion has been described for three of six SNPs that increase strength of natural splice sites189,190. Numerous common SNPs, which were either deemed neutral or predicted to affect splicing, have not been confirmed experimentally14,22,25,34,52,94,99,131,133,144,145,148,151,165,166,168,179,183,185,188,198–208. Polymorphisms with significant information changes should be investigated, as they may not be completely benign and can have a significant impact on mRNA splicing.\n\n\nInference of variant pathogenicity by IT analysis\n\nRecently, American College of Medical Genetics and Genomics recommendations for reporting incidental findings in sequencing have suggested that bioinformatic predictions are not sufficient to declare clinical significance209. Preceding the publication of these guidelines, numerous peer-reviewed articles suggested variants analyzed by IT to be causative/pathogenic/disease-causing, without confirmation of the predicted splicing effect101,135,137,150,160,167,178,205,210–218. Other authors have qualified the interpretation of bioinformatic results with less certain terms (i.e. ‘suggest’ and ‘likely’ pathogenic)110,112,176,219–223. Leclerc et al. (2002)165 state that a predicted variant confirmed to affect splicing is likely deleterious, but could not be unequivocally shown to cause the observed phenotype. Although IT predictions can relate a sequence change to the resultant phenotype, caution should be exercised when deeming a predicted splicing variant as pathogenic in the absence of other functional evidence. The high level of concordance between IT mutation analysis and experimental findings indicates that this approach, in conjunction with other evidence, can be used to detect splicing effects, which may be used to explain disease phenotypes.\n\n\nComparisons to other software programs\n\nThere are now over a dozen other publically available splicing prediction tools, some using strategies similar (MaxEntScan [MES]) and others, which are quite different (NNsplice) that are compared with IT224,225. Vreeswijk et al. (2009) assessed the applicability of different splice prediction programs to diagnose BRCA1/2 variants. These authors recommended that the outcome of 3 programs was sufficient for analysis, unless all three predictions were discordant from one another (2 for false positive predictions). Despite the obvious appeal of consensus between different analytical methods, a major caveat in using polling strategies for mutation assessment is that these approaches are prone to both systematic and sampling errors40.\n\nWe summarize results of 36 publications that used both IT-based prediction tools and one or more alternate prediction tool (14 for 5´ and 3´ splicing, six for splicing regulatory proteins) to assess mutations23,39,97,99,103,111,114–117,123,130,132,141,147,156,158,165,166,171,179,185,189,197,210,218,226–235. The analysis performed by the authors allowed us to compare the similarity of predictions to those programs and IT in Table 2a and Table 2b. Those most commonly used for 5´ and 3´ splice sites (NNsplice, MES, NG2, HSF and SSF) were highly concordant for natural sites (85.4% for donor and 77.6% for acceptor sites; Table 2a). Discordance of acceptor predictions may be due to methodologies that do not analyze the complete acceptor site (HSF analyzes only 14 intronic nucleotides upstream of acceptor splice sites)236. Some programs (SSF, HSF) exhibit greater concordance with IT for cryptic splice site prediction (96% for donor and 76.9% for acceptor sites). The level of discordance between IT and other commonly used software programs (59.5% for donor and 60% for acceptor sites) may be attributable to the empirically-derived scoring thresholds and the validation sets used to predict mutated splice sites. Models that are typically built (or trained) using known natural splice sites may be less sensitive for differentiating true cryptic splice forms from decoys in the genome, which tend to be weaker than natural splice sites. Tools are highly consistent when analyzing variants expected to be neutral with respect to splicing (100%; N = 71). Colombo et al. (2013) compared nine programs to evaluate accuracy in predicting mRNA splicing effects and reported that ASSA, along with HSF, demonstrated 100% informativeness and specificity.\n\nASSEDA has also been used to analyze RNA binding proteins that enhance or silence exon recognition (Table 2b). ESEfinder was used for 42.2% of these mutations in one or more regulatory binding sites237,238. However, variants predicted by ESEfinder to have deleterious effects are discordant with some IT predictions (6 of 15; Table 2b). The discordance with ESEfinder may be associated with differences in the respective analytic methods, as several of the models (SF2/ASF, SC35, SRp40) used by ASSA and ESEfinder were created from the same source of experimental data87,239. While the majority of the discordant results were cited in a single study114 (5/6 variants), the small size of the dataset (ranging from 28–34 sites) may artificially exacerbate differences between these results. In multiple instances, ASSA has been used to analyze SR proteins, but other programs were used to analyze 5´ and 3´ splice site mutations23,99,115. This was surprising, since the donor and acceptor Ri values are generated by default by ASSA and ASSEDA. The advantage of performing both constitutive and regulatory splice site analysis with IT is that all results are reported on the same scale, and the strengths of all interactions, and effects of mutations are directly comparable to one another.\n\nConcordance was assessed from the analysis of variants from 36 publications which used IT-based tools and a secondary predictive method. Each value corresponds to the number of variants that were concordant with IT-based tools versus the total number of variants for each category. 1 – includes Vreeswijk et al. (2008), which may not have properly reported predicted cryptic sites, as they did not report any cryptic sites predicted by ASSA beyond the default window size (54 nt) from the mutation. 2 – predictions made using the SSF-like algorithm in the Alamut splicing prediction module were combined with the SSF category (SSF is no longer supported). 3 – Aissat et al. (2013) contributes highly to the discordance of these programs, and may be due to improper reporting/analysis. 4 – Mutations predicted by alternate program to affect SR protein to which ASSEDA has no model (i.e. 9G8) were not included in statistics.\n\nMES – MaxEntScan; BDGP – Splice Site Prediction by Neural Network, NNSplice; NG2 – NetGene2; HSF – Human Splice Finder; SSF – Splice Site Finder; SSqF – Splicing Sequences Finder; GS - GeneSplicer; SV – SpliceView; SP – Splice Predictor; SS - Shapiro-Senapathy; GenS – GenScan; ASD - ASD-Intron analysis; GeneS – GeneScan; GM – GeneMark; PESX - Putative Exonic Splicing Enhancers/Silencers.\n\n\nOther applications of information theory-based splice site analysis\n\nThe use of IT to analyze splicing is not limited to sequence variant analysis. The natural and alternative splicing of several genes have been characterized using this method107,200,240. Khan et al. (2002) scanned all natural sites in the XPC gene and found a weak acceptor (-0.1 bits), and with RT-PCR found that this exon (exon 4) was skipped to a greater extent than another (exon 7), which possessed a strong acceptor, illustrating a relationship between the information content of a natural splice site and its level of alternative splicing. IT has also been used in genetic engineering in the design and alteration of binding sites, and has been used in the design of constructs for transgenic animal models241–243. Thus, IT-based splice site analysis can be adapted for other important molecular genetic applications.\n\n\nGuidelines for information theory-based splicing mutation analyses\n\nOur comprehensive review of the use of IT in splicing mutation analysis has led us to propose general recommendations, which we formulate as guidelines. Adoption of these guidelines should ensure the accurate and comprehensive results from IT analyses of VUS and other pathogenic variants that alter mRNA splicing.\n\nWhen analyzing a variant with ASSEDA, the user is prompted to select an mRNA isoform (GenBank or RefSeq accession) from the gene in question. When entering the same variant (in either IVS or c. notation) for different isoforms, either the variant will parse one but not the other representation, or the variant syntax will be processed for both. In the first situation, the user is prompted to verify the position and substitution, which may elicit the realization that the incorrect isoform had been selected. However, in the case where the variant can still be parsed (despite being incorrectly entered for the isoform selected), an incorrect nucleotide may coincidentally have the same sequence, and the user may not necessarily realize that the intended position is not being analyzed. We were unable to reproduce results for several variants, because the mRNA or gene isoform was not reported. This issue could be resolved by comparing the genomic sequence in papers where the context of the mutation was included50,95,141,179,244–246. Where flanking sequences were unavailable, the location of the mutation was inferred from either descriptions in the text, the corresponding Ri value of the splice site, or relative coordinate numbering144,247,248. Although we attempted to reproduce all the results, this was not always possible if the specified sequence was ambiguous or the source was deprecated (GenBank accession numbers, BAC clones, etc.)48,97,172,179,180,208,227,232,249,250.\n\nWe note that ASSA/ASSEDA cannot account for genes with redacted exons, where the exon numbering or sequence in the original mRNA accession has not been corrected. A well-known example is BRCA1, for which the constitutive isoform lacks the exon designated as number 4. IVS notation beyond this point in this gene must be reduced by one intron. Alternatively, one of the HGVS-approved methods can be used to input variants, or the variant can be designated with the genomic coordinate (g.) format. Review of ASSA/ASSEDA output (coordinates and/or the sequence walker20) is a prudent approach to confirm that the correct region has been analyzed.\n\nTo eliminate ambiguity, we recommend that reported variants be accompanied by the accession number used in its analysis (consistent with HGVS notation36) and the genomic coordinates with the corresponding reference genome build. The table of results from ASSEDA or Shannon pipeline output could also be included as supplementary published material. This will ensure that reported results can be reproduced and compared to other experimental or in silico results.\n\nThe results generated by IT software provide Ri,initial, Ri,final, and ΔRi for donor and acceptor sites by default, and for all other ribl matrices selected. Reporting these values along with the interpretation improves the clarity of said interpretation. Several publications have not reported Ri, and instead only the interpretation of these values125,138,146,212,227,251,252. This presumes that the analysis was performed correctly, and accurately interpreted. In one instance, our reanalysis differed from the published interpretation138. Other publications provide Ri values, but were incorrectly reported, which resulted in misinterpretations48,122,253. Simply reporting ΔRi itself does not provide sufficient information about the context of the mutation or possible cryptic splice sites, which is necessary to fully appreciate the resultant effect on splicing136,245,254. We recommend Ri values be provided for each variant analyzed. We also suggest that the specific donor and acceptor ribl used for variant analysis be indicated, because of the differences obtained using the genome-wide and original PWMs in IT analysis30,33. The distinction can also be significant, when the Ri,final value of a mutated splice site approaches Ri,min.\n\nMissense and synonymous mutations can alter natural splicing, create cryptic sites, and alter crucial ESE and ESS binding sites255. IT tools have been employed to analyze exonic variants that strengthened or create exonic cryptic sites, which were also confirmed experimentally25,39,41,43,98,105,116,124,130,149,151,178,256,257. Similarly, IT tools can predict potential effects on strengths of SR and hnRNP protein recognition sites23,117. There is no justification for cataloguing intronic and exonic variants, but only assessing splicing effects for the intronic variants or those within natural splice sites119,132,175,186,208,210,214,215,248,258,259. We recommend that IT-based analysis should evaluate all variants within a gene for potential splicing mutations.\n\nMany studies have reported only coding changes and the results of IT (or other in silico) analyses without experimental validation. Our review indicated that IT-based splicing predictions are highly concordant with validation results (87.9%) Nevertheless, the discordant mutations support the need for robust post-prediction validation, since even a single discordant result can lead to misdiagnosis. We do not detect any consistent pattern amongst the discordant predictions to provide guidance as to which IT analyses will be erroneous. Experimental verification will mitigate incorrect interpretations of IT predictions and has been recommended by others26.\n\nASSA/ASSEDA allows the user to alter size of sequence window range surrounding the mutation. The default window range has been set to maximize the speed of analysis, which is to some degree dictated by the number of matrices and the length of the sequence analyzed. Arbitrary abbreviation of the sequence analysis window can result in the failure to detect activated intronic or exonic cryptic sites, which can in some instances significantly lengthen (eg. 231 and 313 nucleotide extensions, respectively166,171) or shorten the corresponding natural exon. Therefore, we suggest expanding this window if one wishes to assess the possibility that long range, pre-existing cryptic splice sites may be activated.\n\nWe note that unequivocal prediction of cryptic splice site use in large exons (> 1000 nt) can be challenging due to the reliance of these gene regions on splicing enhancers, silencers, and other regulatory elements to prevent ectopic splice site use and ensure fidelity of splicing260. Di Leo et al. (2007) determined a variant abolishing the natural acceptor for exon 26 of APOB (7572 nt long), which activated a weak cryptic site 1180 nt downstream261. There are several other stronger candidate cryptic splice sites that occur between the natural and cryptic splice site, but there is no evidence that any are used in the individual carrying this mutation.\n\nComplex insertions and deletions in IVS or c. notation may occasionally be parsed to the wrong coordinates within a gene. Indels will parse properly when genomic coordinates are used. If IVS or c. notation is used, it is suggested that users confirm that the expected alteration of the mutation is correct by reviewing the sequence walker display generated by ASSEDA for all insertions, deletions and duplications.\n\n\nData availability\n\nF1000Research: Dataset 1. Dataset for mRNA splicing mutations in genetic disease, 10.5256/f1000research.5654.d38248262\n\n\nSoftware availability\n\nThe Splicing Mutation Calculator (SMC) is available at http://splicemc.cytognomix.com.\n\nhttps://github.com/F1000Research/splicemc\n\nhttp://dx.doi.org/10.5281/zenodo.12422263\n\nGNU GPLv3", "appendix": "Author contributions\n\n\n\nPKR conceived, coordinated, and directed this study. NGC compiled the literature and determined which articles were eligible for inclusion. NGC and EJM summarized the articles. NGC, EJM and PKR wrote the manuscript, which has been approved by all authors.\n\n\nCompeting interests\n\n\n\nPKR is the inventor of US Patent 5,867,402 and other patents pending, which underlie the prediction and validation of mutations. He is one of the founders of Cytognomix, Inc. which is developing software based on this technology for complete genome or exome splicing mutation analysis.\n\n\nGrant information\n\nPKR is supported by the Canadian Breast Cancer Foundation, Canadian Foundation for Innovation, Canada Research Chairs Secretariat and the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant 371758-2009). NGC received fellowships from the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit, and the CIHR Strategic Training Program in Cancer Research and Technology Transfer Program.\n\n\nAcknowledgements\n\nWe would like to gratefully acknowledge the efforts of Shannon Brown and Ben Shirley for creating Splicing Mutation Calculator software (SMC), which has been deposited in Zenodo (DOI: 10.5281/zenodo.12422).\n\n\nSupplementary Material\n\nSupplementary file containing Supplementary Figure 1 and Supplementary Bibliography.\n\n\nReferences\n\nKan Z, Rouchka EC, Gish WR, et al.: Gene structure prediction and alternative splicing analysis using genomically aligned ESTs. Genome Res. 2001; 11(5): 889–900. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nModrek B, Resch A, Grasso C, et al.: Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res. 2001; 29(13): 2850–2859. 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PubMed Abstract \| Publisher Full Text\n\nPagani F, Baralle FE: Genomic variants in exons and introns: identifying the splicing spoilers. Nat Rev Genet. 2004; 5(5): 389–396. PubMed Abstract \| Publisher Full Text\n\nWadt K, Choi J, Chung JY, et al.: A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma. Pigment Cell Melanoma Res. 2012; 25(6): 815–818. PubMed Abstract \| Publisher Full Text\n\nTiteux M, Mejía JE, Mejlumian L, et al.: Recessive dystrophic epidermolysis bullosa caused by COL7A1 hemizygosity and a missense mutation with complex effects on splicing. Hum Mutat. 2006; 27(3): 291–292. PubMed Abstract \| Publisher Full Text\n\nHertecant JL, Ben-Rebeh I, Marah MA, et al.: Clinical and molecular analysis of isovaleric acidemia patients in the United Arab Emirates reveals remarkable phenotypes and four novel mutations in the IVD gene. Eur J Med Genet. 2012; 55(12): 671–676. PubMed Abstract \| Publisher Full Text\n\nKang DH, Lee DH, Hong YH, et al.: Identification of a novel splicing mutation in the ARSA gene in a patient with late-infantile form of metachromatic leukodystrophy. Korean J Lab Med. 2010; 30(5): 516–520. PubMed Abstract \| Publisher Full Text\n\nBolisetty MT, Beemon KL: Splicing of internal large exons is defined by novel cis-acting sequence elements. Nucleic Acids Res. 2012; 40(18): 9244–9254. PubMed Abstract \| Publisher Full Text\n\nDi Leo E, Magnolo L, Lancellotti S, et al.: Abnormal apolipoprotein B pre-mRNA splicing in patients with familial hypobetalipoproteinaemia. J Med Genet. 2007; 44(3): 219–224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaminsky N, Mucaki E, Rogan P: Dataset 1. Dataset for mRNA splicing mutations in genetic disease. F1000Research. 2014. Data Source\n\nBrown S, Shirley B, Caminsky N, et al.: Splicing Mutation Calculator (splicemc.cytognomix.com): Initial release. Zenodo. 2014. Data Source" }	[ { "id": "6917", "date": "18 Dec 2014", "name": "Matthias Titeux", "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\nThe manuscript by Caminsky et al. reviews the use of Information Theory (IT) based tools to predict splicing and splicing defects and their possible consequences on the matured transcripts. It is well written and well organized to guide the reader.Following an introduction covering the basics of the splicing mechanism and signals on the pre-mRNA used by the spliceosome to define the exonic and intronic sequences, the authors described the mathematics behind in silico prediction and then focus on the use of their tools (ASSEDA, SMC and Shannon pipeline) and their evolution over the past decade. They review the possibilities and limitations of such tools and compare them to other splicing prediction softwares (HSF, SSF, NNsplice, ESEFinder, RESCUE-ESE…).Over the years, IT-based splicing predictions have made progress and the overall rate of predictions concordant with experimental validations is around 88%. It has thus become a valuable tool for geneticists and molecular biologists. The authors also list the most common mistakes made by researchers while using their tools, and the ways to avoid them. They also stress the difficulties in predicting the consequences of splicing defect in particular cases due to poorly defined ESE/ESS sequences, combinatorial effects of splicing regulatory proteins (SR proteins and hnRNPs) and large exonic sequence which contains a large number of cryptic donor and splice sites and thus their definition is dependent on the binding of these regulatory proteins.The manuscript is therefore of great importance for people that use such splicing prediction software as it presents their possibilities, limitations and the best way to report the results. Experimentally validated variants, associated with their predictions(should the authors properly report how the prediction was performed) will help to refine the tools.Such in silico tools are even more valuable in a genomic era where large number of variants are identified by deep sequencing (exomes, whole genome sequencing...) some of which being of unknown significance. Adding better splicing defects prediction (apart from the 2 bp most conserved in the natural splice sites) to the filters used in the prioritization pipeline of next generation sequencing projects should be considered.I therefore recommend the manuscript for indexation without reservations, if small minor issues listed below can be addressed.Minor issues :Figure 5 is not called in the text.Since the journal uses a numbered formatting style for the references, please add the number of the reference in sentences like “Smaoui et al. 2004 described….” (page 17), since it is easier to find the given reference among over 260.In the supplementary table 2, in the column “concordance (Y/N)” there is in some cases a “P” indicated whose meaning is not clear.", "responses": [ { "c_id": "1240", "date": "17 Mar 2015", "name": "Peter Rogan", "role": "Author Response", "response": "We have included a paragraph describing Figure 5 (p. 12, right panel).We have included a numerical reference at the end of each sentence that contains a cited reference in the text.Tables that contain the entry “P” in the “Concordance” column has been altered. The column header “Concordance (Y/N/P)*” is described by the key,“Y: Yes; N: No; P: Partial,” in the legend." } ] }, { "id": "7225", "date": "09 Feb 2015", "name": "Klaas Wierenga", "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\nThe paper by Caminsky et al. is a welcome and timely review of the complexities of pre-mRNA splicing, the relationship between splicing mutations, detection thereof by IT and/or laboratory, and new challenges posed by next generation sequencing.It is a rather lengthy and somewhat intimidating review, and I can imagine that many readers, even interested ones, may not make it all the way to the end, certainly not in one session. On the other hand, the review paper is likely to reside on the desk of molecular laboratory directors and and other genetics professionals with an interest in the molecular aspects of genetics.The review is well written, and the order to topics discussed is logical. Maybe the introduction to splicing is a little short, e.g. little space is dedicated to discussing the spliceosome. The review of the various splice 'sensing' software, and the technology underlying these was in depth.The relationship between IT predicting splice mutations and laboratory studies to confirm the actual results of aberrant splicing was very well done, and the discussion of NMD and other causes of technical issues relating to demonstrating mutant mRNA resulting from splicing mutations was delightful.The discussion about laboratory standards (also related to ACMG recommendations) regarding splicing was excellent.Lastly, the discussion of the impact of splicing mutations and IT in the era of large datasets, including NGS was concise and accurate.In summary, this review ought to be mandatory reading for all genetics professionals in molecular laboratories, incl. those involved in whole exome/genome sequencing.The figures were well-selected, and the tables were helpful.One minor remark: I would mention PLP1 as the gene associated with Pelizaeus-Merzbacher disease (p6, R, middle para).", "responses": [ { "c_id": "1239", "date": "17 Mar 2015", "name": "Peter Rogan", "role": "Author Response", "response": "While we agree that a detailed understanding of the mechanisms underlying splicing is important to include, we have opted to guide the reader to other pertinent reviews on the spliceosome and relevant mechanisms, so as to not extend the length of this already lengthy article (modifications have been made to the Introduction – p.3 second paragraph on left panel). We do not discuss this in detail later on in the review. We have chosen to provide more details on components and processes of splicing that we later discuss in our review, such as splicing regulatory factors, donor and acceptor sites, the branch-point, etc.We have stated that PLP1 is the gene associated with Pelizaeus-Merzbacher disease (p6, R, middle para). This prompted us to do the same for other diseases and genes that were mentioned throughout the text including: NF1 (neurofibromatosis type 1 disease – p.3 and p.13), MSH2 (hereditary nonpolyposis colon cancer – p.3), XPC (xeroderma pigmentosum – p.9), MYBPC3 (hypertrophic cardiomyopathy – p.9), COL7A1 (recessive dystrophic epidermolysis bullosa – p.15), CFTR (cystic fibrosis – p.15), BRCA1 (hereditary breast/ovarian cancer – p.15), CGI-58 (Dorfman-Chanarin syndrome – p.16), APOA5 (hypertriglyceridemia – p.16), CYP46A1 (Alzheimer’s disease – p.17), APOB (familial hypobetalipoproteinaemia – p.20)." } ] } ]	1	https://f1000research.com/articles/3-282
https://f1000research.com/articles/3-170/v1	25 Jul 14	{ "type": "Research Article", "title": "Combining low sampling frequency smartphone sensors and video for a Wearable Mobility Monitoring System", "authors": [ "Hui-Hsien Wu", "Edward Lemaire", "Natalie Baddour", "Hui-Hsien Wu" ], "abstract": "A proof-of-concept Wearable Mobility Monitoring System (WMMS) was developed to identify daily activities and provide environmental context, using integrated BlackBerry Smartphone low sensor and video data. Integrated accelerometer data were used to identify mobility changes-of-state (CoS) in real-time, trigger BlackBerry video capture at each CoS, and save activity outcomes on the Smartphone. System evaluation involved collecting WMMS output and (separate) camcorder video under realistic conditions for five able-bodied subjects. The subjects each performed a consecutive series of mobility tasks; including, walking, sitting, lying, stairs, ramps, elevator, bathroom activities, kitchen activities, dining activities and outdoor walking. Activity, timing and contextual information were obtained from the camcorder for comparison. Sensitivity results for sensor-based CoS identification were 97-100% for standing, sitting, lying and taking an elevator; 67-73% for walking-related CoS (stairs, ramps); 40-93% between walking and small movements (brushing teeth, etc.); and below 27% for daily living activities. False positives occurred in less than 12% of all activities, with less than 5% false positives for half the measures. Better classification results were achieved when using both acceleration features and Smartphone integrated video for all activities except sitting. The evaluation demonstrated that the WMMS algorithm and BlackBerry platform were effective for detecting mobility activities, even with low sampling rate sensors. The combined sensor and video analysis enhanced mobility task identification and contextual information.", "keywords": [ "Wearable", "activity monitoring", "mobility", "change of state", "smartphone", "accelerometer", "video" ], "content": "Introduction\n\nUnderstanding how people move within their daily lives is important for healthcare decision-making. Typically, a person’s mobility status is self-reported in the clinic, thereby introducing error and increasing the potential for biased information. Functional scales can be administered to help gain an understanding of a person’s mobility status1–4. However, these tests do not measure how a person moves when leaving the clinic. A quantitative method of characterizing mobility in the community would provide valid and useful information for healthcare providers and researchers.\n\nWearable systems have been developed to evaluate mobility in any environment or location. These portable systems are worn on the body and collect mobility information within the home and community. Examples include accelerometer-based systems5–8 portable electromyographs (EMG)9, and foot pressure devices10. Some wearable systems can be used for long-term and low effort monitoring.\n\nSmartphones provide an ideal interface for mobility assessment in the community since they are small, light, easily worn, and easy to use for most consumers. These phones are multitasking, computing platforms that can incorporate accelerometers, GPS, video camera, light sensors, temperature sensors, gyroscopes, and magnetometers11,12.\n\nAs shown in Table 1, cell phones have been used to recognize multiple activities by analyzing the phone’s accelerometer or external sensor data. However, most systems only used the phone as a wireless data transfer device, not as a wearable computing platform. These systems used an external computer for analysis of the data.\n\n1Location = sensor location on the person\n\n2Freq = data acquisition frequency\n\nWearable video-sensor systems have also been developed to log digital memories. Video, GPS, electrocardiogram, and acceleration were used to record location and context of a person’s daily life16–18. These Lifelogs had several limitations, including information retrieval, synchronization, light quality, low visual resolution, artifacts, and automatic annotation.\n\nA previous project showed that sensors located in a “smart-holster” could be combined with a BlackBerry Smartphone to provide image-assisted mobility assessment5. An external accelerometer, temperature sensor, humidity sensor, light sensor, and Bluetooth were integrated into the Smartphone holster. Software was written for the BlackBerry 9000, which had an embedded camera and GPS. A hierarchical decision tree combined with a double threshold algorithm classified signals to recognize changes-of-state (CoS) and activities. The BlackBerry then automatically took a digital picture at each CoS. This preliminary work confirmed that a wearable mobility monitoring system (WMMS) could use acceleration and pictures to identify walking movements, standing, sitting, and lying down, and additionally provide context for these activities.\n\nSubsequent research19 used the BlackBerry 9550 Smartphone, with an integrated accelerometer, and revised algorithms to detect changes of state (CoS) and classify activities of daily living. The low sampling rate of the BlackBerry 9550 required some of the algorithms for the first WMMS to be revised. Having all sensors and computing power within the Smartphone provides a broadly accessible platform for wearable activity monitoring. A single subject case study resulted in an average sensitivity of 89.7% and the specificity of 99.5% for walking-related activities, sensitivity of 72.2% for stair navigation, and sensitivity of 33.3% for ramp recognition. Ramp results were poorer since accelerations for ramp gait were similar for walking gait.\n\nSince it was demonstrated that new BlackBerry Smartphones can identify CoS in real-time19, and Smartphone video capture is available on these devices, a preliminary evaluation of the usefulness of wearable video for improving activity classification and context identification is needed. The present study builds on this prior work and presents a proof-of-concept evaluation of a new BlackBerry-based WMMS that uses internal sensors and cell phone video to identify a range of mobility and daily living activities by using the CoS to trigger the acquisition of short video clips. A proof-of-concept evaluation is a necessary step before a large scale evaluation with people with disabilities. This study is the first to integrate sensor and (non-automated) video analysis for wearable mobility monitoring.\n\n\nMaterials and methods\n\nThe prototype WMMS was developed for BlackBerry OS 5.0 on the Storm2 9550 Smartphone. The WMMS used the BlackBerry’s integrated accelerometer (3-axis, ± 2g), GPS, camera, and SD memory card for mobility data collection. The 3.15 MP camera had a maximum video resolution of 480x352 pixels and a video frame rate of 30 frames per second20. During video capture, no accelerometer or GPS data were available. With video control active, the accelerometer sampling rate was approximately 8 Hz21. For the WMMS, GPS data were sampled at 1 Hz, but GPS data were not used in the algorithm presented in this paper. The WMMS was worn in a holster on the right, front pelvis.\n\nThe new WMMS software developed in22, sampled time, acceleration, and GPS location and then saved the raw data to a 16Gb SD-card. Accelerations were processed to calibrate the axis orientation, using the gravity rotation method4. The processed acceleration was input into a feature extraction algorithm19 for analysis within 1-second data windows. Following feature extraction, the features were analyzed in a decision tree to determine if a CoS had occurred. If a CoS was identified, a three-second video clip was captured. A second decision tree was used to categorize the activity19. Time, features, and activity classification were saved to an output file on the SD card.\n\nAcceleration features were identified that were sensitive to changes in mobility status19. These included mean Y-axis acceleration, standard deviation (STD) in X,Y,Z axes (1), Y-axis range (Range_Y) (2), Sum of Ranges (SR) (3), Signal Magnitude Area (SMA) of SR (4), difference of Sum of Ranges (DiffSR) (5), and range of X and Z (Rxz) (6). The inclination angle (7) could distinguish sitting, lying, and standing for the classification of static activities. While the GPS provides useful location information, GPS was not required in this study because accelerometer-based recognition of vehicle riding showed good results19.\n\nSTD_Y=1N∑i=1N(yi−my)2 (1)\n\nRange_Y = (MaxY – MinY) (2)\n\nSR = (Range_X + Range_Y + Range_Z) (3)\n\nSMA_SR=∑i=1NSRi (4)\n\nDiffsr = (SR2 – SR1) (5)\n\nRxz = (Range_X + Range_Z) (6)\n\nInclination Angle = arctan (mzmy)(°) (7)\n\nIn equations (1) to (7), my is the mean Y-acceleration, yi is the individual acceleration value, N is the samples per data window, SR2 is the sum of ranges in current window, SR1 is the sum of ranges in previous window, and finally mz is the mean Z-acceleration.\n\nThe CoS algorithm described in19 used pre-set thresholds for feature analysis. Each feature was independently analyzed using single or double-thresholds and scored as true or false. These Boolean values were combined to recognize the activities reported in19 (static state, taking an elevator, walking-related movements) and also to identify changes of state for small movements during activities of daily living (ADL) (meals, hygienic activities, working in the kitchen). CoS classification ran in real-time to enable accurate Smartphone video recording.\n\nThe decision tree for activity classification, described in19, used the same features and thresholds as the CoS algorithm to classify eight activities: sitting, standing, lying, riding an elevator, small stand-movements, small sit-movements, small lie-movements, and walking. The small movement categories included ADL and movements while sitting and standing. Following data collection, the video clips were manually reviewed by a human operator to help classify activities. Clips were played back on a BlackBerry 9700 phone. Video and audio were available for qualitative assessment of the current activity.\n\nA convenience sample of five able-bodied subjects (four males and one female, age: 35.2 ± 8.72 years, height: 174.58 ± 10.75 cm, weight: 66.46 ± 9.7 kg) were recruited from the Ottawa Hospital Rehabilitation Center (TOHRC, Ottawa, Canada) for the proof-of-concept evaluation. A form that included informed consent, information sheet, and media agreement was obtained for each subject. Ethical approval for the study was obtained from the Office of Research Ethics and Integrity, University of Ottawa, File Number: A08-12-01. Subjects who had injuries or gait deficits were excluded.\n\nData collection took place within the TOHRC (hallways, elevator, one bedroom apartment, stairs, Rehabilitation Technology Laboratory) and outside on a paved pathway. Participants wore the WMMS on their right-front waist, in a regular BlackBerry holster attached to a belt, with the camera pointing forward. No additional instructions were given for WMMS positioning. The participants were asked to follow a predetermined path and perform a series of mobility tasks, including, standing, walking, sitting, riding an elevator, brushing teeth, combing hair, washing hands, drying hands, setting dishes, filling the kettle with water, toasting bread, a simulated meal at a dining table, washing dishes, walking on stairs, lying on a bed, walking on a ramp, and walking outdoors. Each activity in the sequence took approximately 10 to 20 seconds to complete. Each trial had 41 changes of state. Three trials were captured on the Smartphone and on a digital video camcorder.\n\nActivity timing was obtained from the camcorder recording for comparison with the WMMS output. Start and end points of each trial were identified by shaking the Smartphone for 2 seconds. The camcorder also provided contextual information for analysis.\n\nData were imported from the Smartphone SD card into Microsoft Excel for statistical analysis. The Smartphone video clips were reviewed by two independent evaluators to qualitatively classify activities and assess video quality. The evaluators had access to the WMMS activity classification results during video classification.\n\n\nResults\n\nThe results were analyzed in terms of CoS and activity classification. For CoS identification at the transition point between activities (Table 2), sensitivities for standing, sitting, lying, and taking an elevator were between 97% and 100%. Walking-related CoS, such as stairs and ramps, had 67% to 73% sensitivity. Sensitivity5 related to CoS between walking and small movements, such as brushing teeth, were between 40% and 93%. The CoS results for daily living activities were poorer (below 27%) since the continuous series of small movements produced similar acceleration features.\n\n1TP = average true positives\n\n2FN = average false negatives\n\nTable 3 shows the specificity5 results for activity classification using the CoS algorithm19. These classifications are compared between data windows for CoS identification. The number of false positives was less than 12% for all measures, with half the measures reporting less than 5% false positives. Walking produced the most false positives, identifying a walking-CoS when no CoS occurred, for 324 out of 2700 cases (12%). Large standard deviations were found for some measures due to timing differences (i.e., people who walked slower took more time and had more data points for analysis, leading to more true negatives).\n\n1FP = average false positives\n\n2TN = average true negatives\n\nBetter activity classification results were achieved when using both acceleration features and video clips for all activities except sitting, as compared to using the accelerometer only (Table 4). Acceleration-only analysis with the activity classification algorithm19 had a greater sensitivity than acceleration-and-video for sitting because one CoS was missed in one trial, resulting in no associated video data. Using the accelerometer only, none of the ADLs were identified; however, no false positives were reported, resulting in a high specificity.\n\n1Acc. = accelerometer\n\n2SE = sensitivity\n\n3SP = specificity\n\nSince accelerometer data were unavailable during video recording, no acceleration could be recorded for a minimum of 2.9 seconds after a CoS was identified. For example, if a person sits in a chair, stands up, and sits down again within 4 seconds (3 seconds of video capture and 1-second data window), the CoS will not be detected or identified. However, digital video would be available during this period for activity classification, enabling the evaluator to see the additional movements during post-processing. The average accelerometer sampling frequency was 7.88 ± 1.39 Hz. Video analyses became difficult in some circumstances, in particular when video images were dark.\n\nThe BlackBerry 9550 included a light sensor, but the sensor output was unavailable with Java API 5.0. Light intensity level could be beneficial for recognizing indoor and outdoor environments.\n\nAs shown in Figure 1, the extracted features were used to recognize small movements, identified using a combination of SR and STD_Y. Discrete small movements were easily distinguished; however, a CoS could be missed for a continuous series of small movements that has similar accelerometer features. For example, brushing teeth and then combing hair.\n\nSum of Ranges to distinguish small movements: brushing teeth (BT), combing hair (CH), washing hands (WH), drying hands (DH), moving dishes (MD), moving a kettle (MK), toasting bread (TB), preparing a meal (PM), and washing dishes (WD).\n\nThe sum of ranges was more sensitive than the STD_Y for distinguishing mobility states (Figure 2). Further, the SMA-SR curves were smoother than the sum of ranges curves. Smooth SMA-SR curves were better at defining classification thresholds for climbing stairs and walking.\n\nTop: Sum of ranges (SR) and STD_Y to distinguish walking and static states (BT=brushing teeth, CH=combing hair, WH=washing hands, and DH=drying hands).\n\nBottom: SMA-SR to identify climbing stairs and walking.\n\nRecording a video was a valuable medium for evaluating mobility activities since audio information was available and multiple-images could be used in the analysis (Table 5). The sense of motion also provided useful information for categorizing the activity and context.\n\nActivity recognition for static and walking states was confirmed by video-clip evaluation. Occasionally, the video showed a moving-hand during walking, as the arm swung during locomotion. Since the phone was located on the right waist, the video usually showed more of the right side than the left. For ramp navigation, the Smartphone video was not able to show the ramp, however, the video did allow the evaluator to determine if the person was ascending or descending. Similarly, the video was unable to show the stairs when descending, but the downward movement and sound of the footfalls on the stairs allowed the evaluator to categorize the stair descent activity.\n\nThe elevator CoS was usually triggered by a transition from walking to standing, rather than from elevator movement. The activity was classified using the video by seeing the elevator door or the inside of the elevator in the video-clip. The elevator inside was unclear and dark, but the elevator sound was clearly defined. Further, the bathroom, kitchen, and dining room areas were clearly identified from video analysis.\n\nSmall movements, such as brushing teeth, combing hair and moving plates could not be identified because these activities were out of the Smartphone video field. Some clips could clearly identify moving a kettle, toasting bread, meal preparation, and washing dishes (Figure 3). Furthermore, drying hands and meal preparation needed continuous video images to identify the activities (i.e., single images would not be useful). Moreover, the video-based activity classification was hampered for people with shorter lower bodies when a tabletop or kitchen counter obstructed the phone. For example, moving a kettle and toasting bread were not visible in the video field for these shorter participants.\n\nImages from BlackBerry video for small movements: a) kitchen cabinet and oven, b) bathroom sink, c) dining table and meals, d) moving a kettle, e) toasting bread, f) meal preparation, g) washing dishes, h) towel and towel rack, i) drying hands.\n\n\nDiscussion\n\nThis study demonstrated that BlackBerry accelerometer signal analysis and cell phone video assessment can be combined to identify many mobility activities and the context of these activities. Since a readily available Smartphone was worn in a typical manner, at the waist in a holster, and no external sensors or hardware was required, the WMMS could be easily implemented in the community.\n\nThe ability to appropriately identity a CoS is critical for a WMMS that uses video, with accurate and real-time CoS identification needed to trigger the acquisition of video clips at the appropriate time to enable post-processing. The WMMS successfully recognized CoS for both static and walking activities. By taking the initial activity classification from the sensor data and refining the classification decision using the BlackBerry video, superior activity recognition results were obtained.\n\nStatic activity (sitting, standing, lying) classification accuracy improved from outcomes in the literature (below 94.6%,5,13,23) to 97.3%. The results would have been 100% except one CoS was missed during a walk-to-sit CoS of one of the trials. More thorough biomechanical analysis is required to identify the movement pattern that created this outlying error. Standing and lying down were recognized with 100% accuracy.\n\nThe WMMS produced better sensitivity results for walking than the previous Smartphone-smart-holster system5. While various studies in the literature had better walking accuracy (above 96%7,23), these studies only distinguished differences between very distinct mobility states, such as running, jumping, and no-movement. Accuracy of these systems would likely reduce if they were categorizing similar activities, such as running on level ground and running up an incline.\n\nStair navigation identification improved from 60% in the literature5,13 to 75% with the new WMMS. However, the result did not reach the 95% target. Acceleration-based categorization remains difficult for stairs since the signal patterns between walking on level ground and stairs are similar, and the stair slope may not greatly change accelerations at the waist for able-bodied people.\n\nCoS identification for ramp walking was enhanced from 43.3% for the previous system5 to 66% because the new WMMS used a specific ramp judgment within the decision tree. Ramp classification also improved from 16.6%5 to 43.5%. Accelerometer signals from able-bodied people do not always change when moving from level ground to an incline, however, accelerometer signals from people with mobility disabilities may be sufficiently distinct to enable consistent ramp CoS triggering. During ramp descent, classification errors may occur if a walking CoS is triggered before the person finishes descent and the waist-mounted video does not show the ramp bottom. An altitude sensor may help detect changes in body vertical position during ramp and stair navigation, thereby providing more accurate CoS triggering and sensor-based classification.\n\nSpecificity is also important for a WMMS that incorporates video since identifying a CoS when no change actually occurs (i.e. a false positive) results in inappropriate video capture that affects storage capacity, battery life, and increases the workload for video post-processing. Most prior research did not report specificity or false positive results13–15. For the two studies that reported false positives, the maximum false positive rate was above 12% for walking-related activities5,24. Improved threshold calibration methods that are specific to the individual may help to decrease false positives and false negatives.\n\nThe BlackBerry device was able to process the features and algorithms in real-time and save outcome data for each 1-second window. Most mobility research used cell phones to collect raw data and then analyzed features and algorithms offline13–15,25. For the new WMMS, threshold settings, decision trees, feature extraction and timing were executed on the Smartphone in real time.\n\nThe combination of accelerometer and video camera was superior to using the accelerometer data alone to provide mobility information for activities of daily living. Bathroom, kitchen, and dining room activities were recognized by videos in 63% to 94% of the cases. Although the combination of accelerometer and video could not consistently identify small ADL movements, this method had better accuracy than the accelerometer only, which could not categorize any of these ADLs. Further, CoS for small movements could not be clearly identified. A higher accelerometer sampling rate may allow for more complex signal analysis that could improve small movement CoS identification. Additional features, such as signal magnitude area, skewness5, energy, correlation26, and kurtosis27, might help to recognize the small movement CoS, but these features need greater sampling rates and/or greater accelerometer range.\n\nThe video was better than still images for refining activity classification and recognizing context, such as flooring type, bathroom/kitchen, and outdoors. From previous research5, still images had limitations in dark areas such as elevators, but video clips with audio were helpful for identifying an elevator and other states. Since videos had continuous images to distinguish upward and downward movement, these clips were useful for stairs and ramp navigation. Although video cameras could potentially recognize small ADL movements, the waist-mounted Smartphone location limited the camera to activities occurring about waist height.\n\nWhile BlackBerry video proved useful, the current implementation requires human time to review and classify the activities. Manual video assessment could be used for research and specific clinical applications; however, automated video analysis would make the system more efficient and promote widespread use of video data for activity classification. Further research into automated video analysis is required to achieve this goal.\n\n\nConclusions\n\nBuilding on previous work that demonstrated the BlackBerry’s ability to identify changes of state in real-time, a new WMMS was presented that uses only the Smartphone’s accelerometer and video for unsupervised and ubiquitous mobility analysis for research and healthcare applications. This study was the first to integrate sensor and video analysis for wearable mobility monitoring. This sampling rate for the phone sensors was lower than used in previous studies, demonstrating that a standalone WMMS can be implemented even for older-hardware phones. This standalone WMMS was designed for independent community ambulation and has minimal space requirements and setup time. Furthermore, the context of a person’s mobility activities can be identified, including the environment in which mobility takes place.\n\nWhile monitoring mobility, the new system saves raw sensor data and features. The raw data could be analyzed by other researchers or used in post-processing to improve activity classification. Novel multi-feature algorithms were developed to recognize activities under lower accelerometer sampling rates and range. By combining and weighting sum, range, and covariance statistics, the WMMS was able to recognize standing, sitting, lying, riding an elevator, walking on level ground, ramps, stairs, and ADL (washing hands, drying hands, setting dishes, moving a kettle, toasting bread, preparing a meal, and washing dishes). Static activities, walking on level ground, walking on stairs, walking on a ramp, and riding an elevator had higher sensitivities than previous studies, but the overall CoS identification and activity classification performance could be improved by further research.\n\nAdding additional sensors, increasing the accelerometer sampling rate/sensitivity, and adding new user-specific threshold calibration methods can be considered in future work. The classification of other small movement ADL activities also requires further research to increase sensitivity and specificity. While evaluation with able-bodied participants was warranted for this proof-of-concept study, further evaluation research with various patient populations is required before this WMMS can be accepted for use by people with mobility disabilities.\n\n\nConsent\n\nInformed written consent to publish the results of this study was obtained from each participant.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for combination of smartphone sensors and video for a wearable mobility monitoring system, 10.5256/f1000research.4790.d3253828", "appendix": "Author contributions\n\n\n\nEL conceived the WMMS. HW, EL and NB designed and developed the WMMS and its evaluation protocol. HW wrote the WMMS code, performed the data collection and analysis. HW, EL and NB and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was jointly funded by the Natural Sciences and Engineering Research Council of Canada (NSERC, #CRDPJ 408109) and Research in Motion.\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 would like to thank Shawn Millar for assistance with data collection and Whitney Montgomery for statistical review. This project was funded by the Ontario Centers of Excellence, Natural Sciences and Engineering Research Council of Canada (NSERC), and BlackBerry. The study sponsors had no involvement in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication.\n\n\nReferences\n\nHowe JA, Inness EL, Venturini A, et al.: The Community Balance and Mobility Scale--a balance measure for individuals with traumatic brain injury. Clin Rehabil. 2006; 20(10): 885–895. PubMed Abstract\n\nBlum L, Korner-Bitensky N: Usefulness of the Berg Balance Scale in stroke rehabilitation: a systematic review. Phys Ther. 2008; 88(5): 559–566. PubMed Abstract \| Publisher Full Text\n\nHerman T, Inbar-Borovsky N, Brozgol M, et al.: The Dynamic Gait Index in healthy older adults: the role of stair climbing, fear of falling and gender. Gait Posture. 2009; 29(2): 237–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeaby L, Torrance G: Reliability of a physiotherapy functional assessment used in a rehabilitation setting. 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Publisher Full Text\n\nWu H, Lemaire ED, Baddour N: Data for combination of smartphone sensors and video for a wearable mobility monitoring system. F1000Research. 2014. Data Source" }	[ { "id": "6335", "date": "24 Oct 2014", "name": "Paul McCullagh", "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\nThe paper: “Combining low sampling frequency smartphone sensors and video for a Wearable Mobility Monitoring System”, examines the feasibility of combining the accelerometry and video, available in a commercial smart phone for classifying activities of living in the home.As can be anticipate some activities are easy to classify; others with movement overlap are more difficult. Data are provided which can allow the work to be progressed by other researchers. Whilst the individual elements of the work are not novel (accelelerometry for activity monitoring and video for life logs and reminiscence), the combination and the superior performance of the sensor modalities provide a useful contribution.Abstract:This provides good orientation for the reader and cites the headline results. The sensitivity of 27% for daily living activities is low and show the challenge of higher level classification. Overall activity and mobility (changes of state, CoS) may however be more relevant features anyway (in a subsequent referral). The sample number (N=5) is small and is described as a convenience sample, for this initial feasibility study.Material and methods:The WMMS system is based on a Blackberry OS5.0. There is no mention as to whether this can be easily implemented on other smart phones (Android and IOS). Accelerometer sampling rate of 8Hz is appropriate but may miss faster transitions. The feature extraction algorithm and decision tress rely on cited literature. These are key components of the activity classification used for CoS detection. The use of thresholds usually makes it difficult to generalize to the wider population (male/female, height, weight etc.) for accelerometry.Results:“Better activity classification results were achieved when using both acceleration features and video clips for all activities except sitting, as compared to using the accelerometer only” This is the crux of the paper and the results underpin this. Of course this is a relative measure. It’s difficult to determine what would be a clinically useful measure. The figures are useful and inform the reader as to the difficulty of differential classification between some of these activities. There is some useful reflection on the use of video. The time required to manually classify the video is a limiting feature.Conclusions:The conclusions are realistic and pave the way for further research questions. In particular it may be possible to improve the classification algorithms and the publication of the data sets is advantageous.The Blackberry has a restricted market and the evolution of smartphone hardware and software continues to advance rapidly. Porting of the algorithms to other operating systems and provision source code would enable further refinement of the approach. There is little comment on the acceptability of the device, particularly with regard to privacy issues. These are not apparent in simulated environments but would occur if such a device is to be deployed, particularly in a multi-person home environment.", "responses": [ { "c_id": "1056", "date": "31 Oct 2014", "name": "Natalie Baddour", "role": "Author Response", "response": "Thank you for a thorough and thoughtful review. Work on the WMMS continues on an ongoing basis; we have developed the WMMS for the BlackBerry Z10, which has an average sampling rate of 50Hz. However, based on the work in this paper, it is interesting to note that reasonable results can be obtained even with a much lower sampling rate. The WMMS with the Z10 has been evaluated with larger populations and we are working on writing up those results.We are also currently working on porting the WMMS algorithms to android, with an eye to developing them for the iOS in the near future also.Thank you once again for taking the time to write a thoughtful and thorough review." } ] }, { "id": "7747", "date": "27 Feb 2015", "name": "Alan Godfrey", "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 investigates the use of a smart phone as a wearable mobility monitoring system. However, it is of moderate quality and there are a number of issues that warrant attention for the reader. The study could have been simplified, shortened and presented in a more clear and concise manner. Instead it is often vague and illogical in most parts, and introduces concepts/methods that are unsuitable. In brief:The title should make reference to the fact that it is a feasibility / proof of concept study. Throughout the manuscript several unsubstantiated claims are made without any real justification.Introduction:The authors remark that several video-sensor systems have been used in the past, with various limitations. While this study uses a smart phone as the system, the same limitations were encountered with no insight as to how those limitations may be overcome to progress this type of monitoring.Materials and Methods:This section is poorly written. For example the authors introduce GPS and later say it wasn't used. Therefore the text can be misleading in parts and could have been reduced and made concise and easier to read.A flow diagram of the data processing and activity recognition used from previous work would have been useful to explain the flow.Accelerometer features:Use of the inclination angle distinguished sitting from standing - this is true for their slim (66kg) young adults - how successful do the authors think the method will be for older and/or heavier subjects? How did the authors define sitting, i.e. what was the trunk angle? Would the method work if the participants were sat upright with a trunk orientation similar to standing?Test procedure:The evaluators were not blinded during video analysis. This is not discussed as a (major) limitation: bias (favoring correct classification) may have been introduced during classification. Some of the activities performed by the participants seems a bit arbitrary in what is basically a feasibility study. With a device worn on the hip, its not feasible to detect brushing teeth or combing hair (as shown by poor sensitivity values) nor is it worthwhile, at this stage. (brushing occurred outside video field - discussion). It would have been more suitable to concentrate on physical capability tasks relating to whole body movement. Results:\"Better classification results were achieved when using acceleration and video\" - of course as video was manually analysed. Periods of video capture hinders accelerometer logging - how is this system likely to be useful in the example of a postural transition? For example: this limits the use within pathology and more specifically falls, where most falls can occur during sitting to standing transition. Use of the acceleration data is key to detecting a transition and subsequent fall.It appears that the accelerometer data could distinguish very little of the activities with manual video observations proving key and the only suitable means to distinguish tasks.Discussion: In a few instances the authors refer to \"sounds\" - perhaps this might be a better tool to help distinguish activities in an automated system?", "responses": [ { "c_id": "1241", "date": "16 Mar 2015", "name": "Natalie Baddour", "role": "Author Response", "response": "Thank you for taking the time to read the manuscript. In response to comments:The abstract and introduction both make reference to the fact that this is a proof-of-concept study.We have uploaded a new version of the manuscript removing the reference to the GPS in materials and methods.The new version of the manuscript also includes a figure of the high-level overview of the WMMS algorithm.Although it was requested by reviewer #2, we have not included the figure of the decision tree used in the WMMS algorithm in the new version of the manuscript. Such a figure would not be meaningful without explanation, which would greatly lengthen the manuscript. The details of the algorithm are given in reference #19, the focus of which was the algorithm. The focus of this manuscript was on the evaluation of the algorithm.Correspondingly, the circuit used to evaluate the algorithm was designed to include activities from \"real life\" such as hair-combing and teeth-brushing. The intent was not for the a waist-worn WMMS system to identify these activities but rather to see how the WMMS algorithm would perform once these everyday (small movement) activities were included. We felt that this would be be a more realistic test of the proof-of-concept WMMS, rather than investigating the performance of the algorithm with only a limited set of activities. The comment about the use of the inclination angle to distinguish sitting from standing likely causing problems with older or heavier adults is quite astute. This was, in fact, our experience and an algorithm to correct for the larger girth of some waists has been developed and incorporated into subsequent versions of the WMMS. M. Tundo, E. Lemaire, N. Baddour, Correcting Smartphone Orientation for Accelerometer-Based Analysis, Proceedings of the IEEE International Symposium on Medical Measurements and Application, Gatineau, Canada, May 2013. dx.doi.org/10.1109/MeMeA.2013.6549706." } ] } ]	1	https://f1000research.com/articles/3-170
https://f1000research.com/articles/4-66/v1	13 Mar 15	{ "type": "Opinion Article", "title": "The culture of scientific research", "authors": [ "Catherine Joynson", "Ottoline Leyser", "Ottoline Leyser" ], "abstract": "In 2014, the UK-based Nuffield Council on Bioethics carried out a series of engagement activities, including an online survey to which 970 people responded, and 15 discussion events at universities around the UK to explore the culture of research in the UK and its effect on ethical conduct in science and the quality of research. The findings of the project were published in December 2014 and the main points are summarised here. We found that scientists are motivated in their work to find out more about the world and to benefit society, and that they believe collaboration, multidisciplinarity, openness and creativity are important for the production of high quality science. However, in some cases, our findings suggest, the culture of research in higher education institutions does not support or encourage these goals or activities. For example, high levels of competition and perceptions about how scientists are assessed for jobs and funding are reportedly contributing to a loss of creativity in science, less collaboration and poor research practices. The project led to suggestions for action for funding bodies, research institutions, publishers and editors, professional bodies and individual researchers.", "keywords": [ "Research", "culture", "science", "integrity", "misconduct", "ethics", "Nuffield Council on Bioethics" ], "content": "Project origins\n\nIn December 2014, the Nuffield Council on Bioethics, an independent body that explores ethical issues in biology and medicine, published the findings of a series of engagement activities that explored the culture of scientific research in the UK. There were several motivations behind the project. First, two of the Council’s previous inquiries considered the factors that affect how and what scientific research is carried out, and identified a need for further examination of the issues. Emerging biotechnologies: technology, choice and the public good1 looked at the factors affecting the direction of research, such as funding sources, interest in the economic and societal impact of research and public expectation, and found that these can lead to ‘overpromising’ by researchers that risks undermining public trust in science and misleading national policy. Novel neurotechnologies: intervening in the brain2 looked at the pressures on academic scientists to demonstrate the practical and economic impacts of their work and the potential for these pressures to encourage premature emphasis upon commercial applications, or publication bias towards positive or newsworthy findings. Second, the Council was aware of a wider public debate about integrity and misconduct in research, sparked perhaps by several high profile cases of research misconduct, and felt it to be an area where the Council might usefully contribute. It commissioned a background paper on scientific research integrity to inform the Council’s deliberations3.\n\nAlthough the Council recognised that there were already a number of important initiatives that were promoting rigour and integrity within the research community4,5, it suspected that aspects of the culture in which research is funded, practised and communicated may be undermining efforts to raise the conduct of science to the highest ethical standards. After consulting organisations that work closely with the scientific community, it appeared that the Council’s concerns were shared by others, and that there was a lack of evidence about the experiences of individual researchers in terms of the pressures and challenges they faced in the course of their work. The Council decided to embark upon a series of engagement activities to explore the issues further.\n\n\nProject methods\n\nIn October 2013 the Council set up a Steering Group, which included members and staff of the Council, as well as staff of the Royal Society, Society of Biology, Institute of Physics, Royal Society of Chemistry and Academy of Medical Sciences, to design and oversee a project exploring the culture of research. The aim of the project was “to foster constructive debate among all those involved in scientific research about the culture of research in the UK and its effect on ethical conduct in science and the quality, value and accessibility of research; and to advance current debate through wide dissemination of the outcomes of these discussions”. ‘Science’ and ‘scientific research’ were not strictly defined in order to allow anyone involved in science or a related discipline to take part in the activities.\n\nThe activities of the project included:\n\nAn online survey that was open from March to July 2014 and received 970 responses (Supplementary file 1 lists the questions used in the online survey and an analysis of the online responses is available at: http://nuffieldbioethics.org/wp-content/uploads/The_culture_of_scientific_research_survey_analysis_for_web.pdf).\n\nFifteen discussion events co-hosted with universities around the UK between June and September 2014, involving around 740 speakers and participants.\n\nEvidence-gathering meetings with funding bodies, publishers and editors of scientific research, and academics from the social sciences with expertise in the practice and culture of scientific research.\n\nA report summarising the views expressed in the survey responses and by those who took part in the events and meetings was published on 4 December 20146. The following documents were published on the same day (www.nuffieldbioethics.org/research-culture):\n\nA detailed analysis of the survey responses carried out by the research consultancy Research By Design.\n\nA summary of the university discussion events written by the Nuffield Council on Bioethics Secretariat.\n\nA background paper covering recent research, policy developments and current debate relevant to the culture of scientific research in the UK, also written by the Nuffield Council on Bioethics Secretariat.\n\nIt should be noted that the people who took part in the survey and discussion events were self-selecting participants and therefore cannot be assumed to be representative of the wider researcher population. Most of those who took part in our activities were involved or interested in research being undertaken by higher education institutions (HEIs), which carry out around a quarter of UK research and development. A large proportion of the survey respondents and event participants work in either bioscience or medicine and are early-career researchers, which is reflective of the demographics of the HEI research community. Notwithstanding these limitations in the data, we believe some important themes and ideas emerged during the project that are relevant to many areas of academic research.\n\n\nProject findings\n\nA full account of the project findings can be found on the Nuffield Council on Bioethics website. The key points are outlined below.\n\nIn order to frame later questions about the effects of research culture on the production of high quality science, it was important to establish at the beginning of the survey what respondents regarded to be ‘high quality science’. When respondents were asked to select five words from a list that best describe their understanding of high quality research, the five most frequently selected words were:\n\n1 Rigorous\n\n2 Accurate\n\n3 Original\n\n4 Honest\n\n5 Transparent\n\nDuring the project activities it emerged that several other components are thought to be particularly important in the production of high quality science, namely: collaboration, multidisciplinarity, openness and creativity. For example, increased collaboration was the most common answer given when survey respondents were asked what feature of the UK research environment is having the most positive effect on science. The respondents (a quarter) who raise this think collaboration is leading to better communication between researchers, greater sharing of data and methodologies, less competition between different research teams, and reduced feelings of isolation among researchers.\n\nPositive attitudes towards openness were expressed in answer to several of the survey questions. For example, 61 per cent of survey respondents thought that the move towards open access publishing is having a positive or very positive effect overall on scientists in terms of encouraging the production of high quality research. In addition, almost two thirds of respondents believe data sharing policies in the UK are having a positive or very positive effect overall. Respondents believe increased transparency and data sharing are facilitating the dissemination of results, enabling research to be accomplished more quickly and cost effectively, and allowing greater scrutiny of research findings. A greater proportion of respondents aged under 35 think data sharing policies are having a very positive effect overall than those aged over 45 (15 per cent vs. 7 per cent).\n\nThe description of high quality science by the participants of our project has notable similarities with the five themes that were identified during the UK Government’s consultation for its science and innovation strategy, also published in December 2014. The themes are excellence, collaboration, agility, place and openness7.\n\nThe motivations of scientists provide additional insights into how they view research, and the majority of the survey respondents clearly chose a career in science in order to find out more about the world around them. When respondents were asked to rank phrases to describe what they believe motivates them in their work, the top three were:\n\n1 Improving my knowledge and understanding\n\n2 Making scientific discoveries for the benefit of society\n\n3 Satisfying my curiosity\n\nHigh levels of competition in scientific research emerged as a strong theme running through all the project activities. Applying for funding is thought to be very competitive by the majority of the survey respondents (94 per cent), as is applying for jobs and promotions (77 per cent). Around nine in ten think making discoveries and gaining peer recognition is quite or very competitive.\n\nHigh levels of competition for jobs and funding in scientific research are believed by survey respondents both to bring out the best in people and to create incentives for poor quality research practices, less collaboration, and headline chasing. For example, behaviours such as rushing to finish and publish research, employing less rigorous research methods and increased corner-cutting in research were raised by 29 per cent of survey respondents who commented on the effects of competition on scientists.\n\nWhen asked which features of the UK research environment are having the most negative effect, the most common answer given by survey respondents (31 per cent) was the lack of funding available. In addition, concerns were expressed about a loss of creativity and innovation in science caused by strategically-directed funding calls, short-term funding, and trends towards funding of safer research projects and established research centres. For example, when asked what they would like to change about the UK research environment, over 42 per cent of respondents comment on funding issues, with some expressing a desire for more funding for ‘riskier’ projects. There is a feeling that funding bodies have become more conservative and favour safer research projects, where results are almost guaranteed in advance, but this approach, respondents believe, can hamper scientific development.\n\nWhen we tried to verify whether concerns about funding trends had a factual basis, we found that funding bodies offer a wide range of research grants, fellowships, studentships, training and other programmes, and support many different types of research. However, detailed information about trends in levels of funding and types of research being funded over time was not readily available from most funding bodies.\n\nIn a competitive system, the criteria used to assess the quality and value of science influences what science is pursued and how scientists behave. Peer review is the mainstay of most research assessment processes, including those carried out by journals to assess which work to publish, by funding bodies to determine which proposals to fund or how much core funding to allocate, and by institutions to decide who to appoint or promote to academic positions.\n\nPublish or perish. Throughout the project we heard repeatedly that publishing in high impact factor journals is still thought to be the most important element in determining whether researchers gain funding, jobs and promotions, along with article-level metrics such as citation numbers. This has created a strong pressure on scientists not only to ‘publish or perish’, but to publish in particular journals. Given that acceptance of a paper in a prominent journal typically requires that the findings represent a major, and possibly newsworthy, advance in knowledge, this trend is believed to be resulting in important research not being published, such as research with negative findings or research that replicates or refutes others’ work. Assessment processes that focus on publications in particular journals are also thought to be creating disincentives for multidisciplinary research, authorship issues and a lack of recognition for non-article research outputs.\n\nPeer review. Seventy-one per cent of the survey respondents believe the peer review system in the UK is having a positive or very positive effect overall on scientists in terms of encouraging the production of high quality science. However, concerns were raised about unconstructive reviewer comments and shortages of peer reviewers. Participants at several of the events raised the need for a review of the way in which peer review is carried out in the sciences. In particular, we heard support for both double-blind and open peer review as alternatives to the current system. The importance of peer reviewers being given training, time and recognition for their work was emphasised.\n\nThe REF. In 2013, the UK higher education funding bodies undertook a process for assessing research quality, the Research Excellence Framework (REF), to inform the allocation of core funding to HEIs from 2015. The process involved peer review of each institution on the basis of 1) the outputs of research (such as journal publications, datasets and patents), 2) the impact of past research on the economy, society and culture, and 3) the vitality and sustainability of the research environment. The REF was a frequent topic of discussion during the project activities. We heard that the REF is thought to be a key driver of the pressure on researchers to publish in high impact journals, with many unaware or untrusting of the instructions given to REF panels not to make any use of journal impact factors in assessing the quality of research outputs. When asked for their views specifically on the REF, almost 40 per cent said they think the REF is having a negative or very negative effect overall on scientists in terms of encouraging the production of high quality science (with 25 per cent saying they believe it is having a positive or very positive effect). It was raised in several of the discussion events that the REF may be disadvantaging multidisciplinary work.\n\nImpact. The assessment by the REF of the impact of research beyond academia has been controversial, as has similar attempts by other funding bodies to consider the impact of research, such as the Research Councils’ ‘Pathways to impact’ section of grant applications. We found that the assessment of the societal and economic impact of research are welcomed by some, but others believe it is creating a culture of short-termism, pushing aside interest in curiosity-driven research, and resulting in researchers exaggerating the potential applications of research in grant proposals. The Research Councils we spoke to believe they have a duty to explain to the public and the Government the impact of public investment in science. They emphasised that this is done mostly retrospectively, and applicants are not expected to be able to predict at the application stage the economic or societal impacts that research will achieve. The results of the REF, published on 18 December 2014, found that the vast majority of submissions demonstrated impacts that were outstanding (4) or very considerable (3)8. We await with interest the findings of research being undertaken by the Higher Education Funding Council for England (HEFCE) to evaluate the strengths and weaknesses of the REF process, as well as a review of the role of metrics in research assessment9.\n\nRecognising other professional activities. Almost half of the survey respondents believe provision of professional education, training and supervision in the UK is having a positive or very positive effect overall on scientists in terms of encouraging the production of high quality science. However, although staff development, PhD awards and research collaboration are already recognised by the REF in the ‘Environment of research’ category and often feature in university promotion criteria, there was a clear perception among the event participants that this kind of activity is undervalued. It was suggested during the discussion events that research organisations should pay closer attention to and value the hard-to-measure and often invisible ways in which researchers contribute to the production of high quality science. This may include mentoring, training, teaching, peer review, university administration, public engagement and contributing to the work of national bodies and policy makers.\n\nThere is a wide range of activities that could be considered to constitute research misconduct or poor quality research practice. This includes data fraud, poor experimental design, corner-cutting in research methods, inadequate replication of research, ‘cherry picking’ results, inappropriately slicing up data to create several papers, authorship issues, plagiarism, over-claiming the significance of work in grant proposals and papers, and carrying out poor quality peer review.\n\nResearch integrity came up frequently at the discussion events. Participants noted that honesty and trust is fundamental to science, and high profile cases of research misconduct may be undermining public trust in science. The view was expressed that high levels of competition for scarce resources put scientists under immense pressure which means that scientists are “bound to behave less well”.\n\nThis was confirmed by the survey findings. Fifty-eight per cent of respondents to the survey are aware of scientists feeling tempted or under pressure to compromise on research integrity and standards, although evidence was not collected on any behaviour associated with these findings. Twenty-six per cent of respondents have themselves felt tempted or under pressure to compromise on research integrity and standards. A higher proportion of respondents aged under 35 years (33 per cent) stated they had felt tempted or under pressure in comparison with those aged above 35 years (21 per cent). Thirty-eight percent of the survey respondents who comment on research integrity and standards think the ‘pressure to publish’ can encourage the fabrication of data, altering, omitting or manipulating data, or ‘cherry picking’ results to report. Thirty-one per cent of respondents think there is pressure to focus on and report positive results, rather than negative results, and that researchers rushing to publish results may not conduct appropriate replications and scrutiny of their work.\n\nThe first sector-wide research guidance for universities, The Concordat to Support Research Integrity, was published in 201210. Sixty per cent of survey respondents think that initiatives that promote integrity in science in the UK, such as codes of conduct, are having a positive or very positive effect overall on scientists in terms of encouraging the production of high quality science. In addition, over half of survey respondents think ethical review processes in the UK are having a positive or very positive effect. This view is especially prevalent among respondents from social science, psychology, medicine and bioscience, and respondents note that ethical standards in the UK are thought to be high in comparison to other countries.\n\nSuggestions for improving research integrity in the UK were made by event participants. Universities, they suggested, have a responsibility to create conditions to support ethical research conduct and demonstrate clearly the consequences of poor research practice. Training in good research practice was thought to be important in this regard, particularly for PhD students, but time pressures on senior scientists might be preventing this from happening at the moment. Universities might also be more open about how individual cases are resolved.\n\nThe number of academic staff across all disciplines employed in English HEIs has risen from around 105,000 in 2003–04 to around 126,000 in 2012–1311. Around 30 per cent of science PhD graduates go on to post-doctoral research positions, but only around four per cent of science PhD graduates proceed to permanent academic posts with a significant research component12.\n\nWhen asked if there is anything they would like to change about the UK research environment, more than a third of survey respondents cite issues related to career structure and progression. The following concerns were frequently mentioned by survey respondents and event participants:\n\nShort-term contracts and job insecurity for post-doctoral researchers\n\nReliance on external funding for job retention, which drives the ‘pressure to publish’\n\nPressure to progress but high competition for jobs and funding\n\nThe need to keep relocating in order to take up the next position\n\nLimited opportunities for women in particular to have career breaks\n\nHeavy workloads and long hours\n\nHigh ‘drop out’ rates\n\nAlmost twice as many female survey respondents as male respondents raise issues related to career progression and the short-term culture within UK research when asked which features of the research environment are having the most negative effect on scientists. In addition, fifty-four per cent of respondents think the way scientists are assessed for promotion during their career is having a negative or very negative effect overall on scientists in terms of encouraging the production of high quality science, compared to 22 per cent who think it is having a positive or very positive effect.\n\nIn terms of how issues relating to careers and workloads affect the production of high quality science, survey respondents believe that they contribute to a culture of short-termism, high levels of stress, a lack of time to think and the loss of talented individuals from academia, which in turn results in a loss of creativity and innovation. Respondents also raise the possibility that high levels of competition for jobs may encourage poor quality research practices.\n\nSuggestions for ways of addressing some of these concerns were raised during our discussions. For example, mentoring of early career scientists and the provision of appropriate career advice was suggested at several of the events as a possible way to help mitigate anxieties and help researchers be realistic about their prospects for a career in scientific research. Mentoring and advice may also help people to plan and develop their career paths at an earlier stage, and ensure they gain experience that will be transferrable to other sectors. PhD students are already encouraged by some funders to spend time in other sectors in order to expand their skills and experience, and the Royal Society recently published guidance on how doctoral students’ careers expectations can be best managed so that they, and their supervisors, understand the wide range of careers to which a PhD can lead, both inside and outside of scientific research13. However, the funders we spoke to reported less progress in this area among post-doctoral researchers.\n\nThere are a number of other existing initiatives that aim to improve researcher careers. The Concordat to Support the Career Development of Researchers14, for example, was highlighted during the project as a positive development in improving the way in which researchers are promoted and recruited. A recent report on progress with the implementation of the Concordat found that, although significant transformation has been achieved, further challenges remain, for example with regards to researchers taking more responsibility for their own professional and career development15.\n\nParticipants at some of the events noted that the number of women in science has increased and that the introduction of formalised research assessment systems may have helped to tackle gender biases, which may have formerly influenced decisions about funding allocation and career progression. The Athena SWAN Charter, a national scheme that recognises good employment practice for women working in science, was mentioned at a number of events and is seen as having a positive influence on diversity in science. However, a majority (57 per cent) of those who responded to a call for evidence that formed part of HEFCE’s review of metrics in research assessment were negative about increasing the use of metrics, and common among their concerns was that a further use of metrics could disadvantage under-represented groups: early-career researchers, women, those with disabilities, and black and minority ethnic (BME) academics16. A workshop on equality and diversity organised by HEFCE as part of the review highlighted problems associated with ‘implicit bias’ and the possible unintended consequences of research assessment regimes17. These concerns emphasise the importance of diversity in assessment methods.\n\n\nObservations and suggestions for action\n\nScientists told us they are driven by the desire to improve their knowledge and understanding, to make discoveries for the benefit of society and to satisfy their curiosity. High quality research was described as: rigorous, accurate, original, honest and transparent; and collaboration, multidisciplinarity, openness and creativity are thought to be important for the production of high quality science. Within this context, the findings of the project led us to make some general observations:\n\nIn some cases the culture of scientific research does not support or encourage scientists’ goals and the activities that they believe to be important for the production of high quality science.\n\nA highly competitive environment with a narrow range of assessment methods are thought to be the main risk factors.\n\nThere seem to be widespread misperceptions or mistrust among scientists about the policies of those responsible for the assessment of research.\n\nAmong all the relevant stakeholders, concerns about the culture of research are often attributed to matters that they think are outside their control or are someone else’s responsibility.\n\nWe believe there is a collective obligation for the actors in the system to do everything they can to ensure the culture of research supports good research practice and the production of high quality science. As such, we provide a number of suggestions for action for funding bodies, research institutions, publishers and editors, professional bodies and individual researchers (see Figure 1). Key examples are:\n\nFunders: ensure funding strategies, policies and opportunities, and information about past funding decisions, are communicated clearly to institutions and researchers; and provide training for peer reviewers to ensure they are aware of and follow assessment policies.\n\nResearch institutions: cultivate an environment in which ethics is seen as a positive and integral part of research; ensure that the track record of researchers is assessed broadly; and provide mentoring and career advice to researchers throughout their careers.\n\nPublishers and editors: consider ways of ensuring that the findings of a wider range of research meeting standards of rigour can be published; consider ways of improving the peer review system; and consider further the role of publishers in tackling ethical issues in publishing and in promoting openness among scientists.\n\nResearchers: actively contribute to the adoption of relevant codes of ethical conduct and standards for high quality research; use a broad range of criteria when assessing the track record of fellow researchers; and engage with funders, publishers and learned societies to maintain a two-way dialogue and contribute to policy-making.\n\nLearned societies and professional bodies: promote widely the importance of ensuring the culture of research supports good research practice and the production of high quality science; and take account of the findings of this report in relation to guidelines for members on ethical conduct and professionalism.\n\n\nReaction so far\n\nThe Steering Group hopes that the findings of this project provide useful evidence that will advance future debate on the culture of scientific research in HEIs. At this early stage, the findings appear to have been received very positively.\n\nThe Presidents of five major science organisations (the Nuffield Council on Bioethics, the Royal Society, Society of Biology, Royal Society of Chemistry and Academy of Medical Sciences) jointly wrote a Foreword for the report of the project18. In the Foreword, the Presidents welcomed the report, recognised the integrated view that the culture-wide approach of this project provides, and committed to consider the report’s suggestions for action in the context of their own communities.\n\nTo raise awareness of the findings among policymakers, a launch event was held in the Houses of Parliament on 4 December 2014. The event was hosted by Andrew Miller MP, Chair of the House of Commons Science and Technology Committee and around 45 invited individuals attended, including representatives of funding bodies, journals and publishers, professional bodies and learned societies, universities, select committees, and others such as government departments and NGOs. Participants noted the many positive aspects of the project findings, for example that researchers clearly care about doing good research and the challenges they face. In addition, many positive developments were also highlighted, such as trends in publishing towards open peer review and the publication of a larger range of research outputs. All agreed that it was a complicated area and a collective and co-ordinated effort to tackle the issues was required. Throughout 2015, the Nuffield Council on Bioethics, working in particular with the organisations represented on the project steering group, will be encouraging all relevant stakeholders to do just that. If you are interested in getting involved, please contact Catherine Joynson on cjoynson@nuffieldbioethics.org.", "appendix": "Author contributions\n\n\n\nOL chaired the Steering Group that devised and oversaw the project from 2013–2014. CJ was employed by the Nuffield Council on Bioethics to support the Steering Group and implement the activities of the project. Both contributed to the drafting of the report of the project findings summarised in this paper. CJ prepared the initial draft of this paper, which describes and reproduces the project findings. Both authors were involved in the revision of the paper and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe project that is reported in this paper was funded and carried out by the Nuffield Council on Bioethics as part of its core work programme.\n\n\nAcknowledgments\n\nThe authors would like to acknowledge the contributions of the members of the Steering Group of the project described in this paper. The Steering Group over saw the design and implementation of the project, provided connections with the scientific community, and contributed to the drafting of the report of the project findings.\n\nSupplementary file 1: Questions used in the online survey. Doi: 10.5256/f1000research.6163.s43527.\n\n\nReferences\n\nNuffield Council on Bioethics. Emerging biotechnologies: technology, choice and the public good. London: Nuffield Council on Bioethics. 2012. Reference Source\n\nNuffield Council on Bioethics. Novel neurotechnologies: intervening in the brain. London: Nuffield Council on Bioethics. 2013. Reference Source\n\nJacob AM: Scientific Research Integrity: Background Paper for the Nuffield Council on Bioethics. 2013. Reference Source\n\nUniversities UK. The concordat to support research integrity. 2012. Reference Source\n\nEuropean Science Foundation. Good scientific practice in research and scholarship. 2000. Reference Source\n\nNuffield Council on Bioethics. The findings of a series of engagement activities exploring the culture of scientific research in the UK. 2014. Reference Source\n\nHM Treasury. Our plan for growth: science and innovation. 2014. Reference Source\n\nThe Higher Education Funding Council for England. Research Excellence Framework 2014: The results. 2014. Reference Source\n\nThe Higher Education Funding Council for England. Independent review of the role of metrics in research assessment. 2015. Reference Source\n\nUniversities UK. The concordat to support research integrity. 2012. Reference Source\n\nHigher Education Funding Council for England. A briefing on Staff employed at HEFCE-funded HEIs: Trends and profiles. 2014. Reference Source\n\nThe Royal Society. The Scientific Century: securing our future prosperity. 2010. Reference Source\n\nThe Royal Society. Doctoral students’ career expectations principles and responsibilities. 2014. Reference Source\n\nVitae. Concordat to Support the Career Development of Researchers. An Agreement between the Funders and Employers of Researchers in the UK. 2008. Reference Source\n\nVitae. Progress in implementing the Concordat principles. 2014. Reference Source\n\nThe Higher Education Funding Council for England. Independent review of the role of metrics in research assessment. Summary of responses submitted to the call for evidence. 2014. Reference Source\n\nThe Higher Education Funding Council for England Blog. Metrics for all? by Jude Hill. 2015. Reference Source\n\nNuffield Council on Bioethics. The findings of a series of engagement activities exploring the culture of scientific research in the UK. 2014. Reference Source" }	[ { "id": "7955", "date": "19 Mar 2015", "name": "Curt Rice", "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\nThe opinion article The culture of scientific research contributes to a debate focused on the tension between doing research and holding researchers and research institutions accountable. I find that the piece does not particularly surprise me, but it's contribution is nonetheless important as it offers a somewhat less anecdotal treatment of the issues, as well as offering constructive suggestions about how to move forward.The article reports on the results of a survey done among scientists in the UK, with 970 responses, and on the results of a series of 15 \"discussion events\" having around 740 speakers and participants.Both the survey and the focus groups considered the nature of scientific careers and the circumstances in which scientists currently work and related these things not only to each other but to the nature and quality of research being carried out.It is heartening to learn that scientists report their primary motivations as being the quest for knowledge and the application of that knowledge in the service of society. We want to understand stuff. That's what we do. And counting pages published and impact factors is a distraction. But it's more than that.Some of the results of the study reveal the belief that our current infrastructure for funding moves us away from riskier curiosity-driven projects, towards more conservative research.The perniciousness of impact factor is seen as pressuring scientists to try for particular journals, as well as leading researchers away from reproducibility projects or the examination of negative results.The structure of careers -- especially with strings of post-docs and temporary positions -- yields a culture of short-termism. I think this is a very serious point. If early career scientists have to apply for renewal or new positions every 2-3 years, they have to get results within the first year of a position to have something published by the time they apply for their next job. This is not good for the quest for knowledge and understanding, and I think this point is under-appreciated in the debate about the nature of an academic career. Another important topic the the article addresses is the pressure to take ethical short cuts. This in turn is one of the core issues in the authors' suggestions for improving our system. They stress the importance of training in ethical norms and practices and advocate for explicit engagement with this aspect of researcher training and practice.In addition to encouraging us to nurture ethical training and practice, the authors also would like to see more training to do peer review and broader evaluation in connection with hiring, advancement and the awarding of grants.The nature of research is being manipulated and damaged by the nature of the context in which research is carried out. This article investigates that claim more thoroughly, provides evidence, and offers constructive suggestions about what could be different.Read this piece. And talk about it with your research communities.", "responses": [] }, { "id": "7956", "date": "23 Mar 2015", "name": "Cornelia Lawson", "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 report provides a summary of the findings of the Nuffield Council on Bioethics’ “The culture of scientific research” project. The report provides insights into the conditions faced by scientists in the UK and makes a number of constructive suggestions at all levels of the academic enterprise that could help improve the culture of research and ensure continued high quality research. The work done by the Council has already been of great importance for the discussion of scientific culture in the UK.I have some issues with the structure and focus of the report that prevent me from accepting without reservations. First, the report summarises the items that are considered particularly important for high quality science: collaboration, multidisciplinarity, openness and creativity. The discussion throughout the report and especially the concluding chapters would benefit from referring back to these four elements and how they can be fostered. For example, it is not clear how the suggestions for action will improve the conditions for multidisciplinary research.Second, the subsections in ‘project findings’ are not clearly divided. While the first two subsections look at what research is and why people do it, the remainder is dedicated to the various elements that have a negative effect on the culture of research and that need to be improved. I would therefore suggest to split the section into two. The competition subsection could then serve as an introduction to the other elements (funding, assessment, careers etc.) as it is defined by them. Competition is seen as bad due to the problems with funding, assessment and careers and not because it is bad per se. In fact the report finds that half the survey respondents see competition as a good thing, because it is a driver for high quality research. A clearer split of the section and a better discussion of the role of competition would therefore benefit the report as a whole. Finally, the box headings in Figure 1 should correspond to the sub-section headings in ‘project findings’ (which they partially already do).Finally, I have one very specific comment regarding the funding section. At the end of the section it says that information on trends in levels of funding and themes supported was not available. However, considering that most of the survey respondents come from the biosciences a look at BBSRC funding data, available on their website, can give a first indication of a time trend. There it becomes clear that the number of funded project did not increase over time (unlike the number of academic staff) which may result in more funding competition.", "responses": [] }, { "id": "7957", "date": "26 Mar 2015", "name": "Kate Bullen", "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\nThis article reports the rationale and findings of a major piece of work undertaken by the Nuffield Council on Bioethics in 2014. The changes to the funding of research in the UK, due to recessionary pressures, and the increased emphasis on \"research with impact\" by major funders and the REF2014 process, provides the context for the work. The importance of the article lies in the way in which it provides a context and narrative to these wider changes. Government and funders develop and impose strategic policy decisions in a 'top-down' process; this article reports on how those decisions are experienced from the 'bottom-up'. The article presents a range of fundings derived from an extensive and comprehensive data collection process. The authors identify the limitations of the data set (self-selected participants for example), but nonetheless the study provides a rich source of stimulus material for discussion and debate. Some of the key findings include:The age differential in assessments of the worth of data sharing (15% of <35 year olds positive compared to 7% of >45 year olds). The importance of social benefit and the collective good as fundamental to scientific research. The tension between achieving collective good whilst promoting and supporting individual career progression. The dual role of competition as a spur to delivery of 'best work' versus the pressure to take short cuts to achieve outcomes.These findings, in conjunction with the others reported, are clearly suggestive of a body of researchers who increasingly aware of the pressure to perform to an ever higher level. The article reports the tension experienced by researchers who are striving to generate work of value, but are also aware of the temptations of \"cutting corners\" to achieve. For this reason the article acts as a catalyst for further debate, and generates more questions than it answers. What are the unintended consequences of increasingly stringent research ethics review processes? What is the real impact of the 'impact agenda' on research? Do REF type exercises discriminate between researchers and their work in positive or negative ways? How effective is peer review, and is it crumbling under the weight of research being generated? These questions, together with the important issue of gender equality being raised through the ECU Athena SWAN award and the emphasis on gender in Horizon 2020, and the changing nature of higher education in the UK, are all importantly raised by this article. The authors end with a series of sensible suggestions for future development. The value of the article lies in the fact that these suggestions are made on the basis of evidence generated from the scientific community. How these suggestions, and variations of them, are adopted and assessed will be an important, and as yet undetermined, outcome of this report.", "responses": [] } ]	1	https://f1000research.com/articles/4-66
https://f1000research.com/articles/4-65/v1	13 Mar 15	{ "type": "Research Article", "title": "Effects of mutations on the molecular dynamics of oxygen escape from the dimeric hemoglobin of Scapharca inaequivalvis", "authors": [ "Kevin Trujillo", "Tasso Papagiannopoulos", "Kenneth W. Olsen", "Kevin Trujillo", "Tasso Papagiannopoulos" ], "abstract": "Like many hemoglobins, the structure of the dimeric hemoglobin from the clam Scapharca inaequivalvis is a “closed bottle” since there is no direct tunnel from the oxygen binding site on the heme to the solvent. The proximal histidine faces the dimer interface, which consists of the E and F helicies. This is significantly different from tetrameric vertebrate hemoglobins and brings the heme groups near the subunit interface. The subunit interface is also characterized by an immobile, hydrogen-bonded network of water molecules. Although there is data which is consistent with the histidine gate pathway for ligand escape, these aspects of the structure would seem to make that pathway less likely. Locally enhanced sampling molecular dynamics are used here to suggest alternative pathways in the wild-type and six mutant proteins. In most cases the point mutations change the selection of exit routes observed in the simulations. Exit via the histidine gate is rarely seem although oxygen molecules do occasionally cross over the interface from one subunit to the other. The results suggest that changes in flexibility and, in some cases, creation of new cavities can explain the effects of the mutations on ligand exit paths.", "keywords": [ "Hemoglobin", "Scapharca inaequivalvis", "molecular dynamics", "oxygen escape" ], "content": "Introduction\n\nUnderstanding the ligand escape pathways in the hemoglobin of Scapharca inaequivalvis (HbI) can provide useful insight into how structural fluctuations help facilitate ligand migration to and from the internal heme pocket. This dimeric hemoglobin has long served as a good model for studying allosteric binding due in part to its small size of 33 kDa1,2. The native clam dimeric hemoglobin structure3 is shown in Figure 1a. The dimer contacts are through the E and F helices, bringing the heme groups near the subunit interface3,4. This arrangement is significantly different from tetrameric vertebrate hemoglobins5. The clam dimer exhibits cooperativity with a Hill coefficient of 1.5 for oxygen binding but no response to hetrotropic allosteric ligands like bisphosphoglycerate, carbon dioxide or protons2. The structural changes that occur upon ligand binding are due to intra-subunit rearrangements along the E-F helices and the displacement of interface water clusters3,6. Meta-analysis of a number of structures7 produced a mechanism in which the E and F helices had strong interactions across the dimer interface allowing a small rotation but no translation within the interface during the allosteric transition. Time-dependent Raman scattering studies have established that the tertiary and quaternary changes occur simultaneously and raise questions as to how such structural changes are brought about in a concerted manner to ensure proper protein function8. A combination of NMR and molecular dynamics studies on HbI1 indicate that backbone flexibility contributes to both the free energy of ligand binding and the allosteric subunit rotation in the interface.\n\nA. Crystal structure of HbI obtained from the protein data bank (ID: 3SDH). B. The helices of HbI are colored as follows: A-blue, B-red, C-ochre, D-orange, E-green, F-pink, G-cyan and H-purple. The residues shown in space-filling representation were found by implicit ligand sampling10 to be on the major ligand escape. His69 is at the subunit interface on the E alpha helix, and the rest of the residues form a channel between the B and G helices; including Ile25, Asn32, Ala35, Leu36, Val121, Ser124, and Lys125. The E and F helices are involved in intra-subunit communication. The BG pathway is adjacent to the Xe4 cavity (Figure 2B), allowing escape between these two helices.\n\nA. The trajectories for 14 oxygen molecules during a 10 ns simulation of the wildtype HbI are shown in blue. The main escape pathways are between the B and G and the E and F helices. One oxygen crossed over the subunit interface and another remained in the interface. B. The internal cavities are revealed through the presence of the dense trajectory paths. The B, Xe4, Xe2, and Xe1 cavities are shown.\n\nHbI mutants have provided insight about how local alterations in protein structure can affect ligand binding and cooperativity. The residue Phe97 was first identified as undergoing the largest conformational change upon ligand binding by being displaced from the distal heme pocket to the subunit interface9. The mutants F97V and F97L were shown to remain in the heme pocket even upon ligation due to their smaller size, while the F97Y mutant remained in the subunit interface. These experiments initially demonstrated that keeping this functionally important residue packed within the distal heme pocket leads to a significant increase in oxygen affinity9. The binding rates of Phe97 mutants demonstrated that ligand induced structural changes help to facilitate ligand binding. These results could also reflect changes in structural fluctuations.\n\nThe distal histidine gate hypothesis has been proposed to explain how an oxygen molecule can enter the closed heme cavity10. His69 is on the E helix and Phe97 is on the F helix, suggesting that the structural movement of Phe97 upon ligation may be initiated by swinging of the histidine gate and may also account for the mechanism of cooperativity through the E-F helices10. Ligand docking sites in internal cavities were first identified in myoglobin and proposed as a means of enhancing ligand escape11. Several internal cavities have been identified in HbI using xenon binding experiments to help elucidate plausible ligand migration pathways12. The role of the internal cavities on ligand binding pathways has been studied in mutants of HbI. I25W and I114F were prepared in order to block ligand docking along the Xe4 and Xe2 cavities, respectively. The I25W decreased ligand escape into the solvent and had higher geminate rebinding than HBI. The Xe4 site could not be confirmed as the sole migration route because ligand escape was not completely blocked. The I114F mutant also did not completely block ligand escape. Implicit ligand sampling of the wild type structure simultaneously revealed a major pathway between the B and G helices and a minor pathway between the G and H helices that provides a direct route between bulk solvent and the internal heme pocket that depends on the structural dynamics10. Although previously identified internal cavities have been dismissed from being along primary escape pathways10, blocking such cavities decreased ligand escape13. The residues associated with the major pathway found by implicit ligand sampling are highlighted in Figure 1b.\n\nThe position of the heme group has been demonstrated as fundamental for stable R-state interactions and is dependent on ligand-induced subunit rotation14. Previous studies on HbI10 have suggested the distal histidine gate as the major ligand escape pathway. Time-resolved crystallography showed that photodissociated CO rapidly bound to the distal B site and to the Xe2 and Xe4 cavities. In addition, blocking the Xe4 cavity with dichloroethane did not affect CO rebinding, suggesting that this cavity was not on a major escape route. Since the crystal lattice has a substantial effect on ligand escape, it may be that a significant conformational change or, at least, a change in conformational flexibility would be needed to allow the ligand to exit. These factors would be consistent with the histidine gate path but could be explained by other paths as well.\n\nPrevious computational studies on globins have suggested that ligand migration pathways are not conserved within this family of homologous proteins. Cohen and Schulten15 used implicit ligand sampling to demonstrate different pathways in a variety of globins. Heroux, Mohan and Olsen16 showed that a point mutation in a truncated hemoglobin could change the oxygen escape routes. In this study we apply locally enhanced sampling molecular dynamics17 (LESMD) to visualize ligand binding in wildtype and six mutant structures of HbI. Since the bound ligand in HbI is effectively trapped in a closed bottle, the dynamics of the structure are critical to understanding ligand escape from the protein. It is useful to study the binding pathways in the clam dimer mutants compared to the native structure because mutations have been known to alter the flexibility of the protein and, therefore, may alter possible oxygen binding pathways18. The crystal lattice could be limiting the flexibility of the protein in crystallographic studies, thus restricting necessary quaternary movements for ligand entry. Previous studies have argued that the crystal lattice would not affect the BG escape helices and that the protein has been found to be a very rigid structure overall, thus minimizing the effect of performing binding studies in a crystal10,19. Using molecular dynamics to study ligand escape, it was possible to visualize how the flexibility of the protein would permit a sample of oxygen molecules to explore the most energetically favorable escape pathways in the absence of crystal lattice restraints. We have been able to show how the previously identified internal cavities could facilitate ligand escape between the B and G helices, the G and H helices or other novel escape routes.\n\n\nMethods\n\nThe atomic coordinates for the native clam dimer structure and the six mutants studied were obtained from the Protein Data Bank20. The crystallographic structure of the carbon monoxide bound native clam dimer at 1.7 Å was used (PDB: 3SDH4). The carbon monoxide bound mutants F97L, F97V, M37V, M37F, I114F, and I25W were also obtained from the Protein Data Bank (PDB: 2AV011, 2AUQ11, 2GRH21, 2R4W12, 1JWN22, 2R4Z12). The histidines were protonated appropriately according to their environments in the protein structures. The carbon monoxide was replaced with an O2 ligand. The iron was parameterized as Fe (II) and bonded to the O2 during the equilibration of the system23. For the LESMD simulations, fourteen copies of oxygen molecules were used (seven in each protein subunit). The oxygen molecules do not interact with each other and interact with the rest of the system at a scaling factor of 1/14. The protein was immersed in a water box with 0.15 M NaCl added to neutralize the charge. The cutoffs for nonbonding interactions were 12 Å. The switch distance was 10 Å, and a 1.0 1–4 scaling factor was used. The protein was equilibrated at a constant temperature of 310 K and a pressure of 1 atm using a procedure described earlier16. Production simulations were performed for 10 ns at constant pressure and temperature with the 14 O2 ligands unbound from the iron. All LESMD calculations were performed using NAMD version 2.6 and the CHARMM27 all atom force field, while the data analysis was accomplished using Visual Molecular Dynamics software (VMD)23–25.\n\n\nResults\n\nNative Protein: Analysis of the oxygen trajectories in the native structure revealed movement through internal cavities and several different escape pathways (Figure 2). Of the 14 oxygen molecules, 5 escaped between the B and G helices, 4 between the E and F helices, 2 between the B and E helices, and 1 between the C and G helices. Two oxygen molecules went into the subunit interface through similar paths near Phe97. One of these oxygen molecules crossed completely into the other subunit in the subunit interface, while the other remained in the subunit interface for the remainder of the simulation.\n\nThe trajectories for the O2 ligands are shown in Figure 2. The presence of internal cavities within the protein is clearly visible, and the important residues are highlighted in Figure 2b. Three internal cavities were confirmed as migration sites within the protein. Furthermore, the oxygen molecules are largely confined within the internal cavities throughout the simulation before escaping. Prior to escape, seven oxygen molecules were in the Xe4 cavity, two were in the Xe2 cavity, two were in the Xe1 cavity, and one was in the B cavity. Before crossing into the subunit interface, one oxygen molecule was in the Xe4 cavity and one oxygen molecule was in the Xe2 cavity.\n\nPhe97Mutants: Due to the large structural transition of Phe97 upon ligation, there was profound effect on the oxygen escape pathways and the internal movement of the oxygen molecules within the protein cavities as a result of the structural alteration of Phe97. In the F97L simulation (Figure 3a and Figure 4a), 3 escape pathways were identified. Six oxygen molecules escaped between the C and G helices, five between the B and G helices, and one between the A and F helices. Two oxygen molecules did not escape during the 10 ns simulation. One oxygen molecule went into the subunit interface near Leu97 before reentering the subunit from which it came. The O2 trajectories did not use the Xe1 cavity but more often went into the Xe2 cavity compared to the paths in the native structure. In addition, an entirely new cavity was formed adjacent to the B cavity and is bordered by the residues Tyr50, Leu40, Glu46 and Glu110. This pocket is occupied by several oxygen molecules on their exit routes but it is a dead-end and all of the ones that enter eventually return to the distal pocket before leaving the protein via another route.\n\nThe trajectory for a particular oxygen molecule was recorded until it escaped the protein, “escape” being defined as more than 5 Å from the protein surface. The trajectories for oxygen molecules that failed to escape were recorded for the entire 10 ns. A. F97L B. F97V C. M37V D. M37F E. I114F F. I25W\n\nIn the F97V simulation (Figure 3b and Figure 4b), twelve oxygen molecules escaped between the B and G helices. No other escape path was evident. Two oxygen molecules failed to escape. Oxygen transport via the Xe1 cavity was attenuated, and the Val97 mutation created an additional docking site for the oxygen molecule by expanding the size of the Xe2 cavity.\n\nA. F97L trajectories highlighting the internal cavities. B. F07V trajectories highlighting the internal cavities. The internal cavities in each structure can be compared with the B, Xe4, Xe2, and Xe1 cavities shown in Figure 2B\n\nMet37 Mutants: Met37 is in the heme pocket. M37V and M37F were investigated how Met37 mutations affect oxygen escape. Several escape pathways were identified in M37V (Figure 3c and Figure 5a). Five oxygen molecules escaped between G and H helices, three between C and G helices, two between A and F helices, and one each at the FG corner and between the C and E helices. Two oxygen molecules failed to escape, and one oxygen molecule rapidly went into the A subunit from the B subunit before crossing back over again and exiting the protein. The crossing was near Phe97. Lastly, the M37V mutation resulted in a larger B cavity, causing the oxygen molecules to spend more time in this cavity. In addition, movement along the Xe4 and Xe2 cavities was increased, while movement along the Xe1 cavity was nearly absent. There is also a novel cavity in M37V lined by the residues Gly46, Thr47, Lys113, and Ile114. One oxygen molecule was present in this cavity prior to crossing over to the other subunit and subsequently exiting between the G and H helices near the novel cavity.\n\nA. M37V trajectories showing the internal cavities used for oxygen escape. B. M37F trajectories reveal the internal cavities for ligand transport. The relative position of each internal cavity can be compared to the B, Xe4, Xe2, and Xe1 cavities in Figure 2B.\n\nThree escape pathways were identified in the M37F simulation (Figure 3d and Figure 5b). The major escape pathway was between the B and G helices, with eleven oxygen molecules escaping through this path. One oxygen molecule escaped between the D and E helices, one between the G and H helices, and one oxygen molecule failed to escape. Two oxygen molecules entered into the subunit interface near Phe97. One of these from the B subunit ultimately escaped through that subunit, while the one from the A subunit crossed into the B subunit through the Phe97 gate. Additionally, a novel internal cavity was created near the subunit interface lined by the residues Thr72, Lys96, and His69. A few oxygen molecules lingered in this new cavity, but they were unable to cross into the subunit interface despite the cavity’s proximity to the subunit interface. Lastly, the Xe1 cavity does not appear to be an important internal migration route in the M37F mutant. Greater flexibility was also observed with respect to the residues of the B and G helices compared to the native structure, consistent with the observed structural escape pathways.\n\nI114F Mutant: Ile114 is located along the path between the B cavity and the Xe4 cavity. Mutation to a larger residue such as phenylalanine has been proposed to restrict movement along this pathway22. Four escape pathways were observed in the I114F simulation (Figure 3e and Figure 6a). Six oxygen molecules escaped between the B and G helices, three between the D and G helices, two between the C and G helices, and one between the D and F helices. Additionally, the size of the Xe2 cavity was expanded to allow escape via the residues Val94, Ala7, Leu90, and Leu10. Migration into the Xe4 and Xe2 cavities was still observed, but migration though the Xe1 cavity was absent. Escape between the CG and DG helices was enhanced by the creation of an internal cavity lined by the residues Asn44, Lys113, Leu40, Tyr47, and Glu46. Lastly, only one oxygen molecule moved into the subunit interface. It migrated from the Xe4 cavity of the A subunit to rapidly escape between the B and G helices of the B subunit.\n\nA. I114F trajectories showing the oxygen escape paths in pink. B. I25W trajectories showing the oxygen escape paths in red. The labeled residues can be compared to the internal cavity residues of the B, Xe4, Xe2, and Xe1 cavities shown in Figure 2B.\n\nI25W mutant: The effect of mutation Ile25 to a larger residue such as tryptophan had previously been expected to restrict ligand docking in the Xe4 cavity12. The molecular dynamics simulation of I25W (Figure 3f and Figure 6b) showed that migration through the Xe4 cavity was restricted due to the tryptophan residue. The simulation revealed five escape pathways in total. Five oxygen molecules escaped between the C and G helices, five between the G and H helices, two between the B and E helices, and one each escaped between the C and D helices and the A and F helices. Oxygen migration became more prominent through the Xe2 and Xe1 cavities, as well as through a novel cavity lined by the residues Asn44, Lys113, Leu40, Tyr47, and Glu46.\n\n\nDiscussion\n\nAnalysis of the native structure of the dimeric clam hemoglobin showed the presence of internal cavities10. The molecular dynamics simulations reported here confirm that these internal cavities are important stops on the oxygen escape pathways. As shown in the oxygen trajectories (Figure 2–Figure 6), the ligands spent most of the trajectory in the cavities in the native and mutant proteins. Structural alterations due to mutations cause alterations in ligand escape. New internal cavities created in several of the mutants produced novel potential escape pathways not seen in the native structure. Restricted movement within a cavity or along a tunnel due to the mutation to a larger residue also changed the potential pathways. This computational result modifies previous studies which assumed that a structural mutation to a larger residue would make the internal cavity completely unable to function22. Our LESMD simulations suggest that alterations to the internal cavities as a result of the mutations alter their function rather than eliminate them.\n\nPrevious studies have used implicit ligand sampling (ILS) to identify the potential escape pathway between the B and G helices10. Although this pathway has been largely ignored in favor of the histidine gate path10, the LESMD simulations showed that its proximity to the Xe4 cavity allows for oxygen transport, making it a potentially important ligand escape pathway. Since ILS and LESMD use very different algorithms to predict potential pathways, the agreement of these two methods is significant. The computational results suggest pathways that are not immediately obvious from the crystal structure of HbI. Elber compared the benefits of simulations and experiments on globins26 and found that simulations identify the possible escape paths, while experiments reveal the likely escape paths. The paths found in our simulations resulted from routes readily accessible between internal cavities and bulk solvent. Implicit ligand sampling studies have also shown an active role of internal cavities as important docking sites in myoglobin27,28.\n\nCrystallographic and molecular dynamics studies have revealed that a water cluster of 17 molecules at the interface rearrange in the ligated structure that could serve to enhance vibrational energy transport between subunits29–32. The stable interface interactions can be viewed as a means of transferring information and enhancing intra-subunit communication. We were able to confirm that the existence of a stable, hydrogen bonded water network within the subunit interface. This network is not destroyed by the mutations presented here. For example, at least ten water molecules stayed in the interface throughout the simulation for the F97V mutant (Figure 7). Although the F97V had a particularly stable hydrogen-bonded water network, similar networks are found in the other proteins also. The existence of such a stable water network may explain why the oxygen molecules were not observed leaving the dimer through the interface rather than towards the bulk solvent. We did observe oxygen molecules crossing between subunit in many of our simulations. This, however, is a much rarer event in the LESMD simulations than escape between pairs of helices. The stable hydrogen bonding network of the interfacial water molecules may provide a tunnel for directing ligands across the interface. LESMD and other studies have demonstrated that globins are known to use tunnels to enhance ligand transport31,33,34.\n\n\nConclusion\n\nOur molecular dynamics studies have revealed that local structural alterations can result in fluid alterations in protein transport pathways. The studies presented here suggest that multiple exit paths for oxygen exist in the wildtype and most, but not all, of the mutant HbI proteins. Thus, the interpretation of experimental changes in oxygen binding due to mutations must be done with caution. LESMD has revealed that the interfacial water clusters and hydrogen bonding network may establish a channel through which oxygen molecules may flow between subunits. LESMD has also elucidated possible escape pathways, with escape between the B and G helices being among the most common escape route. The histidine gate did not seem to be an important escape route in any of the simulations. Importantly, we have demonstrated that the internal cavities functionally enhance oxygen transport in simulations of the dimeric hemoglobin of Scapharca inaequivalvis.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of molecular dynamics trajectories, 10.5256/f1000research.6127.d4352835", "appendix": "Author contributions\n\n\n\nKWO selected the problem and the methodology, KT, TP and KWO did the simulations and the data analysis, KT and KWO wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe Loyola University Chicago McNair scholars program is acknowledged for funding and support to KT.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nLaine JM, Amat M, Morgan BR, et al.: Insight into the allosteric mechanism of Scapharca dimeric hemoglobin. Biochemistry. 2014; 53(46): 7199–210. 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Biophys J. 2007; 93(10): 3591–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeroux MS, Mohan AD, Olsen KW, et al.: Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis. IUBMB Life. 2011; 63(3): 214–20. PubMed Abstract \| Publisher Full Text\n\nGolden SD, Olsen KW: Identification of ligand-binding pathways in truncated hemoglobins using locally enhanced sampling molecular dynamics. Methods Enzymol. 2008; 437: 459–75. PubMed Abstract \| Publisher Full Text\n\nBolognesi M, Rosano C, Losso R, et al.: Cyanide binding to Lucina pectinata hemoglobin I and to sperm whale myoglobin: an x-ray crystallographic study. Biophys J. 1999; 77(2): 1093–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoffi A, Verzili D, Chiancone E, et al.: Stereodynamic properties of the cooperative homodimeric Scapharca inaequivalvis hemoglobin studied through optical absorption spectroscopy and ligand rebinding kinetics. Biophys J. 1994; 67(4): 1713–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerman HM, Westbrook J, Feng Z, et al.: The Protein Data Bank. Nucleic Acids Res. 2000; 28(1): 235–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnapp JE, Pahl R, Srajer V, et al.: Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin. Proc Natl Acad Sci U S A. 2006; 103(20): 7649–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnapp JE, Gibson QH, Cushing L, et al.: Restricting the ligand-linked heme movement in Scapharca dimeric hemoglobin reveals tight coupling between distal and proximal contributions to cooperativity. Biochemistry. 2001; 40(49): 14795–805. PubMed Abstract \| Publisher Full Text\n\nMackerell AD Jr, Feig M, Brooks CL 3rd, et al.: Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J Comput Chem. 2004; 25(11): 1400–1415. PubMed Abstract \| Publisher Full Text\n\nPhillips JC, Braun R, Wang W, et al.: Scalable molecular dynamics with NAMD. J Comput Chem. 2005; 26(16): 1781–1802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHumphrey W, Dalke A, Schulten K, et al.: VMD: visual molecular dynamics. J Mol Graph. 1996; 14(1): 33–38. PubMed Abstract \| Publisher Full Text\n\nElber R: Ligand diffusion in globins: simulations versus experiment. Curr Opin Struct Biol. 2010; 20(2): 162–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen J, Arkhipov A, Braun R, et al.: Imaging the migration pathways for O2, CO, NO, and Xe inside myoglobin. Biophys J. 2006; 91(5): 1844–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomita A, Sato T, Ichiyanagi K, et al.: Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin. Proc Natl Acad Sci U S A. 2009; 106(8): 2612–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPardanani A, Gambacurta A, Ascoli F, et al.: Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity. J Mol Biol. 1998; 284(3): 729–39. PubMed Abstract \| Publisher Full Text\n\nZhou Y, Zhou H, Karplus M: Cooperativity in Scapharca dimeric hemoglobin: simulation of binding intermediates and elucidation of the role of interfacial water. J Mol Biol. 2003; 326(2): 593–606. PubMed Abstract \| Publisher Full Text\n\nOrlowski S, Nowak W: Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin. J Mol Model. 2007; 13(6–7): 715–23. PubMed Abstract \| Publisher Full Text\n\nGnanasekaran R, Agbo JK, Leitner DM: Communication maps computed for homodimeric hemoglobin: computational study of water-mediated energy transport in proteins. J Chem Phys. 2011; 135(6): 065103065103/1, 065103/10. PubMed Abstract \| Publisher Full Text\n\nWinter MB, Herzik MA Jr, Kuriyan J, et al.: Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain. Proc Natl Acad Sci U S A. 2011; 108(43): E881–E889, SE881/1–SE881/7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalter MD, Blouin GC, Soman J, et al.: Determination of ligand pathways in globins: apolar tunnels versus polar gates. J Biol Chem. 2012; 287(40): 33163–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlsen K, Trujillo K, Papagiannopoulos T, et al.: Dataset 1 in “Effects of mutations on the molecular dynamics of oxygen escape from the dimeric hemoglobin of Scapharca inaequivalvis”. F1000Research. 2014. Data Source" }	[ { "id": "8021", "date": "18 Mar 2015", "name": "Emilia Chiancone", "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\nThis is a nicely written manuscript that provides a fairly complete ligand binding Molecular Dynamics frame to Scapharca inaequivalvis HbI and several functionally relevant mutants. Trajectories of biatomic ligand molecules have been carefully examined in mutated proteins and have highlighted the contributions of “native” or “mutant induced” cavities to the overall dynamics of ligand escape. Such contributions are proposed to profoundly affect the trajectories in a non-trivial manner, by the demonstration that filling cavities with bulky amino acid side chains does not automatically result in ligand exclusion from the cavity itself. At the same time, the simulation thus provided appears to tune down the role of a distal histidine gate that would not represent a significant escape route for the bound ligand in Scapharca HbI.In this respect, the discussion section could and should be improved. It might gain a wider and more general impact by including a comparison with similar molecular dynamics studies on myoglobin in which histidine rotamers appear to exert an effect upon ligand escape dynamics.", "responses": [] }, { "id": "7958", "date": "23 Mar 2015", "name": "Andrea Mozzarelli", "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 mansucript by Trujillo et al. reports on the investigation of the escape pathway of oxygen in HbI from Scapharca inaequivalvis carried out exploiting computational tools. Wild type and selected mutants were investigated to evidence the effects of mutations on the escaping routes as well as on the relevance of cavities where ligands can transiently bind. The robustness of molecular simulation results is difficult to assess. One key element for this assessment is the agreement with experimental data whenever they are available. In this case, previous kinetic studies using CO as a ligand on wild type and mutants (quoted in the manuscript), as well as oxygen binding in solution (quoted in the manuscript) and in the crystalline state (Mozzarelli et al.,1996), are available. Authors should re-write the Discussion by comparing computational and experimental data, and pointing to agreements and disagrements. This approach was followed by Bisht et al. (2011), Spyrakis et al. (2011) and Abbruzzetti et al. (2011) for escaping routes in AHbI from Arabidopsis thaliana.\n\nFurther points:Authors should indicate i) how many times the simulations with the 14 oxygen molecules were carried out, ii) which is the statistics on the different escaping routes, offering a visual indication from the most probable to the less probable escaping route.As shown by Mozzarelli et al. (1996) the cooperativity is fully conserved in HbI crystal as in solution, contrary to human Hb where crystallization prevent tertiary and quatrenary transitions. This makes very interesting the computational analysis here reported because the structure used in the investigation is functionally active.", "responses": [] }, { "id": "7959", "date": "25 Mar 2015", "name": "Karin Nienhaus", "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 very interesting molecular dynamics study on ligand migration within and escape from the dimeric wild-type Scapharca inaequivalvis HbI and various mutants. The study suggests that multiple ligand exit pathways co-exist and that the histidine gate does not serve as an important escape route in the simulations. Interestingly, very similar conclusions were drawn by Cohen et al. (ref. 27), who studied ligand escape from myoglobin by molecular dynamics and also found a large number of exit routes.Experimentally, there is no direct evidence identifying the entry/exit portal for ligands into Mb or other globins. Perutz and Matthews (1966) speculated early on that rotation of the distal histidine side chain could form a channel between the heme pocket and the solvent. Later on, crystal structures of Mb with bulky ligands bound to the heme iron showed that an exit channel can be pried open by these ligands (Ringe et al., 1984; Johnson et al., 1989). However, there are also reports arguing in favor of multiple pathways, including a high-throughput flash photolysis experiment on more than 1,500 Mb variants (Huang and Boxer, 1994). The strongest experimental evidence of ligand entry and escape via the ‘histidine gate’ presently comes from a time-resolved x-ray crystallography study on the L29W myoglobin mutant (Schmidt et al., 2005). In the mutant, ligands that became trapped in the Xe1 cavity could only escape from this cavity upon rare fluctuations of the W29 side chain that opened the path into the distal pocket. The enormously long residence time in Xe1 clearly proved that the CO in L29W had no alternative pathway available to leave the Xe1 cavity, at least on time scales relevant for the ligand binding reaction.Flash photolysis experiments on CO-ligated wild-type Scapharca inaequivalvis HbI and various mutants and dioxygen equilibrium binding studies suggested that the histidine gate mechanism may hold also for this dimeric protein (Nienhaus et al., 2007). In the experiment, the internal cavities served as transient docking sites, as also seen in the simulations, but were not way stations on the exit route. Notably, in the H69L mutant and especially the H69V-I114M double mutant, the fraction of ligands that rebound geminately was significantly enhanced and bimolecular rebinding was very fast. Therefore, for me as an experimentalist, these mutants seem to be very promising candidates for additional ligand dynamics simulations because I would expect pronounced differences compared to the wt protein. I suggest to include these simulations in the present manuscript and to extend the discussion section to provide a more detailed view on the ‘histidine gate problematics’.", "responses": [] } ]	1	https://f1000research.com/articles/4-65
https://f1000research.com/articles/4-64/v1	12 Mar 15	{ "type": "Research Article", "title": "Interactive lectures: Clickers or personal devices?", "authors": [ "Lesley J. Morrell", "Domino A. Joyce", "Domino A. Joyce" ], "abstract": "Audience response systems (‘clickers’) are frequently used to promote participation in large lecture classes, and evidence suggests that they convey a number of benefits to students, including improved academic performance and student satisfaction. The limitations of these systems (such as limited access and cost) can be overcome using students’ personal electronic devices, such as mobile phones, tablets and laptops together with text message, web- or app-based polling systems. Using questionnaires, we compare student perceptions of clicker and smartphone based polling systems. We find that students prefer interactive lectures generally, but those that used their own device preferred those lectures over lectures using clickers. However, device users were more likely to report using their devices for other purposes (checking email, social media etc.) when they were available to answer polling questions. These students did not feel that this distracted them from the lecture, instead, concerns over the use of smartphones centred around increased battery usage and inclusivity for students without access to suitable technology. Our results suggest that students generally preferred to use their own devices over clickers, and that this may be a sensible way to overcome some of the limitations associated with clickers, although issues surrounding levels of distraction and the implications for retention and recall of information need further investigation.", "keywords": [ "Classroom response system", "Interactive teaching", "Interactive lecture", "Personal response system" ], "content": "Introduction\n\nAudience response devices, also known as electronic voting systems or ‘clickers’ have a strong body of evidence supporting their use in Higher Education (Caldwell, 2007; Kay & LeSage, 2009; reviewed in Keough, 2012). Clickers have been found to be useful for engaging students in large lecture classes, promoting participation, facilitating reflection and formatively (and anonymously) testing understanding. Lecturers can adjust their teaching in real time in response to difficulties that students may be having with particular concepts, to promote understanding. Further benefits of using clickers include increased attendance, improved attention spans and positive outcomes such as higher levels of academic performance and student perception of satisfaction (reviewed in Barnett, 2006; Kay & LeSage, 2009; Keough, 2012; Salemi, 2009; Sutherlin et al., 2012).\n\nHowever, there are a number of drawbacks to using clickers. There is a time investment involved in preparing interactive lectures that may not pay off if access to hardware is limited because of lecturer demand versus investment at an institutional level. Additionally, handing out and collecting hardware can eat into teaching time (Dunn et al., 2013; King & Robinson, 2009). However, many students now carry with them personal devices such as mobile phones, tablets and laptops that allow for similar interactive lectures styles using these technologies (Brett, 2011; Dahlstrom & Bichsel, 2014). Text-message based interaction has already received attention in the literature (Voelkel & Bennett, 2014). Text-based systems allow similar multiple-choice responses to lecturer questions, but have the added benefit of allowing students to post questions to a ‘text-wall’ or similar, something that is not possible with most clickers. The drawbacks of a text-based system, however, centre around the cost to the student (Voelkel & Bennett, 2014): each message carries a financial obligation on the part of the student (although some students will have bundles offering unlimited texts, many will have a limited number and some will pay per message). This additional cost to the students may be considered unacceptable, particularly where students pay fees for their education.\n\nWeb-based (or app-based) response systems are increasingly available and carry with them the benefits of both clickers and text-message systems (Dunn et al., 2013), with options for multiple choice, numerical answer and text-based questioning. Their use is less well studied, tending to focus on the evaluation of particular systems (e.g. Dunn et al., 2013; Méndez & Slisko, 2013; Voelkel & Bennett, 2014; Wash, 2014). Web-based systems that connect via a University’s wireless network will carry no additional cost to the student. Possible drawbacks centre on a student’s likelihood of distraction, particularly by social networks while online (Dunn et al., 2013). This could pose a problem because there is evidence to suggest that individuals are not as skilled at multi-tasking as they might think (Sanbonmatsu et al., 2013). Multi-tasking is particularly difficult when the tasks are similar (i.e. use the same cognitive channels), such as simultaneously viewing lecture slides on a screen and reading social network status updates (Rubinstein et al., 2001; Trafton & Monk, 2007). Text-message interruptions to a lecture can reduce learning (Conard & Marsh, 2014; Rosen et al., 2011), and laptop usage (Fried, 2008), particularly long browsing sessions (Grace-Martin & Gay, 2001) and behaviours unrelated to academic work (Gaudreau et al., 2014), such as using Facebook can reduce academic (test) performance (Junco, 2012; Junco & Cotten, 2012). Distraction or disruption can also result in students being less able to apply their knowledge flexibly in new situations (Foerde et al., 2006). In addition, recent work suggests that checking social networks during lectures can also be distracting to other students for whom the screen is visible, and can have implications for information retention, measured by lower test scores (Sana et al., 2013).\n\nHere, we report the results of a web-based system trial, using students’ own devices, and compare the costs and benefits to both the use of standard clickers in lectures, and to lectures with a similar level of interaction but no technology. We use paper-based questionnaires and focus groups to analyse students’ perception of enjoyment, understanding, ease of use and distraction in the three types of lectures.\n\n\nMethods\n\nWe carried out the trial in two level 5 (year 2 of a standard UK 3 year Bachelor degree) modules in the School of Biological, Biomedical and Environmental Sciences at the University of Hull, in lectures by two different lecturers (Behavioural Ecology, LJM, 104 registered students; Evolutionary Biology, DAJ, 38 registered students). There was some overlap in the students taking the modules. In each module, at least one lecture used institutionally-held clickers (Turning Technologies Response Card RF with Turning Point; ‘clicker lectures’), at least one used an app-based response system (eInstruction Flow (http://www.einstruction.eu/downloads/)), since acquired by Turning Technologies; ‘device lectures’), and at least one used ‘hands-up’ or ‘shout-out’ interaction (‘no-technology lectures’). We chose the eInstruction app-based system because it was free for the students to use, being costed on a per-instructor basis. Also, to avoid any potential disadvantage to students who were unable or unwilling to download the app for the ‘device lectures’ (available for Android and iPhone), we purchased a small number of “crickets” – clicker hardware devices that are integrated, allowing both the app and clickers to be used simultaneously. Students were advised in advance to download the app, but those that did not, or did not possess suitable personal technology, were able to collect a cricket at the start of the device lectures.\n\nBetween four and eight multiple choice questions were devised for each lecture, and the type of lecture assigned randomly throughout the semester. Lecture slots were either 50 minutes (Evolutionary Biology) or 110 minutes with a break mid-way through the session (Behavioural Ecology). Students answered the questions using the technology available in that particular lecture. After the final lecture, students were asked to complete a paper-based questionnaire (Supplementary Material) asking a range of questions about the technology they possessed, their perception of their enjoyment, understanding and informativeness of the different types of lecture, their likelihood of distraction and preferred approach to interactive lectures. Students who used their own technology to answer the questions in the device lectures completed a version of the questionnaire comparing all three types. Students who did not use their own technology answered a modified version of the questionnaire comparing the no-technology lectures with the clicker lectures. We used two follow-up semi-structured focus group interviews with ten participants in total (one group of eight, one group of two) to obtain a more nuanced understanding of student perceptions of using their own technology in lectures. Focus groups primarily concentrated on the distraction element of the students using their own technology as response systems, and all participants were volunteers.\n\nThe proportions of device and clicker users giving particular responses were compared using proportion tests (prop.test in Rv2.13.0; R Development Core Team, 2011). Exact binomial tests were used to assess whether the proportion of participants giving a particular response was greater than 0.5.\n\nThe School of Biological, Biomedical and Environmental Sciences and the Faculty of Science and Engineering ethical approval committees approved the project and questionnaires prior to any work commencing. Questionnaire data (including student contact details) was input and anonymised by student interns, who also carried out and transcribed focus group interviews. Lecturing staff were unaware of student identity in data analysis. Consent information (Supplementary Material) was included with the questionnaire: participation was voluntary and students were free to withdraw at any time.\n\n\nResults\n\nSeventy-eight students completed the questionnaire. Of these, 44 (56.4%) used their own device, while 34 (43.6%) used only clickers. Throughout, we refer to those that used their own devices as ‘device users’ and those that used the clickers we provided as ‘clicker users’ (device users used clickers in the clicker-only lectures). Age and gender of the students reflected the make-up of the cohort: 53.9% male, 43.6% female (two students did not answer), and 65 students (88.3%) were aged 18–23, with five (6.4%) aged between 24 and 29, five (6.4%) between 30 and 35 and three (3.9%) students aged 36 or over.\n\nAlmost every student owned at least one type of device that could potentially be used, with a large proportion owning multiple device types. Sixty-seven students (85.9%) owned a smartphone, with 76.9% owning a laptop, 32% owning a tablet, and 9% owning a netbook or ‘other’ technology (including iPods and non-smart mobile phones). While 21 students (26.9%) owned only one type of technology, the remainder owned more than one (2:42.3%, 3:26.9%), with two students declaring ownership of four types of devices that could potentially be used as part of an audience response system (a smartphone, laptop, netbook and tablet). One student did not possess any type of technology that could be used.\n\nAs clickers are used in other modules the students may have taken (notably a level 4 module Ecology and Evolution), students were asked if they had previous experience of using clickers in lectures: 67.9% (53 students) had, while 32.1% (25 students) had not.\n\nOf the 44 students that used their own device, 37 (84.1%) used a smartphone and five (11.4%) used a tablet, with the remaining two students using either a laptop or other type of technology. In terms of operating system, 59.1% used iOS, 38.6% used Android, and one student (2.3%) used Windows (on a laptop). Of the 34 students who did not use their own device to participate, 23 (67.7%) already had a device that they would be willing to use, while eight (23.5%) did not have a device that they would be willing to use (although all owned at least one device that could potentially be used). The remaining three students (8.8%) did not currently possess a device, but were thinking of getting one. These three students included the one who did not currently own any types of technology and two possessing only a laptop.\n\nWe asked students which type of interactivity was more enjoyable, informative, understandable, and made lectures seem to pass more quickly. Figure 1 summarises the responses. In general, device users felt that interactive lectures (either using their own devices or using clickers) enhanced their enjoyment and understanding slightly more than clicker users did, although these differences were not significant. Device users who preferred interactive lectures were divided in whether they preferred using clickers, their own devices or had no preference between the two types of technology (see Figure 1). These three preference categories have been grouped together as ‘interactive lectures’ in the analysis that follows. Overall, very few students felt the no-technology lectures scored most highly on any of the four measures.\n\nStudent perceptions of the three different types of lecture (no interactive technology, clicker and personal devices as response systems), in terms of their a) Enjoyment, b) Understanding, c) Informativeness and d) perception of the speed at which the lecture passes (fast = good), for students that used their own technology (device users) and those that used clickers (clicker users).\n\nIn terms of enjoyment (Figure 1a), 77.3% of device users (34 students) preferred lectures with interaction via either clickers or their own technology, compared to 55.9% of clicker users (proportion test: χ2 = 3.107, df = 1, p = 0.078) preferring interactive lectures over the no-technology lectures. Significantly more than half of the device users preferred interactive lectures (binomial test: p < 0.001), but this was not true for clicker users (p = 0.608). None of the device users and only 5.9% of the clicker users (2 students) felt that the no-technology lectures were the most enjoyable (Figure 1a).\n\nFor levels of understanding (Figure 1b), 70.5% of device users (31 students) and 52.9% of the clicker users (18 students; χ2 = -1.825, df = 1, p = 0.179) felt that the interactive lectures were more understandable. This represents significantly more than half of the device users (p = 0.010) but not the clicker users (p = 0.864). Two students (4.5%) who used their own technology and two students who used clickers (5.9%) felt that the no-technology lectures were more understandable (Figure 1b).\n\nStudents expressed less clear overall preferences in terms of the informativeness of the lectures (Figure 1c) or the speed at which they passed (Figure 1d). 61.4% of device users (27 students) and 55.9% of clicker users (19 students; χ2 = 0.598, df = 1, p = 0.439) felt that the interactive lectures were more informative than the non-interactive ones, but this was not significantly more than half (device users: p = 0.174, clicker users p = 0.608). Again, a few students (four device users and three clicker users) felt the no-technology lectures were the most informative (Figure 1c). In terms of the speed at which the lecture appeared to pass (Figure 1d), almost half the students (45.5% of device users and 44.1% of clicker users) had no preference with two device users and four clicker users feeling the no-technology lectures passed more quickly. The remaining 50% of device users (22 students) and 44.1% of clicker users (15 students) felt that the interactive technology lectures passed more quickly (proportion test: χ2 = 0.083, df = 1, p = 0.774, binomial tests, device users: p = 1, clicker users: p = 0.608; Figure 1d).\n\nWhen asked whether having their device ready to answer questions distracted them from the lecture, 36 of the device users (81.8%) answered in the negative, while seven (16.9%) said that it did. A mixed response was highlighted in the focus groups:\n\n“It was easier to me like, ah, I’ll just reply to this text quickly”\n\n“I think I’d be more tempted because it’s like well I could just say I’m doing it for this purpose [answering the questions] when really I wasn’t”\n\n“I don’t think it [having your device ready] makes a difference at all”\n\nHowever, when asked which, if any of a number of web-based and interactive activities (including checking email, using social media, browsing the web and texting or sending a message) they carried out during each type of lecture, much higher numbers reported using their devices. Overall, 70.5% of device users admitted to using their devices for at least one other activity during the device lectures, compared to only 25% in clicker lectures and 43.2% in the no-technology lectures (Figure 2a). Students were significantly more likely to self-report using their device for other purposes in the device lectures than the other two lecture types (proportion tests: device lectures vs clickers lectures, χ2 = 16.443, df = 1, p < 0.001, device lectures vs no technology lectures, χ2 = 5.604, df = 1, p = 0.018), but there was no difference in self-reported usage between clicker lectures and no technology lectures (χ2 = 2.478, df = 1, p = 0.115). Thus, students are most likely to use their devices for other purposes when they are also using them as audience response devices.\n\na) Likelihood of device users using their device for other purposes during each of the three types of lecture (with no interactive technology, clickers or personal devices as responses systems). b) Number of students using their device for each broad category of activity (email, social media, browsing the web, sending or receiving text or other messages, and other activities) during each of the three types of lecture (with no interactive technology, clickers or personal devices as responses systems).\n\nStudents used their devices for checking email, using social media, browsing the web, sending and receiving messages as well as other activities (Figure 2b), with the majority of students reporting using their device for multiple purposes. When asked in the focus groups about the sort of activities they used their devices for during lectures, students in the first focus group suggested that it was mostly socialising rather than study-related, although the second focus group highlighted the academic activities that they might engage in, such as checking the Virtual Learning Environment (VLE) for assignments, going through the slides or using Google to search for information about unfamiliar topics. Both focus groups identified Facebook as being the primary “culprit”. Other activities included online shopping for groceries and other items.\n\nTwenty seven device users (61.4%) and 21 clicker users (61.8%) reported being aware of others using their devices for other purposes during a normal lecture. In the device lectures, this proportion increased slightly among clicker users (to 79.4%) but not device users (61.4%). When students in the focus groups were asked whether they noticed others around them using their devices for other purposes, students again answered affirmatively, but suggested that this was no more than usual. They did find that it could be distracting:\n\n“I did notice a few people like on Facebook and things”\n\n“I know a few times I’ve been in lectures where people next to me have been on a game and I’ll be like oh I’ll just go on that for a minute and then you kind of get into it and I’ve spent like a full 2 hours on a game”\n\n“If they’re on the phone it’s not distracting. But if they’re talking then it will distract me”\n\n“People are sometimes playing games and that’s distracting. You can see it out of the corner of your eye.”\n\nInterestingly, two students mentioned that they actively avoided taking their laptops and tablets to lectures because they find them much more distracting than their smartphones:\n\n“I never take my laptop in… got an increased chance of getting distracted, because it’s there with a little button that says ‘internet’ on”\n\nHowever, despite admitting that they were more likely to get distracted, students felt that the interactive element of the lecture meant it held their attention more:\n\n“I’m actually trying to think about the questions and actually answer it whereas before I’d just be sat there like… [tails off]”\n\n“I was actually paying more attention to try and get the right [answer]”\n\n“…we actually had a discussion about the question, before we pressed it, so we were all a bit more attentive to the lecture”\n\nThey also highlighted that factors other than having their devices available made them more likely to use them. Primarily, they suggested that they would be more likely to use their devices once they were already disengaged with the lecture:\n\n“…I’m getting bored and I have to go on my phone to keep awake”\n\n“I don’t think I am distracted by it. I tend to turn to it when I’m bored or not engaged with lectures”\n\nThey were also more likely to get distracted in environments (lecture theatres) that are perceived as being of lower quality:\n\n“I’m more likely to use my phone in there” [a lecture theatre identified by other students in the group as being “awful” and having issues with echoes].\n\nOf the 44 students who used their own device (device users), 39 answered the question “were you happy to use your own devices in this way?” with a positive response (88.6%) while five students (11.4%) would prefer not to, but would if it was the only option. No students responded “no” to this question. Of the device users 32 (72.7%; significantly more than half: p = 0.004) preferred the lectures using student-owned technology as response devices, while 12 (27.3%) preferred using clickers (Figure 3). Of the 34 students who did not use their own devices (clicker users), 76.5% (26 students) preferred the clicker lectures (significantly more than half: p = 0.003), while 14.7% (five students) preferred the lectures with no interactive technology (Figure 3; two students had no preference and one did not answer). No student who was unable to participate using their own device reported feeling disadvantaged in those lectures.\n\nThe proportion of students who preferred lectures using their personal devices, clickers, and no interactive technology, by the type of response system they used in the device lectures (personal devices or clickers).\n\nSome other general themes emerged from the interviews and free text comments. Students mentioned that they would be unwilling to use their own devices if they had to pay for text messages or use up their data allowances, and highlighted software glitches as a potential concern for the wider use of the technology. Several students mentioned that battery usage would act as a barrier to their participation, particularly (as seemed to be the case with the software we used) if the device remained active rather than going into standby when the app was active, and required participants to log in again if the app was minimised. One student highlighted that their battery reserve decreased by 20–25 percentage points during a 1-hour lecture.\n\nAlthough all the students who agreed to participate in the focus groups used their own devices, they identified a number of barriers to participation in their peers, including app availability (not available on Windows phones), the fact that those who did not have suitable technology would feel disadvantaged, particularly if compatible clickers were not available. One student suggested that this may lead to disengagement with the lecture:\n\n“…you put some people at a disadvantage if they don’t have a smartphone or whatever…”\n\n“… a few people said they didn’t feel involved in the lecture they just sit there and they’re like, well I can’t do anything so I’m just going to sit there, I’m not going to pay attention and then they’re probably more likely to go on their phones and actually like do something completely different just because like they couldn’t interact properly”\n\nStudent participation is likely to vary widely depending on the software used, and as alternatives become available, some of the barriers highlighted here may disappear.\n\n\nDiscussion\n\nOur results reflect previous findings that students prefer interactive lectures using audience response systems (Addison et al., 2009; Keough, 2012; Sutherlin et al., 2012). Both the students who used their own technology and students who used only clickers overwhelmingly preferred these lecture types to the traditional ‘hands-up’ or ‘shout-out’ interactive lectures, with the students using their own technology preferring this option to clickers (Figure 3). Device users, in particular, were more likely to find these lectures enjoyable and felt that they increased their understanding (Figure 1). Interactive lectures (and interactive teaching styles) are widely perceived as resulting in better student outcomes (Freeman et al., 2014) and thus the use of students’ own technology represents and effective way of overcoming some of the shortfalls associated with clickers, such as limited availability and the time associated with handing out and collecting them in (Dunn et al., 2013; King & Robinson, 2009).\n\nWe were particularly interested in whether having their devices ready on the desk to answer the interactive questions meant that students were more likely to use their devices for other purposes, thus becoming distracted from the lecture. Although 81.6% of device users reported that they were not distracted, 70.5% admitted that they used their device for a non-academic purpose during the lecture (particularly during the device lecture). This suggests that students did not equate using their devices for other purposes with being distracted from the lecture. The focus groups highlighted that in general, some students felt they used their devices for non-academic purposes when they were already distracted from (or bored by) their lectures. It is possible that, as highlighted by Sanbonmatsu et al. (2013), students who are multitasking by remaining engaged with the lecture and using their devices for non-academic purposes, do not feel distracted despite the fact that their cognitive performance maybe compromised. Further analysis using test scores and/or knowledge retention could examine this.\n\nPrevious work has shown that students use their phones or laptops (McCoy, 2013) in class for a wide range of academic and non-academic activities that are outside the direct remit of the class, which may lead to reduced ability to remember lecture content (Hembrooke & Gay, 2003). This has led to some calls for these technologies to be banned from the classroom, or for students to be banned from using them (Shirky, 2014; Sørensen, 2014) including by the students themselves (McCoy, 2013). Our results support the suggestion that encouraging students to use their technology for one purpose (e.g. as an audience response system) might also encourage them to use it for other purposes, and in this way, perhaps stand-alone clicker systems are more beneficial than using students’ own technology. Indeed, in this study, using a clicker system lead to a decrease in use of personal technology for non-academic purposes.\n\nAlthough students may feel that they are able to multitask (simultaneously using their device for another purpose, yet not feeling they are distracted from the lecture), evidence suggests that people are not as good at multitasking as they think. In fact, individuals who most frequently multitask tend to be those who are least cognitively able to effectively carry out multiple tasks simultaneously (Ophir, et al., 2009; Sanbonmatsu et al., 2013), and tend to be those who are less able to block out distractions and focus on a single task (Sanbonmatsu et al., 2013). This said, how much of an issue is it if students are distracted from lecture material? In the focus group interviews, several students suggested that being distracted in lectures was not necessarily a problem, as it is University policy that all lectures are placed on the VLE:\n\n“If I’ve missed something… I can just go on eBridge [VLE] and go back through the slide and go through what I’ve missed”\n\n“…being distracted within the lecture isn’t so much of a deal if you can then go back to it and read it later and do wider reading.”\n\nDespite the increased distraction, there are a number of benefits to using web-based systems (outlined in the introduction), but awareness of the small number of students who do not possess the right technology, or do not want to use their own technology is necessary. Inclusivity in teaching is an important issue, and staff need to ensure that all students have access to appropriate technology that they are willing to use. The use of a system that allows the integration of apps and stand-alone clickers is one solution to this problem. Alternatively departments or Universities could provide compatible devices that could be used (e.g. iPods or tablets; Finkelstein et al., 2013). Although the system we chose to test was cost-free to the students, another issue highlighted by the students was the use of battery life: opportunities for students to recharge their devices during the day are limited, particularly if they are moving between lecture theatres and classrooms. Widespread adoption of personal devices-as-clickers, such that students were using their devices in multiple classes each day, would exacerbate this issue.\n\nAdopting personal technology to enhance student learning is an attractive prospect, in terms of both staff and student experience. However, the development of systems that minimise the barriers to use that we highlight is key. Additionally, an awareness of the potential for distraction is important and a discussion with students is recommended so that such distractions can be managed accordingly. Ultimately, interactive engagement during lectures enhances enjoyment and understanding and facilitating this method of engagement is valuable.\n\n\nData availability\n\nF1000Research: Dataset 1. Demographic data, 10.5256/f1000research.6207.d44001 (Morrell & Joyce, 2015).\n\nF1000Research: Dataset 2. Device Users, 10.5256/f1000research.6207.d44002 (Morrell & Joyce, 2015).\n\nF1000Research: Dataset 3. Clicker Users, 10.5256/f1000research.6207.d44003 (Morrell & Joyce, 2015).\n\n\nConsent\n\nAll participants agreed to the use of their anonymised data (see ‘Morrell & Joyce – Consent and Questionnaire.pdf’ in the Supplementary Information).", "appendix": "Author contributions\n\n\n\nLJM & DAJ designed and carried out the study, analysed the data and prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was funded by a grant from the University of Hull Innovations in Student Learning Fund to LJM and DAJ.\n\nI confirm that the 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 indebted to our undergraduate interns Khia Dobbinson and Matthew Walker for inputting the questionnaire data and to Khia, Matthew and Julie Furnell for carrying out the semi-structured focus group interviews and producing the transcripts. Graham Scott and Liz Pearce provided helpful feedback throughout the project.\n\n\nSupplementary information\n\nContents of data files & key to entries. Click here to access the data\n\nConsent Form and Questionnaire. Click here to access the data\n\nhttp://dx.doi.org/10.5256/f1000research.6207.s44005\n\nFree text from questionnaires. Click here to access the data\n\nhttp://dx.doi.org/10.5256/f1000research.6207.s43985\n\nTranscripts from semi-structured interviews. Click here to access the data\n\nhttp://dx.doi.org/10.5256/f1000research.6207.s43986\n\n\nReferences\n\nAddison S, Wright A, Milner R: Using clickers to improve student engagement and performance in an introductory biochemistry class. Biochem Mol Biol Educ. 2009; 37(2): 84–91. PubMed Abstract \| Publisher Full Text\n\nBarnett J: Implementation of personal response units in very large lecture classes: Student perceptions. Australas J Educ Technol. 2006; 22(4): 474–494. Reference Source\n\nBrett P: Students’ experiences and engagement with SMS for learning in Higher Education. Innov Educ Teach Int. 2011; 48(2): 137–147. Publisher Full Text\n\nCaldwell JE: Clickers in the large classroom: current research and best-practice tips. CBE Life Sci Educ. 2007; 6(1): 9–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConard MA, Marsh RF: Interest level improves learning but does not moderate the effects of interruptions: An experiment using simultaneous multitasking. Learn Individ Differ. 2014; 30: 112–117. Publisher Full Text\n\nDahlstrom E, Bichsel J: ECAR Study of Undergraduate Students and Information Technology, 2014. Research report. Louisville, CO: EDUCASE Centre for Applied Research. 2014. Reference Source\n\nDunn PK, Richardson A, Oprescu F, et al.: Mobile-phone-based classroom response systems: Students’ perceptions of engagement and learning in a large undergraduate course. Int J Math Educ Sci Technol. 2013; 44(8): 1160–1174. Publisher Full Text\n\nFinkelstein A, Winer L, Buddle CM, et al.: Tablets in the forest: Mobile technology for inquiry-based learning. EDUCAUSE Review. 2013. Reference Source\n\nFoerde K, Knowlton BJ, Poldrack RA: Modulation of competing memory systems by distraction. Proc Natl Acad Sci U S A. 2006; 103(31): 11778–11783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman S, Eddy SL, McDonough M, et al.: Active learning increases student performance in science, engineering, and mathematics. Proc Natl Acad Sci U S A. 2014; 111(23): 8410–8415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFried CB: In-class laptop use and its effects on student learning. Comput Educ. 2008; 50(3): 906–914. Publisher Full Text\n\nGaudreau P, Miranda D, Gareau A: Canadian university students in wireless classrooms: What do they do on their laptops and does it really matter? Comput Educ. 2014; 70: 245–255. Publisher Full Text\n\nGrace-Martin M, Gay G: Web browsing, mobile computing and academic performance. Educ Technol Soc. 2001; 4(3): 95–107. Reference Source\n\nHembrooke H, Gay G: The laptop and the lecture: The effects of multitasking in learning environments. J Comput High Educ. 2003; 15(1): 46–64. Publisher Full Text\n\nJunco R, Cotten SR: No A 4 U: The relationship between multitasking and academic performance. Comput Educ. 2012; 59(2): 505–514. Publisher Full Text\n\nJunco R: In-class multitasking and academic performance. Comput Hum Behav. 2012; 28(6): 2236–2243. Publisher Full Text\n\nKay RH, LeSage A: Examining the benefits and challenges of using audience response systems: A review of the literature. Comput Educ. 2009; 53(3): 819–827. Publisher Full Text\n\nKeough SM: Clickers in the Classroom: A Review and a Replication. J Manage Educ. 2012; 36(6): 822–847. Publisher Full Text\n\nKing SO, Robinson CL: ‘Pretty Lights’ and Maths! Increasing student engagement and enhancing learning through the use of electronic voting systems. Comput Educ. 2009; 53(1): 189–199. Publisher Full Text\n\nMcCoy B: Digital distractions in the classroom: Student classroom use of digital devices for non-class related purposes. Faculty Publications, College of Journalism & Mass Communications. 2013; Paper 71. Reference Source\n\nMéndez D, Slisko J: Software Socrative and smartphones as tools for implementation of basic processes of active physics learning in classroom: An initial feasibility study with prospective teachers. Eur J Phys Educ. 2013; 4(2): 17–24. Reference Source\n\nMorrell L, Joyce D: Dataset 1 in: Interactive lectures: Clickers or personal devices? F1000Research. 2015. Data Source\n\nMorrell L, Joyce D: Dataset 2 in: Interactive lectures: Clickers or personal devices? F1000Research. 2015. Data Source\n\nMorrell L, Joyce D: Dataset 3 in: Interactive lectures: Clickers or personal devices? F1000Research. 2015. Data Source\n\nOphir E, Nass C, Wagner AD: Cognitive control in media multitaskers. Proc Natl Acad Sci U S A. 2009; 106(37): 15583–15587. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Development Core Team. R: A language and environment for statistical computing. 2011. Reference Source\n\nRosen LD, Lim AF, Carrier LM, et al.: An empirical examination of the educational impact of text message-induced task switching in the classroom: Educational implications and strategies to enhance learning. Psicología Educativa. 2011; 17(2): 163–177. Publisher Full Text\n\nRubinstein JS, Meyer DE, Evans JE: Executive control of cognitive processes in task switching. J Exp Psychol Hum Percept Perform. 2001; 27(4): 763–797. PubMed Abstract \| Publisher Full Text\n\nSalemi MK: Clickenomics: Using a classroom response system to increase student engagement in a large-enrollment Principles of Economics course. J Econ Educ. 2009; 40: 385–404. Reference Source\n\nSana F, Weston T, Cepeda NJ: Laptop multitasking hinders classroom learning for both users and nearby peers. Comput Educ. 2013; 62: 24–31. Publisher Full Text\n\nSanbonmatsu DM, Strayer DL, Medeiros-Ward N, et al.: Who multi-tasks and why? Multi-tasking ability, perceived multi-tasking ability, impulsivity, and sensation seeking. PLoS One. 2013; 8(1): e54402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShirky C: Why I just asked my students to put their laptops away. Medium.com. 2014. Reference Source\n\nSutherlin AL, Sutherlin GR, Akpanudo UM: The effect of clickers in university science courses. J Sci Educ Technol. 2012; 22: 651–666. Publisher Full Text\n\nSørensen BM: Facebook fight: why we banned laptops, iPads and smartphones in lectures. The Conversation. 2014. Reference Source\n\nTrafton JG, Monk CA: Task interruptions. Rev Hum Factors Ergon. 2007; 3(1): 111–126. Publisher Full Text\n\nVoelkel S, Bennett D: New uses for a familiar technology: introducing mobile phone polling in large classes. Innov Educ Teach Int. 2014; 51(1): 46–58. Publisher Full Text\n\nWash PD: Taking advantage of mobile devices: Using Socrative in the classroom. J Teach Learn Technol. 2014; 3(1): 99–101. Publisher Full Text" }	[ { "id": "7938", "date": "20 Apr 2015", "name": "Alan Cann", "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\nThis interesting and timely study raises intriguing questions for future work. While it clearly shows that interactive lectures are popular with many students, there is some evidence of a cohort who prefer their lectures to be free from technology distractions. As the authors correctly note, the big unanswered question here is the potential impact on student attainment, which, as with most technological interventions, is difficult to measure accurately. Nonetheless, this work represents and responds to an area which many universities and staff are currently pondering, and as such it represents a useful contribution. I have no comments on the methodology, statistical analysis or writing which has been carried out at an exemplary level, other than to say it would be useful to see effect sizes calculated where statistically significant differences are claimed.", "responses": [] }, { "id": "9033", "date": "18 Jun 2015", "name": "Chris Buddle", "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\nThis is a nice study that focuses on a very interesting question - about whether students use their own devices differently than 'traditional' clickers (or student response systems). The article is well written and covers the topic well. The results are well presented, convincing, and this is a valuable contribution.The authors may want to weave some additional literature into their paper, e.g., I did come across this overview of a recent paper that shows how texting and tweeting during lecture could be 'beneficial' to students. It certainly aligns with my own thinking on the topic. See: https://www.timeshighereducation.co.uk/news/could-texting-and-tweeting-lectures-be-good-learningI might also suggest the authors discuss a little more about how important the role of the instructor is in the process of using any kind of 'interactive' technology in a classroom. So much depends on the level of comfort of the instructor, and the 'distracted' element of the student responses is related to this. I assume that the level of distraction in this case study is relatively low because the instructor is being a good role model and providing a rich learning environment. The challenges when an instructor shoehorns in \"clickers\" (or devices) without adjusting lecture style, etc. is significant and should be addressed.It's a little unfortunate that the authors did not have a true control - ie., no devices or clickers. However, I also realize this is very difficult to achieve in a teaching and learning context, and when student grades are on the line. However, it does raise the issue of how general the findings might be, based on this case study, and perhaps some additional text to reflect this could be inserted.Overall, however, this is a very good case study and is a good contribution!", "responses": [] } ]	1	https://f1000research.com/articles/4-64
https://f1000research.com/articles/4-48/v1	19 Feb 15	{ "type": "Opinion Article", "title": "FDA approved drugs as potential Ebola treatments", "authors": [ "Sean Ekins", "Megan Coffee", "Megan Coffee" ], "abstract": "In the search for treatments for the Ebola Virus, multiple screens of FDA drugs have led to the identification of several with promising in vitro activity. These compounds were not originally developed as antivirals and some have been further tested in mouse in vivo models. We put forward the opinion that some of these drugs could be evaluated further and move into the clinic as they are already FDA approved and in many cases readily available. This may be important if there is a further outbreak in future and no other therapeutic is available.", "keywords": [ "FDA approval", "repurposed drugs", "antivirals" ], "content": "\n\nAs the Ebola outbreak continues and the costs spiral1 we should perhaps be considering what alternative treatments are close to hand in Africa to complement the public health measures that have been used to date2. Two independent studies funded by the US Defense Threat Reduction Agency in 2013 identified FDA approved drugs worthy of further evaluation. This work now seems prescient although it appears to have not been followed through to any public conclusion.\n\nIn one study, the antimalarials amodiaquine and chloroquine (Figure 1) were found to be active using in vitro cell culture assays and an in vivo mouse model3. Both drugs are cheap, generally safe, and likely readily accessible in Africa. These compounds have also shown relatively broad activity against other viruses in vitro and in vivo in animal models (Dengue, Coronavirus OC43, SARS etc.)4–7. A second study suggested selective estrogen receptor modulators (SERM) clomiphene and toremifene (Figure 1) as inhibitors of Ebola virus8. The latter compounds are likely more accessible in the west and indicates that other FDA or EMEA approved drugs may be worth testing including those with hormonal effects that are SERMs. More recent work from 2014 in Europe identified a further 3 FDA drugs, amiodarone, dronedarone and verapamil (Figure 1) that inhibit filovirus entry at plasma levels attainable in humans9. The mechanism of action for most of these drugs is unknown although, using computational methods we have recently shown that the antimalarials and SERMs may share some pharmacophore features which may be important to infer a potential common target or targets10. To our knowledge likely well over 100 small drug-like molecules have now been identified with activity against the Ebola virus including over 50 FDA drugs derived from a reporter assay at NCATS11–14.\n\nAs we await the development of a vaccine or biologic could we consider assessing the efficacy of the antimalarials or the other ‘FDA approved drugs’, as either treatments or prophylactics to prevent the Ebola virus from spreading further? While there can be no guarantee they will work (perhaps requiring adjusted dosage) they may be a last resort. It is possible there are other “non-antivirals” that are widely used in Africa that may also be effective against Ebola. Another example of where ‘non-antiviral’ FDA approved drugs have been found to have ‘anti-viral activity’ is for Hepatitis Virus B and D where the sodium taurocholate co-transporting polypeptide (NTCP) was identified as a receptor15 and screening produced drugs such as azelastine, pioglitazone, glyburide, irbesartan and ezetimibe that inhibited the transporter and may provide potential treatments16,17. Of these compounds, azelastine has been shown to possess in vitro activity against Hepatitis Virus B to date17.\n\nThe aforementioned screens of ‘FDA approved drugs’3,8,9 for Ebola virus activity, were far from comprehensive, covering only some of the known approved drugs currently in use. In an age where drug repurposing is in vogue18–22 and it can be facilitated by computational methods23–25, it would seem a valuable resource for finding compounds active against the Ebola virus. For example, the recent pharmacophores developed for Ebola10 and virtual screens11 could be used to computationally search larger datasets of FDA approved drugs and prioritize additional compounds for testing in vitro. Alternative treatments may also be found by studying those close to patients who may not have contracted the disease and are taking a drug for another chronic disease. Whether we can find a treatment for Ebola by serendipity is questionable but some of the published studies with known drugs might point us in the right direction of where to look. The opportunity to put already available drugs like those already identified3,8,9,11–14 back on the table may be a useful tool for frontline doctors to have and is worthy of more urgent discussion and research.", "appendix": "Author contributions\n\n\n\nBoth authors contributed to the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNeither author has competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nDr. Christopher D. Southan, Dr. Joel S. Freundlich, Dr. Peter Madrid and Dr. Nadia Litterman are acknowledged for discussions on Ebola.\n\n\nReferences\n\nButler D, Morello L: Ebola by the numbers: The size, spread and cost of an outbreak. Nature. 2014; 514(7522): 284–5. PubMed Abstract \| Publisher Full Text\n\nTrad MA, Fisher DA, Tambyah PA, et al.: Ebola in west Africa. Lancet Infect Dis. 2014; 14(11): 1045. PubMed Abstract \| Publisher Full Text\n\nMadrid PB, Chopra S, Manger ID, et al.: A systematic screen of FDA-approved drugs for inhibitors of biological threat agents. PLoS One. 2013; 8(4): e60579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Wilde AH, Jochmans D, Posthuma CC, et al.: Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture. Antimicrob Agents Chemother. 2014; 58(8): 4875–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeyaerts E, Li S, Vijgen L, et al.: Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrob Agents Chemother. 2009; 53(8): 3416–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVincent MJ, Bergeron E, Benjannet S, et al.: Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J. 2005; 2: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoonyasuppayakorn S, Reichert ED, Manzano M, et al.: Amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity. Antiviral Res. 2014; 106: 125–34. PubMed Abstract \| Publisher Full Text\n\nJohansen LM, Brannan JM, Delos SE, et al.: FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Sci Transl Med. 2013; 5(190): 190ra79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGehring G, Rohrmann K, Atenchong N, et al.: The clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry. J Antimicrob Chemother. 2014; 69(8): 2123–31. PubMed Abstract \| Publisher Full Text\n\nEkins S, Freundlich JS, Coffee M, et al.: A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus. F1000Res. 2014; 3: 277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeljkovic V, Loiseau PM, Figadere B, et al.: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Res. 2015; 4: 34. Publisher Full Text\n\nKouznetsova J, Sun W, Martínez-Romero C, et al.: Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerging Microbes Infect. 2014; 3: e84. Publisher Full Text\n\nLong J, Wright E, Molesti E, et al.: Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry. F1000Res. 2015; 4: 30. Publisher Full Text\n\nLitterman N, Lipinski C, Ekins S, et al.: Small molecules with antiviral activity against the Ebola Virus. F1000Res. 2015; 4: 38. Publisher Full Text\n\nYan H, Zhong G, Xu G, et al.: Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife. 2012; 1: e00049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong Z, Ekins S, Polli JE, et al.: Quantitative NTCP pharmacophore and lack of association between DILI and NTCP Inhibition. Eur J Pharm Sci. 2014; 66C: 1–9. PubMed Abstract \| Publisher Full Text\n\nFu LL, Liu J, Chen Y, et al.: In silico analysis and experimental validation of azelastine hydrochloride (N4) targeting sodium taurocholate co-transporting polypeptide (NTCP) in HBV therapy. Cell Prolif. 2014; 47(4): 326–35. PubMed Abstract \| Publisher Full Text\n\nBlatt J, Farag S, Corey SJ, et al.: Expanding the scope of drug repurposing in pediatrics: the Children’s Pharmacy Collaborative. Drug Discov Today. 2014; 19(11): 1696–1698. PubMed Abstract \| Publisher Full Text\n\nDyall J, Coleman CM, Hart BJ, et al.: Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob Agents Chemother. 2014; 58(8): 4885–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang R, Southall N, Wang Y, et al.: The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Transl Med. 2011; 3(80): 80ps16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOprea TI, Mestres J: Drug repurposing: far beyond new targets for old drugs. AAPS J. 2012; 14(4): 759–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalsh CT, Fischbach MA: Repurposing libraries of eukaryotic protein kinase inhibitors for antibiotic discovery. Proc Natl Acad Sci U S A. 2009; 106(6): 1689–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDudley JT, Deshpande T, Butte AJ: Exploiting drug-disease relationships for computational drug repositioning. Brief Bioinform. 2011; 12(4): 303–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDudley JT, Sirota M, Shenoy M, et al.: Computational repositioning of the anticonvulsant topiramate for inflammatory bowel disease. Sci Transl Med. 2011; 3(96): 96ra76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkins S, Williams AJ, Krasowski MD, et al.: In silico repositioning of approved drugs for rare and neglected diseases. Drug Discov Today. 2011; 16(7–8): 298–310. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7764", "date": "23 Feb 2015", "name": "Raymond Lin", "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\nThis is a different take on the approach to therapeutics for Ebola. The idea of using non-antivirals as potential therapeutics has been broached before, and it is natural to extend that proposition to Ebola. The authors provide a good summary of some candidate agents and the laboratory evidence to suggest it might be worth a try. Although the mechanistic explanation is not available, one mechanism which is common to some of them is by their effect on cell membrane transport through pores. The use of this class of drugs would also largely overcome some ethical issues which pertain to experimental drugs. Of course, in practice, the conduct of clinical trials would be more challenging than might appear. The finding of appropriate cases and controls, and the fact that mortality seems also largely determined by early access to supportive measures like re-hydration- these will complicate the ability to detect an outcome difference. On the subject of re-repurposing of drugs, we note also that some non-Ebola antivirals might be re-purposed for Ebola e.g. favipiravir, which has been approved for influenza in some countries.", "responses": [ { "c_id": "1245", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "Thank you for your review and comments. We also mention favipiravir and other non-Ebola antivirals in our recent review http://f1000research.com/articles/4-38/v1." } ] }, { "id": "7744", "date": "27 Feb 2015", "name": "James Popp", "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 Opinion Article provides an interesting and potentially important view that currently approved drugs may have activity against the Ebola virus that would allow rapid entry into clinical use due to the previous approved status. While this concept is consistent with previous scientific discussions of the potential for “repurposing” of drugs, the focus on the Ebola virus is very germane to the immediate medical crisis and the need for effective therapies related to Ebola infections. The presented opinion provides a high level overview of previously published data identifying agents with potential efficacy and the opinion appropriately expands the concept to using computational approaches to identify other drugs with potential activity. The concept of studying Ebola virus exposed individuals who did not contract the disease as an approach to identify drugs that may have a beneficial effect is very good although fraught with difficulties when such studies may be attempted under “field” conditions. This point should be expanded. The authors are encouraged to give additional thought and provide additional opinion regarding the approach(s) that can or should be taken beyond the identification of drugs that may have potential efficacy in an Ebola outbreak. Since the opinion recommends additional screening of drugs for potential efficacy, how will (should) decisions be made to select specific agents for further evaluation or clinical use? What criteria should be deemed essential to make decisions in a selection process? These are critical issues since limited resources (and they will always be limited) will require decisions as to which molecules will be prioritized in the selection for the next level of evaluation or use.", "responses": [ { "c_id": "1246", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "Thank you for your review. In the latest version, the compounds from figure 1 with known ebola virus activity in vitro and in vivo, were used as a starting point for similarity searching >1300 FDA approved drugs in a mobile app (the same type of approach could likely be taken with other software). This would suggest several FDA approved molecules that could be readily tested and may be accessible in Africa (e.g. additional antimalarials and antimicrobials etc). While its unclear if these have been tested to date this type of approach could be taken on a larger scale. It may also point to the importance of a tertiary amine in these compounds for their mechanism.In our most recent opinion http://f1000research.com/articles/4-58/v1 we discuss using the physician's perspective to group treatments which addresses the reviewers question of what criteria may be essential." } ] } ]	1	https://f1000research.com/articles/4-48
https://f1000research.com/articles/4-61/v1	10 Mar 15	{ "type": "Review", "title": "Military-specific application of nutritional supplements: a brief overview", "authors": [ "Kyle Hoedebecke", "Will Brink" ], "abstract": "The Soldiers of America's military endure numerous physical and mental challenges that demand strict physical fitness regimens, extreme mental agility, and a perpetual readiness to deploy at a moment's notice. The chronicity of these stressors has the potential to dramatically reduce performance - both directly and indirectly. Because of this risk, many Soldiers turn to nutritional supplements with hopes of optimizing performance. Increasing amounts of research have demonstrated that various supplements may enhance overall physical prowess, health, and offer quicker recovery in the face of corporal or psychological extremes. Most individuals, including many medical and nutrition professionals, possess only an elementary comprehension of nutritional supplements and their effect on Soldiers in training or combat environments. Nevertheless, a grasp of these details is required for safety and optimal benefits. Various compounds have been evaluated - to include evidence within the military setting - and found to augment endurance, increase cognitive function, decrease knee pain, or offer hearing or lung protection in the face of high-energy impulses. These efficacious outcomes may serve to augment the health and longevity of these Soldiers; however, continued research is needed for efficacy and long-term safety within specific environments.", "keywords": [ "soldiers", "nutrition", "supplements", "performance", "military" ], "content": "Introduction\n\nThe men and women of America’s military face numerous physical and mental challenges that demand strict physical fitness regimens, extreme mental agility, and a perpetual readiness to deploy at a moment’s notice. The chronic nature of these multiple stressors has the potential to dramatically reduce performance directly or indirectly (see Table 1). Because of this risk, many Soldiers turn to nutritional supplements in an attempt to combat such negative effects. In one instance, the Naval Health Research Center surveyed nutritional supplement usage in the SEAL population and established that nearly 78% of this community uses various nutritional supplements1. Even more perturbing is their reported reliance on fellow operators and bodybuilding magazines for supplement advice – neither of which are considered as accurate or unbiased sources1. Similarly, another study showed that, of 2,215 Americans beginning U.S. Army Special Forces and Ranger training discovered, 85% reported a history of supplement use, 64% stated present use, and 35% stated daily nutritional supplement use secondary to multiple motives2. These findings cohere with the authors’ observations of supplement use in the US Army and it is realistic to believe that corresponding figures may be found in other military branches, though more data is needed for confirmation of such pervasive use.\n\nIncreasing amounts of research continues to demonstrate that various nutritional supplements may augment performance as well as general wellbeing; at the same time, these compounds must still be targeted to specific cohorts and must complete validation testing for safety and effectiveness. Most individuals, including many medical and nutrition specialists, maintain only a basic grasp of these nutritional supplements and their effects in the face of extreme physical and mental demands of military training and combat. Knowledge of both is required for continued advancement in this field. Though many supplements lack the necessary data to support their use, some existing studies demonstrate compelling and relevant positive benefits. What follows is a brief review of specific nutritional supplements that suggest a positive impact on military populations. Literature search was performed using PubMed and Google Scholar performed in March 2014.\n\n\nProtection in the face of high-energy impulse noise\n\nSelect groups of Soldiers face regular and repeated exposures to high-energy impulse noise with the potential for permanent hearing loss as well as a decreased operational effectiveness, long-term health decline, and combat readiness3. Multiple studies have found oral magnesium as an efficacious treatment for the prevention of hearing loss secondary to impulse noise. Even before testing in humans, data in animal models, such as guinea pigs, identified direct correlations between decreased serum magnesium levels and harmful noise-induced permanent hearing threshold shifts4 with subsequent evaluations finding similar associations in humans. A placebo-controlled, double-blind study analyzing 300 healthy military recruits undergoing basic training with multiple exposures to impulse noises compared two groups - one receiving 167mg magnesium aspartate orally and the other with placebo daily – with results showing a significant preservation of baseline hearing in the magnesium cohort when compared to the placebo control5. An additional well-performed study confirmed these results demonstrating the protective nature of magnesium for hearing loss secondary to exposure to high impulse noise6. Though it is preferable to have magnesium prior to any exposure, there is evidence that post-exposure oral magnesium supplementation still offers hearing protection with a therapeutic effect inversely proportional to the length of time elapsed between the exposure and the start of treatment7. Its efficacy may be further improved in the presence of other antioxidants – specifically vitamins A, C, and E. These compounds, all of which are commonly found in standard multivitamin tablets, have shown to augment the efficacy of magnesium and its ability to protect against hearing loss3.\n\nSimilarly, the lungs also have a heightened susceptibility to these types of injuries secondary to abrupt rises in atmospheric pressure via concussive forces from explosions. Additional low-level exposures are associated with significant pathology within various susceptible parts of the body. A study from the Walter Reed Army Institute of Research showed low-level exposures to correlate with pathological pulmonary changes secondary to oxidative damage caused by antioxidant depletion – increasing the risk of long term problems such as respiratory insufficiency and adult respiratory distress syndrome8. Because of the link to oxidative damage, there is a potential benefit from multivitamin supplementation during military training or deployment when the risk of high impulse energy exposure is at its greatest8. Considering the low cost and risk profile, military medical providers should consider recommending that their Soldiers take multivitamins with antioxidants in deployed or training environments.\n\n\nMinimizing musculoskeletal injuries despite prolonged training and operational requirements\n\nThe often unimaginable amounts of stress on both mind and body during simulated or live combat situations often correlate with increased injury rates. Joint pain, fractures, and other pathologies plague recruits, trainees, and veteran operators alike – a worrisome situation where individual and unit readiness are potentially sacrificed. Implementing measures to safely minimize or treat injuries would contribute to combating work-related stressors and subsequently augmenting a unit’s operational effectiveness. One such detractor is chronic pain from degenerative joint disease (DJD) caused by and worsened with repetitive microtrauma. Several combinations of glucosamine, chondroitin, and manganese have been effective for this ailment in both dogs and humans9–12. The Naval Special Warfare Command performed a 16 week randomized, double-blind, placebo-controlled crossover trial with 34 Soldiers suffering from chronic knee or lumbar pain secondary to DJD. Half were placed in a group given glucosamine HCl (1,500 mg/day), chondroitin sulfate (1,200 mg/day), and manganese ascorbate (228 mg/day) while the others received a placebo control. Though not effective for back pain, this supplement combination has proven effective for the treatment of knee pain in this study and multiple other studies9,11–12.\n\nMoreover, Soldiers and trainees experience numerous myalgias, contusions, sprains, and other acute injuries caused by high training and operational tempos. More worrisome is the potential reduction in overall health and immunity, increased susceptibility to infections, and higher likelihood of heat-related injuries. One study evaluated such outcomes in relation to post-exercise protein consumption in Marine recruits. These healthy males were randomly assigned to three treatment arms – placebo, control, and protein supplement – during a basic training period of 54 days. Compared with the other 2 arms of the study, the protein supplement cohort averaged 33% fewer total medical visits, 28% fewer bacterial/viral infections, 37% fewer muscle/joint problems, and 83% fewer visits due to heat exhaustion13. Post-exercise protein supplementation, as seen in this study, offers the ability to positively improve medical outcomes, musculoskeletal resiliency, and tissue hydration during extended levels of physical demand – signifying a potential medical therapy allowing for the avoidance of various health problems found in populations undergoing physical and mental extremes13. These outcomes may further have a positive effect by improving recruit/trainee dropout rates, reducing number of sick days, and decreasing overall medical expenditures.\n\n\nMental and physical maintenance under stress\n\nB-alanine (BA) – a known substrate of carnosine – has been shown to augment prolonged physical performance. This supplement plays an important role helping to buffer hydrogen ions during high-intensity exercise14. A meta-analysis of 15 peer-reviewed publications evaluating the effects of BA – with a median of 179 g BA supplemented – on exercise concluded that there was a significant improvement for exercise lasting 60–240 seconds (P = 0.001) as well as >240 s (P = 0.046) compared to placebo15. Of note, there was no improvement for exertion less than 60 seconds (P = 0.312)15. Similar improvements have also been shown in Soldiers. A group of 20 elite operators were placed in either a BA group or placebo group for a 4 week period and had a variety of military and physical fitness tests before and after supplementation. At the end of the study, the Soldiers in the BA arm showed no improvement in cognition; however, they did show significantly better power performance, marksmanship, and target engagement speed from pre-ingestion levels as compared to placebo15.\n\nCaffeine use has also displayed improvement in various aspects of a Soldier’s performance16–17. A prospective randomized study of 62 trainees during Navy SEAL Hell Week placed the participants into one of four arms: 100, 200, or 300 mg of caffeine or a placebo. The marksmanship of each group was tested at baseline and again after exposure to training stressors and 73 hours of sleep deprivation. The results showed that 200 to 300 mg of caffeine allowed individuals to sight targets and fire faster while maintaining accuracy during periods of high stress and combined sleep deprivation17. Furthermore, a meta-analysis of 41 placebo-controlled double blinded studies found further evidence of caffeine’s ergogenic effects that include improved exertional capabilities, decreased perceived exertion during ruck marches, better reaction time, improved memory and overall cognitive function, better mood, and overall improved performance16. When used in low to moderate doses between 38 to 400 mg caffeine daily, Soldiers and military units can take advantage of the positive operationally pertinent effects of caffeine while minimizing potential side effects such as dehydration or insomnia18.\n\n\nConclusion\n\nThis paper is not meant to be an exhaustive review of every supplement with possible direct military applications, but rather to identify the potential of these compounds through the above specific examples which were conducted with the appropriate population. Limitations of this study include a non-systematic review approach and the possible omission of relevant studies – including those with negative or null results. This topic remains a highly controversial subject though data exists illustrating the potential benefit of many nutritional supplements. The authors suggest that much of this dispute stems from the continued need for additional data, exaggerated marketing claims, and a lack of FDA regulation; however, the military medical community is not taking advantage of the potential positive benefits by choosing to ignore virtually all nutritional supplements. Additional research and a streamlined method of training and information sharing among providers are still necessary and potentially beneficial.\n\nFurthermore, various inexpensive supplements may serve to save the military millions of dollars. For each compound, a cost/benefit analysis must be conducted for recommendations of location, longevity, and amount of supplements use. Areas where the military can benefit monetarily in the short term are through reduced recruit/trainee dropout rates or suppressed rates of injury and in the long term through a decline in known associated medical problems (arthritis, hearing loss, traumatic brain injury (TBI), etc.) and reduced turnover rates by increasing a Soldier’s operational effectiveness and longevity.\n\nUtilizing the latest research would allow for the preparation of dedicated formulas within specific military populations based on the likely stressors that each would encounter; however, no relationship exists between the published benefits of certain supplements and the military’s recommendation of their use – leaving the individual Soldier to make do with information supplied by non-vetted sources. Unit medical providers must be knowledgeable in the area so as to inform their Commanders and Soldiers of proper and suggested use. Unfortunately, too many studies and supplements exist for this short article to review adequately. Regardless, the options discussed above have evidence showing a positive effect in lengthening Soldiers’ operational life span and should be considered in a front-line approach.", "appendix": "Author contributions\n\n\n\nWilliam Brink performed the initial literature search in October 2013, repeated by Kyle Hoedebecke in March 2014. Both authors drafted the manuscript, critically revised it for intellectual content and approved the final version to be published.\n\n\nCompeting interests\n\n\n\nWB has consulted for the nutraceutical industry; he had no financial competing interests relating to the supplements covered in this review at the time of publication. KH had no competing interests to disclose.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSchneider K, Hervig L, Ensign WY, et al.: Use of supplements by US Navy Seals. Med Sci Sports Exerc. 1998; 30(5): S60. Publisher Full Text\n\nArsenault J, Kennedy J: Dietary supplement use in U.S. Army Special Operations candidates. Mil Med. 1999; 164(7): 495–501. PubMed Abstract\n\nLe Prell CG, Hughes LF, Miller JM: Free radical scavengers vitamins A, C, and E plus magnesium reduce noise trauma. Free Radic Biol Med. 2007; 42(9): 1454–1463. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScheibe F, Haupt H, Ising H: Preventive effect of magnesium supplement on noise-induced hearing loss in the guinea pig. Eur Arch Otorhinolaryngol. 2000; 257(1): 10–16. PubMed Abstract \| Publisher Full Text\n\nAttias J, Weisz G, Almog S, et al.: Oral magnesium intake reduces permanent hearing loss induced by noise exposure. Am J Otolaryngol. 1994; 15(1): 26–32. PubMed Abstract \| Publisher Full Text\n\nJoachims Z, Netzer A, Ising H, et al.: Oral magnesium supplementation as prophylaxis for noise-induced hearing loss: results of a double blind field study. Schriftenr Ver Wasser Boden Lufthyg. 1993; 88: 503–16. PubMed Abstract\n\nScheibe F, Haupt H, Mazurek B, et al.: Therapeutic effect of magnesium on noise-induced hearing loss. Noise Health. 2001; 3(11): 79–84. PubMed Abstract\n\nElsayed NM, Gorbunov NV: Interplay between high energy impulse noise (blast) and antioxidants in the lung. Toxicology. 2003; 189(1–2): 63–74. PubMed Abstract \| Publisher Full Text\n\nLeffler CT, Philippi AF, Leffler SG, et al.: Glucosamine, chondroitin, and manganese ascorbate for degenerative joint disease of the knee or low back: a randomized, double-blind, placebo-controlled pilot study. Mil Med. 1999; 164(2): 85–91. PubMed Abstract\n\nMcCarthy G, O'Donovan J, Jones B, et al.: Randomised double-blind, positive-controlled trial to assess the efficacy of glucosamine/chondroitin sulfate for the treatment of dogs with osteoarthritis. Vet J. 2007; 174(1): 54–61. PubMed Abstract \| Publisher Full Text\n\nClegg DO, Reda DJ, Harris CL, et al.: Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med. 2006; 354(8): 795–808. PubMed Abstract \| Publisher Full Text\n\nCohen M, Wolfe R, Mai T, et al.: A randomized, double blind, placebo controlled trial of a topical cream containing glucosamine sulfate, chondroitin sulfate, and camphor for osteoarthritis of the knee. J Rheumatol. 2003; 30(3): 523–528. PubMed Abstract\n\nFlakoll PJ, Judy T, Flinn K, et al.: Postexercise protein supplementation improves health and muscle soreness during basic military training in Marine recruits. J Appl Physiol (1985). 2004; 96(3): 951–956. PubMed Abstract \| Publisher Full Text\n\nHobson RM, Saunders B, Ball G, et al.: Effects of β-alanine supplementation on exercise performance: a meta-analysis. Amino acids. 2012; 43(1): 25–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffman JR, Landau G, Stout JR, et al.: β-alanine supplementation improves tactical performance but not cognitive function in combat soldiers. J Int Soc Sports Nutr. 2014; 11(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTharion WJ, Shukitt-Hale B, Lieberman HR, et al.: Caffeine effects on marksmanship during high-stress military training with 72 hour sleep deprivation. Aviat Space Environ Med. 2003; 74(4): 309–314. PubMed Abstract\n\nLieberman HR, Tharion WJ, Shukitt-Hale B, et al.: Effects of caffeine, sleep loss, and stress on cognitive performance and mood during US Navy SEAL training. Psychopharmacology (Berl). 2002; 164(3): 250–261. PubMed Abstract \| Publisher Full Text\n\nRuxton CHS: The impact of caffeine on mood, cognitive function, performance and hydration: a review of benefits and risks. Nutrition Bulletin. 2008; 33(1): 15–25. Publisher Full Text" }	[ { "id": "7906", "date": "10 Mar 2015", "name": "Douglas S. Kalman", "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\nThe authors did a good job of presenting background, rationale and motivation for their brief overview.If magnesium aided for protecting hearing or reducing loss of hearing in those exposed to impulse noise, how come there is not much data on use in hearing loss outside those in this setting?One area that appears overlooked and worthy for inclusion is the aspects of military training, correlation with injury and or illness (e.g., during basic training or elite training). Meaning, research by Flakoll, McClung, Tharion, Etzion-Daniel, Lappe and others appears not covered but yet worthy of at least a paragraph.Article acceptable with minor edits in that authors add to their writing using the above names and leads.", "responses": [ { "c_id": "1260", "date": "11 Mar 2015", "name": "Will Brink", "role": "Author Response", "response": "Dr. Kalman, Thank you for your considered response to our paper. To your Q:\"If magnesium aided for protecting hearing or reducing loss of hearing in those exposed to impulse noise, how come there is not much data on use in hearing loss outside those in this setting?\"An excellent question to which we do not have an adequate answer. As you can see, the data is quite supportive and compelling of the protective effects of magnesium to hearing due to high impulse noise in animal and human models. Seemingly, another example a lack of awareness of the potential benefits of this non toxic and inexpensive mineral that could be of benefit in many industrial settings where hearing damage so common, especially where people are unable to wear hearing protection.\"One area that appears overlooked and worthy for inclusion is the aspects of military training, correlation with injury and or illness (e.g., during basic training or elite training). Meaning, research by Flakoll, McClung, Tharion, Etzion-Daniel, Lappe and others appears not covered but yet worthy of at least a paragraph.\"We do cover briefly in various locations in the paper, as well as Table 1, some of the negative effects of training and combat on immunity, performance and other effects. Do you feel it's a lack of depth on that particular issue that should be added? Perhaps some objective numbers, via the authors you supplied, on rates of injury and drop out rates." }, { "c_id": "1264", "date": "12 Mar 2015", "name": "Douglas S. Kalman", "role": "Reviewer Response", "response": "Links for articles that I think are worthy if possible of a mention:Vit D - http://www.ncbi.nlm.nih.gov/pubmed/25005834Iron - http://www.ncbi.nlm.nih.gov/pubmed/24188143 Tyrosine - http://www.ncbi.nlm.nih.gov/pubmed/17078981An \"in-general\" study on baseline nutrition of \"military\" (sub-par to start with) - http://www.ncbi.nlm.nih.gov/pubmed/18849866 Vitamin D - Calcium and military stress fractures: http://www.ncbi.nlm.nih.gov/pubmed/18433305 andhttp://www.ncbi.nlm.nih.gov/pubmed/11305081If any of the above links can be added GREAT, as helpful for the picture you share." }, { "c_id": "1270", "date": "16 Mar 2015", "name": "Will Brink", "role": "Author Response", "response": "Excellent information, thank you. I have some of that data, but we didn't want to make the review overly lengthy but it might make sense to add additional sources. The Tyrosine data has always been of particular interest. A mention of military baseline nutrition being less than optimal would assist the overall message of the review." } ] }, { "id": "7905", "date": "20 Apr 2015", "name": "Tim N. Ziegenfuss", "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 applaud the authors for their efforts on this paper. The topic is extremely important, and the information is accurate and well written. I only wish they had delved deeper into the related literature as there are many other studies on the supplements they reviewed as well as a vast array of nutritional supplements that have the potential to improve military readiness and/or performance. That said, both of these limitations have been acknowledged by the authors. I encourage them to publish several \"follow up\" papers to address them.", "responses": [] } ]	1	https://f1000research.com/articles/4-61
https://f1000research.com/articles/4-9/v1	12 Jan 15	{ "type": "Review", "title": "Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity", "authors": [ "Tomas Koltai" ], "abstract": "Objective: To review the mechanisms of anti-cancer activity of nelfinavir and other protein inhibitors (PIs) based on evidences reported in the published literature.Methods: We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed.Conclusions: The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death.PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors.Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment.The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes.", "keywords": [ "Nelfinavir", "protease inhibitor", "cancer", "endoplasmic reticulum stress", "unfolded protein response", "Akt" ], "content": "Abbreviations\n\nNFV: Nelfinavir\n\nPI: HIV Protease Inhibitors\n\nERS: Endoplasmic reticulum stress\n\nUPR: Unfolded protein response\n\nBCRP: Breast cancer resistance protein\n\nFAS: Fatty Acid Synthase\n\n\nIntroduction\n\nIn March 1997, the United States Food and Drug Administration (FDA) approved Nelvinavir (NFV, brand name Viracept) for HIV treatment in humans1. NFV is a safe, orally available, and potent drug against HIV-1 and HIV-22. This protease inhibitor (PI) was developed by the private pharmaceutical sector and was a big success in the treatment of AIDS in association with other anti-retroviral drugs3. The introduction of PIs combined with HIV reverse transcriptase inhibitors started the era of HAART (highly active anti-retroviral treatment) and is nowadays the standard of care in HIV-AIDS4.\n\nPIs inhibit HIV-1 and HIV-2 proteases (which are aspartate proteases), impeding virus replication and release of infecting viral particles from diseased cells. The mechanism of action of protease inhibitors involves competitive binding to the enzyme5.\n\nNFV is being progressively displaced from HIV therapeutics by second generation HIV PIs, but has shown interesting off target actions in cancer.\n\nThe possible use of anti-HIV drugs against cancer is not new: in the 1990s AZT (zidovudine or azidothymidine) was proposed as anti-neoplastic drug, but clinical trials did not confirm the preliminary good results obtained in vitro6.\n\nThat HIV PIs target other molecules besides the HIV protease is quite evident if we examine adverse effects like insulin resistance and lipodystrophy. These and other evidences such as inhibition of tumor cell production of cytokines, anti-angiogenesis, induction of apoptosis and others, suggest off targets effects for PIs, and hints to the concept of a new class of drugs against cancer with multiple anti-cancer effects6.\n\nNFV, the most important anti-cancer drug of the PI family, if repurposed for cancer treatment, would have an important advantage: it has been used for more than 15 years in HIV treatment and its safety, pharmacokinetics, and adverse events are well known. Serious adverse events are not common with the exception of diarrhea when used at high doses.\n\nResearch on NFV as a potentially useful drug for cancer treatment6 started in 2009.\n\nIn this article, we thoroughly review the literature published in this matter and analyze mainly the anti-cancer mechanisms of action of NFV.\n\nCertain controversies regarding NFV activity in lipid metabolism will be considered in depth.\n\n\nEvidences of nelfinavir anti-cancer activity\n\nA partial response of Kaposi’s sarcoma patients to PIs was published in 19987 and good results with regression (six complete responses out of 10 patients)8. In 1999 Niehues et al.9 published complete regression of Kaposi’s sarcoma in a child treated with highly active anti-retroviral therapy (HAART). Sgadari et al. (2003) described also the inhibition of Kaposi’s sarcoma with protease inhibitors and they also mention that these drugs can antagonize vital properties of tumor cells like growth, invasion, tissue remodelling, angiogenesis and survival. They consider these effects to be a consequence of inhibition of invasion, matrix metalloprotease, proteasome and NF-κB signaling10. The possible mechanisms of PIs off target activity on tumor cells were described by pioneering work of Schmidtke et al. in 199911: they observed that ritonavir was a modulator of proteasomal activity, allowed normal proliferation when used at low concentrations, but affected protein degradation when present at higher concentrations, and cell cycle was arrested.\n\nIkezoe et al.12 described that protease inhibitors increased cellular growth inhibition of all transretinoic acid (ATRA) on cell cultures of myelocitytic leukaemia lines. Protease inhibitors also increased differentiation of acute myeloid leukemia cell lines.\n\nIn 2004, Ikezoe13 described the mechanisms involved in anti-cancer activity of protease inhibitors in myeloma cells.\n\nThe mechanisms involved in PIs anti-cancer activity are summarized in chronological order on Table 1.\n\n\nNelfinavir and the ERS-UPR pathway\n\nNFV inhibits the proteases S1P and S2P that are involved in SREBP-1 maturation and other proteases necessary for protein maturation and folding (yet not fully identified) in the endothelial reticulum49.\n\nActivation of the unfolded protein response (UPR) starts in the ER when abnormal accumulation of protein is detected57. This was investigated thoroughly in yeasts where detection of abnormal protein occurs through Ire1p/Ern1p-mediated signaling from the ER (in mammals there are three sensor proteins IRE1α, PERK and ATF658). UPR activation leads to the specific removal of 252 nucleotides intron from a precursor mRNA of the transcription factor HAC-1p, and the resulting mature mRNA HAC-1p is translated to produce active HAC-1p. This transcription factor translocates to the nucleus and promotes the transcription of chaperones like GRP78 that facilitates removal of abnormal proteins from the ER through retrotranslocation and final disposal by the ubiquitin-proteasome pathway59.\n\nHAC1 precursor mRNA is constitutively expressed but not translated until Ire1p/Ern1p sensor removes the necessary nucleotides.\n\nThus the UPR is an intracellular signaling pathway where the ER “informs” the nucleus on the need to increase the levels of molecular chaperones and folding enzymes in order to maintain the ER homeostasis. Therefore UPR keeps unfolded proteins in the ER until they are correctly folded before they can go to their final destination. NFV seems to produce cellular stress by accumulation of misfolded or abnormal proteins in the ER, overwhelming the normal ER protein folding machinery60. Chaperones bound to unfolded proteins in the ER initiate protein kinase cascades that inhibit translation, reverse translocation, activate ubiquitination enzymes, induce autophagia, and when stress is extreme, induce apoptosis.\n\n\nMechanism of action of Nelfinavir in cancer\n\nERS: Endoplasmic reticulum stress.\n\nFor this figure the model of nelfinavir in liposarcoma was used.\n\nFor activation of SREBP it is necessary that luminal S1P (a protease) cleaves first, followed by intramembrane S2P (another protease) to liberate the transcriptionally active amino-terminal segments of nSREBP. NFV inhibits S1P and S2P, so that transcriptionally active SREBP is not produced. Accumulation of inactive SREBP is one of the UPR initiators.\n\nSaquinavir-NO has been tested in human melanoma cells with good results77.\n\n*It is necessary to underscore the finding that cancer stem cells expressing embryonic genes like Oct4, Sox2 and others, are particularly prone to apoptosis when PIs are used, particularly iopinavir (nelfinavir and saquinavir are also effective in this matter).\n\n\nInteractions of NFV and PIs with other drugs\n\nIndinavir and NFV increase anti-malarial action of artemisinin in vitro on Plasmodium falciparum78, but artemisinin has also an off target anti-cancer activity. Thus it is reasonable to raise the question: may the association of artesiminin with NFV increase anti-cancer activity?\n\nAnother research team70 has included both, NFV and artesiminin, in a multidrug repurposed protocol (CUSP 9) for the treatment of relapsed glioblastoma.\n\nCelecoxib is an ER stressor that may enhance NFV anti-tumor activity79.\n\nChloroquine and hydroxicloquine are autophagy inhibitors and may work synergistically with NFV, down-regulating autophagy and increasing apoptosis70,80.\n\nNelfinavir may produce overproduction of mcl1 through upregulation of Erk ½, which would reduce apoptosis. The problem can be solved adding sorafenib81,82.\n\nIn breast cancer cells, tamoxifen enhances anti-cancer activity of NFV83. This synergism was independent of the estrogen receptor status so that the authors consider that the association of NFV and tamoxifen may be advantageous even in patients with no hormone responsive tumors.\n\nSaquinavir has an interesting off target effect: it decreases intracellular ATP in adipocytes56. If this effect is similar in tumor cells, an association with metformin and 2-deoxyglucose may produce anti-cancer activity84–86. There is growing interest on metabolic perturbators in cancer therapy and saquinavir may play a role in this field.\n\n\nClinical trials\n\nA phase I dose escalation trial performed in 201487 established a MTD (maximum tolerated dose) of 3125 mg twice daily and described that 45% of patients with solid tumors treated with this dose decreased AKT activity and increased ERS indicators. This indicated a possible benefit in neuroendocrine tumor patients and also established that dose limiting toxicity consists in neutropenia.\n\nThe dose (3125 mg bid) is more than twice the dose used in HIV treatment. But lower doses, in the range of those used in HIV treatment have been tested, combining nelfinavir with chemoradiotherapy in pancreatic cancer with evidence of efficacy88. No control group was used in this research, so comparison was established with known data from previous publications, mainly the favourable possibility of tumor resection after treatment.\n\nEven lower doses (625 mg and 1250 mg bid) were tested in a phase I trial of NSCLC in stages IIIA/IIIB combined with chemoradiotherapy89. In nine out of 12 patients a PET scan was available post-treatment with 100% overall response (56% complete response and 44% partial response). Unfortunately, in this trial there was no control group; 50% of the patients (six out of 12) lived for more than 22 months after treatment; 25% (three out of 12) lived without disease for more than 32 months. The results may be considered favourable, even without a control group.\n\nBuijsen et al.75 recommend a dose of 750 mg NFV bid for a phase II trial of this drug in combination with chemoradiotherapy in locally advanced rectal cancer.\n\nOngoing phase II trials are mainly in myeloma (associated with bortezomib or lenalomide), glioblastoma patients (associated with chemoradiation), pancreas (associated with gemcitabine and radiation) and lung. (See http://www.clinicaltrials.gov).\n\nThe first results of clinical trials did not show a meaningful improvement in the outcome of patients with refractory adenoid cystic carcinoma which is a malignant salivary gland tumor which usually has a poor prognosis. In this case NFV was used as monotherapy76.\n\n\nWhy nelfinavir has a role to play in cancer therapy?\n\nAkt activation is an important step in cancer phenotype and is a key player in acquisition and maintenance of cancer hallmarks. Akt is a nodal regulator of cellular survival pathways90. There are no drugs at the present time that can inhibit this protein with a good safety profile. Wortmannin, perifosine and other chemicals designed for PI3K/Akt inhibition were too toxic for clinical use or have shown disappointing results, so they did not enter the medical practice. Insulin stimulation of Akt phosphorylation was reduced by 55% at achievable doses20. At the same time there is clear evidence that it favours apoptosis and growth inhibition at clinically tolerable and achievable doses.\n\nThis anti-Akt activity of NFV can be reinforced by concomitant mTOR inhibition which results in synergistic cytotoxicity63. This may be due to the fact that mTOR inhibition without Akt inhibition eliminates a negative biofeedback loop on Akt, producing increased phoshorilation of Akt91. According to Sarbassov92 this negative feedback is born in the mTORC2 complex.\n\nAccording to Carracedo93, this negative feedback loop goes as far as PI3K (Figure 6).\n\nmTOR inhibitors have become a new and important tool against cancer, for example in renal cell carcinoma. But the negative biofeedback loop on Akt must be solved to achieve really good results. NFV could be the clue.\n\nAt the left is depicted the pathway under normal or pathological circumstances; in the middle drawing mTOR inhibition is counterbalanced by Akt activation due to loss of the negative biofeedback circuit; on the right, inhibition of both, mTOR and Akt may result in increased anti-cancer results.\n\nBut the most important anti-tumor activity of NVR is not limited to Akt inhibition but ER stress and UPR which may be one of the pathways leading to apoptosis94.\n\nAdditional features of NFV and other protease inhibitors are\n\n1) the ability to sensitize cancer cells to chemoradiotherapy,\n\n2) anti-angiogenesis by decreasing VEGF/HIF expression,\n\n3) decreased expression of FAS (fatty acid synthase),\n\n4) the combination of radiation and PIs is well tolerated39,94,\n\n5) NFV cancer cell killing ability can easily be enhanced with other ER stressors like celecoxib79. Cho et al. found enhanced killing of chemoresistant breast cancer cells after celecoxib treatment that aggravated ER stress; perillyl alcohol is another stress aggravator that has been used with that purpose96,\n\n6) In head and neck cancer related to HPV, NFV produced down-regulation of Akt and radiosensitization97,\n\n7) NFV not only down-regulates Akt but also MAPK (in adenoid cystic cancer)98, and retards oral cell proliferation including normal keratinocytes and squamous cell cancer99,\n\n8) There are evidences, at least in pancreatic cancer, that NFV dependent down-regulation of Akt is independent of the mutational status of K-ras100,\n\n9) There is clear evidence (in glioblastoma) of the relation between NFV and apoptosis through the following pathway46:\n\nNFV------ER stress--------CHOP------up regulation of TRAIL receptor DR5\n\n10) Down-regulation of MMP-9 (reduced expression and secretion of MMP-9 by human preadipocytes)64,101,\n\n11) Increased apoptosis by NFV when associated with anti-autophagy drugs like chloroquine or hydroxychloroquines, particularly in triple negative breast cancer cells102.\n\n\nPossible controversies\n\nThe SREBP pathway for regulation of fat metabolism is initiated through proteolytic cleavage of precursor forms of the SREBPs (125 Kd protein) in ER membranes. When cells are in need of sterol, the precursor SREBPs are hydrolyzed by a 2-step mechanism involving membrane-bound serine protease S1P and a metalloprotease S2P. The N-terminal fragment of SREBP (nSREBP) is a 68 Kd protein that translocates to the nucleus where it works as a promoter-enhancer, binding to sterol regulatory elements located in DNA and activates gene transcription (Figure 4). The nuclear SREBP can be rapidly degraded by a proteasome-mediated mechanism. This provides regulation of gene transcriptional activities103.\n\nTransgenic mice over-expressing the constitutively active nuclear forms of the SREBPs (nSREBPs) revealed that overexpression of SREBP-1 or SREBP-2 leads to activation of genes involved in the cholesterol and fatty acid biosynthesis cascades. These transgenic mice displayed the classical features of generalized lipodystrophy, similar to those found in patients under PI therapy104.\n\nRiddle et al. in 2001105 found that PI therapy (they used ritonavir) induced the accumulation of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues. As a consequence, fatty acid and cholesterol biosynthesis were increased in these tissues. The authors consider that lipodystrophy, hyperlipidemia, and insulin resistance, are the consequence of activated SREBP-1 and SREBP-2 accumulation in the nucleus of liver and adipose tissues. The possible mechanism for these events, according to their criteria is PI suppression of activated SREBP degradation in the nucleus. In summary, Riddles’s study showed that ritonavir induced lipid metabolism abnormalities through stabilization of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues.\n\nThese findings are in contrast with those of Guan49,106 where NFV inhibited the nuclear translocation of the sterol regulatory element binding protein-1 (SREBP-1) in castration resistant prostate cancer and liposarcoma through inhibition of S1P. This led to accumulation of unprocessed SREBP-1.\n\nRiddle et al. described accumulation of processed SREBP-1 in the liver and adipose tissue while Guan found accumulation of unprocessed SREBP1 in ER and Golgi with no translocation to nucleus in liposarcoma and castration resistant prostate cancer tissue.\n\nThe controversy may be explained in the following way:\n\n1) There are three different isoforms of SREBP: SREBP-1a, SREBP-1c and SREBP-2.\n\n2) SREBP-1a and -1c have different expression profiles: SREBP-1a is highly expressed in proliferating cells, such as cancer cells, while SREBP-1c is the predominant form in normal cells, particularly hepatocytes104.\n\n3) The target genes for the three SREBP isoforms are different.\n\n4) Riddle et al. found increased SREBP-1 and two in the nucleus of liver and adipose tissues; these SREBPs are the active form (they make no difference between SREBP-1a and SREBP-1c).\n\n5) Guan et al. found increased SREBP in Golgi in the inactive form (precursor) of tumor tissues treated with NFV.\n\n6) It is possible that tumor tissues that overexpress SREBP-1a behave in a different way than liver and adipose tissue that overexpress SREBP-1c.\n\n7) Riddle et al. tested ritonavir and Guam et al. tested NFV, so the pharmacological effects between these PIs may differ.\n\nA second controversy that stems from the one described above is on the effect of NFV on FAS:\n\n1) According to Guan et al.106, NFV decreases expression of FAS in liposarcoma cells and castrate resistant prostate cancer as was depicted in Figure 3.\n\n2) According to Lenhard et al. 2000107, NFV increases expression of FAS in HepG2 cells (which show many of the normal biochemical functions of non tumor liver parenchymal cells).\n\nMay this difference be due to tissue-specific effects of NFV? Does NFV have different effects in tumor tissues and normal tissues?\n\nTo definitely solve these controversies, it is necessary to proceed with further experimental research, but the findings described above necessarily raise the doubt that mechanisms that work in tumor cells might be slightly different from those working in hepatocytes and adipocytes.\n\n\nPossible negative aspects of PIs in cancer\n\nDespite the anti-cancer activity of NFV and PIs, these drugs do not reduce the risk of developing cancer in HIV population108 and also exert certain depression of immunological functions, interfering with the differentiation program of monocytes into dendritic cells109.\n\nPIs increase the expression of P-glycoprotein (ABCB1) in Kaposi’s sarcoma cell lines increasing the multidrug resistance phenotype110. At the same time ABCB1 expression depends on Akt activation111 and NFV inhibits partially Akt. The final result of the two antagonistic aspects requires further research.\n\nThere are well known undesirable side effects with HIV PIs, like hyperlipidemia, insulin resistance and lypodystrophy (peripheral fat wasting and excessive central fat deposition). One of the main responsible mechanisms of these side effects is the suppression of the breakdown of SREBP in the liver and adipose tissues resulting in increased fatty acid and cholesterol biosynthesis. SREBP accumulation in adipose tissue causes lipodystrophy.\n\nPIs suppress proteasome-mediated breakdown of nascent apolipoprotein (apo) B, resulting in the overproduction of triglyceride. Finally, PIs also suppress the inhibition of the glucose transporter GLUT-4 activity in adipose tissue and muscle. This contributes directly to insulin resistance and diabetes112.\n\nHepatomegaly and hepatic steatosis are direct consequences of the metabolic alterations explained above105.\n\n\nNew PIs with anti-cancer activity\n\nIn 2010 You et al.113 synthesized a new indinavir analogue with remarkable anti-cancer activity, similar to NFV: CH05-10. This drug achieved similar cytotoxity to NFV but at lower concentrations, against leukaemia, melanoma, ovarian and prostate cancer cell lines.\n\nIn 2009 Saquinavir-NO was introduced114; it showed interesting anti-cancer properties in melanoma xenografts with significantly lower toxicity than saquinavir.\n\n\nConclusions\n\nThe most relevant mechanisms of PIs anti-cancer activity are Akt inhibition and ER stress.\n\nFollowing our exhaustive analysis of the current medical literature we conclude that NFV anti-cancer activity is mainly dependent on ER stress-UPR.\n\nAkt inhibition plays also a very important role but is not the unique or main source of anti-cancer effects.\n\nThe evidences that support these conclusions are:\n\n1) Even at very high doses of NFV (3,125 mg bid), Akt achieved a level of inhibition around 55% in cell culture20.\n\n2) NFV is at the same time a strong ER stressor and an Akt inhibitor.\n\n3) Anti-cancer activity can be achieved at much lower doses than those necessary for Akt inhibition.\n\n4) Increasing ER stress by adding Celecoxib to NFV enhances cytotoxicity.\n\n5) Autophagy, which is one of the mechanisms cells use to survive increasing ER stress115, is inhibited by adding chloroquine or hydroxychloroquine to NFV. In this case, apoptosis is significantly enhanced80,102.\n\n6) The PIs with anti-cancer activity like NFV, ritonavir116, saquinavir117, and the experimental drug CH05-10113 are strong ER stressors. Amprenavir is a PI that induces no ER stress and its anti-cancer activity is significantly weaker than that of NFV, although it has Akt inhibiting effects.\n\n7) Ritonavir, which is ER stressor, shows anti-cancer activity although it does not down-regulate Akt at concentrarions usually found in HIV patients23.\n\n8) Inhibition of proteasome with bortezomib has a synergistic effect with NFV apoptotic activity118\n\n9) PIs can be classified regarding anti-cancer activity at clinically achievable concentration in patients in\n\nA) Akt inhibitors only: e.g. amprenavir\n\nB) ER stressors only: e.g. ritonavir\n\nC) Akt inhibitors and ER stressors: examples NFV and experimental PI CH05-10. Of course this is the group that shows stronger anti-cancer activity.\n\nThere is enough evidence of NFV anti-cancer effects and there is adequate knowledge of how this activity works, so that NFV deserves well designed phase II clinical trials, as adjunct cancer therapy.\n\nAssociations with proteasomal inhibitors, celecoxib and other cell stressor should also be investigated in the clinical setting due to possible synergy. Tamoxifen with NFV may show interesting results in breast cancer.\n\nAlthough a large amount of publications, including reviews, have been written on NFV and other PIs in cancer, none has been dedicated to a thorough examination and analysis of the mode of action of these pharmaceuticals as off target drugs (with the exception of the review by Gantt et al.2). It is hoped that this review will encourage an increment adequately powered and well-designed clinical trials in the various cancer types, beyond the phase I trials that have been recently performed, and specifically trials where these compounds may be tested in association with other known anti-cancer pharmaceuticals like NFV associated to bortezomib and hydroxychloroquine in myeloma, or mTOR inhibitors with NFV in HNSCC and many other possible combinations where the dual feature of NFV, ER stressor and Akt inhibitor, are required.\n\nIn myeloma NFV increases proteasome inhibition by bortezomib and may overcome resistance to proteasomal inhibitors. This is an action exclusive of NFV and not shared with other PIs67, with the additional advantage that NFV shows the highest cytotoxic activity against primary myeloma cells.\n\nIf apoptosis is described as a cascade, then apoptosis stimulator drugs like NFV should be viewed as enhancers of this cascade. An initiator of the cascade is still necessary, for example chemoradiotherapy. After this initial step, apoptosis stimulator drugs increase the amount of cells entering this pathway. This might be one of possible reasons why nelfinavir alone has shown poor results in a clinical trial used as monotherapy.\n\nThis does not mean that NFV cannot act as an initiator, but the evidences show that it is prone to be an enhancer of apoptosis rather than an initiator.\n\n\nFuture directions\n\nAll the evidences presented in this review reinforce the concept that NFV is a useful drug in cancer treatment. It should be considered in association with chemoradiotherapy in the design of new protocols for diseases like multiple myeloma (in association with bortezomib and hydroxychloroquine) and prostate, pancreas and lung cancer where clinical trials are ongoing. New PIs are being developed with better anti-cancer profile like CH05-10 and saquinavir-NO77 and further development of new PIs with stronger anti-cancer activity, will probably go on in the future.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPai VB, Nahata MC: Nelfinavir mesylate: a protease inhibitor. Ann Pharmacother. 1999; 33(3): 325–339. PubMed Abstract \| Publisher Full Text\n\nGantt S, Casper C, Ambinder RF: Insights into the broad cellular effects of nelfinavir and the HIV protease inhibitors supporting their role in cancer treatment and prevention. Curr Opin Oncol. 2013; 25(5): 495–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlexner C: HIV-protease inhibitors. N Engl J Med. 1998; 338(18): 1281–1292. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "7500", "date": "10 Feb 2015", "name": "Adali Pecci", "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\nThe article by Koltai reviews the extensive literature concerning Nelfinavir as an anti-cancer drug. Overall the study makes a good update of the evidences of nefilnavir anti-cancer activity and its role in controlling ERS-UPR pathway. It would be interesting to include in the discussion information about the different trials that are currently carry on with Nelfinavir in different tumors.", "responses": [] } ]	1	https://f1000research.com/articles/4-9
https://f1000research.com/articles/4-44/v1	13 Feb 15	{ "type": "Observation Article", "title": "Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral", "authors": [ "Lei Jiang", "Xin-Ming Lei", "Sheng Liu", "Hui Huang", "Lei Jiang", "Xin-Ming Lei", "Sheng Liu" ], "abstract": "Fusion of embryos or larvae prior to metamorphosis is rarely known to date in colonial marine organisms. Here, we observed for the first time the embryos of the broadcast spawning coral Platygyra daedalea could fuse during blastulation and further develop into conjoined larvae, and the settlement of conjoined larvae immediately resulted in inborn juvenile colonies. Considering the frequent record of synchronous spawning events and spawn slicks in the field, fusion of embryos might be a naturally occurring phenomenon, and act as an adaptive strategy to form pre-metamorphic united larvae and larger recruits, thereby promoting early survival. However, whether fusion of embryos is common in spawning corals and its implications deserve further investigation.", "keywords": [ "fusion", "conjoined larvae", "spawn slicks", "inborn colonies", "Platygyra daedalea" ], "content": "Introduction\n\nIn sessile colonial marine invertebrates (e.g., sponges, cnidarians, bryozoans and ascidians), fusion among conspecifics during early ontogeny could immediately lead to a marked increase in juvenile size, thereby enhancing the performance in growth, survival and competition1,2. In addition, the allogenic fusion is expected to form chimeras which possess greater genetic variability and wider ranges of physiological resistance1. Larvae of colonial marine organisms tend to settle in a gregarious manner3–7 and their juveniles often come into physical contact through growth and then fuse8–10. These life history traits increase the opportunities for fusion, and important rates of chimerism due to allogenic fusion have been detected in wild natural populations of corals and ascidians11,12. Nevertheless, fusion of embryos or larvae during planktonic and dispersive phase (i.e. prior to settlement and metamorphosis) is rarely known to date.\n\nModular marine invertebrates like sponges and cnidarians usually spawn in a high synchrony and the embryos also tend to aggregate after release13–15, thus also providing the chance of contact and fusion among embryos or larvae. For instance, sticky eggs released by the oviparous sponge Cliona celata were found to adhere to each other and form flattened egg mass, within which larvae fused in twos or threes. The compound larvae metamorphosed into sponges with single oscula, indicating the cytomictical fusion among embryos or larvae13. More recently, larvae of viviparous sponge Haliclona sp. have been demonstrated to fuse and generate swimming chimeras16. Furthermore, sexually produced embryos of a non-colonial sea anemone Urticina feline were observed to fuse naturally during internal brooding, generating pre-metamorphic cytomictical and sectorial chimeras17,18. These findings suggested that the direct contact between embryos and larvae would facilitate fusion either during internal brooding or pelagic phase.\n\nFor broadcast spawning corals, synchronous spawning events usually result in billions of naked embryos floating at the sea surface in the form of spawn slicks19,20. The direct contact between naked embryos highlights the possibility of fusion of coral embryos while sticking together in slicks. Moreover, previous studies have demonstrated there is a window in ontogeny, before allorecognition system matures, when newly settled polyps can fuse21. Time for allorecognition maturation in reef corals varied from 4 months following settlement in brooding species22, to 1–3 years in spawning species9,10. This further supports the possibility of fusion at embryonic stage when allorecognition may be weak in corals. As yet, the possible occurrence of fused embryos and conjoined larvae in broadcast spawning corals has not been investigated.\n\nHere, we happened to test this unexplored probability of fusion of embryos in broadcast spawning reef corals. We experimentally mimicked spawn slicks using gametes collected from 4 mature colonies of Platygyra daedalea, and followed the fate and development of embryos within lab-generated slicks.\n\n\nMaterials and methods\n\nTen gravid colonies of P. daedalea (20–30 cm in diameter) were collected at depth between 2–4 m from Luhuitou fringing reef in Sanya, China (18°12′N, 109°28′E). Corals were maintained in an outdoor tank with flowing sand-filtered seawater in Tropical Marine Biological Research Station in Hainan, Sanya. Four colonies spawned around 22:00 on May 18, 2014 (5 nights after full moon). Egg-sperm bundles were collected using pipettes, then mixed and gently agitated to facilitate bundle disintegration and cross-fertilization. Fertilization was allowed to take place for about 2 hours, after which eggs (ca. 300, 000) were washed two times with fresh seawater and suspended in a 15 cm-diameter jar. Because of the logistical constraints, eggs were left undisturbed and they formed dense slicks on the seawater surface. The next morning around 08:30, embryos were inspected under a dissecting microscope and we accidentally discovered that some embryos fused. Embryos were washed and seawater was changed twice daily thereafter. Two days after fertilization, 500 larvae were randomly sampled to count the proportion of each type of fusion. Seven days after fertilization, chips of crustose coralline algae Hydrolithon onkodes were used to induce the settlement of larvae and the recruits were reared in the lab at 28°C until June 26.\n\n\nResults\n\nEmbryos became bowl shaped (cushion stage) 8 h after fertilization. Notably, some embryos fused (Figure 1A) and a substantial proportion even stuck together into dense aggregates (Figure 1B). It could be deduced that fusion of embryos took place some time during blastulation. Mortality of embryos within the first 2 days was extremely high (>50%) and the dense aggregates all died and decomposed. Unitary larvae became pear-shaped and began to rotate actively 20 h after fertilization, while conjoined larvae were highly variable in shape. Bi-fused larvae were dominantly peanut-shaped, and multi-fused larvae were arranged in chains or triangles, or in the form of the letter “T” or “L” (Figure 1C, D).\n\n(A) Fused embryos (arrows point to the fusing areas). (B) A dense aggregate comprising more than 20 embryos. (C–G) Unitary (asterisks) and conjoined larvae. (H, I) Inborn colonies. (J) Single settlers. (K) Incomplete settlement of perpendicularly bi-fused larvae, with the left partner being parallel to the substrate. (L) An inborn colony 26 days post-settlement. Roman numbers indicate visible individuals within inborn colonies. Scale bars 250 μm.\n\nOf the 500 randomly sampled larvae, 174 (34.8%) were conjoined with 2–4 partners. Conjoined larvae clearly showed their spatial arrangement after elongation and fusion was apparently without polarity. Larvae either joined at the aboral end (Figure 1E, F), or united side by side (Figure 1G), or even fused perpendicularly (Figure 1K). Furthermore, 56 out of the 174 conjoined larvae (32.2%) united at the aboral extremity and only these larvae were potentially competent to metamorphose normally into inborn colonies (Figure 1H, I), which were prominently larger in size than the single settlers (Figure 1J). In contrast, perpendicularly bi-fused larvae settled incompletely, with one partner metamorphosing and firmly attaching while the other still being parallel to the substrate and not able to settle (Figure 1K), ultimately leading to the death of the whole entity 3 days later. Since the coralline algae provided here was not suitable for the settlement of P. daedalea larvae, only 12 inborn colonies were obtained in total and they persisted for 26 days post-settlement when the study ended (Figure 1L).\n\n\nDiscussion\n\nThe present study documented for the first time the fusion of embryos, the subsequent formation of conjoined larvae and inborn colonies in a broadcast spawning coral. Fusion of P. daedalea embryos was spontaneous, resulting simply from the aggregation and contact of embryos in mimicked slicks, which was analogous with that in sponge C. celata13. While unlike the cytomictical compound larvae in sponge C. celata, partners within the conjoined P. daedalea larvae still retained a degree of individuality, suggesting sectorial fusion of coral embryos and supporting the assumption that corals typically exhibit sectorial fusion1.\n\nCorals often spawn their gametes during seasonally calm periods and low-amplitude tides19,20,23 and spawn slicks extending up to few km in length were often observed in the field20,24. Given that slicks remained aggregated 1–2 d after spawning19,20 and embryos can fuse during embryogenesis within 8 h post-fertilization, fusion of coral embryos is highly favored in situ. On the other hand, although mass coral spawning events usually involved several species, significant temporal differences in spawning to ensure fertilization and reproductive isolation have been demonstrated for many sympatric species25–27, which considerably increase the encounters between embryos of the same species in slicks. Taken together, fusion of coral embryos might be a natural phenomenon. However, the density of embryos here was 1700 cm-2 and likely to be much higher than that in the field. Therefore, the extent to which fusion of embryos occurs in natural slicks and whether there is a density effect on the formation of conjoined larvae remain to be fully evaluated.\n\nAt last, approximately 12 percent of all the larvae had the potential to form inborn colonies, which persisted for one month and exhibited no sign of rejection. The inborn colonies here originated from fusion of embryos and settlement of conjoined larvae, contrasting the traditional concept that the asexual budding of the primary polyp leads to the formation of a young coral colony28, and thus fusion of embryos could be an unexpected shortcut to colony formation in reef corals. These facts raise questions as to the ecological implications of inborn colonies formed as a consequence of fusion of embryos in corals. Firstly, larger coral colonies composed of multiple fused partners are known to yield remarkable gained benefits, such as enhanced survival and growth5,8. Hence, the larger initial size and the status of multi-polyp at settlement may confer these inborn colonies better capacities to compete for space and survive partial mortality.\n\nFusion of coral embryos also shed new light on the chimerism in scleractinian corals, which was often attributed to fusion of gregariously settling larvae5,7, or of juveniles that come into contact through growth7,9,10. However, our study documented fusion between individuals in P. daedalea occurred at the embryonic stage, earlier than any other corals studied to date. It should be pointed out that though embryos here were produced sexually from 4 parent colonies, it did not denote they were genetically distinct. Therefore, the possibility of isogenic fusion can not be totally ruled out7. However, it is reasonable to assume the presence of allogenic fusion, suggesting fusion of embryos might add a novel mechanism to form chimeric larvae and colonies in scleractinian corals. In that case, the increased genetic diversity within these inborn colonies may translate into versatile physiological qualities, thus enabling them to better cope with environmental changes unless negative interactions occur1,29,30.\n\nOverall, this is the first report of fused embryos and pre-metamorphic conjoined larvae in reef corals. Fusion of coral embryos could be an adaptive strategy to form larger and chimeric recruits, thereby promoting growth and survival during the vulnerable early stages5,8. Clearly, future studies are required to explore whether fusion of embryos is common in spawning corals and fully evaluate its biological and ecological implications.\n\n\nEthics statement\n\nCoral sampling was permitted by the Administration of Sanya Coral Reef National Nature Reserve, the Department of Ocean and Fisheries of Hainan Province.", "appendix": "Author contributions\n\n\n\nLJ conceived and performed the study. All authors wrote the manuscript and gave final consent for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Public Science and Technology Research Funds Projects of Ocean (201305030-3) and the National Natural Science Foundation of China (41306144 and U1301232).\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\nRinkevich B, Weissman IL: Chimeras in colonial invertebrates: a synergistic symbiosis or somatic-and germ-cell parasitism? Symbiosis. 1987; 4(1–3): 117–134. Reference Source\n\nBuss LW: Competition within and between encrusting clonal invertebrates. Trends Ecol Evol. 1990; 5(11): 352–356. PubMed Abstract \| Publisher Full Text\n\nKeough MJ: Kin-recognition and the spatial distribution of larvae of the bryozoan Bugula neritina (L.). Evolution. 1984; 142–147. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun Z, Hamel JF, Mercier A: Marked shifts in offspring size elicited by frequent fusion among siblings in an internally brooding marine invertebrate. Am Nat. 2012; 180(5): E151–E160. PubMed Abstract \| Publisher Full Text\n\nGilmour JP, Smith LD, Brinkman RM: Biannual spawning, rapid larval development and evidence of self-seeding for scleractinian corals at an isolated system of reefs. Mar Biol. 2009; 156(6): 1297–1309. Publisher Full Text\n\nOliver JK, Willis BL: Coral-spawn slicks in the Great Barrier Reef: preliminary observations. Mar Biol. 1987; 94(4): 521–529. Publisher Full Text\n\nRinkevich B: Allorecognition and xenorecognition in reef corals: a decade of interactions, in Coelenterate Biology 2003, D. Fautin, et al., Editors. Springer Netherlands, 2004; p. 443–450. Publisher Full Text\n\nFrank U, Oren U, Loya Y, et al.: Alloimmune maturation in the coral Stylophora pistillata is achieved through three distinctive stages, 4 months post-metamorphosis. Proc Biol Sci. 1997; 264(1378): 99–104. Publisher Full Text \| Free Full Text\n\nvan Woesik R: Calm before the spawn: global coral spawning patterns are explained by regional wind fields. Proc Biol Sci. 2010; 277(1682): 715–722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrison PL: Coral spawn slicks at Lord Howe Island, Tasman Sea, Australia; the world’s most southerly coral reef. Coral Reefs. 2008; 27(1): 35. Publisher Full Text\n\nVan Oppen MJH, Willis BL, Van Rheede T, et al.: Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in corals. Mol Ecol. 2002; 11(8): 1363–1376. PubMed Abstract \| Publisher Full Text\n\nFukami H, Omori M, Shimoike K, et al.: Ecological and genetic aspects of reproductive isolation by different spawning times in Acropora corals. Mar Biol. 2003; 142(4): 679–684. Publisher Full Text\n\nLevitan DR, Fukami H, Jara J, et al.: Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex. Evolution. 2004; 58(2): 308–323. PubMed Abstract \| Publisher Full Text\n\nHarrison PL, Wallace CC: Reproduction, dispersal and recruitment of scleractinian corals. In Ecosystems of the world: coral reefs, Z. Dubinsky, Editor. Elsevier: Amsterdam, 1990; 133–207. Reference Source\n\nPineda-Krch M, Lehtilä K: Costs and benefits of genetic heterogeneity within organisms. J Evol Biol. 2004; 17(6): 1167–1177. PubMed Abstract \| Publisher Full Text\n\nRinkevich B, Yankelevich I: Environmental split between germ cell parasitism and somatic cell synergism in chimeras of a colonial urochordate. J Exp Biol. 2004; 207(Pt 20): 3531–3536. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7666", "date": "16 Feb 2015", "name": "Andrew H. Baird", "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\nThis is well written article and an interesting observation although some of the literature cited is a little dated (e.g. Harrison & Wallace 1990). To my knowledge this is first time that anyone has followed these embryonic chimeras through to settlement and that fact that they can settle and grow is an important observation. However, embryonic chimera formation is an artifact of the experimental conditions i.e. the very high densities of embryos in still water in the bowls. Indeed, embryonic chimera formation is quite common, particularly when working with larvae of merulinds at higher temperatures. It is very rare to see chimeras in spawn slicks or on settlement tiles, at least on soaks of short duration, although this has yet to be quantified. Therefore, embryonic chimera formation is highly unlikely to be of much ecological or evolutionary significance. Indeed, a phenonomen of much more ecological significance is the fact that many embryos break up during development under conditions likely to prevail in the wild (see Heyward and Negri, 2012).", "responses": [ { "c_id": "1237", "date": "05 Mar 2015", "name": "Lei Jiang", "role": "Author Response", "response": "Spawn slicks are commonly observed in the field and more importantly, they remained aggregated 1-2 days after spawning (Oliver et al., 1987; Gilmour et al., 2009). It is true that these conditions are favorable for the formation of embryonic chimeras. Moreover, the high density and absence of water turbulence in this study might trigger the fusion of embryos. Under similar experimental conditions, larvae of A. millepora tended to settle in aggregation (Puill-Stephan et al., 2012), and larvae of T. coccinea formed swimming polyp clusters (Mizrahi et al. (2014)). Therefore, though fusion of coral embryos has never been reported in the wild, we speculate it might occur in the field and remains to be determined especially corals often broadcast spawn their gametes during calm periods and low-amplitude tides." } ] }, { "id": "7679", "date": "17 Feb 2015", "name": "Baruch Rinkevich", "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 short research article by Jiang et al. is timely, adds to our understanding on the magnitude of natural chimerism, and meets research standards with methodological details that made available to allow others to replicate this study. I recommend indexation pending responding on the below minor editorial/clarification points:Abstract (line 2)- An unclear sentence. Suggest revising from: ‘Here, we observed for the first time the embryos….’ to ‘Here, we document for the first time that embryos…’. See also below the note appended to the citation of Mizrahi et al., 2014 and define your statement, ’for the first time’. ‘Conjoined larvae’ (as from Abstract line 4 and along the manuscript). This term and the following terms ‘chimera’ and ‘fusion’ are mutually used, irrespective to the somehow different biological statuses they present (for example, ‘conjoined’ is a very sterile term indicating being, coming, or brought together so as to meet, touch, overlap, or unit). I would suggest to use the term ‘Conjoined larvae’ in the Results section when first describing the process of larval joining and then use the terms ‘fusion’ and ‘chimerism’ in all other parts of the manuscript to state the biological outcome. Related to the aforementioned- it will be most valuable to show evidence for the ‘fusion’ outcomes, such as histological sections, or that no disjointing between the conjoined larvae occurred following the employment of physical force.Introduction and Discussion- There are four papers that I would suggest the authors to add and to cite. The first and the most important is the study by Mizrahi et al. (2014). In this paper the authors showed that Tubastraea coccinea offspring can metamorphose and aggregate/fuse in groups of up to eight polyps in the water column, before settlement. This may also change the way Jiang et al. refer to their finding (statements such as ‘the first time’ should be revised). The second paper is the study on sponge chimeras by Gauthier and Degnan (2008). This paper reveals the potential intermixing of cells between fused partners. The third paper is the old report by Duerden, the first observation documenting ‘aggregated colonies’ (Duerden,1902). It will also be valuable to cite Rinkevich (2011). Introduction 2nd paragraph, 1st sentence: ‘Modular marine invertebrates …. tend to aggregate after release’ is redundant to the former introduction text. Delete or rephrase. M & M: ‘… then mixed and gently agitated…’ Specify if the mixed eggs/sperm were from the same genotype or from all/several genotypes. M & M: ‘…to count the proportion of each type of fusion’. Which ‘types’ of fusion the authors imply to? Detail/explain. Discussion, below Fig 1: ‘…P. daedalea larvae still retained a degree of individuality…’. What is the meaning of ‘individuality’ here? What is the meaning of ‘a degree’? Morphologically? Physiologically? Define and rephrase. Discussion, the sentence: ‘It should be pointed out that though embryos here were produced sexually from 4 parent colonies, it did not denote they were genetically distinct’. Something is wrong with the sentence. If larvae are sexually produced they should be genetically distinct from each other. Rewrite.", "responses": [ { "c_id": "1236", "date": "05 Mar 2015", "name": "Lei Jiang", "role": "Author Response", "response": "We revised the sentence in abstract as suggested. Our observation presented the embryonic chimeras as a result of fusion of embryos in a broadcast spawning coral, whereas the results of Mizrahi et al., (2014) revealed that the larvae of brooding coral Tubastraea coccinea could metamorphose and aggregate in swimming groups. Thus we did document for the first time the fusion between individuals at the embryonic stage in reef corals. Moreover, we followed the settlement and growth of these chimeric larvae. Overall, it is proper and discreet to state “for the first time”. We have followed the reviewer’s advice to use “conjoined larvae” in the Results and Abstract sections where described the larval joining. This was only an accidental observation and it was a pity that we did not preserve samples for histological sections, nor did we employ physical force to see whether the conjoined larvae would disjoint. As aforementioned, we did observe the embryonic chimeras and follow the settlement and post-settlement growth of chimeric larvae in a broadcast spawning coral for the first time. Mizrahi et al. (2014) revealed that the swimming polyp clusters survived longer and did not confirm whether these clusters can settle. Therefore, the findings of Mizrahi et al. (2014) were not contradictory to our statement of “the first time”. We have added the suggested references. But as for Rinkevich (2011), we think it is more suitable to cite the classical reference of Rinkevich et al. (1987) which mainly focused on the chimerism in colonial marine invertebrates. We deleted the latter part of this sentence and made it more specific as to the synchrony in gametes release. We used the word “combined” to show that the bundles from 4 colonies were brought together for cross fertilization. We replaced “each type of fusion” with “chimeric larvae”. We made it more specifically that these conjoined larvae were multi-headed to demonstrate the sectorial fusion. We were sorry for this mistake because we were a little confused at first when referring to the results of Puill-Stephan et al. (2012). They found that fusion between sexually produced larvae resulted in non-chemiric colonies of one genotype. Thus we got a misunderstanding that the sexually produced larvae were not genetically distinct absolutely. However, they only use nine microsatellite loci to genotype. We have corrected and rewritten this part." } ] } ]	1	https://f1000research.com/articles/4-44
https://f1000research.com/articles/2-131/v1	23 May 13	{ "type": "Research Article", "title": "Reliability and reproducibility of spectral and time domain optical coherence tomography images before and after correction for patients with age-related macular degeneration", "authors": [ "Aymen Rashid", "Yasir J Sepah", "Roomasa Channa", "Elham Hatef", "Matthew Shulman", "Diana V Do", "Quan Dong Nguyen", "Aymen Rashid", "Yasir J Sepah", "Roomasa Channa", "Elham Hatef", "Matthew Shulman", "Diana V Do" ], "abstract": "Purpose: To evaluate the reproducibility and reliability of Optical Coherence Tomography scans (OCT) obtained using the Time Domain (TD-OCT) StratusTM OCT, and the Spectral Domain (SD-OCT) SpectralisTM and CirrusTM OCT devices before and after manual correction in eyes with either Neovascular (NV-AMD) or Non-Neovascular (NNV-AMD) Age-related Macular Degeneration.Methods: We conducted a prospective observational study of 36 patients (50 eyes) with NV-AMD or NNV-AMD at a university-based retina practice. OCT scans were taken simultaneously using one TD-OCT and two SD-OCT devices. Macular thickness measurements were assessed before and after correction of the OCT algorithm by constructing Bland-Altman plots for agreement and calculating intraclass correlation coefficients (ICCs) and coefficients of repeatability (COR) to evaluate intraclass repeatability.Results: The Spectralis device had the highest number of images needing manual correction. All machines had high ICCs, with Spectralis having the highest. Bland-Altman plots indicated that there was low agreement between both Cirrus™ and Stratus™ and Spectralis™ and Stratus™, while there was good agreement between the Cirrus™ and Spectralis™ devices. The CORs were lowest for SpectralisTM and similar with each other and had higher values for CirrusTM and StratusTM. Agreement, CORs, and ICCs generally improved after manual correction, but only minimally. Conclusion: Agreement is low between devices, except between both SD-OCT machines. Manual correction tends to improve results.", "keywords": [ "age related macular degeneration", "neovascularization", "optical coherence tomography", "spectral domain", "time domain" ], "content": "Introduction\n\nOptical Coherence Tomography (OCT) is a non-invasive imaging modality that allows acquisition of cross-sectional images of the retina. OCT is useful in monitoring and evaluating retinal thickness in many retinal disorders. One example is Age-related Macular Degeneration (AMD), a progressive, blinding disease that is mostly Non-Neovascular (NNV-AMD) but can be associated with choroidal Neovascularization (NV-AMD). Currently, OCT is also being employed as an outcome measure in many multicenter clinical trials of AMD with Time Domain OCT (TD-OCT) devices being the most common1,2. Spectral Domain OCT (SD-OCT) is a newer technology that obtains high-resolution scans of the retina.\n\nAs this technology is increasingly being utilized by many ophthalmologists to evaluate and monitor patients and guide treatment decisions2, it is important to understand the reliability and accuracy of thickness measurements obtained with the various devices currently available. Recently, studies have shown that in patients with AMD, there is a high frequency of errors in automated retinal thickness measurements due to incorrect segmentation of the retina in the TD-OCT machine, specifically in NV-AMD2,3. Using a Spectral Domain OCT (SD-OCT) device Menke et al. found that retinal thickness measurements in NNV-AMD cases had fewer errors than in NV-AMD cases, mostly due to the pathology of the former disease resulting in retinal pigment epithelial (RPE) layer changes4.\n\nManual correction of the OCT algorithm is an option in newer generations of the OCT review software and as more devices are coming to the market, it is important to understand the clinical importance of manual correction of OCT algorithms and the agreement of thickness measurements from different machines before and after correction. To date, no other study has examined the effects of manual correction of the thickness algorithm in SD-OCT and TD-OCT machines in eyes with AMD. In our study, we evaluated the intra-session repeatability and agreement in retinal thickness measurements for patients with NV-AMD and NNV-AMD before and after manual correction using three different OCT devices: Stratus™ TD-OCT and two SD-OCTs, Spectralis™ and Cirrus™.\n\n\nMethods\n\nInstitutional Review Board (IRB)/Ethics Committee approval was obtained and HIPAA guidelines were followed for the study. Written Informed consent was obtained from study subjects.\n\nPatients with a confirmed diagnosis of AMD were enrolled in the study. Two senior retina specialists (QDN and DVD) made the diagnosis of AMD. Patients under treatment with intravitreal injections of anti-Vascular Endothelial Growth Factor (VEGF) agents were also allowed to participate in the study.\n\nPatients were scanned twice by certified OCT operators on a TD-OCT device (Stratus™ OCT) and two SD-OCT devices (Spectralis™ and Cirrus™ OCT) machines in random order and with 5–10 minutes between each device. The same operator performed all the scans on any given patient. Scans on a single device were performed consecutively and 5 minutes apart from each other.\n\nOne TD-OCT machine, Stratus™ (software version 4), and two SD-OCT machines, Spectralis™ (software version 5.01) and Cirrus™ (software version 5.0.0.326) were used. Stratus™ is a TD-OCT machine that uses a super luminescent diode with a wavelength of 820 nm. It provides an axial resolution of 10 µm and image acquisition speed of 400 A-scans/second. Using the Stratus™, two Fast Macular Thickness Maps (FMTM) were acquired from each eye. The FMTM is created through acquiring six radial B-scans, each consisting of 512 A-scans, and at an angle of 30° from each other with the point of intersection centered on the fovea.\n\nSpectralis™ uses a super luminescent diode with a wavelength of 870 nm. It provides axial resolution of 4 µm and image acquisition speeds of up to 40,000 A-scans per second. Two volume scans were acquired from each eye using a raster scan of 19 lines covering 20×15° of the fundus. Using the TruTrack™ functionality of the Spectralis™ OCT, each line was averaged 15 times or more. Cirrus™ HD-OCT also uses a super luminescent diode with a wavelength of 840 nm. It provides images with an axial resolution of 5 µm and acquisition speeds of 27,000 A-scans per second. We acquired two 512×128 macular cube scans (128 B-scans and 512 A-scans, covering a retinal area of 6.0×6.0 mm) from each eye.\n\nScans from each of the three devices were reviewed at the Retinal Imaging Research and Reading Center at the Wilmer Eye Institute by independent graders. Incorrect identification of inner and outer retinal boundaries by automated algorithms in Spectralis™ and Cirrus™ devices was manually corrected. Stratus™ images could not be corrected due to the lack of editing capabilities in the operating system provided with the machine at the time the study was conducted. Only five patients required corrections and were excluded from the analysis. The proprietary software identifies retinal boundaries for measurement of retinal thickness that are specific to each device. Whereas each device identifies the inner limiting membrane (ILM) as the inner boundary of retina, identification of the outer boundary is different for each device. Stratus™ identifies the junction between the inner and outer segments of photoreceptors (IS/OS) as the outer boundary, Spectralis™ identifies the posterior border of the retinal pigment epithelium (RPE), and Cirrus™ identifies the inner border of the RPE as the outer retinal boundary.\n\nWhenever the foveal center could be identified, grids were repositioned for scans with off-center positioning of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. However, in some cases, morphological changes associated with the advanced disease made identification of the foveal center unreliable. Adjustment of grid position was not possible for Stratus™ OCT. Scans were excluded from analysis only if identification of retinal layers and determination of the retinal thickness was not possible. OCT scans from which extraction of thickness data for the central 1 mm sub-field was not reliable, due to missing data in the image or the scan being out of range, were also excluded from analysis.\n\nThe retinal thickness measurements of the nine standard ETDRS subfields (Figure S1 illustrates the nine-subfield abbreviations) were recorded from each device before and after correcting the errors in the scans’ algorithm.\n\nBland-Altman plots were constructed to determine agreement between devices; both 95% confidence intervals and limits of agreements were calculated. Reproducibility of measurements was determined by calculating the coefficients of repeatability (COR) for each machine. Intraclass correlation coefficients (ICCs) were used to determine the reproducibility for each device. The statistical significance of difference in thickness before and after correction of images across devices was determined via the student’s t-test with α = 0.05 with Bonferroni correction for multiple comparisons. STATA version 10 and Microsoft Excel 2007 were used for data management and analysis. The statistical analysis was performed before and after any manual corrections were made to the algorithm errors described above.\n\n\nResults\n\nFifty eyes from 36 patients were included in the study; 29 eyes had NV-AMD and 21 eyes had NNV-AMD. The mean age of the study subjects was 76.6 years. Males had a mean of 76.3 with a range of 61 to 90 years. Females had a mean of 76.83 with a range of 53 to 90 years.\n\n\nExclusion and corrections\n\nScans from four eyes could not be recovered from the database and scans from three eyes had algorithm errors with incorrect identification of retinal boundaries and were excluded from analysis. Scans were not corrected for off-center positioning of the scan as moving the ETDRS grid was not possible with the available software version.\n\nScans in six eyes scanned first and eight eyes scanned second were corrected either for off-center fixation of the eye or for incorrect automated identification of retinal boundaries. The thickness measurements before and after correction were not statistically significant (p<0.05) for any of the subfields and also when stratified by diagnosis.\n\nThirty-three scans among the first set and 32 among the second set were corrected. The inner inferior subfield for NV-AMD was the only subfield that was statistically significant before and after correction. Figure 1 plots the frequency of the differences before and after correction for the central subfield for all scans. 77% of the differences were less than 48 μm and 50% were less than 10 μm.\n\nThe mean (+SD) of the macular thickness of all of the subfields, including the central 1 mm subfield (foveal thickness; FTH) for Stratus™, Cirrus™, and Spectralis™ devices before and after manual correction of scans, stratified by diagnosis of NV-AMD and NNV-AMD, is shown in Table 1. For NV-AMD, the FTH values for the central 1 mm were 375 µm (+129 µm), 253 µm (+74 µm), 312 µm (+110 µm) for Spectralis™, Stratus™, and Cirrus™ respectively. After correction, the values were 335 µm (+106 µm) for Spectralis™ and 318 µm (+110 µm) for Cirrus™. On the other hand, the FTH values for NNV-AND in the central 1 mm before correction were 298 µm (+87 µm), 193 µm (+32 µm), and 229 µm (+30 µm) for Spectralis™, Stratus™, and Cirrus™ respectively. Spectralis™ was the only device to have a different FTH value (248 µm +56 µm) after correction. Overall, Spectralis™ had the highest retinal thickness values (range: 280 to 372 µm), depending on the subfield. The retinal thickness measurements obtained via Cirrus™ were slightly less (range: 230 to 320 µm), while Stratus™ had the lowest values, ranging from 190 to 270 µm. There were no significant (p<0.05) differences between the mean FTH of the first and second scans for each of the three devices.\n\nSpectralis™ vs. Cirrus™ before correction: for Neovascular Age-related Macular Degeneration (NV-AMD), T1, S1, and I2 (p<0.05); and for Non-Neovascular (NNV-AMD), C1, T1, N1, and I2. Spectralis™ vs. Cirrus™ after correction (p<0.05): for NV-AMD, every field except S2, and I2 were not significant; and for NNV-AMD, the inner subfields were not significant (p>0.05). Spectralis™ vs. Stratus™ after correction: for NV-AMD, C1 (p<0.05).\n\nThe central subfield ICC values for all three machines were very high at 99.6%, 97.2% and 96.4% before correction for Spectralis™, Stratus™, and Cirrus™ respectively, and 99.4%, and 97.4% after correction for Spectralis™ and Cirrus™. The ICC values were greater than 95% for all subfields and both diagnoses except the outer inferior field for NNV-AMD for Spectralis™. Stratus™ values ranged from 78.9% to 99.2% for NV-AMD and 94.7% to 99% for NNV-AMD, before and after correction, respectively. Cirrus™ values ranged from 88.5% to 99.9% and 99.1% to 99.8% for NV-AMD before and after correction, respectively. The values for NNV-AMD for Cirrus™ ranged from 99.3% to 99.9% and 71.4% to 99.7% before and after correction, respectively. Table 2 shows the ICC values between images for all three machines before and after correction, both combined and stratified by diagnosis. It should be noted that all of the machines had ICC values >90% for the central subfield while the Spectralis™ had no subfields less than 99% after correction. In the central subfield, Spectralis™ had a COR of 20 µm NV-AMD which increased to 23 µm; both Cirrus™ and Stratus™ had relatively larger CORs of 64 µm (reduced to 49 µm after correction) and 35 µm, respectively. For NNV-AMD, the COR for the central subfield was 15 µm for both Cirrus™ and Spectralis™, and was 24 µm for Stratus™. After correction, the value decreased for Spectralis™ to 12 µm and increased to 36 µm for Cirrus™. The COR of all subfields for each device before and after correction of algorithms and stratification by disease are given in Table 3.\n\nOverall Spectralis™ had the lowest COR, with values ranging from 5–30 µm. Cirrus™ and Stratus™ had similar values ranging from 5–70 µm, even after correction. The COR for Cirrus™ increased by 15–40 µm after correction for NNV-AMD. Also, Cirrus™ COR values were 10–30 µm higher than Stratus™ values for both NV-AMD and NNV-AMD. Agreement between machines was poor, except between Spectralis™ and Cirrus™ after correction. Table 4–Table 5 show 95% confidence intervals and limits of agreement of the Bland-Altman plots between devices before and after manual correction.\n\nFigure 2A–F show Bland-Altman plots with 95% confidence intervals for the FTH comparison of the machines before and after correction. Before correction, the mean difference between the machines was 32 µm for Spectralis™ vs. Cirrus™, 52 µm for Cirrus™ vs. Stratus™, and 84 µm for Spectralis™ vs. Stratus™. Manual correction reduced the differences, with it being 15 µm for Spectralis™ vs. Cirrus™, 51 µm for Cirrus™ vs. Stratus™, and 67 µm for Spectralis™ vs. Stratus™. When stratified by diagnoses, the values were 34 µm and 29 µm for Spectralis™ vs. Cirrus™, 53 µm and 47 µm for Cirrus™ vs. Stratus™, and 88 µm and 79 µm for Spectralis™ vs. Stratus™ for NV-AMD and NNV-AMD respectively, before correction. After manual correction, the values reduced to 17 µm and 14 µm Spectralis™ vs. Cirrus™ and 70 µm and 61 µm Spectralis™ vs. Stratus™ for NV-AMD and NNV-AMD, respectively. The confidence interval widths, on average, were 5–10 µm smaller than when comparing between an SD-OCT and a TD-OCT machine. The average interval width decreased between 5–10 µm after correction for any disease and comparison, except for the Cirrus™ vs. Stratus™ comparison.\n\nA: Spectralis™ vs. Cirrus™ before correction. B: Spectralis™ vs. Cirrus™ after correction. C: Cirrus™ vs. Stratus™ before correction. D: Cirrus™ vs. Stratus™ after correction. E: Spectralis™ vs. Stratus™ before correction. F: Spectralis™ vs. Stratus™ after correction.\n\n\nDiscussion\n\nThe advent of OCT has revolutionized the way patients with retinal disorders are evaluated and monitored. However, like every new device, the current devices employing time- or spectral domain technology have certain limitations. One such common and clinically relevant issue is the presence of random errors in the identification of the inner and outer boundaries of the retina by the OCT algorithm. With respect to AMD, studies have shown that lesions such as fibrotic scars, choroidal neovascularization disrupting the RPE, and subretinal fluid would produce errors in the automated segmentation algorithms because the software would not correctly delineate the outer retinal boundary3,5. In our study, we found that 66% of the Spectralis™, 14% of the Cirrus™ and 6.5% of the Stratus™ scans had algorithm errors. Giani et al. reported similar results; for Cirrus™, they reported 25% and 16% algorithm error rates for NNV-AMD and NV-AMD, respectively. However, for Spectralis™, they reported 16.67% and 57.6% algorithm error rates and 8.33% and 62.5% rates for Stratus™ for NNV-AMD and NV-AMD, respectively5. Other studies have reported Stratus™ outer boundary algorithm errors of approximately 43% for both forms of AMD and 60% for NV-AMD3,6.\n\nReasons for differences in our error rates compared to previous studies include the lack of standard definition of an algorithm error. Rather than having an exact definition of an algorithm error, which may not be clinically significant5, in our study, the decision was made by two masked observers who determined if the correction would be important. In addition, even though Spectralis™ segments the outer border of the RPE, a study by Jaffe et al. reported that it may also be including the Bruch’s membrane in its calculation, thus including sub-RPE pathology such as drusen when segmenting the outer border of the retina7. These differences may be due to the fact that our study was prospective and, while acquiring scans, the operators tried their best to ensure no errors occurred during scan acquisition. Lastly, we did not exclude scans if the signal strength was low or if the machine gave a low analysis confidence message, as other studies have done8–10.\n\nAfter correction the thickness measurements for the Spectralis™ and Cirrus™ scans were not significantly different. This may be due to the fact that the majority of the scans required minor corrections. For example, most of the Spectralis™ scans resulted in a 10 µm or less change in the central subfield thickness. Retinal thickness measurements were similar in both SD-OCT machines and were greater than Stratus™. Correction reduced the difference of the thickness measurements between the two SD-OCT devices to less than 20 µm; in some cases as noted above, the difference was no longer statistically significant. Other studies in normal and pathologic eyes including Diabetic Macular Edema (DME) and macular degeneration have also demonstrated that the difference in retinal thickness between the SD machines can be attributed to the differences in segmentation of the automated algorithms7,10,11.\n\nDespite the large numbers of scans with algorithm errors, the COR of Spectralis™ was lower for every subfield than that of Stratus™ or Cirrus™. The COR of Cirrus™ was equal to or larger than Stratus™ for both forms of the disease. In all three devices, the COR was generally better for NNV-AMD when compared to NV-AMD, especially after correction. This difference between diseases can be attributed to the pathology of NV-AMD disrupting the outer border, which makes it difficult for the automated algorithm to accurately segment the retinal layers12,13. At this point, we are not aware of any previous study looking at the repeatability of Spectralis™ images in AMD. Previous studies on normal eyes have reported a high repeatability of measurements with Spectralis™, with differences between repeated measurements being within 1 µm11,14. For Stratus™ OCT images, other studies have found central subfield repeatability values in patients with NV-AMD to be 50 µm and 32–35 µm for NNV-AMD patients after correction/exclusion of scans with errors8,15; our study confirms this finding. There has been one other published study looking at the repeatability of Cirrus™ OCT in NV-AMD, which found a central subfield repeatability value of 42 µm before correction and 27 µm after exclusion16. The difference between this study and our measurements may be associated with our smaller sample size. In addition, we chose not to exclude any poor quality scans, which may cause larger differences.\n\nIn addition to a lower COR, Spectralis™ also had the highest ICC values for both NV-AMD and NNV-AMD, before and after correction. For NV-AMD, Cirrus™ had higher coefficients after correction, and for NNV-AMD, Cirrus™ had lower coefficients compared to Stratus™. While no previous studies have reported ICC values for AMD patients, Pierro et al. found comparable results in normal eyes, with Cirrus™ ICC values ranging from 83–97% and Stratus™ ICC values from 72–95%17. The most likely reason for the low repeatability and high ICC values for Spectralis™ is the eye-tracking capability, which ensures that artifacts due to eye movement are minimized and the machine scans only when the tracking software identifies the same position on the fundus14.\n\nBland-Altman plots indicate that there is agreement between SD-OCT machines. Correcting images also influenced agreement between machines. We found that 95% confidence intervals were narrower compared to an SD-OCT and TD-OCT and correcting the algorithm errors further narrowed the intervals. The mean difference between machines indicates that the lowest differences were between Spectralis™ and Cirrus™, especially after correction. This is mostly likely due to the effects of manually correcting the Spectralis™ images and the fact that both machines have similar scanning technologies. The limits of agreement were similarly very wide for all three machines, and were narrower after correction of images, especially for the two SD-OCT machines. Jaffe et al. reported similar results looking at NV-AMD, with limits of agreements being approximately 225 µm between a SD-OCT and TD-OCT7. The poor agreement suggests that clinicians should exercise caution when trying to use the data from different machines interchangeably.\n\nOur study is not without its limitations. First, the software version for the Stratus™ images would not allow correction of images. However, very few images needed to be corrected. In addition, two people independently manually corrected the images, resulting in inaccuracies in segmentation line correction. Finally, the images were only taken at one imaging center, which could have resulted in bias.\n\nIn summary, we found that although Spectralis™ had the highest frequency of errors in AMD patients, correction of images did not result in significant changes in retinal thickness due to the errors being very small. Spectralis™ had the lowest coefficient of repeatability values. Thus Spectralis™ may be the best suited for examining minute morphological and thickness changes. Also, because of the wide Bland-Altman 95% intervals, there is not much agreement between the SD-OCT and TD-OCT machines. Based on our findings, we recommend that scans be carefully analyzed at reading centers before the thickness values are accepted as reliable.", "appendix": "Author contributions\n\nAR and YJS and RC conceived the study. AR, RC, and EH and MS carried out the research. AR, YJS, RC and MS prepared the first draft of the manuscript. DVD and QDN supervised the project. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe current manuscript was partially presented at the ARVO annual meeting, Fort Lauderdale, Florida in 2010.\n\n\nSupplementary figure\n\nThe following Early Treatment Diabetic Retinopathy Study grid depicts the abbreviations for the nine subfields. C1–Central 1 mm; N1–Inner nasal; S1–Inner superior; T1–Inner temporal; I1–Inner inferior; N2–Outer nasal; S2–Outer superior; T2–Outer temporal; I2–Outer inferior.\n\n\nReferences\n\nRitter M, Elledge J, Simader C, et al:Evaluation of optical coherence tomography findings in age-related macular degeneration: a reproducibility study of two independent reading centres. Br J Ophthalmol. 2011; 95(3): 381–5.\n\nRay R, Stinnett SS, Jaffe GJ: Evaluation of image artifact produced by optical coherence tomography of retinal pathology. Am J Ophthalmol. 2005; 139(1): 18–29.\n\nDomalpally A, Danis RP, Zhang B, et al:Quality issues in interpretation of optical coherence tomograms in macular diseases. Retina. 2009; 29(6): 775–81.\n\nMenke MN, Dabov S, Knecht P, et al:Reproducibility of retinal thickness measurements in patients with age-related macular degeneration using 3D Fourier-domain optical coherence tomography (OCT) (Topcon 3D-OCT 1000). Acta Ophthalmol. 2011; 89(4): 346–51.\n\nGiani A, Cigada M, Esmaili DD, et al:Artifacts in automatic retinal segmentation using different optical coherence tomography instruments. Retina. 2010; 30(4): 607–16.\n\nKrebs I, Haas P, Zeiler F, et al:Optical coherence tomography: limits of the retinal-mapping program in age-related macular degeneration. Br J Ophthalmol. 2008; 92(7): 933–5.\n\nHan IC, Jaffe GJ: Comparison of spectral- and time-domain optical coherence tomography for retinal thickness measurements in healthy and diseased eyes. Am J Ophthalmol. 2009; 147(5): 847–58, 858 e1.\n\nPatel PJ, Chen FK, Ikeji F, et al:Intersession repeatability of optical coherence tomography measures of retinal thickness in early age-related macular degeneration. Acta Ophthalmol. 2011; 89(3): 229–34.\n\nYehoshua Z, Rosenfeld PJ, Gregori G, et al:Progression of geographic atrophy in age-related macular degeneration imaged with spectral domain optical coherence tomography. Ophthalmology. 2011; 118(4): 679–86.\n\nForooghian F, Cukras C, Meyerle CB, et al:Evaluation of time domain and spectral domain optical coherence tomography in the measurement of diabetic macular edema. Invest Ophthalmol Vis Sci. 2008; 49(10): 4290–6.\n\nWolf-Schnurrbusch UE, Ceklic L, Brinkmann CK, et al:Macular thickness measurements in healthy eyes using six different optical coherence tomography instruments. Invest Ophthalmol Vis Sci. 2009; 50(7): 3432–7.\n\nKrebs I, Hagen S, Brannath W, et al:Repeatability and reproducibility of retinal thickness measurements by optical coherence tomography in age-related macular degeneration. Ophthalmology. 2010; 117(8): 1577–84.\n\nJoeres S, Tsong JW, Updike PG, et al:Reproducibility of quantitative optical coherence tomography subanalysis in neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2007; 48(9): 4300–7.\n\nMenke MN, Dabov S, Knecht P, et al:Reproducibility of retinal thickness measurements in healthy subjects using spectralis optical coherence tomography. Am J Ophthalmol. 2009; 147(3): 467–72.\n\nPatel PJ, Chen FK, Ikeji F, et al:Repeatability of stratus optical coherence tomography measures in neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2008; 49(3): 1084–8.\n\nParravano M, Oddone F, Boccassini B, et al:Reproducibility of macular thickness measurements using Cirrus SD-OCT in neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2010; 51(9): 4788–91.\n\nPierro L, Giatsidis SM, Mantovani E, et al:Macular thickness interoperator and intraoperator reproducibility in healthy eyes using 7 optical coherence tomography instruments. Am J Ophthalmol. 2010; 150(2): 199–204.e1." }	[ { "id": "3258", "date": "03 Feb 2014", "name": "Ilse Krebs", "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 reproducibility of retinal thickness measurements with different OCT devices in eyes with age-related macular degeneration before and after error correction is presented. This is a topic of high interest and actuality. The title and Abstract are appropriate.\"To date, no other study has examined the effects of manual correction of the thickness algorithm in SD-OCT and TD-OCT machines in eyes with AMD.\" \"At this point, we are not aware of any previous study looking at the repeatability of Spectralis™ images in AMD.\"This is not really true, and I want to refer to publications dealing with this topic (Krebs et al. 2012; Krebs et al., 2011; Krebs et al., 2009). The results of these studies should be discussed, as some of the results are confirmed by the results of the current study. Most of the studies focus only on the central 1000µm area, whereas in this study also more peripheral areas were examined. This should be discussed a little bit more because this might be interesting: were the failures only in the central part, or also in the periphery (the pathology of neovascular AMD is located centrally therefore pathology related failures should occur only in the central area.) It is mentioned in the discussion section, but it should also added to the methods: how many observers assessed the segmentation errors, and performed the error correction? The lack of significant differences before and after correction might be due to the small number of examinations requiring correction. The sample size seems to be quite low, was there any calculation when planning this study? A possible bias of including both eyes in a part of patients should be mentioned.", "responses": [ { "c_id": "720", "date": "28 Feb 2014", "name": "Yasir Sepah", "role": "Author Response", "response": "Dear Dr. Krebs,Thank you for your valuable comments. We agree that our study is not the only study that has tried to deal with this topic. We will make changes to the manuscript to clarify this statement.We will add the results and analysis of the peripheral areas to the discussion in the revision.No sample size calculation was performed before the conduct of the study. Two eyes of the same patient were included because of difference in the severity of the disease between the two eyes of the same patient. The control group patients contributed only one eye to the analysis." } ] }, { "id": "4688", "date": "12 May 2014", "name": "Igor Kozak", "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\nThis study reports on reliability and reproducibility of optical coherence tomography (OCT) scans before and after manual correction in eyes with age-related macular degeneration (AMD). It concludes that manual correction improves automated segmentation and that the agreement, as evidenced by intraclass correlation coefficients and coefficients of repeatability, is better between spectral-domain OCT instruments. The study brings important numerical comparisons of measurements of central foveal thickness using different instruments. Such comparisons are crucial especially with transitioning from time-domain to spectral-domain OCT technology in ongoing and future clinical trials. The paper is well written. Minor comments for authors:The same operator acquired OCT scans of the same eyes on the same instruments. It would be useful to know how many graders and how independently performed manual correction at the Reading Center. The Spectralis system via TruTrack provides excellent ability to perform follow-up scans from the exact retinal areas. How was this dealt with using other two systems in order to avoid sampling error? Misidentification of inner retinal later is a common artifact and has been found in a large number of scans including the instruments used in this study. The authors are encouraged to cite some of those studies such as Ho et al. (2009). Another useful study to mention with respect to comparing time-domain and spectral-domain OCT instruments: Mylonas et al. (2009). Based on a study of reproducibility in Stratus OCT, any artifact resulting in an error that is more than 50μm is clinically significant, suggesting 50 μm as a cutoff for retreatment of neovascular AMD patients (Patel et al., 2009). In another study any artifacts resulting in automated segmentation errors of more than 10% of the actual (manually measured) ETDRS center subfield thickness were considered clinically significant (Browning et al., 2008). In this study, some of the variations after manual correction surpassed these margins. Maybe some comment in Discussion regarding this issue.", "responses": [] } ]	1	https://f1000research.com/articles/2-131
https://f1000research.com/articles/4-57/v1	27 Feb 15	{ "type": "Opinion Article", "title": "The role of globalization in drug development and access to orphan drugs: orphan drug legislation in the US/EU and in Latin America", "authors": [ "Renée J.G. Arnold", "Lida Bighash", "Alejandro Bryón Nieto", "Gabriela Tannus Branco de Araújo", "Juan Gabriel Gay-Molina", "Federico Augustovski", "Lida Bighash", "Alejandro Bryón Nieto", "Gabriela Tannus Branco de Araújo", "Juan Gabriel Gay-Molina", "Federico Augustovski" ], "abstract": "Compared to a decade ago, nearly three times as many drugs for rare diseases are slated for development. This article addresses the market access issues associated with orphan drug status in Europe and the United States in contrast to the legislation in five Latin American (LA) countries that have made strides in this regard--Mexico, Brazil, Colombia, Chile and Argentina. Based on the success of orphan drug legislation in the EU and US, LA countries should strive to adopt similar strategies with regard to rare diseases and drug development. With the implementation of new targeted regulations, reimbursement strategies, and drug approvals, accessibility to treatment will be improved for people afflicted with rare diseases in these developing countries.", "keywords": [ "market access", "orphan drugs", "Latin America", "rare disease" ], "content": "Introduction\n\nMedications for the approximately 7,000 rare diseases in the world account for less than 10% of global pharmaceutical spending. Although significant, the contribution is small compared to pharmaceuticals for common diseases. The definition of a rare disease varies, but it is generally said to affect <1 in 2000 people in the European Union or <200,000 people in the United States (Acta Pediatra). Given the low incidence and prevalence of these diseases, they individually reach only a small percentage of the global population; together, however, they affect between 6% and 8% (or 420 million to 560 million people), thus imposing a significant global burden. Of this total, approximately 6 million patients affected by rare diseases are in Mexico, 13 million in Brazil, and 3 million in Argentina. México, Argentina and Colombia use the EU definition of rare diseases, while Brazil, Péru and Chile have bills under consideration, but still have not defined rare diseases or orphan drugs.\n\nOrphanet defines an orphan drug as a “drug not developed by the pharmaceutical industry for economic reasons but which responds to public health need”. This includes products to treat rare diseases as well as products withdrawn from the market for economic/therapeutic reasons, e.g., thalidomide, and products that have not yet been developed1. To further complicate matters, certain drugs may be designated as “orphan” only in subpopulations (e.g., the elderly) within a particular non-rare disease indication, such as cancer. Or, a single drug may be developed for a rare disease, but then further developed for non-rare variants of that therapeutic category. Sometimes this last designation is taken even further in that a non-rare disease can be \"aggressively\" segmented into multiple rare diseases to achieve an orphan designation for a drug2. The lack of clear regulations surrounding orphan drugs in Latin America (LA) is concerning, given the millions of Latin Americans who have heterogeneous access to treatment for their rare diseases. Although the recognition of the special status of rare drugs and diseases is generally regarded as an accepted, positive label that will result in improved health for people in need of care, the controversy lies with exactly how people with orphan diseases in the US and EU, as well as in LA, should be covered in terms of legislation, drug approvals, reimbursement, and investment, especially given the disparate incentives given to the drug industry for the development of orphan drugs.\n\nApproximately 80% of rare diseases have a genetic origin3. The remainder are the result of bacterial and viral infections, allergies or degenerative conditions4. Most rare diseases (75%) are manifested early in life and affect children from 0 to 5 years of age5. They also contribute significantly to morbidity and mortality in the first 18 years of life. The primary challenge is to balance patient demand with the rising costs in the drug development industry due to scientific and technological advancements.\n\n\nOrganizations\n\nSeveral organizations have been involved in the development of legislation and advocacy for rare disease research, in addition to being advocates for patients with rare diseases. Some of these organizations are discussed below:\n\nOrphanet was established in France in 1997 with the support of the French Ministry of Health; its current membership includes 35 countries. Orpha.net is a reference portal for information about orphan drugs and rare diseases. The Orphanet annual report for 2012 states that “Negotiations were initiated with Argentina, Brazil, China, Chile, Japan and Russia” for the purpose of joining the organization. Interestingly, Mexico and Brazil are among the top 10 countries to access data from the orpha.net website1.\n\nThe International Rare Diseases Research Consortium (IRDiRC) was launched in April 2011 by the European Commission and the US National Institutes of Health to foster international collaboration in rare diseases research. IRDiRC brings researchers and organizations investing in rare disease research together in order to achieve two main objectives—to deliver 200 new therapies for rare diseases, and to develop the means to diagnose most rare diseases by the year 2020 (IRDiRC).\n\nEurordis is a non-governmental alliance of patient organizations representing 590 rare disease patient organizations in 54 countries3.\n\nThe International Conference on Rare Diseases and Orphan Drugs (ICORD) is a not-for-profit group comprised of academics, patient advocacy groups, regulatory authorities, public policy professionals, and people from the healthcare services industry. The group is primarily involved in policy advocacy. In 2010, a conference convened in Buenos Aires calling for globalization of research on rare diseases and drug development6,7. The most recent conference took place in Ede, The Netherlands, with the main theme of “The Societal Value of Prevention, Diagnosis, and Treatments of Rare Diseases (ICORD).\n\nThe European Commission Expert Group on Rare Diseases (previously EUCERD, the European Union Committee of Experts on Rare Diseases) has the mandate to aid the \"European Commission with the preparation and implementation of community activities in the field of rare diseases, in cooperation and consultation with the specialized bodies in Member States, the relevant European authorities in the fields of research and public health action and other relevant stakeholders acting in the field\" (EUCERD).\n\n\nLegislation\n\nAs shown in Figure 1, Latin American countries have come relatively \"late to the game\" in terms of legislation surrounding rare diseases. The state of the legislation will be addressed below and grouped according to the presence or absence of a definition for rare diseases in each country.\n\nMéxico. A change to a general health law in Mexico was enacted at the beginning of 2012, Article 224; the law was amended to recognize both orphan diseases and the orphan drugs to treat them8. The Mexican Pharmacopeia previously provided a definition of an orphan drug, but it is now officially a Federal Law. However, the new regulations do not provide an “exhaustive body of law” for orphan drugs, and there is no regulation for “exclusivity”.\n\nFurthermore, Seguro Popular is a public health insurance policy initiated in 2003 that expands the accessibility of comprehensive health care services to millions of previously uninsured Mexicans. As of 2011, four rare diseases had been incorporated into the Seguro Popular scheme and seven drugs had been incorporated into the Basic Formulary (COFEPRIS). Additionally, seven new drugs received the “Orphan Drug Commercialization License” in 2013 and a total of eight rare diseases are now incorporated in the scheme.\n\nOf note, in September 2014 a patient suffering from paroxysmal nocturnal hemoglobinuria filed a lawsuit against the Instituto Mexicano del Seguro Social (Mexican Social Security Institute) or IMSS, claiming that IMSS had the constitutional responsibility to pay for his treatment (eculizumab). However, the court ruled in favor of IMSS, saying that they were only obliged to pay for the treatment if the drug was already included in the Basic Formulary but emphasized that the hospital could submit a request to the General Health Council for the evaluation of the inclusion of eculizumab in the Basic Formulary (Comunicados de Prensa). This case illustrates that the Basic Formulary defines what can and cannot be acquired by the public healthcare institutions; unfortunately, certain treatments for rare diseases are still not included in the formulary. Thus, there remains a need for regulatory incentives that promote investment for research and development of orphan drugs.\n\nArgentina. Argentina enacted its first national law (Law 26.689) for the healthcare of people with rare diseases in June 2011, largely as a result of advocacy led by the Geiser Foundation (Grupo de Enlace, Investigación y Soporte - Enfermedades Rares), a regional initiative created in 2001 to pool rare disease resources. The law defines rare diseases using the EU definition and requires the health system and public/private social security schemes to provide patient support. The law also mandates the establishment of a national patient registry for neonatal screening programs and the creation of a central coordinating committee9,10. Although the law was enacted in 2011, it is still not regulated; thus, the law provides a general framework for progression in the arena of rare diseases but has little enforcement power in practice. In 2012 the drug and food regulatory agency (ANMAT) created a commission for the evaluation and authorization of drugs for special conditions, using the same definition as the aforementioned law. Among other mandates, the decree requires intensive post-marketing surveillance for drug approval (decree 4622/2012).\n\nIn 2014 the superintendent of health, the body that regulates the provision of health technologies for the social security and private sectors as well as the reimbursement of selected technologies, created a mentoring system (Sistema de Tutelaje de Tecnologias Sanitarias Emergentes) of conditional reimbursement where information about patient-relevant outcomes is required for reimbursement (Ministerio de Salud). Though not specific for orphan status, some rare drugs and diseases are included (i.e., galsulfase for type IV mucopolysaccaridosis and eculizumab for nocturnal paroxysmal hemoglobinuria). Additionally, there is an ongoing legislation project for the creation of a specific fund for rare/catastrophic diseases. Similar to other countries in the region where health is considered a universal right, many of the coverage decisions are enacted through litigation in the judicial system11.\n\nChile. After 15 years of effort, Chile finally passed a law pertaining to the financing of medications for rare diseases on January 15, 2015 (La Ley Ricarte Soto). Specifically, the proposal seeks to find a sustainable approach to healthcare coverage for 20 million patients who are affected by rare, costly diseases. Over four years, the government will carry out this grand initiative with an investment of 200 billion pesos. This is 10 times greater than the amount of money that is currently dedicated to such coverage.\n\nAdditionally, with the support of the Chile Regional Chapter of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR), the HTAnetLatAm 2014 Roundtable took place at the Chile Institute of Public Health—Ministry of Health in Santiago, Chile in September of 2014 (HTAnetLatAm). The roundtable discussion focused on high-cost drugs for rare diseases in LA.\n\nColombia. In 2010, Colombia passed the Orphan Disease Law and hosted the 2nd National Forum of Orphan Diseases9. Specifically, the 2010 Law 1392 recognizes orphan diseases as a significant issue in healthcare due to their low prevalence and high cost to the health system12. As a result, the government has enacted rules to ensure social protection for people with rare diseases. Additionally, through the 1438 Law adopted in 2011, the government confirmed that a rare disease is labeled as an “orphan disease” if the disease is chronically debilitating, life-threatening, and its prevalence is less than 1 in every 5,000 people13. Stemming from these laws, the government made significant regulatory actions within the healthcare information system14. Furthermore, in 2013 the Ministry of Health, through the Colombian Fund for High Cost Diseases (High Cost Account), conducted the first census of patients with orphan diseases, in which 13,168 cases were reported (Cuenta de Alto Costo). Currently, workshops are being conducted to define models of care in these diseases to ensure adequate access to healthcare that is especially required for this population.\n\nBrazil. A review of health litigation in Latin American countries revealed that, in Brazil, fewer lawsuits are filed against drugs in the Exceptional Circumstance Drug Dispensing Program (13–31%) as compared to basic medications in the Unified Health System (SUS) (50%), suggesting that a foundation for a solid legal framework for drugs for orphan diseases may already be in the works (Rev Panam Salud Publica).\n\nIn 2013 a public consultation took place regarding the need for the adoption of a “National Policy for Rare Diseases” in Brazil15, and in January 2014 a Rare Diseases National Attention Policy was established (MOH Ordinance No. 199).\n\nThis innovative policy adopts a broader perspective regarding rare diseases, taking into consideration the established Brazilian National Humanization Policy (PNH) and, thus, the following needs:\n\n1) Comprehensive and multidisciplinary healthcare for people with rare diseases;\n\n2) Standards for the qualification of Specialized Care Services and Reference Services for Rare Diseases in the Brazilian public health system; and,\n\n3) A definition for the scope of activity of Specialized Care Services and Reference Services for Rare Diseases in the Health System, in addition to establishing technical quality standards for proper performance of their duties in the context of the healthcare network. This also takes into account the assistance required by the health care managers in regulating the access, control and evaluation of assistance for people with rare diseases in the SUS.\n\nAdditionally, this new national policy establishes the guidelines for patients with rare diseases and the program financial funding for SUS. As defined by the policy, a rare disease in Brazil is one that affects up to 65 people in every 100,000 individuals, or 1.3 per 2,000 individuals. Brazil strives to achieve certain milestones through this law, which include the following: ensure the universality, completeness and fairness of health services for individuals with rare diseases, with a subsequent reduction in morbidity and mortality for patients with such diseases; establish set guidelines for people with rare diseases care in all the SUS care levels; provide and expand equal and regulated healthcare access for people with rare diseases; ensure that people with rare diseases have timely access to available diagnostic and therapeutic methods; and establish care for people with rare diseases.\n\nAfter the establishment of the policy, a new public consultation took place to create a prioritized list of rare disease protocols, as established by the SUS (Public Consultation No. 20). The National HTA commission (CONITEC) had an intensive scientific collaboration on this process and will be responsible for the creation of 12 new guidelines in 2015 (CONITEC Consultas Públicas). Currently, 35 treatments for rare diseases are covered by the SUS. It is important to remember that, in Brazil, the public health system is a right for all 202 million citizens living in the country.\n\nPeru. Currently, no definition for rare diseases exists in Peru. However, in 2011 the country passed legislation promoting treatment and a national strategy for rare diseases, which includes diagnosis, surveillance, prevention, care and rehabilitation for such conditions9,10.\n\n\nSpecific recommendations for harmonization (based on suggestions of the international biotechnology industry organization [BIO])\n\nThe Biotechnology Industry Organization, a not-for-profit trade association representing more than 1,100 companies, academic centers and research institutions in over 30 countries globally, constructed a letter commenting on the Brazilian Ministry of Health's proposed Standards for Enabling Specialized Care Services and Reference Centers for Rare Diseases in the SUS and Guidelines for Integral Care for People with Rare Diseases in the SUS15. These recommendations, while not universally appropriate for/specific to all LA countries, are useful as a benchmark for many developing nations. They, in conjunction with others from ICORD (Acta Pediatra), are abstracted below:\n\nAdopt the EU definition of a rare disease;\n\nIdentify a subset of rare diseases as “ultra-rare”— (Although there is no internationally agreed upon definition of an ultra-rare disease, the UK National Institute for Health and Care Excellence (NICE) defines an ultra-rare disease to be one that affects less than 1000 people in the UK. Other regional-specific definitions may be appropriate as well)16.\n\nDo not categorize the diseases based on their etiology, but only by their prevalence, to reduce restrictions in patient access;\n\nAllow for different types of authorizations, (e.g., the EU has three approval pathways: full approval/authorization, conditional marketing authorization, and authorization under exceptional circumstances; the US has accelerated approval regulations based on surrogate endpoints followed by post-market confirmatory studies);\n\nInstitute incentives for industry, such as tax credits (US) for R&D, fee waivers (US/EU), and market exclusivity (US/EU);\n\nCreate early access/compassionate use programs, such as the French Temporary Use Authorization17;\n\nEstablish a federally-funded Coordination Center to act as a central information resource, a patient/researcher support system, and a promoter of public awareness;\n\nEstablish a special technical subgroup for rare diseases within existing technology appraisal agencies so that the health technology assessment (HTA) processes (such as cost-effectiveness analysis/budgetary impact modeling) typically used are evaluated with an eye towards the unique challenges of rare diseases, e.g., very high cost/quality-adjusted life year (QALY) but low budgetary impact because of small patient populations. Include other factors besides cost, such as lack of alternative treatments, equity of care, fairness of process, and society’s willingness-to-pay;\n\nAdopt a comprehensive approach, including education, prevention, diagnosis, care and treatment; in addition, support social, basic and clinical research;\n\nInvolve patient advocacy groups in identifying patients, making genetic testing available, providing resources for screening. Additionally, have the groups involved with informed consent and autonomous decision making, advisory groups and expert panels; and,\n\nUse a multi-stakeholder approach.\n\n\nDiscussion\n\nThis article focused largely on legislation in order to address some of the inadequacies relevant to making drugs available for orphan diseases; of course, there are many mitigating factors surrounding the debate on market access. For example, as indicated in a recent European review of EU orphan drug policies18, society’s values in regards to funding these drugs, which would never be considered as cost-effective as drugs for more prevalent diseases, need to be clarified in LA. Similarly, consideration needs to be given to the revision of pricing and reimbursement policies in LA. Certain questions remain unanswered: does there need to be a cap on profits that can be generated by a drug with an orphan designation before it “loses” that designation? What types of analyses, e.g., budgetary impact modeling, need to be undertaken to demonstrate the relative cost-effectiveness of these agents in their respective populations? In addition, it is suggested that determining and revealing specific research priorities, e.g., for specific orphan diseases as a group, would incentivize companies to undertake research in those areas.\n\nRecommendations for the harmonization of orphan drug development in LA with that of developed nations include adopting the EU definition of rare disease, instituting incentives for industry, and establishing a special technical subgroup for rare diseases that considers the unique challenges of rare diseases, among other techniques. Implementation of some or all of these approaches, which have been used somewhat successfully in the US, EU and other developed countries, will play a large role in improving access of orphan drugs to patients in LA and other emerging markets with rare diseases. Considering the scarcity of resources and trade-offs involved for decisions pertaining to which rare diseases to address and how to address them, it is incumbent upon all stakeholders to promote harmonization and advances in legislation, regulation and market access schemes, with clear rules and incentives on orphan disease definitions and drug development. Indeed, Drummond and Towse suggested using a more collaborative manifesto amongst countries to address these issues, similar to the global business case employed by the pharmaceutical companies in development of these and other agents18. This methodology, in conjunction with discussions about specific countries’ societal values and affordability issues, will help to address the needs of this special patient population.", "appendix": "Author contributions\n\n\n\nEach author was responsible for the information about rare diseases within their country of residence. Dr. Augustovski reviewed and commented on the entire manuscript and Ms. Bighash researched and reviewed references, translated some of the original reference material and revised the manuscript as necessary. This work was based on a workshop Renée JG Arnold conceived and moderated at the ISPOR 4th Latin America Conference in Buenos Aires, September 2013, on the same subject. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nhttp://www.orpha.net/consor/cgi-bin/index.php [Accessed August 1, 2013].\n\nDenis A, Mergaert L, Fostier C, et al.: Issues surrounding orphan disease and orphan drug policies in Europe. Appl Health Econ Health Policy. 2010; 8(5): 343–50. PubMed Abstract \| Publisher Full Text\n\nwww.eurordis.org [Accessed August 1, 2013].\n\nSharma A, Jacob A, Tandon M, et al.: Orphan drug: Development trends and strategies. J Pharm Bioallied Sci. 2010; 2(4): 290–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInterfarma. Rare Diseases: Contributions for a National Policy. 2013. Reference Source\n\nhttp://www.icord.se [Accessed August 4, 2013].\n\nForman J, Taruscio D, Llera VA, et al.: The need for worldwide policy and action plans for rare diseases. Acta paediatr. 2012; 101(8): 805–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttp://orphandruganaut.wordpress.com/2013/01/30/orphan-drugs-in-mexico/ [Accessed August 2 , 2013].\n\nWong-Rieger D: State of the art of rare disease activities around the world: overview of the non-European landscape. 6th European Conference on Rare Diseases and Orphan Products. Brussels, Belgium: Orphanet J Rare Dis. 2012; 7(Suppl 2): A2. Free Full Text\n\nhttp://www.checkorphan.org/grid/news/people/argentina-and-peru-making-progress-with-new-rare-disease-laws-legislated-in-both-countries [Accessed August 2, 2013].\n\nRonconi LM: The right to health: a model for determining the contents of the minimum core and the periphery. Salud colect. 2012; 8(2): 131–49. PubMed Abstract \| Publisher Full Text\n\nLey 1392 de 2010. 2010. Reference Source\n\nLey 1438 de 2011. 2011. Reference Source\n\nColombia avanza en la identificación de los pacientes con enfermedades huérfanas. 2014. Reference Source\n\nRe: Public Consultation N°7 of April 10, 2013. 2013.\n\nNice Citizens Council Report: Ultra Orphan Drugs. 2004. Reference Source\n\nUrbinati D, Masetti L, Toumi M: Early Access Programmes (EAPS): review of European system. 2012. Reference Source\n\nDrummond M, Towse A: Orphan drugs policies: a suitable case for treatment. Eur J Health Econ. 2014; 15(4): 335–40. PubMed Abstract \| Publisher Full Text\n\nSistema de tutelaje de de tecnologias sanitarias emergentes. Superintendencia de servicios de salud. Resolución Nº 2206/2013. Reference Source\n\nReveiz L, Chapman E, Torres R, et al.: Right-to-health litigation in three Latin American countries: a systematic literature review. Rev Panam Salud Publica. 2013; 33(3): 213–22. PubMed Abstract \| Publisher Full Text\n\nBrazil. Ministry of Health. Ordinance No. 199, 30 of January of 2014. Reference Source\n\nBrazil. MoH. Secretary of Science, Technology and Strategic Inputs. Public consultation No. 20, 26 of September of 2014. Reference Source\n\nBrazil. MoH. Secretary of Science, Technology and Strategic Inputs. Department of Management and Merger of Health Technology. Prioritization Protocols and Therapeutic Guidelines for Integrated Management of People with Rare Diseases. 2014. Reference Source\n\nMedicamentos Huérfanos. COFEPRIS: Comisión Federal para la protección contra Riesgos Sanitarios. 2011 and 2013. Reference Source\n\nComunicados de Prensa No. 142/2014. México D.F. 17 September 2014. Reference Source" }	[ { "id": "7856", "date": "03 Mar 2015", "name": "John Forman", "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\nThe authors add to the public domain valuable information about the legislative and policy steps in selected Latin American countries to deal with rare disorders. This is welcome in the context of the emerging globalisation of health polices modelled on the success of pioneering policies elsewhere, particularly in the United States, Japan and the European Union. The work describes actual policy initiatives in Latin America, and avoids the frequent limits of much discussion on this topic which often concentrates primarily on the US and EU experience and simply advocates greater adoption of such policies elsewhere. Here we see the real experience of the globalisation process in its impact in several countries in a specific region.It is notable that the authors briefly describe some aspects of diagnosis, prevention, healthcare, and rehabilitation in their discussion. But the article begins and concludes with strong emphasis on discovery, regulation and reimbursement of novel drugs for rare diseases. While drug discovery and access are of great importance to rare disease patients and their families, so too is the rest of their journey through life in the health and disability system. This and future articles on the topic could be enhanced by greater commentary on how these other important matters are also addressed in various national plans.The work highlights the need for similar investigation of rare disease policies in other world regions, to add to the global knowledge set.", "responses": [ { "c_id": "1273", "date": "17 Mar 2015", "name": "Renee Arnold", "role": "Author Response", "response": "We very much appreciate the reviewer's comments and agree that facilitating the patient journey through the healthcare maze is of paramount importance. It is through organizations, such as the reviewer's, that involve patients with rare diseases in the legislative process that patients will be able to influence the healthcare system to work to their/their children's benefit." } ] }, { "id": "7869", "date": "16 Mar 2015", "name": "Wendy Lipworth", "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\nThis article addresses an important issue: what countries can do to ensure that patients with rare diseases have access to safe, effective and affordable medicines. It is essentially a descriptive (rather than critical/analytic) article, that would primarily be of interest to those involved in orphan disease policymaking.A few points for improvement:It is not clear how the first part of title \"The role of globalization in drug development and access to orphan drugs\" relates to the article. The article is a description of orphan drug-related laws and policies in a series of countries, so it is not clear what is meant by \"the role of globalisation\" The introduction could be organised better by discussing orphan drugs in general, and then giving the facts and figures regarding Latin America (rather than weaving the two together as is currently the case). It would be helpful to know a bit more about the health systems in the countries discussed so that the descriptions of orphan drug provisions can be placed in context. Finally, it would be helpful to know why the countries were divided according to the definition of rare disease that they have adopted. Was there some expectation that different definitions would lead to different policies and laws?", "responses": [ { "c_id": "1274", "date": "17 Mar 2015", "name": "Renee Arnold", "role": "Author Response", "response": "We thank the reviewer for her thoughtful comments. Specific discussion to the critique follows:1. The role of globalization is meant to show how orphan diseases are a global concern and that developing countries, such as those in Latin America, would do well to follow the lead of countries in the developed world in their approach to improving access to treatments for patients with these diseases.2. While we understand the reviewer's point here, this was not meant to be an exhaustive review on orphan drugs, but an opinion piece on how legislation is Latin America is currently progressing along the lines of countries whose policies on orphan diseases/drugs are more established.3. We agree, but that is beyond the scope of this opinion article.4. The countries were arbitrarily divided according to their current policies or lack thereof for reader ease of understanding the current progression, by country, of policy development." } ] }, { "id": "7855", "date": "18 Mar 2015", "name": "Durhane Wong-Rieger", "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\nOverall: This is a very valuable document providing an update on legislative and access policies and programs for orphan drugs/rare diseases in Latin America. It is helpful to see all of this brought together. The following comments are provided to suggest ways in which the data may be further analyzed and discussed to provide a more fulsome understanding and implications for the future for rare diseases/orphan drugs in this region.The appropriateness of the title: The title is somewhat misleading in that the “role of globalization” is not really addressed, that is, there is limited demonstration of how the various legislative, regulatory, and assessment policies are influenced by countries’ referencing each other or global standards. Perhaps most notable commonality is the similarity to the EU prevalence definition for orphan drug/rare disease. Indeed the uniqueness of each country’s approach and what is contained in legislation or other policy appears to be grounded in their healthcare policies, system, infrastructure, and priorities, beyond rare.Abstract as an adequate summary of the article: The abstract provides an overarching statement about strides in five countries but does not really summarize the information presented. Nor does it summarize broadly the strides being made, or the challenges in market access. Rather, there are statements about the need for and potential benefit of adopting similar strategies and need for implementation of targeted methods, which are undoubtedly valid points but not driven by the data provided about each country’s approach, success, and limitations. Why would harmonized strategies be beneficial?If there is a comprehensive explanation of study design, methods and analysis, and their suitability to the investigation: This is mostly a descriptive study and, as such, seems to source the information from the legislation and policies in each country. There is no reference to how the information has been validated as to the actual implementation or the progress made in adopting these. Nevertheless, one can accept on face validity the up-to-date information about the status of orphan drug/rare disease legislation, policies, strategies, and practices in each of the five countries referenced. As such, the documentation is very useful. It would be useful if there were more analysis, which might shed some light on how these approaches are similar, and how they are different, and what each strategy implies for the healthcare providers and patients in each country. There are also rather loose transitions between drug legislation and market access and policies and practices targeting other areas of healthcare, such as newborn screening, comprehensive care, and social services. In Mexico and to some degree Argentina, legislation appears to target orphan drugs and access while other countries like Colombia and Brazil have legislation and programs that cover other areas of healthcare. It is less clear the degree to which Chile and Peru are addressing healthcare, including diagnosis and prevention, as well as drug access. It would be interesting to explore how and why these differences have occurred at this time.It would be useful to understand the degree to which each country does a complete regulatory review of a submitted orphan drug or relies upon review done by other countries. Several have referenced HTA specifically for rare diseases/orphan drugs; is there sufficient information to comment on whether these approaches are unique or collaborative. Is HTAnetLA taking a role (following the Roundtable), or is each country’s HTA likely to following unique strategies?Whether the conclusions are balanced and justified on the basis of the results: It would be helpful to have more discussion of the information presented here that could lead to the proposal that harmonization and collaborative approaches are valuable and feasible. It may be helpful to use the BIO recommendations (presented at the end) as a framework for assessing each country’s current status, implications for impact, and proposals for each country for next steps .", "responses": [] } ]	1	https://f1000research.com/articles/4-57
https://f1000research.com/articles/4-56/v1	26 Feb 15	{ "type": "Research Article", "title": "In vitro retention of a new thermoplastic titratable mandibular advancement device", "authors": [ "Marc Braem" ], "abstract": "Oral appliance (OA) therapy with a mandibular advancement device (OAm) is a non-invasive, alternative approach to maintaining upper airway patency. The main requirement for an OAm to be effective is the adequate retention on the teeth while the patient is asleep. We evaluated the retentive forces of a new low-cost, customizable, titratable, thermoplastic OAm (BluePro®; BlueSom, France). Dental impressions and casts were made for one patient with complete upper and lower dental arches including the third molars and class II bite proportions. A setup based on Frasaco ANA-4 models was also used. Two protrusive positions of the mandible were investigated: 3 mm and 8 mm, representing respectively 25% and 65% of the maximal protrusion. The forces required to remove the BluePro® device from the carriers were recorded continuously over 730 cycles (=365 days, twice a day) to simulate 1 year of clinical use. At 8 mm protrusion the BluePro® device showed retentive forces of ~27N. There was a slight but non-significant decrease in retentive forces in the tests on the epoxified carriers which was not found on the ANA-4 carriers. There were no significant differences between the carriers as a function of protrusion. The BluePro® device tested in the present study possesses sufficient retention forces to resist initial jaw opening forces and full mouth opening forces estimated to be ~20N. It could therefore broaden the indications for use of thermoplastic OAms. It could provide a temporary OAm while a custom-made OAm is being manufactured or repaired. Patients could be provided with a low-cost try-out device capable of reliable titration, providing an indication of effectiveness and of patient acceptance of an OAm, although the effect of device shape and size on therapeutic outcome is not yet known. Finally it could provide an affordable OAm solution in resource-restricted healthcare settings.", "keywords": [ "mandibular advancement device", "obstructive sleep apnoea", "oral appliance", "retention force" ], "content": "Introduction\n\nOral appliance (OA) therapy with mandibular advancement devices (OAm) is becoming the main non-invasive and alternative approach to continuous positive airway pressure. By advancing the mandible and tongue, upper airway patency is favoured during sleep thereby preventing upper airway collapse1. Although many types of OAm are available2–5, each requires retention on the teeth to maintain protrusion of the mandible during sleep. Lack of retention causes loosening of the OAm and may result in reduced efficacy of treatment, patient complaints about poor fit and an increased risk of side effects4,6,7.\n\nThe main disadvantages of custom-made OAm are not only the cost, but also, more importantly, the time required to manufacture the device, while the obstructive sleep apnoea (OSA)-patient is not being treated. A comparable situation arises when the OAm of a patient under treatment needs to be repaired in the dental laboratory. Furthermore, clinical practice demonstrates that patients are often not willing to leave the OAm for repair because they have become dependent on the device for a good night’s sleep.\n\nThe primary requirement for an OAm to be effective is the adequate retention on the teeth while the patient is asleep. Recently, in vitro testing of such retentive characteristics of different types of OAm, including thermoplastic ones, was reported in the literature8, demonstrating that generalization of the retentive characteristics with respect to the design and type of an OAm is not pertinent.\n\nThe present report compares the retentive forces of a novel customizable titratable thermoplastic low-cost OAm to other devices reported previously in the literature.\n\n\nMaterials and methods\n\nAn experimental customizable titratable thermoplastic oral appliance commercialized for use by healthcare professionals under the name BluePro®, was tested (BlueSom, France) (Figure 1).\n\nDental impressions and casts were made (Impregum Penta Soft Medium, 3M ESPE AG, Germany) for one patient with complete upper and lower dental arches including the third molars and Class II bite proportions. A setup based on Frasaco ANA-4 models (Frasaco, Germany) was also made.\n\nThe protrusion to be incorporated into the BluePro® was measured from the vestibular rim of the incisive edge of the upper left central incisor, in a horizontal plane up to the lingual rim of the incisive edge of the lower left central incisor. These distances represent clinically relevant protrusive positions as noted during actual OA therapy in patients. Two protrusive positions were investigated in the present tests: 3 mm and 8 mm of protrusion, representing 25% and 65% of the maximal protrusion in the particular patient.\n\nSamples of the BluePro® were fitted onto the epoxified casts (n=10 for each protrusion) as well as on the ANA-4 models (n=10 only for 8mm protrusion, see further) according to the manufacturer’s instructions (BluePro® user leaflet). The exact procedure was as follows:\n\nBoiling water was poured into a flat-bottomed polypropylene box. The upper and lower parts of the BluePro® were then placed into the hot water until the inner material in both parts became fully transparent. A timer was started and the upper part was taken out of the water and put to one side to cool down for 30s. The same procedure was then repeated for the lower part and the upper part was immediately shaped onto the upper carrier, either the epoxified cast or ANA-4 carrier, depending on the test required.\n\nThe carrier was pressed firmly into the upper part of the BluePro®, sliding it a bit forward to create an optimal fit into the tray of the BluePro®. Finger pressure was then applied and the softened material on the inside of the tray was immediately flattened. The softened material was also pressed into shape at the outside of the tray to fit it tightly around the carrier. Both the carrier and the BluePro® were put on a flat table, the BluePro® facing down, and a dead weight was placed on top to simulate gentle biting and to immobilize the device in the required position, without popping or creeping out. This procedure was completed within 30s, then was repeated for the lower jaw.\n\nBoth parts were allowed to cool down for 5 min. The thermoplastic material inside the tray was then checked to see if it had become cold and opaque again and the BluePro® was removed from the carrier and rinsed in cold water. If necessary, sharp edges of superfluous softened material were removed, as well as any material that had crept into the rails or locks. The device was then dried and stored in the original packing at room temperature prior to testing.\n\nThe casts and ANA-4 models were mounted in average value inclination of the occlusal plane and centred in a hydraulic cyclic test machine (Dartec HC10, Testbench Dartec 9600 Controller; Dartec, UK) as described previously8.\n\nWhen mounting the casts, stubs were added to these to facilitate removal and reinsertion. Both upper and lower casts were mounted at a fixed position, giving a protrusion of 3mm or 8mm, depending on the test.\n\nWhen mounting the ANA-4 carriers into the fatigue machine, first a bite-registration was made with silicone on a plastic spoon with a protrusion of 8mm compared to the habitual occlusion. Using this bite registration as a reference, the ANA-4 carriers were fixed in a reproducible way so that all BluePro® samples were tested in an identical protrusion. Due to technical limitations of this version regarding the length of the rim for the clasps, it turned out that the 3mm protrusion could not be tested. Next, for the fitting of the OAm-TP on the ANA-4 carriers a specific procedure was followed.\n\nFirst the upper and lower parts of the BluePro® were inserted onto the carriers but without interconnecting (Figure 2A). The upper carrier together with the OAm could now move up and down freely as the piston moved. The lower model stayed fixed to the bottom of the machine. The upper carrier with the BluePro® was then lowered until it touched the lower carrier establishing the zero-position calibration for the stroke. Next, both the upper and lower parts of the OAm were connected by sliding the locks onto the rails, but without locking and firmly seated thereafter by finger pressure. The locks were then closed and the BluePro® was positioned correctly in the machine for the given protrusion (Figure 2B). In this position, there appeared to be a gap in the frontal region preventing the correct repositioning at reinsertion during the fatigue test. To avoid this phenomenon, a passive splint in silicone putty was inserted (Figure 2C). The upper piston was moved upwards until the BluePro® came loose from either of the ANA-4 carriers and with the sample now hanging free and unloaded, the load (force) was set to zero so that both the stroke (displacement) and load (force) were calibrated correctly. The fatigue test was then started.\n\n(A) positioning of the BluePro® device on the ANA-4 carriers that are connected to the pistons of the servo-hydraulic fatigue machine, prior to tightening of the titration clips; (B) situation after tightening of the titration clips, joining the upper and lower part of the BluePro® device; (C) identical situation as in ‘B’ but on the epoxified gypsum carriers.\n\nThe forces required to remove the BluePro® from the carriers were continuously recorded during 730 cycles (=365 days, twice a day) at 35°C in a dry environment to simulate 1 year of clinical use. Data were stored in CSV format for export to Excel (Microsoft, USA, see Dataset 1 and Figshare Dataset).\n\nThe test was setup as a stroke-controlled test with a triangular waveform, thus the piston repeatedly moved up and down with a given stroke and frequency. The piston speed was 7.5mm/s and was kept constant during all tests. The stroke length was set to 7.5mm, back and forth from 0.0mm to 7.5mm. This stroke was chosen carefully so that the BluePro® came loose from the carrier, although not completely so that re-entrance could be carried out without misalignment at reinsertion during the next loading cycle. The test frequency was 7.5mm/s at the optimal stroke, resulting in a test frequency of 0.5Hz.\n\nGraphs of the waveforms were captured every 36 cycles, starting at cycle 10 and ending at cycle 694. On the load graphs, the point at which the BluePro® came loose from the carrier was detected since the load dropped to zero and stayed at zero for the rest of the upward trajectory. At this point the load needed to give a flat line at zero, also indicating that the load-channel had been calibrated to zero correctly.\n\nMeasurements were compared using a paired Student’s t-Test (Statistica Release 6, StatSoft Inc. Tulsa, USA) at a significance level of 0.05.\n\n\nResults\n\nThe removal forces (N) expressed as a mean (± SD) for both protrusion positions (3 mm and 8 mm) at the beginning and end of the tests revealing the effect of test duration are shown in Table 1: there was a slight but not significant decrease in retentive forces for the tests on the epoxified carriers, not to be found for the ANA-4 carriers. There were no statistical differences between the different test conditions and carriers as a function of the protrusion for the BluePro®.\n\nInspection of the inner surfaces of the test samples revealed the presence of some wear particles and tear characteristics as shown in Figures 3A and 3B.\n\n(A) Almost none to (B) moderate signs of wear due to the repetitive loading, presenting itself under the form of small wear particles that are torn off the thermoplastic material as a consequence of the cyclic loading.\n\n\nDiscussion\n\nThe setup used in this study extends the experimental setup used previously8 by introducing a more standardized set of carriers with respect to tooth anatomy and shape of the tooth arcs. The results indicate that the measurements did not differ significantly between the two types of carriers, namely epoxified casts versus ANA-4 Frasaco models.\n\nIn OAm therapy for OSA it has not yet been defined to what extent variations in OAm design may affect clinical efficacy, side effects and patient compliance9. Design features may also dictate retention of the OAm during sleep. Knowledge of such retentive characteristics is therefore essential for selection of an effective OAm for clinical use. It has previously been shown that it is feasible to evaluate in vitro retentive characteristics of monobloc types of OAm8 and the results of the present study indicate that a duo-bloc OAm can also be tested in a fixed protrusive position, thereby resembling the monobloc setup. The present findings also indicate that, compared to the previously published results, the experimental OAm currently tested at 8mm protrusion showed retentive forces of ~27N as shown in Table 1. These findings further support the hypothesis8 that introducing a soft thermoplastic body inside a more rigid tray improves retention of a thermoplastic type of OAm by improving rigidity and fit of the OAm. The present conclusion also supports recent findings with another prefabricated thermoplastic OAm that is also contained in a rigid shell7.\n\nIn the present study, the retention forces measured for the thermoplastic OAm did not change significantly over time (Table 1) and running-in wear and wear due to repetitive insertion and removal did not significantly influence the results. An increased amount of protrusion also did not cause higher retention forces (Table 1), indicating that its retentive capacities are independent of mandibular protrusion for the protrusive range studied. Additional research and clinical reports are needed to further study these effects.\n\nJaw opening forces10 at an interincisal distance of about 10 mm range from 2N to 9N and increase thereafter to an average of 19.9 ± 4.5N at an interincisal distance of about 49 mm. Looking at these values from the literature, it becomes clear that the thermoplastic OAm tested in the present in vitro study possesses sufficient retention forces to resist initial jaw opening as well as full mouth opening forces.\n\nOften customizable thermoplastic OAms are less expensive than custom-made appliances in the dental technical lab. This additional element could broaden the indications for use of such devices. Since titration is now considered an inevitable prerequisite for obtaining success in OA treatment, better selection of appropriate patients for OAm therapy is essential. One approach to the selection of patients could be to provide the patient with a low-cost try-out device that is capable of reliable titration. Such a diagnostic tool would not only provide an evaluation of the effectiveness of an OAm in an at-home situation, but would also help check whether or not the patient is willing to use an OAm. It could also provide a temporary OAm while a custom-made OAm is being manufactured or repaired. Finally it could bring an OAm solution to certain resource-restricted healthcare settings in which custom-made appliances are unaffordable. Although these indications seem promising, it should be noted that some elements regarding the design of an OAm and its effects are not yet known. This includes the bulkiness of the OAm and its effect on the position of the tongue for example.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data of MRA-fatigue tests, 10.5256/f1000research.6061.d4312011\n\nFigshare: Data of retentive forces of mandibular advancement device BluePro®. 10.6084/m9.figshare.130656212", "appendix": "Competing interests\n\n\n\nThe author is the promotor of a SomnoMed Research Grant. The author has no competing interest relating to BlueSom.\n\n\nGrant information\n\nBlueSom France provided funding for this project including the supply of BluePro® devices free of charge and payment for final manuscript editing by Newmed Publishing Services.\n\n\nReferences\n\nDieltjens M, Vanderveken OM, Heyning PH, et al.: Current opinions and clinical practice in the titration of oral appliances in the treatment of sleep-disordered breathing. Sleep Med Rev. 2012; 16(2): 177–85. PubMed Abstract \| Publisher Full Text\n\nde Almeida FR: Complexity and efficacy of mandibular advancement splints: understanding their mode of action. J Clin Sleep Med. 2011; 7(5): 447–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLettieri CJ, Paolino N, Eliasson AH, et al.: Comparison of adjustable and fixed oral appliances for the treatment of obstructive sleep apnea. J Clin Sleep Med. 2011; 7(5): 439–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanderveken OM, Devolder A, Marklund M, et al.: Comparison of a custom-made and a thermoplastic oral appliance for the treatment of mild sleep apnea. Am J Respir Crit Care Med. 2008; 178(2): 197–202. PubMed Abstract \| Publisher Full Text\n\nHoffstein V: Review of oral appliances for treatment of sleep-disordered breathing. Sleep Breath. 2007; 11(1): 1–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsuda H, Almeida FR, Masumi S, et al.: Side effects of boil and bite type oral appliance therapy in sleep apnea patients. Sleep Breath. 2010; 14(3): 227–32. PubMed Abstract \| Publisher Full Text\n\nFriedman M, Hamilton C, Samuelson CG, et al.: Compliance and efficacy of titratable thermoplastic versus custom mandibular advancement devices. Otolaryngol Head Neck Surg. 2012; 147(2): 379–86. PubMed Abstract \| Publisher Full Text\n\nVanderveken OM, Van de Heyning P, Braem MJ: Retention of mandibular advancement devices in the treatment of obstructive sleep apnea: an in vitro pilot study. Sleep Breath. 2014; 18(2): 313–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan AS, Cistulli PA: Oral appliance treatment of obstructive sleep apnea: an update. Curr Opin Pulm Med. 2009; 15(6): 591–6. PubMed Abstract \| Publisher Full Text\n\nPeck CC, Sooch AS, Hannam AG: Forces resisting jaw displacement in relaxed humans: a predominantly viscous phenomenon. J Oral Rehabil. 2002; 29(2): 151–60. PubMed Abstract \| Publisher Full Text\n\nSpray I: Dataset 1 in “In vitro retention of a new thermoplastic titratable mandibular advancement device” F1000Research. 2015. Data Source\n\nSpray I: Data of retentive forces of mandibular advancement device BluePro®. Figshare. 2014. Data Source" }	[ { "id": "7860", "date": "05 Mar 2015", "name": "Timothy Quinnell", "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\nThe title accurately conveys the article’s content. The abstract is a good summary of the work being reported. The author clearly describes the application of an in vitro method for measuring the retentive forces of mandibular devices, to a new titratable thermoplastic device. This novel method was recently reported by the author and colleagues (citation 8). It seems to be a useful in vitro tool for assessing the ability of mandibular devices to resist displacement by jaw opening – an important failing of many thermoplastic devices is poor oral retention. The technique naturally has its limitations, which have been discussed in detail in citation 8. It would be worth the author briefly referring to them in this article including: lack of lubrication; linear carrier movement vs. non-linear, ‘least resistance’ pathway of jaw opening in vivo; the use of carriers from only 1 patient. It is not clear how 730 cycles represents a year’s use. If it refers to jaw opening at the end of the night to remove the device then should this not be 365? Nonetheless the data are credible and provide useful insight (alongside citation 8) into the varying in vivo retentive properties of different devices; and device durability; and further demonstrate a potentially straightforward means of in vitro OA testing. The protrusions used were clinically relevant. I agree with the author’s conclusions, that the results suggest thermoplastic OAms (with adequate retentive properties) could have a role in: titration; temporary custom made device substitution; and longer term treatment. However I disagree that titration is now a prerequisite for successful OA treatment, because there is strong evidence that non-titratable devices do work (Thorax 2014;69:10 938-945); and the superiority of titration has not been proven by rigorous head-to-head trials. Finally, device effectiveness depends as much on comfort and tolerability (influencing adherence) as on oral retention.", "responses": [] }, { "id": "7832", "date": "30 Mar 2015", "name": "Julia Cohen-Levy", "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\nThis is a well designed in-vitro study, supported with adequate experimental data and statistical analysis. Results are accurately presented and discussed, in a clear and concise manner. The conclusions are justified on the basis of the presented results.Some minor revisions could be positively added to the manuscript, in the discussion part. The flaws of the 'in-vitro' study of the retention of a thermoplastic oral device should, in my opinion, be also discussed as the human mouth motion has very little to do with the testing machine, in relation to lateral movements of the mandible. The influence of saliva, and maybe of mouth temperature might have an influence on the material properties with time.", "responses": [] } ]	1	https://f1000research.com/articles/4-56
https://f1000research.com/articles/4-54/v1	26 Feb 15	{ "type": "Case Report", "title": "Case Report: Radical prostatectomy without prostate biopsy in PI-RADS 5 lesions on 3T multi-parametric MRI of the prostate gland", "authors": [ "Andrew Keller", "Boon Kua", "Boon Kua" ], "abstract": "Objective: Current practice mandates a prostate biopsy for histological confirmation of prostate cancer prior to a radical prostatectomy. Prostate biopsy, whether performed trans-rectally or trans-perineally, is an invasive procedure which typically involves an anaesthetic and has the risks of urosepsis, bleeding and haematoma. Post-biopsy inflammatory changes can also obliterate natural tissue planes thereby potentially compromising the quality of a nerve sparing procedure and increasing positive margin rates.3T-Multi-Parametric Magnetic Resonance Imaging of the Prostate (3T mpMRI-P) is gaining increasing acceptance in the identification and localisation of prostate cancer. In experienced centres, the positive predictive value has been reported to be as high as 95%.Methods: Two patients with rising and elevated age- adjusted PSAs and palpable malignant prostate nodules on Digital Rectal Examination (DRE) underwent 3T mpMRI-P. Both patients had Prostate Imaging-Reporting and Data System (PI-RADS) 5 lesions in their peripheral zones corresponding to palpable nodules. Prostate biopsies were offered but declined by both patients. Both were satisfied that there was sufficient evidence on their PSA, DRE and 3T mpMRI-P for a diagnosis of prostate cancer without prostate biopsies and both elected to proceed to a Da Vinci Robotic Assisted Laparoscopic Radical Prostatectomy (RALRP).Results: Unilateral nerve sparing RALRPs were performed on both patients without complication. Histology demonstrated Gleason 4+4=8 and 4+3=7 prostate adenocarcinomas, with tumour volumes of 14.92cc and 4.5cc respectively.Conclusions: In appropriately counselled patients who have a high pre-test probability of prostate cancer (rising and elevated PSA, malignant nodule on DRE and a corresponding PI-RADS 5 lesion on 3T mpMRI-P), it may be appropriate to proceed to a radical prostatectomy without a tissue diagnosis if the patients have strong reservations about prostate biopsy.", "keywords": [ "Magnetic Resonance Imaging", "Prostate cancer", "biopsy", "diagnostic test accuracy" ], "content": "Introduction\n\nCurrent practice mandates a prostate biopsy for histological confirmation of prostate cancer prior to a radical prostatectomy. Prostate biopsy, whether performed trans-rectally or trans-perineally, is an invasive procedure which usually requires an anaesthetic and has the inherent risks of urosepsis, urinary retention and haematoma1,2.\n\nPost-biopsy inflammatory changes can also obliterate natural tissue planes there by potentially compromising the quality of a nerve sparing procedure and increase positive margin rates3. For this reason prostatectomy is usually delayed by at least 6 weeks to allow for a reduction in the peri-prostatic inflammatory change that follows any biopsy procedure4.\n\nThe use of 3T mpMRI-p is gaining increasing acceptance for both the diagnosis and localisation of prostate cancer. In experienced centres, the positive predictive value of Prostate Imaging-Reporting and Data System (PI-RADS) 5 lesions has a specificity of 97–100% in biopsy naïve patients5–8. We describe two case reports of radical prostatectomy without tissue diagnosis. The patients involved both had objections to confirmative biopsy, PI-RADS 5 lesions on mpMRI-p and high pre-test probabilities of prostatic malignancy. We believe this report will be of interest for urologists dealing with the dilemma of patients with a high risk of prostatic malignancy, positive mpMRI-p and patient refusal of biopsy procedures.\n\n\nCase 1\n\nA 56 year old man was referred to our service with elevated Prostate Specific Antigen (PSA) titres, which had risen progressively from 1.7 ng/mL to 4.6 ng/mL over a five year period. He had a positive family history of prostate cancer (PC), with his brother having undergone radical prostatectomy 5 years earlier. His digital rectal exam (DRE) showed a malignant nodule in his right lobe and a firm contralateral lobe.\n\nWe requested a 3-Tesla Multi-parametric Magnetic Resonance Imaging of the prostate (mpMRI-p) which identified a Prostate Imaging-Reporting and Data System (PI-RADS) 5 lesion in the right lobe of the prostate with probable extra-prostatic extension (Figure 1). A smaller lesion was present in the left mid peripheral zone (Figure 2) with possible left external iliac node involvement. Staging Computerised Tomography (CT) of the abdomen and pelvis and Tc-99m bone scan showed no radiological evidence of metastatic spread other than the previously mentioned borderline enlarged left external iliac nodes.\n\nThe results of the investigations (PSA titres, DRE) and the chance of a false positive mpMRI-p result of approximately 5% was explained to the patient. The patient was adamantly against undergoing confirmatory prostate biopsy as he was concerned about biopsy related sepsis. He also reported pre-existing anxiety regarding PC since his brother had been diagnosed and treated.\n\nThe patient subsequently underwent a bilateral incremental nerve sparing Robot Assisted Laparoscopic Radical Prostatectomy (RALRP) with left sided extended pelvic lymph node dissection.\n\nThe Operative specimen’s histology demonstrated Gleason 4+3=7 primary tumour with tertiary pattern 5 present (Figure 3) with extra-prostatic extension (EPE) present. Peri-neural invasion was present (Figure 4). A focally positive margin at region of EPE was present over a 0.5 mm base. The specimen’s histological stage was T3a (AJCC 7th Edition 2010).\n\nCribiform glands with comedonecrosis represent Gleason pattern 5.\n\nThe patient made an uneventful recovery from surgery. Post-operative incontinence was mild at 6 weeks, using a single safety pad during the day only. He is trialling sildenafil for his post-operative erectile dysfunction. His PSA at 5 weeks was low at 0.033 ng/mL. His follow-up is ongoing.\n\n\nCase 2\n\nA 64 year old with no family history of prostate cancer and an elevated serum PSA of 9 ng/mL was refereed to our service. His DRE revealed a right sided palpable prostate nodule with extension into the ipsilateral seminal vesicle.\n\nmpMRI-p was requested and confirmed a PI-RADS 5 lesion in the right base and mid-zone of the gland (Figure 5). The lesion extended across midline into the left lobe. Diffusion restriction was present on ADC map (Figure 6). Right-sided seminal vesicle invasion was also demonstrated radiologically (Figure 7).\n\nThe patient was advised to undergo confirmatory prostate biopsy but declined, being satisfied with his diagnosis based on PSA, DRE and Mp-MRI-p findings. CT and Tc-99m bone scans were negative for metastatic spread.\n\nThe patient underwent RALRP. Due to his seminal vesicle invasion a wide non-nerve sparing approach was taken on the right side, with the contralateral nerve bundle spared due to the patient being sexually active. A right sided obturator node dissection was performed concurrently.\n\nHistology demonstrated Gleason 4+4 with tertiary pattern 5 (Figure 8). Tumour volume was 14.92cc. Right neurovascular bundle invasion and bilateral seminal vesicle invasion was present (Figure 9). All resection margins were uninvolved and 0/3 resected nodes were infiltrated by malignancy. His tumours histologic stage was T3b (AJCC 7th Edition 2010).\n\nThe patient made a good recovery following his surgery. Post-operatively his early urinary incontinence was very mild, using a single safety pad per day. His erectile dysfunction is successfully managed with sildenafil.\n\nInitially, at 2 months post-operatively his PSA was undetectable, however, at 5 months it had risen to 0.03 ng/mL, and by 9 months post-operatively it had risen further to 0.14 ng/mL. He was subsequently referred to radiation oncology for salvage radiotherapy for biochemical failure.\n\n\nDiscussion\n\nBoth patients in our small case series were diagnosed and their tumours correctly localised by mpMRI-p. Whilst we strongly recommended both patients proceeded to confirmative prostate biopsies, they both declined further investigation. Both patients had high pre-test probabilities of malignancy even prior to their positive mpMRI-p findings and were satisfied that their diagnoses were correct, and had concerns about the risks of prostate biopsy. They were both informed of and willing to accept the small risk of a false positive diagnosis.\n\nIt is important to note that radical prostatectomy without tissue diagnosis is not recommended as standard practice by the authors, even with a PI-RADS 5 lesion on mp-MRI-p. The case studies presented here are exceptional cases where both patients had strong opposition to biopsy despite counselling otherwise. Whilst mpMRI-p has demonstrated great sensitivity and specificity for identification of high risk prostate cancer, there remains the possibility of over diagnosis, and consequent overtreatment if a confirmatory biopsy is not also obtained pre-operatively. With further development of this imaging technology it is plausible that in the future, high risk patients with PI-RADS 5 lesions on mpMRI-p could undergo a radical prostatectomy without the need for a prostate biopsy. While we have successfully performed two prostatectomies without pre-operative biopsy we do not advocate this as a standard approach.\n\n\nConclusion\n\nIn exceptional circumstances and with appropriately counselled patients who have a high pre-test probability of prostate cancer (rising and elevated PSA, malignant nodule on DRE and a corresponding PIRADS 5 lesion on mpMRI-P), it may be appropriate to proceed to a radical prostatectomy without a prostate biopsy. Further advancements in mpMRI-p imaging techniques may negate the need for routine biopsies in high risk lesions prior to prostatectomy, but this cannot be recommended as routine practice with currently available imaging protocols.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.", "appendix": "Author contributions\n\n\n\nAK writing and literature review. BK concept of article and proofing of article. Both authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNorberg M, Holmberg L, Haggman M, et al.: Determinants of complications after multiple transrectal core biopsies of the prostate. Eur Radiol. 1996; 6(4): 457–61. PubMed Abstract \| Publisher Full Text\n\nLange D, Zappavigna C, Hamidizadeh R, et al.: Bacterial sepsis after prostate biopsy--a new perspective. Urology. 2009; 74(6): 1200–5. PubMed Abstract \| Publisher Full Text\n\nShah JB, McKiernan JM, Elkin EP, et al.: Prostate biopsy patterns in the CaPSURE database: evolution with time and impact on outcome after prostatectomy. J Urol. 2008; 179(1): 136–40. PubMed Abstract \| Publisher Full Text\n\nMartin GL, Nunez RN, Humphreys MD, et al.: Interval from prostate biopsy to robot-assisted radical prostatectomy: effects on perioperative outcomes. BJU Int. 2009; 104(11): 1734–7. PubMed Abstract \| Publisher Full Text\n\nThompson JE, Moses D, Shnier R, et al.: Multiparametric magnetic resonance imaging guided diagnostic biopsy detects significant prostate cancer and could reduce unnecessary biopsies and over detection: a prospective study. J Urol. 2014; 192(1): 67–74. PubMed Abstract \| Publisher Full Text\n\nNi Mhurchu E, O'Kelly F, Collins CD, et al.: Pi-RADS in Practice - The Predictive Value of Pi-RADS Scoring in Targeted Prostate Biopsies for Patients with Elevated PSA, and Previous Negative Biopsies. Radiological Society of North America 2013 Scientific Assembly and Annual Meeting, December 1 - December 6, 2013, Chicago IL Accessed July 22, 2014. 2013. Reference Source\n\nGrey AD, Chana M, Popert R, et al.: Diagnostic accuracy of magnetic resonance imaging (MRI) prostate imaging reporting and data system (PI-RADS) scoring in a transperineal prostate biopsy setting. BJU Int. 2014. PubMed Abstract \| Publisher Full Text\n\nPokorny MR, de Rooij M, Duncan E, et al.: Prospective study of diagnostic accuracy comparing prostate cancer detection by transrectal ultrasound-guided biopsy versus magnetic resonance (MR) imaging with subsequent MR-guided biopsy in men without previous prostate biopsies. Eur Urol. 2014; 66(1): 22–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7952", "date": "13 Mar 2015", "name": "M. Hammad Ather", "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\nIt is an interesting series of two cases. Indeed the basic premise of the authors' for proceeding with treatment of prostate cancer due to complications related with TRUS guided biopsy. Treating prostate by radical surgery without prior biopsy confirmation is challenging the age old dictum in oncology that cancer should not be treated until histologically proven.MRI imaging of the pelvis has improved significantly with the introduction of DW and MP technology. However, confirmation and final diagnosis of cancer remains a histologist domain. I have following observations on the current submission for the authors' to commentThe reference cited 1, 2 are relatively old, better pain control and improved in the TRUS technology and understanding the potential causes of infective complications are better understood now. the insidence of complication related to biopsy has decreased considerably. Although patients have given an informed consent there is a small possibility of not detecting cancer in the final pathology. Are there any medico legal implications.", "responses": [ { "c_id": "1279", "date": "23 Mar 2015", "name": "Andrew Keller", "role": "Author Response", "response": "Dr. Ather, thanks for your comments. I agree with you that while the new imaging technologies are exciting that diagnosis should remain, for the most part histological. These two cases were the exceptions to the rule, and due to patient, rather than surgeon preference. In regards to your comments, whilst pain can certainly be controlled in the TRUS setting with use of twilight sedation, our experience is that even with more intensive regimens of antibiotics the rates of TRUS sepsis are increasing, mostly due to increased prevalence of ciprofloxacin-resistant E. Coli. Secondly, both patients did give informed consent for the prostatectomy, including very clearly being told that there was a chance of a false positive diagnosis from the MR scanning. There are numerous examples of legal action in spite of informed consent of risks and I can see certain patients suing in the event of benign pathology." } ] }, { "id": "9397", "date": "13 Jul 2015", "name": "Uri Lindner", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report the authors describe two cases of patients highly suspicious for prostate cancer based on PSA levels, physical examination and MRI who underwent surgery and the final pathology coincided with the presumed diagnosis.It is not clear what is the benefit of undergoing major surgery without confirmed diagnosis, on the contrary, today we are moving forward and advocating for tissue diagnosis prior to oncological surgery.If the MRI is highly suspicious for tumor one can advocate for MRI/US fusion biopsy and reducing the number of cores taken to a minimum.", "responses": [ { "c_id": "1461", "date": "13 Jul 2015", "name": "Andrew Keller", "role": "Author Response", "response": "Thanks for your comments Dr. Lindner. Whilst there are a few potential benefits for undertaking major surgery without first obtaining histo-pathological diagnosis (zero risk of biopsy related sepsis, no obliteration of tissue planes by post-biopsy inflammation) the authors do not argue that those benefits outweigh the risk of a false diagnosis. Indeed, the benefits of tissue confirmation of malignancy prior to a potentially morbid procedure such as prostatectomy cannot be understated. Instead, this article describes two cases where both patients had significant anxiety regarding their potential diagnosis of prostate cancer. Both patients were counselled on the potential false positive rate of mp-MRI-p and were counselled to have their clinical diagnosis confirmed via biopsy but both declined. Such was their anxiety despite reassurances from the surgeon, that both patients insisted on prostatectomy despite being warned of the risk of potentially benign operative histology. It is possible that even negative pre-operative biopsy might not have allayed their concern. It must be emphasized that the authors do not advocate prostatectomy without biopsy as standard practice. The current specificity of mp-MRI-p is insufficient for confident diagnosis without confirmatory biopsy. In these cases, however, where due to patient factors biopsy was not possible, mp-MRI-p was invaluable in correctly identifying tumour location in order to facilitate prostatectomy." } ] } ]	1	https://f1000research.com/articles/4-54
https://f1000research.com/articles/4-53/v1	26 Feb 15	{ "type": "Review", "title": "A brief review of recent Charcot-Marie-Tooth research and priorities", "authors": [ "Sean Ekins", "Nadia K. Litterman", "Renée J.G. Arnold", "Robert W. Burgess", "Joel S. Freundlich", "Steven J. Gray", "Joseph J. Higgins", "Brett Langley", "Dianna E. Willis", "Lucia Notterpek", "David Pleasure", "Michael W. Sereda", "Allison Moore", "Nadia K. Litterman", "Renée J.G. Arnold", "Robert W. Burgess", "Joel S. Freundlich", "Steven J. Gray", "Joseph J. Higgins", "Brett Langley", "Dianna E. Willis", "Lucia Notterpek", "David Pleasure", "Michael W. Sereda", "Allison Moore" ], "abstract": "This brief review of current research progress on Charcot-Marie-Tooth (CMT) disease is a summary of discussions initiated at the Hereditary Neuropathy Foundation (HNF) scientific advisory board meeting on November 7, 2014. It covers recent published and unpublished in vitro and in vivo research. We discuss recent promising preclinical work for CMT1A, the development of new biomarkers, the characterization of different animal models, and the analysis of the frequency of gene mutations in patients with CMT. We also describe how progress in related fields may benefit CMT therapeutic development, including the potential of gene therapy and stem cell research. We also discuss the potential to assess and improve the quality of life of CMT patients. This summary of CMT research identifies some of the gaps which may have an impact on upcoming clinical trials. We provide some priorities for CMT research and areas which HNF can support. The goal of this review is to inform the scientific community about ongoing research and to avoid unnecessary overlap, while also highlighting areas ripe for further investigation. The general collaborative approach we have taken may be useful for other rare neurological diseases.", "keywords": [ "Charcot Marie Tooth", "rare disease", "CMT1A", "biomarkers", "drug discovery" ], "content": "Introduction\n\nSeveral recent reviews have focused on the need for discovery of therapies for rare diseases1 as well as the importance of increased collaboration2,3. There are few approved treatments for the approximately 7000 rare diseases that affect ~6–7% of the population of the developed world1. Advances in technology are changing how rare diseases are discovered4 and deepening our understanding of them5. While suggestions on how to increase the number of drugs developed for rare conditions have been made1,6,7, it is unlikely that we are going to see a dramatic change unless there is a wholesale shift in the process of drug discovery and development, combined with increased collaboration between academics, industry, government labs and research foundations in the rare disease arena. Perhaps one of the ways this situation can best be changed is if patients and advocates work with the scientists they fund to identify the critical issues that need addressing4,8. This may enable developments that are most likely to show some direct benefit for the disease (and patients) to move research from the lab bench to the clinic.\n\nSome rare diseases have recently been in the spotlight for their ability to inspire the raising of large sums of money, which can hopefully accelerate the search for a cure. Most notable is Amyotrophic Lateral Sclerosis (ALS) which became very high profile in 2014 with the ALS ice bucket challenge9. For several years there has been a considerable discussion in this scientific community about difficulties translating the many preclinical studies for ALS from the mouse to the very costly human clinical trials10,11. It is now apparent that there are several factors that can impact the success of drug discovery even from the very earliest stages, which can in turn influence the transition of new therapeutics into the clinic. When targeting the development of new therapeutics, it is important to consider problematic issues that have prevented success in other disease areas early in the discovery progress. These broader issues can complicate the discovery of new drugs and include: target validation12, artifacts such as promiscuous compounds in small molecule screens13–15, false positives16, how liquids are moved17, leaching from plastics used in labware18, compound aggregation19, the solvent effects20, detergent effects21, data reproducibility21, data quality22,23, data access and standards24, data handling25, and biases introduced by filtering libraries26. Even the recent shift of drug discovery from pharmaceutical laboratories to academic screening centers has issues27 and major gaps have been identified28. Many rare disease foundations or researchers are not even aware of these potential complications, which may ultimately impact the outcome and potential clinical feasibility of the work they fund or pursue, respectively.\n\nWe will briefly summarize recent developments in one rare disease, Charcot-Marie-Tooth disease (CMT), and propose further, currently untapped, opportunities as well as ultimately list what we believe should be the priorities for the field. While CMT may not be representative of every rare disease, this example may inspire other groups to consider not only the research they are funding but to go beyond the current dogma and consider what type of research needs to be funded to enable compounds to come to the clinic in the short, mid and long term. Drug discovery is not an overnight process as it usually takes well over a decade to go from a discovery in the laboratory to a product that is approved for clinical use. The success transitioning from each stage is variable for drugs with orphan designation (with phase 1 and phase 2 success rates above average at 86.8 and 70%, respectively, while the phase 3 success rate at 66.9% was comparable with all indications)29. This would suggest that if we could slightly improve the success of each stage we would see far more drugs approved. We hope that by producing such a summary of recent work and priorities, there will be similar collaborative discussions between researchers and patient organizations for other rare diseases.\n\n\nDiagnosis and mutations\n\nOur particular focus is on CMT, a rare disease which has multiple genetic causes30 and is classified into nine genetic subtypes (CMT1, CMT2, CMT3, CMT4, CMT5, CMT6, CMTDI, CMTRI and CMTX). Research in many areas is redefining our view of this disease and the complexities involved. CMT affects approximately one in 2500 Americans31, who usually have distally pronounced muscle weakness, resulting in difficulty walking and later also gripping objects. Typically, CMT patients display foot deformities, decreased reflexes, and bilateral foot drop32. Recent efforts and progress on CMT emphasize that we are steadily and impressively improving our understanding of the complex underlying biology. At the time of writing, there were over 3800 papers in PubMed on this disease. There is however no treatment for any of this group of disorders encompassed by CMT for which symptoms usually present in the first two decades of life33, so this presents a huge untapped opportunity to improve the lives of the many patients with this debilitating disease.\n\nAccurate diagnosis of CMT is important if we are to identify patients for future clinical trials with treatments for the disease. Currently a tiered approach to genetic testing is used and recommended by clinicians and relies on nerve conduction velocity assessment, disease inheritance pattern and population frequency34. This approach is however laborious, costly and based on recent studies may be ripe for reappraisal. A study in Norway looked at diagnostic laboratory testing for CMT and the spectrum of gene defects in that country35. In total, 549 patients were studied. Nearly 96% of these patients had mutations in just four genes (PMP22, MPZ, GJB1 and MFN2) linked to CMT. These genetic findings are in accordance with what has also been observed in other countries. In the United Kingdom a study of 1607 patients showed mutations or rearrangements in the same four genes in over 90% of the samples36. In these two studies, patients without a mutation in these four genes were then considered for referral31,37. A more recent study in the United States30 has evaluated over 17,000 patients using a variety of gene testing methods. The scale of this study is at least 10 times larger than previous analyses and reproduced the finding that almost 95% of patients had mutations in just four genes. Specifically, it showed that 78.6% of those tested were positive for copy number variations of PMP22. The genes GJB1, MPZ and MFN2 were present in 6.7%, 5.3% and 4.3%, respectively. These combined studies point to an opportunity for changing the algorithm for CMT diagnosis with initial focus on testing just these 4 genes, and patients that are negative for these can then be evaluated further with nerve conduction velocity testing. Early diagnosis has important benefits as the downstream costs of not treating CMT are considerable, and it can prevent unknowingly exacerbating the disorder by avoiding drugs that are contraindicated.\n\nNew CMT mutations are also continually being sought which may help with diagnosis and biological understanding of the disease. This is particularly important in CMT2, in which, unlike CMT1A, the most common mutations (MFN2 and GDAP1) account for only 25% of the total. Next generation sequencing (NGS) was used to screen CMT2 genes in 15 patients in whom MFN2 and GDAP1 mutations were excluded38. A new mutation in HSPB1 was identified, a c.404C>A transversion resulted in p.(Ser135Tyr) amino acid change. A previous p.(Ser135Phe) HSPB1 mutant was found to impact cell viability and neurofilament assembly in cultured cell experiments38. It is highly likely that the newly discovered mutation has a similar if not identical role. It was suggested that NGS is a tool for efficient mutation detection and exclusion in CMT238.\n\nThe role of aminoacyl-tRNA synthetases (ARS) has recently been reviewed as housekeeping enzymes39. That is to say ARS have a key role in ensuring accurate transfer of information in the genetic code. Mutations in GARS (glycyl-tRNA synthetase gene), KARS (lysyl-tRNA synthetase gene), AARS (alanyl-tRNA synthetase gene) and YARS (tyrosyl-tRNA synthetase gene), all are definitively associated in causing axonal CMT40–43. Furthermore, evidence supports the additional association of lysy’-tRNA synthetase, methionyl-tRNA synthetase and histidyl-tRNA synthetase in peripheral axon degeneration44,45. For example CMT2D is an axonal neuropathy characterized by a phenotype that is more severe in the upper extremities. Mutations in the gene encoding GARS have been implicated in CMT2D. Mutations in GARS show loss-of-function features46,47, suggesting that tRNA-charging deficits play a role in disease pathogenesis, but animal studies support a gain of function mechanism47,48. Yet the linkage between mutations and subsequent pathology is unclear. It is difficult to reconcile the mutations in these enzymes with CMT, and numerous possible etiologic mechanisms from loss-of-function to gain-of-function including many that may be outside their usual role49. The latter may be outside their usual role. Yao and Fox39 also describe the role of the ARS proteins in other organisms such as yeast and bacteria as well as drug targets (e.g. antitumor). This should be considered in light of possible complications one sees with other chemotherapeutic agents precipitating CMT. This would be a very important consideration if ARS were to be targeted for modulating various diseases and hints at the potential of how biological discoveries in one area like a rare disease can shed light on many other diseases at little extra cost.\n\n\nBasic research: recent Charcot-Marie-Tooth preclinical research\n\nThe progress of CMT has been described in several steps recently50. Genetic defects in myelinating Schwann cells (e.g. PMP22 duplication, CMT1A30,51 or overexpression leads to missorting or the accumulation of these mutated/overexpressed proteins. In addition to subsequent demyelination, the malfunctioning Schwann cells then fail to sustain axonal support which results in progressive axonal and neuronal loss. The clinical phenotype of CMT is ultimately determined by the resulting neurogenic muscle atrophy.\n\nSeveral recent papers provide some encouraging news in the quest for treatments for CMT1A, which is a primary dysmyelinating disease, and have identified compounds that have reached preclinical or clinical stages (Table 1). In addition to these, one study described how the CMT1A rat model (in early postnatal development) could be treated with a recombinant human growth factor called neuregulin-1 which controls myelin thickness52. In CMT rats this appears to enhance the reduced signaling of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)–v-Akt murine thymoma viral oncogene homolog 1 (Akt) and lower augmented mitogen-activated protein kinase kinase 1 (Mek)-mitogen –activated protein kinase (Erk), and is able to improve the differentiation of Schwann cells in CMT1A. Thus, neuregulin-1 can counter the effect of PMP22 overexpression on downstream signaling. Neuregulin-1 was found to be less effective in treating older animals. This approach may be useful in children where it could be applied before disease onset. This work opens the door for using compounds that modify signaling pathways and the kinases involved.\n\nA second recent paper used genome editing to create an assay for high throughput screening to expand the targets for drug discovery in CMT1A53. The main result of this work was the identification of the protein kinase C modulator bryostatin (Table 1) which lowers PMP22 expression. Interestingly this compound was not identified in previous screens by the group which had delivered the proteasome inhibitors such as bortezomib54 (Table 1). In summary, two independent groups have now focused on the role of kinases in pathways that control PMP22. This work may foster a broader consideration of the many compounds already available that modulate different kinases.\n\n(All structures were extracted from ChemSpider (www.chemspider.com)).\n\nAlternative approaches in ameliorating the disease phenotype in CMT1A have focused on protein quality control mechanisms, specifically autophagy and chaperones. Rapamycin, a calorie restriction mimetic was shown to improve myelination in neuron-Schwann cell explant cultures from neuropathic mice, however this drug did not improve neuromuscular performance in mice in vivo55. The differential response of skeletal muscle and peripheral nerve tissue to rapamycin is hypothesized to be responsible for the lack of functional improvement in neuropathic mice. Additional efforts in identifying therapeutic target pathways for CMT1A neuropathies involve studies on the normal function of PMP22 in myelin. A novel role for PMP22 has been found in the linkage of the actin cytoskeleton with the plasma membrane, possibly through regulating the cholesterol content of lipid rafts56.\n\nOther targets of interest for CMT include histone deacetylase 6 (HDAC6)57 as the inhibition of this can promote survival and regeneration of neurons. It was more recently shown that an increase of α-tubulin acetylation induced by inhibition of HDAC6 corrected axonal transport defects caused by HSPB1 mutations, rescued the CMT phenotype of mutant HSPB1 mice58.\n\nOne of the ways that some CMT patients first become aware of their disease is when they are given a drug treatment for another disease. This is termed chemotherapy-induced neurotoxicity. Drugs such as paclitaxel and the vinca alkaloids that are widely used in cancer treatment cause severe peripheral neuropathy and in some patients, this exacerbates CMT, revealing it perhaps for the first time. There have been recent developments in this area that may protect CMT patients in the future. A synthetic antioxidant called ethoxyquin59, which was approved by the US Food and Drug Administration (FDA) 50 years ago, appears to protect against neurotoxicity in both cell and animal studies. This treatment does not appear to impact chemotherapy and also modulated a protein called heat shock protein 90 (HSP90). This work paves the way for further studies of the neuroprotective ability of this and other compounds and possibly clinical trials for patients with pre-existing CMT that need to undergo chemotherapy and for which there are few options. At the same time it may help reveal the mechanisms of neurotoxicity resulting from these chemotherapeutics. A recent study described how inducible HSP70 prevented aggregation and enhanced the processing of PMP22. The authors also proposed the further study of HSP90 inhibitors in models of PMP22-linked neuropathies60.\n\n\nBasic research: new animal models\n\nThe creation of mouse models for CMT can be used to understand the mechanisms of the various types of this disease and for drug discovery61. Three mouse models of CMT1X, CMT1B and CMT1A were recently developed to study Schwann cells and show that they display a heterogeneous pattern of developmentally regulated molecules62. These could be useful for diagnostic purposes. They also described an inflammatory reaction as a common disease modulator in the mouse and possibly humans with CMT1. LRSAM1 is a E3 ubiquitin ligase and mutations in LRSAM1 have recently been shown to cause CMT. Mouse mutations in Lrsam1 were created for a form of CMT (CMT2P) which had only a very mild neuropathy phenotype with age but was more sensitive to the neurotoxin acrylamide, causing axon degeneration63. LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi, but its cellular function is not yet understood. However, the phenotype of the Lrsam1 mutant mice is so mild that it is of questionable utility as a disease model, and similar findings in genes such as Hint1 highlight the challenges of creating valid mouse models of axonal neuropathies64.\n\nWhile we frequently see groups use mice and rat as animal models of diseases, sometimes they are unsuitable for knocking out genes as they can result in embryonic lethality. The zebrafish has been described as a model of CMT2A which affects the distal axons of motor and to a lesser extent, sensory neurons in humans65. The study assessed the phenotypic effects of mitofusin 2 (MFN2) mutation in zebrafish. The Mfn2 mutant zebrafish do not develop abnormalities until later stages. Previously it had been shown by others that knocking out the MFN2 gene in mice resulted in embryonic lethality. Zebrafish created with this mutation in MFN2 developed normally, however they showed a progressive motor dysfunction as the fish were unable to swim correctly between 100 and 200 days65. Some patients with mutation of the MFN2 gene also show this progressive motor dysfunction. Fishes were monitored in the study65 by video in their aquarium and those swimming at an angle of more than 30 degrees to the horizontal were specifically recorded65. In addition to these studies, in vitro cell culture was utilized to measure the mitochondrial transport in the neurons from the MFN2 knock out zebrafish and it was found that retrograde transport was decreased. Obviously, while humans and zebrafish appear dissimilar, their MFN2 proteins are very similar. This study therefore presents a potentially very useful animal model of CMT2A that can then be explored to test potential future drugs for their effect on mitochondrial dynamics and axonal transport in order to see if they can ameliorate the phenotype (swimming at an angle of more than 30 degrees) observed.\n\nWhen we think of the different animal models for diseases like CMT, the fruit fly (Drosophila melanogaster) is likely the furthest from our mind. Yet the fruit fly has quite possibly overturned our basic understanding of the disease mechanism behind CMT2B, by becoming the first animal model for this disease. CMT2B was long thought to be a “gain of function” disease. A recent study however showed that neurons lacking a gene for rab7 result in neuropathy, while addition of Rab7 proteins could restore function66. Fundamental insights like this can affect how we approach a treatment or cure for CMT2B. Perhaps experimental methods to increase Rab7 protein levels, while opposite to the previous dogma, may actually be correct. There are still multiple hurdles to overcome because the fruit fly may not be a perfect disease model for human CMT2B. A second paper also suggests that the human Rab7 mutation mimics CMT2B in the fly67. In this study, a second fly model was developed and demonstrated attributes of the human disease. Flies have also been successfully used to model neuropathy associated with tRNA synthetases and to identify genetic modifiers of these diseases68,69. This model could be useful for screening compounds as potential therapies and understanding of the mechanism of the disease. This also represents another case of flies being useful of humans. The increasing utilization of different animal models of various CMTs suggests that their role will likely increase in importance and may lead to useful insights and help identify therapeutics.\n\n\nBasic research: gene therapy and stem cells\n\nThere have been relatively few studies assessing gene therapy for CMT. One group recently used neurotrophin-3 (NT-3) gene therapy via adeno-associated virus (AAV) delivery to muscle70. In the Trembler-J model of demyelinating CMT, this gene therapy led to measurable NT-3 secretion and improved motor function, histopathology, and electrophysiology of peripheral nerves.\n\nOther hereditary neuropathies have also been the subject of gene therapy. For example Giant axonal neuropathy (GAN) is caused by loss of function of the gigaxonin protein. In cells this is seen as intermediate filament (IF) aggregation, and leads to a progressive and fatal peripheral neuropathy. GAN mice received an intracisternal injection of an AAV9/GAN vector to globally deliver the GAN gene to the brainstem and spinal cord. These mice showed clearance of peripherin IF accumulations suggesting that gigaxonin gene transfer can reverse the pathology71. Other dominant CMTs may be best approached with allele-specific knockdown methods to try to eliminate the expression of the mutant mRNA. Piloting such approaches in CMT represents another way in which CMT translational research could have a broader impact for other rare diseases.\n\nInduced pluripotent stem cells (iPSCs), which offer an unlimited supply of cells derived from adult patient cells, offer a unique opportunity for human disease modeling and investigation. After reprogramming, iPSCs can be differentiated into many different cell types including neurons and glia, which has led to important findings for understanding disease mechanisms and therapeutic approaches72–75. For example, the derivation of iPSCs from three GAN patients with different GAN mutations was recently reported and key pathological phenotypes observed. These cells were used to support the feasibility of gene replacement therapy76. iPSC-derived motor neurons from axonal CMT patients identified common pathophysiological mechanisms in axonal CMT2A and CMT2E77. Differentiation of CMT iPSCs into Schwann cells, which are most affected cell type in CMT, will be an important next step. Despite progress recapitulating Schwann cell developmental stages, improvement of differentiation protocols is still required to achieve mature, functional Schwann cells with high efficiency78–80.\n\n\nBasic research: improving collaboration and using computational approaches\n\nWe have previously described how research collaborations may enhance drug discovery and how computational approaches can be used to facilitate secure sharing of data between collaborators81,82. The NIH Blueprint Neurotherapeutics Network83 is one example which provides support for small molecule drug discovery and development, access to NIH-funded contract research organizations (CROs), and access to consultants with expertise in various aspects of drug discovery and development. At the center of this is secure collaborative software so that molecules and screening data with intellectual property IP are securely shared between the groups of collaborators84. With the various screening efforts undertaken for CMT1A to date53,54 perhaps it is also worth considering using computational approaches to help identify additional compounds to test using machine learning models built with the data85. Also considering some of the “molecule-centric” issues in drug discovery, the potential compound libraries to be tested could be filtered before HTS for potential PAINS13, false positives, aggregators, etc. As there has been considerable investment in developing chemical probes from various screens of the NIH MLSMR library, assessment by an experienced medicinal chemist has created a score which has been used to derive a computational model86. This could also be used to provide some idea that the molecule may also be reasonable to a medicinal chemist’s perspective.\n\n\nClinical research: CMT biomarkers and natural history of disease\n\nNew clinical biomarkers are needed for CMT. Recently a novel magnetization transfer ratio (MTR) MRI assay of the proximal sciatic nerve (SN) was developed as a potential biomarker of myelin content changes in patients with CMT diseases87. The relationship between MTR and clinical neuropathy scores was assessed. Mean volumetric MTR values were significantly decreased in the SN of patients with CMT1A and CMT2A relative to controls. Skin derived mRNA expression also holds promise to serve as biomarkers in CMT1A patients61.\n\nThe international Inherited Neuropathy Consortium (INC) recently analyzed clinical and genetic data from 1652 patients evaluated at 13 INC centers88. The disease burden of all the mutations was assessed by the CMT Neuropathy Score (CMTNS) and CMT Examination Score (CMTES). Five subtypes of CMT (CMT1A/PMP22 duplication, CMT1X/GJB1 mutation, CMT2A/MFN2 mutation, CMT1B/MPZ mutation, and hereditary neuropathy with liability to pressure palsy/PMP22 deletion) accounted for 89.2% of all genetically confirmed mutations. The study also confirmed that patients could be uniformly assessed between international centers and provides a baseline for future clinical studies.\n\n\nClinical research: clinical trials and outcomes research\n\nWe have recently described some of the compounds for CMT in preclinical52,89 or clinical trials. Two recent publications from Pharnext described the novel combination of three drugs called PXT-3003 and their effect on CMT1A both in the lab90 and in a phase 2 clinical trial91. Initially three drugs approved for other uses (baclofen, naltrexone and sorbitol) were tested separately and were shown to work better at increasing myelination in Schwann cells when combined together in the test tube (at concentrations far lower than their approved doses). The combination of the three drugs was shown to lower Pmp22 expression. In the rat model of CMT1A different measures of effectiveness suggested that PXT-3003 was also promising and likely efficacious. The very low doses of all three components would also negate any adverse side effects. The clinical trial used three dose levels of PXT-3003 in 80 adults with mild to moderate CMT1A. This trial confirmed the safety of the combination drug and the best improvement was seen at the highest dose. No adverse events were observed. Efficacy was also assessed using the CMTNS and Overall Neuropathy Limitations Scale (ONLS). While a relatively modest but statistically significant improvement was observed, this represents the most promising potential treatment to date surpassing the various clinical trials of ascorbic acid (vitamin C), which showed less change from baseline than high dose PXT-3003. There are still many gaps in understanding the mechanism of how PXT-3003 actually exerts its effect. It is hoped that looking at patients over a longer period and possibly treating them earlier before they become clinically affected by the disease, may improve their nerve conduction. PXT-3003 represents the most promising therapeutic for the disease in years but there is still a long way to go (several years at least as PXT-3003 will enter phase 3 clinical trials in 2015) before it may be more widely available as an FDA or European Medicines Agency (EMEA) approved treatment for CMT1A.\n\nWhat do we need for this and other clinical trials to be successful? The dependence on relatively crude outcome measures like the six minute walking test etc. which are still relied on need updating with technologies which track the patient continually92,93, rather than during a visit to the neurologist. New measures or biomarkers could perhaps be developed and applied in clinical trials for CMT94. In addition, more subtle analysis based on surveying the patient and the caregiver may also be instructive.\n\nFor example, if we are to learn more about CMT and the effectiveness of rehabilitation it is worth involving the patient and their caregiver. A recent Italian study described a survey of CMT patients and caregivers and their perspectives and perceptions of rehabilitation efficacy and needs95. This cross-sectional study used several standard questionnaires to survey 123 patients enrolled through clinical and genetic testing. Not surprisingly, the results suggested that patients believe it is important to feel better after physical therapy. There was also a discrepancy between the perception of benefit from rehabilitation for the patient, and the caregiver’s perception of benefit. Such questionnaires might be a useful addition to clinical trials to justify a treatment approval.\n\nPatient reported outcomes are instrumental in getting drugs approved and we are seeing an increase in drugs approved just on quality of life (QoL)96,97. One hundred and eighty-nine CMT1A patients were recently reported to have a QoL that was significantly worse versus the standard population, albeit slightly better than patients with multiple sclerosis98. In this study women had earlier CMT1A onset, higher deterioration of the QoL and when assessed by the ONLS, higher disability of the upper limb98. A recent study of pain in 176 children with CMT used QoL outcomes and other clinical assessments99. It was shown that increased pain correlated with deteriorating QoL scores but not with more severe neuropathy. What could we do to improve QoL of patients with CMT? Clearly, in children steps to alleviate pain would be an improvement.\n\n\nConclusion\n\nWe have briefly summarized some of the developments in CMT research of the last few years. This follows on from a meeting organized by HNF to seek different perspectives which could help us understand what areas may need support and further research. We have also attempted to prioritize areas of CMT research based on the need to bring treatments to market for the waiting patients (Table 2).\n\nCurrently, CMT1A is the only type of CMT for which there is a therapeutic in clinical trials90,91. We need to ensure that this compound is successful because it will benefit the patient and CMT focused scientific community. In order to do this we may want to identify, develop and validate more robust outcome measures for CMT as described earlier. In addition to show the value of treatments and impact on the QoL of patients for insurance companies who would reimburse treatment we need more studies that look into this. Based on our priorities of helping to translate treatments to the clinic and help patients, we perhaps could focus on the development of new clinical endpoints and any efforts to improve and quantify the impact of CMT on patient QoL. If we are to impact patients with CMT it is important to diagnose their disease early and to do this accurately.\n\nHigh to medium level priorities include efforts to find new treatments for other forms of CMT. This may require more collaborations to identify molecules for progression. Encouraging companies to collaborate is also in the best interests of the field but not without its complexities. We can be prepared for this by using software that enables secure collaboration between different parties earlier.\n\nCertainly we still have only a partial understanding of CMT and the search for new mechanisms and targets would be important perhaps for future breakthroughs in treatment. Another important priority is to include recruiting more patients for future clinical trials as we may be underestimating the number of patients with CMT. Registries such as GRIN8 may help in this regard to both identify patients and understand their disease and treatment needs.\n\nIf we are to encourage more research on CMT, animal and cell models, chemicals and reagents need to be available readily and if possible centralized in a repository. Several animal and stem cell models have recently been developed, and it will be important to provide a repository so that other researchers can access them. Knowing which animal and cell models are valid and which are not could help to prevent costly clinical trials (as described for the ALS example earlier). While CMT is not a wide-spread disease and the funds available are limited, we have still to consider the development of such a repository for scientists because it would facilitate compound testing, and such repositories have been successfully developed for other diseases. As compounds like PXT-3003 progress through clinical trials and hopefully show an impact on the disease, other companies will likely show an interest in CMT. These companies will want to screen their libraries in cell and animal models. As we have seen in recent years, preclinical research is happening through collaboration with academic screening centers100. It will be important therefore that as a community we fully characterize these systems and have them available for testing.\n\nWe should also be open to considering biotherapeutic approaches which could complement small molecule drugs. It would appear that there has been relatively little research in gene therapy for CMT. We could certainly leverage the considerable developments that have been occurring for other diseases in this regard. As a small rare disease foundation we have to consider carefully where we put our resources.\n\nClearly there are many other fascinating areas of research that are ongoing or may be required. The clinical trials for CMT recently undertaken are using relatively crude measures for determining efficacy. Longer-term assessments of patients using proteomic and metabolomic approaches may help to identify new biomarkers for CMT. In addition, the development of new scores for the disease101 as well as standardizing them across institutions is important. Developing and accessing approaches that offer a real-time readout on the disease in patients would also be valuable for future clinical trials.\n\nFrom the diagnostic side it would appear that assessing just four genes including PMP22 duplication/deletions, GJB1, MPZ, and MFN, would capture most of the patients with a CMT phenotype. If we are to simplify diagnosis and perhaps reduce costs, a more simplistic algorithm is needed. What other factors may be important for CMT that could modify the disease or the QoL? For example a recent study of meal frequency from studies of animal and human subjects suggests that intermittent energy restriction can improve health indicators and counteract disease processes102. These changes in meal frequency could have an impact on overall health. Should we be studying the effect of this intermittent energy restriction on specific diseases like CMT also?\n\nIn summary, we have drawn attention to some of the most recent advances in CMT research and made suggestions of where funding bodies such as HNF could invest to have maximum short term impact (e.g. ensuring a treatment for CMT1A is approved quickly), as well as long term impact (e.g. prioritizing compounds for other forms of CMT) (Table 2). Our hope is that once a treatment for CMT1A is approved more drug companies will be interested in CMT, investment in research will increase and therefore we have to be prepared for that and the downstream implications on resources, research materials, researchers and ultimately patients themselves.", "appendix": "Author contributions\n\n\n\nSE completed the first draft and coordinated the manuscript preparation. All authors contributed to the writing of the final manuscript.\n\n\nCompeting interests\n\n\n\nSE is a consultant for CDD and employee of HNF, NL is an employee of CDD.\n\nJJH is an employee of Quest Diagnostics.\n\nHNF receives funding from Pharnext to support CMT education and awareness.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThis meeting was funded by the Hereditary Neuropathy Foundation.\n\nSE kindly acknowledges discussions with Teri Rosenbaum-Chou and Blake Gudgel (Modus Health), Richard Harrison and Jennifer Berbaum (Thomson Reuters) as well as the considerable input and suggestions of Dr. Jun Li (Vanderbilt University).\n\n\nReferences\n\nSwinney DC, Xia S: The discovery of medicines for rare diseases. Future Med Chem. 2014; 6(9): 987–1002. PubMed Abstract \| Publisher Full Text\n\nGroft SC: Rare diseases research: expanding collaborative translational research opportunities. 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Reference Source" }	[ { "id": "7842", "date": "17 Mar 2015", "name": "Rhona Mirsky", "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\nThis report reviews recent research on Charcot-Marie-Tooth neuropathies. The report is the result of a recent meeting of the scientific advisory board of the Hereditary Neuropathy Foundation and summarises suggestions made there. The article includes suggestions for improvements to diagnosis, including stratified testing for the most common mutations before screening for the rarer ones, and discussion of new and old animal models that have been developed to study and understand disease mechanisms and for the exploration of possible therapeutic strategies. It reviews other avenues of active research, including selective use of adenoviral vectors and stem cells that are being investigated for possible use in gene therapy for some forms of the disease, for understanding disease mechanisms, and for screening programmes for therapeutic drugs. It highlights the possibility that chemotherapy-induced toxicity may exacerbate some forms of CMT. It suggests that there is room for improved collaboration between different research groups and clinicians and more use of computational approaches. To my knowledge there are already several consortia in addition to the Hereditary Neuropathy Foundation that are already very active in trying to advance therapeutic strategies so I am not sure how much of a deficit there is in this area. On the clinical side a need for reliable biomarkers of the various disease forms is highlighted and the review ends with a discussion of clinical trials and the importance of involving patients and caregivers in the trial outcome measures is emphasised.Overall the article provides an up to date review of the research and possible treatments for this group of relatively rare diseases which will be of use to both researchers and clinicians in the field.", "responses": [] }, { "id": "7845", "date": "01 Apr 2015", "name": "Gerard Said", "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\nThe authors discuss the many issues that can complicate the discovery of new drugs.The authors summarize recent developments in Charcot-Marie-Tooth disease which may be used as an example of what can or should be done to improve development of drugs in rare disease.The vast majority of CMT patients have copy number variations of PMP22. The authors propose a method destined to accelerate the identification of the different mutations associated with the CMT phenotype.The authors briefly review the treatment used in CMT1A rat models and consider their application to humans.Recently association of small doses of baclofen, naltrexone and sorbitol (PXT-3003) seemed promising and well tolerated. Phase 3 study is going to start in 2015, and it will take several years before a significant effect of the medication on CMT1A can be confirmed and the drug approved for treatment.The different methods of evaluation of the benefit of treatment are discussed. In addition, it must be kept in mind that CMT1A is a very slowly progressive disease, with phenotypic variations especially in presentation and course. A large number of patients carrying the same mutation thus need to be included in the studies and followed over many years. It will not be an easy task in a rare disease.This paper is useful in delineating the different steps that can lead to discovery of new drug in rare diseases, emphasizing the need for collaboration between geneticists, biochemists, drug companies and clinicians", "responses": [] } ]	1	https://f1000research.com/articles/4-53
https://f1000research.com/articles/3-315/v1	23 Dec 14	{ "type": "Case Report", "title": "Case Report: Gollop-Wolfgang Complex in a 5 month old baby", "authors": [ "Ihtesham A. Qureshi", "Rohit Kumar Gudepu", "Ravikanth Chava", "Sravya Emmani", "Syed Husain Asghar", "Mohtashim A. Qureshi", "Nimmathota Arlappa", "Ihtesham A. Qureshi", "Rohit Kumar Gudepu", "Ravikanth Chava", "Sravya Emmani", "Syed Husain Asghar", "Mohtashim A. Qureshi" ], "abstract": "Skeletal dysplasias are disorders associated with a generalized abnormality in the skeleton. The Gollop-Wolfgang complex (GWC) is a limb deficiency disorder and an unusual limb malformation with highly variable manifestations. Here we report the interesting case of a 5-month old male baby from India with Gollop-Wolfgang Complex showing bifurcation of the right femur, ectrodactyly of both feet, ectrodactyly of left hand, syndactyly of right hand and unusual presentation of bilateral fibular agenesis and caudal (Sacrococcygeal) agenesis. The etiology of GWC in this 5 month old male baby could possibly be attributed to spontaneous gene mutation due to consanguineous marriage of his parents. The clinical, radiographic findings and the unusual presentation are presented in detail.", "keywords": [ "Skeletal dysplasias", "Gollop-Wolfgang complex", "limb deficiency" ], "content": "Introduction\n\nGeneralized disorders of cartilage and bone have been referred to as skeletal dysplasias and are associated with a generalized abnormality in the skeleton1. Gollop-Wolfgang Complex (GWC) is a rare congenital limb anomaly characterized by tibial aplasia, ipsilateral bifurcation of the thighbone and ectrodactyly2. Ectrodactyly involves the deficiency or absence of one or more central digits of the hand or foot and is also known as split hand/split foot malformation (SHFM)3. Very often, the anomalies of limbs, heart, digestive and urinary tracts and the lumbosacral vertebrae are also affected4.\n\nIn 1980, Gollop et al. described the case two brothers with ectrodactyly and unilateral bifurcation of the femur, absence of both tibiae and monodactyly of the feet5. In 1984, Wolfgang reported a case of right femoral bifurcation and absence of tibia and bilateral central defects of the hand5. Lurie and Ilyina (1986) proposed the eponym GWC for the combination of femoral bifurcation with hand ectrodactyly6. Endo et al. found a total of 12 reported cases and added the case of a Japanese girl with a unique form of this malformation complex. Both hands and feet were involved and the involvement was bilateral2. The etiology of GWC is most likely an error in the complex genetic control of limb development but the exact cause is still unclear7. GWC is listed as a “rare disease” by the United States Office of Rare Diseases [ORD] of the National Institute of Health [NIH] and the approximate incidence is 1 in 1000,0008.\n\n\nCase presentation\n\nA 5-month old male Indian child with normal karyotype (46 XY) born to a 26-year-old primigravida, full term by C-section, presented with limb deformities associated with bilateral ectrodactyly of feet (Figure 1 and Figure 2), syndactyly of right hand (Figure 3) and ectrodactyly of left hand (Figure 4). At the medial distal third of the right femur, a large protrusion was present (Figure 1 and Figure 5). Radiographic images showed bifid femur with fibular agenesis (Figure 6), absence of right 3, 4, 5 metatarsals and phalanges, absence of left 4, 5 metatarsals and phalanges of foot (Figure 7), left lateral X-ray showing caudal (sacrococcygeal) agenesis (Figure 8). Initial diagnosis was made when the parents brought the child to the out-patient department concerned about limb abnormality at the age of 3 months and the final diagnosis was made following admission to the in-patient unit at 5 months, based on both clinical presentation and radiological images. There was no details prenatal history available.\n\nThe parents had documented second degree consanguinity but both did not have any significant family history. Similarly, there was no history of exposure to radiation, prenatal teratogenic medications and infections during pregnancy. The mother did not smoke or drink during pregnancy. The child was breast-fed with good appetite and cry, without any bowel bladder problems, change in skin color or any cleft lip/palate. Echocardiography at the time of admittance revealed no congenital heart defects. The ultrasonography of abdomen and pelvis revealed no visceral or renal abnormalities. Surgical reconstruction treatment was advised but the parents did not give any consent for treatment.\n\n\nDiscussion\n\nIn this presenting case, the parents of the child affected by GWC have a strong documented consanguinity. To the author’s knowledge, the only previously reported case of an Arab Muslim couple who came from a region where other consanguineous families with similarly affected individuals had been reported Kohn et al. in 19899, and the autosomal recessive inheritance seemed evident in the case of a child described by Raas-Rothschild et al. in 199910. In this case report, we report an atypical presentation of GWC with bilateral fibular agenesis and sacrococcygeal agenesis along with pathognomonic features of GWC (bifurcation of femur, syndactyly and ectrodactyly). There were no additional associated abnormalities like cleft lip/palate, tibial agenesis, visceral or cardiac anomalies seen in this patient. In the literature, there is a case reported with distal femoral duplication with fibular agenesis11. The best treatment option for patients with Gollop-Wolfgang syndrome is early knee disarticulation and resection of the protruded bifurcated femur, followed by fitting of a modern prosthesis12. This treatment was discussed with the parents of the patient at 3 months and a follow-up visit was scheduled after 2 months.\n\nThis type of skeletal dysplasia with limb deficiencies could be the result of spontaneous gene mutations and chronic exposure to a toxic substance or infectious agents that results in the disruption of normal skeletal development. History of consanguinity is strongly associated with the developments of congenital anomalies among the newborn babies; there should be pre-marital counseling to discourage and/or to avoid consanguineous marriages and prospective genetic counseling to couple married within the blood relation to prevent conception. Similarly, prenatal diagnosis is one of the appropriate preventive measures for early detection of genetic and fetal anomalies through proper antenatal screening.\n\n\nConsent\n\nInformed written consent for publication of images and clinical details was obtained from the patient’s parents.", "appendix": "Author contributions\n\n\n\nNA, IQ, MQ, RG have performed literature review and manuscript writing. RC helped to make the diagnosis. SA, SE helped in revision of the manuscript. All the authors approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements:\n\nThe authors wish to thank Dr. M. Bharti, Professor at the Department of Radio-diagnosis, Navodaya Medical College, India for providing data.\n\n\nReferences\n\nKrakow D, Rimoin DL: The skeletal dysplasias. Genet Med. 2010; 12(6): 327–341. PubMed Abstract \| Publisher Full Text\n\nBos CF, Taminiau AH: A 5-year follow-up study after knee disarticulation in two cases of Gollop-Wolfgang complex. J Pediatr Orthop B. 2007; 16(6): 409–413. PubMed Abstract \| Publisher Full Text\n\nMoerman P, Fryns JP: Ectodermal dysplasia, Rapp-Hodgkin type in a mother and severe ectrodactyly-ectodermal dysplasia-clefting syndrome (EEC) in her child. Am J Med Genet. 1996; 63(3): 479–81. PubMed Abstract \| Publisher Full Text\n\nErickson RP: Agenesis of tibia with bifid femur, congenital heart disease, and cleft lip with cleft palate or tracheoesophageal fistula: possible variants of Gollop-Wolfgang complex. Am J Med Genet A. 2005; 134(3): 315–317. PubMed Abstract \| Publisher Full Text\n\nWolfgang GL: Complex congenital anomalies of the lower extremities: femoral bifurcation, tibial hemimelia and diastasis of the ankle. Case report and review of the literature. J Bone Joint Surg. 1984; 66(3): 453–458. PubMed Abstract\n\nLurie IW, Ilyina HG: Gollop-Wolfgang Complex in a 3-month-old girl. Am J Med Genetic. 1986; 25: 191–194. PubMed Abstract \| Publisher Full Text\n\nEndo A, Watanabe K, Shimada M, et al.: Bilateral involvement of hands and legs in the Gollop-Wolfgang complex. Am J Med Genet. 1998; 80(5): 529–530. PubMed Abstract \| Publisher Full Text\n\nhttp://rarediseases.info.nih.gov/gard/browse-by-first-letter/G.\n\nKohn G, el Shawwa R, Grunebaum M: Aplasia of the tibia with bifurcation of the femur and ectrodactyly: evidence for an autosomal recessive type. Am J Med Genetics. 1989; 33(2): 172–175. PubMed Abstract \| Publisher Full Text\n\nRaas-Rothschild A, Nir A, Ergaz Z, et al.: Agenesis of tibia with ectrodactyly/Gollop-Wolfgang complex associated with congenital heart malformations and additional skeletal abnormalities. Am J Med Genet. 1999; 84(4): 361–364. PubMed Abstract \| Publisher Full Text\n\nCakir M, Hoefsloot LH, Orhan F, et al.: Distal femoral duplication and fibular agenesis associated with congenital cardiac defect. Indian J Pediatr. 2010; 77(2): 210–211. PubMed Abstract \| Publisher Full Text\n\nWada A, Nakamura T, Fujii T, et al.: Limb salvage treatment for Gollop-Wolfgang complex (femoral bifurcation, complete tibial hemimelia, and hand ectrodactyly). J Pediatr Orthop B. 2013; 22(5): 457–463. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7449", "date": "27 Jan 2015", "name": "Stephen Robertson", "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 report describes a child with a typical presentation of Wolfgang-Gollop complex. The eitiology of this condition is still in doubt although some familial recurrences suggest a genetic cause. The statement in the abstract that a spontaneous mutation is possible owing to parental consanguinity does not make sense and should be deleted. In this discussion there is also a suggestion that consanguineous unions should somehow be discouraged. This is inappropriate and a breach of reproductive autonomy. Such a statement should be removed. Consanguinity is not the strong risk factor for congenital anomalies as the authors imply, conferring approximately a 2 fold enhanced risk of such problems. This assertion needs to be rebalanced.", "responses": [ { "c_id": "1204", "date": "09 Feb 2015", "name": "aatif qureshi", "role": "Author Response", "response": "Really appreciate your comments Dr. Stephen Robertson. I would like to hear back about my newer version of the paper." } ] } ]	1	https://f1000research.com/articles/3-315
https://f1000research.com/articles/4-49/v1	20 Feb 15	{ "type": "Research Article", "title": "Non-contact radiofrequency-induced reduction of subcutaneous abdominal fat correlates with initial cardiovascular autonomic balance and fat tissue hormones: safety analysis", "authors": [ "Jiri Pumprla", "Kinga Howorka", "Zuzana Kolackova", "Eliska Sovova", "Zuzana Kolackova", "Eliska Sovova" ], "abstract": "Background and objective: The non-invasive reduction of subcutaneous abdominal fat became popular in the last decade. Radiofrequency (RF), non-contact, selective-field device Vanquish® has been developed to selectively induce deep fat tissue heating to reduce waist circumference. Our analysis evaluates immediate and sustained effects of this treatment on cardiovascular autonomic function and on selected metabolic parameters.Study design/patients and methods: A retrospective proof-of-concept analysis of RF treatment effects was conducted in 20 individuals with metabolic syndrome, to reduce the subcutaneous abdominal fat. Four 30-minutes treatment sessions (manufacturer´s standard protocol) were performed in 1-week intervals. Vital signs, ECG, lab screening, body composition, subcutaneous fat thickness and spectral analysis of heart rate variability (HRV) have been examined before, after the 1st and 4th treatment, and at follow-up visits 1 month and 3 months after the treatment.Results: The RF treatment led to a significant reduction of abdominal circumference after the 4th session (p<0.001), and during follow-up after 1 and 3 months (p<0.001 and p<0.02, resp.). There was a significant correlation (r=-0.58, p=0.007) between reduction of abdominal circumference and initial very-low frequency (VLF) spectral power at 1 month follow-up. A significant increase of cumulative spectral power in low frequency (p=0.02) and reduction in high frequency (p=0.05) band have been observed immediately (20+14 minutes) after the treatment. On the contrary, no sustained impact on autonomic balance has been recorded 39+18 days after the treatment. A significant correlation between the initial adiponectin values and immediate autonomic response to one treatment was observed in VLF and total spectral bands (r>0.59, p<0.04).Conclusions: Our analysis shows that the selective-field RF treatment is safe and efficient for reduction of subcutaneous abdominal fat. While the treatment increases the immediate sympathetic response of the body to deep tissue heating, no sustained change in autonomic function could be recorded at 1 month follow-up. The observed correlation between initial VLF spectral power and waist circumference reduction at follow-up, as well as the association of initial adiponectin values and immediate autonomic response to the treatment might be instrumental for decisions on body contouring strategies.", "keywords": [ "Metabolic syndrome", "insulin resistance", "selective-field radiofrequency", "body contouring", "subcutaneous fat reduction", "heart rate variability", "autonomic control" ], "content": "Introduction\n\nObesity considerably impairs individual health and aesthetic appearance. In addition to its known health impacts -- reduction of life expectancy and quality of life1 -- obesity leads to numerous problems including disadvantages in employment2, in social interactions and decreased satisfaction with own body image3. These aspects lead to social pressure and subsequently to increased demand for effective procedures for weight reduction, body contouring and beauty enhancement.\n\nCentral obesity is associated with insulin resistance and related components of metabolic syndrome that can be typically treated by nutritional, behavioural and lifestyle changes1. Although the reduction of subcutaneous fat alone does not lead directly to reduction of cardiovascular risk in obese subjects4, there is some evidence that the large-volume liposuction might positively influence the insulinemia5 and thus insulin sensitivity. Furthermore, clinical experience shows that aesthetic procedures leading to improved patient’s self esteem6 often significantly enhance the motivation to further lifestyle changes towards healthier goals.\n\nVarious invasive and particularly non-invasive body contouring procedures for reduction of subcutaneous fat layers have been introduced in the last decade. While the surgical liposuction still counts for the most effective gold-standard procedure in this respect7, due to its invasiveness, downtime and side effects, a bunch of non- or semi-invasive procedures became available as its indirect alternative on the quickly growing (often called “lunch-time-procedure”) market8. However, despite many individual – often only anecdotal – user reports, only a minority of methods is proven according to the evidence-based medicine standards. Such evidence is available for efficacy of chemical lipolysis, based on injection of phosphatidylcholine and deoxycholic acid9, and for selected energy-based technologies using focused ultrasound10, cryolipolysis11 and/or thermal/radiofrequency for lipolysis12. Despite the broad use in the practice, clinical safety data of these aesthetic procedures are scarce, with only a few publications available13. Although these intensive procedures might have a significant impact on autonomic homeostasis and individual health, to our knowledge, no immediate or sustained effects of such treatments on autonomic function have been investigated.\n\nAnalysis of beat-to-beat fluctuations of heart rate (heart rate variability, HRV) is an established tool to non-invasively quantify cardiac autonomic function14. The frequency (spectral) decomposition and quantification of irregular course of heart rate into three main frequency bands allows a detailed view of different domains of the cardiovascular control. The short-term HRV spectral analysis is proven useful for assessment of impact of various physiological stimuli on the body such as food restriction15, endurance physical training16 or guided breathing17. In particular, the very low frequency (VLF) spectral band has been shown to reflect thermoregulatory vasomotor mechanisms, changes in peripheral chemoreceptor activity and fluctuations in renin-angiotensin systems14,18. In this respect, analysis of the VLF band enables the quantification of sympatho-thermogenic autonomic responses related to energy metabolic control, as it has been demonstrated e.g. by an acute cold exposure, spicy food containing capsaicin and green tea extract or low-calorie diet19–22.\n\nThe selective-field radiofrequency device Vanquish®, using electromagnetically induced rapid oscillations of electrical dipoles to heat up the fatty tissue23, is increasingly being used for reduction of subcutaneous abdominal fat. Its efficacy has already been demonstrated24. However, although the reported patient acceptance of these treatments was well to superlative24, no metabolic and/or safety data have been published yet. Our aim was therefore to evaluate the safety and efficacy of this novel technology in a proof-of-concept retrospective data analysis of all clients who attended our clinic and were subjected to the treatment including follow-up within the 5-months time period. This paper focuses on the immediate and sustained effects of the treatment on the autonomic balance of the body and related metabolic values. The analysis is thought as a preparation for a further controlled prospective observation.\n\n\nPatients and methods\n\nA retrospective, uncontrolled, single site proof-of-concept analysis of the impact of selective-field radiofrequency treatment on cardiovascular autonomic control and on selected metabolic data (insulin resistance parameters and fat tissue hormones) was conducted in overweight individuals with components of metabolic syndrome and visually detectable excess of subcutaneous fat who wished to reduce the abdominal circumference. This study was a retrospective proof-of-concept trial and the data have been retrospectively evaluated for the future preparation of a controlled prospective trial, which will require a submission to the local Ethics Committee. For the retrospective data evaluation no approval of IRB was required.\n\nData have been routinely acquired before, at visits immediately after the 1st and 4th treatments, and at follow-up visits in 1-month and 3-months after the last treatment. For assessment of metabolic data, blood sampling was performed before, on the next morning after the 1st and 4th treatments, and 1 and 3 months after the last treatment. Assessment of the intervention effect on the autonomic balance, using the standardized analysis of short-term heart rate variability as obtained during the modified orthostatic load18,25, was performed before, immediately (acute effect) after the 1st treatment, and 1 month (sustained effect) after the last treatment. These data have been acquired during routine services of the clinic.\n\nThe selective-field RF treatment protocol has been offered to all individuals with visually excessive subcutaneous fat wishing to reduce their waist circumference. In the retrospective evaluation of efficacy and safety all patients have been included who accepted the necessity of follow-up investigations. Attendance of follow-up visits was a prerequisite for waiving their treatment fees. No reimbursement or coverage of travel expenses have been offered to these patients.\n\nThe following standard routine criteria of our clinic for exposition to RF treatment were applied: Inclusion criteria were age 20–70 years, both genders, BMI over 25kg/m2, abdominal circumference over 80 and 94 cm in women and men, respectively, with at least 20 mm of abdominal subcutaneous adipose tissue (as measured by calliper at predefined locations), stable weight over the last 6 months and signed informed consent on treatment. Exclusion criteria were pregnancy or insufficient contraceptive methods, surgical liposuction within the last 12 months, insufficiently controlled metabolic disease including diabetes mellitus of both types, untreated hypo- or hyperthyroidism, uncontrolled liver, kidney or cardiovascular disease, implanted pacemaker or metal implant, acute or feverish disease, history of thrombophlebitis, any haematological disease, chronic medication of corticosteroids, beta-blockers, anticoagulants, insufficient treatment adherence or any other clinical or biochemical condition bearing potential to interfere with the treatment targets. Females in child-bearing age were educated about necessary contraceptive methods, and those planning pregnancy in the following 12 months were not subjected to the RF treatment.\n\nThe study population consisted of n=20 (f=18/m=2) subjects with age 47.8±7.2yr, BMI 28.2±3.6 kg/m2, abdominal circumference 96±9 cm, insulin resistance HOMA2 index 1.49±0.80 with insulin sensitivity of 79.8±28.9%, fat percentage in body composition 38±7%, blood pressure 138±12/79±7 mmHg, and with reported insufficient aerobic activity/median 30/Q1=0, Q3=60/min weekly. Chronic treatment of concomitant diseases remained unchanged during the whole treatment period. Six female patients received substitution of hypothyroidism resulting in euthyroid values of TSH (x=1.2±0.8 mU/l), four subjects used antihypertensive medication (ACE inhibitors or sartans) and four subjects had lipid lowering agents (statins). Eight female patients received oral contraceptives. Further details can be found in Table 1.\n\nThe non-contact, selective-field radiofrequency system Vanquish® (BTL Industries) has been used for treatment of subcutaneous fat layers23. All subjects underwent four 30 minutes treatment sessions in the abdominal area, with one week break between the sessions, as recommended in the standard treatment protocol by the manufacturer. Flat multipolar applicator panel was used for emitting the radiofrequency (27.12 MHz) energy for selective generation of deep tissue thermal heating of adipose tissue layers. The unit adjusts the parameters of the emitted energy in real time and shows the instantaneous value on display. This electromagnetic radiation is heating up the adipose tissue much more effectively then surrounding tissues, while limiting potential side effects due to minimized exposition of skin, muscles, or internal organs to this energy23. The treatment procedure consists of placing the emitting panel over abdomen and flanks close to the skin using a spacer which standardizes distance between the panel and the body surface. Once it is in a proper position, treatment can be started while the intensity of the emitted energy is set according to the protocol and to tuning efficiency of the system. The skin temperature is measured before, in 10, 20 and 30 minutes during the treatment, while the subject is frequently asked to give feedback on subjective thermal perception and to immediately report any pain or unpleasant sensations. Operator adjusts the emitted energy intensity close to the tolerable level according to client’s feedback during the treatment procedure and to the measured skin temperature while the safety threshold is set to 42°C.\n\nBlood sampling, autonomic balance evaluated by heart rate variability analysis, clinical assessment including vital signs, casual blood pressure, electrocardiogram (ECG), body composition evaluated by bioelectrical impedance measurements, abdominal circumference in three predefined points and anthropometric assessment by calliper were evaluated at predefined time points as indicated elsewhere.\n\nCasual blood pressure has been measured in accordance with standard recommendations26 in sitting position, using validated oscillometric automated monitor Omron M6 (Omron, Japan). The average from three measurements has been used for data analysis.\n\nA calibrated, computer-assisted system ECG Seiva (Seiva, Czech Republic) has been used for 12-lead surface ECG recordings. Data have been electronically stored and evaluated by a single specialist experienced in ECG readings.\n\nHeight has been measured by validated ultrasound height measuring unit ADE MZ10020 (ADE, Germany) within standardized conditions as set by manufacturer.\n\nTemperature before and during the treatment sessions has been measured by a calibrated non-contact infrared skin thermometer BaseTech IRT-350 (BaseTech, Germany) at predefined locations (around umbilicus and at upper and lower abdominal wall on both sides).\n\nWeight has been measured within the body composition assessment as described below.\n\nBody composition has been assessed by a calibrated scale, Omron BF 511 (Omron, Japan), a 8-sensor, one-frequency (50 kHz, 500 uA) bioelectrical body impedance analysis device, under strictly standardized conditions as set by the manufacturer. The device delivers along with weight and BMI also gender-specific percentage of body fat and muscle mass, basal metabolic rate (in kcal) and amount of visceral fat (arbitrary units). The declared weight measurement accuracy is 1%27.\n\nWaist circumference was measured using a measuring tape with a spring handle (www.netzwerk-lipolyse.de), in order to control for the pressure exerted on the patient’s abdomen. Three measurements in different locations have been performed at the end of gentle expiration, in the standing position: horizontally around the patient’s abdomen at its narrowest part (under the rib cage), at the level of the umbilicus, and 5 cm below the umbilicus. Data were recorded to the nearest millimetre.\n\nAll measurements were performed with the subject in standing position. The measurement points were selected as follows: above the iliac crest in the mid-axillary line, right and left, paraumbilically at 1/3 distance between the iliac crest and umbilicus, right and left, and 5 cm below umbilicus. The skinfold was pinched up firmly between the thumb and forefinger and pulled away from the underlying tissues. The measurements were performed with calibrated calliper of Harpenden type, ie. with a constant measuring pressure 10p/mm2, in accordance with established guidelines28. The results are presented in mm, as average of five subsequent measurements per one point.\n\nA standard blood sampling has been performed in the morning by venipuncture after an overnight 10 hours fasting. After clotting, the serum was separated and immediately explored for most analyses. For fat hormones, the serum was stored at -20°C until analysed. Insulin resistance was evaluated using the HOMA2 calculations based on fasting glycemia and C-peptide values (Homeostatic model assessment as described by Levy et al.29).\n\nA standardized analysis protocol of short-term HRV in time and frequency domain as obtained during a modified orthostatic load (5 minutes supine and 5 minutes in standing position) has been used for quantification of treatment effects on the autonomic control of the body14,18,25. The HRV measurements have been performed using the VariaCardio TF5 system (Advanced Medical Diagnostics Group, UK). The main principle of spectral analysis of HRV is a decomposition (using fast Fourier transform algorithms) of irregular fluctuations of heart rate into regular cycles that represent influences of various domains on the autonomic balance. Such resulting spectral power is then quantified within three standard frequency bands: (1) very-low frequency component (VLF, 0.01–0.04 Hz), its cycles occur with typical frequency of 0.01 Hz, corresponding to wavelength of 100 seconds. The VLF power is related to control of energy metabolism and thermoregulation, changes in peripheral chemoreceptor activity and fluctuations in renin-angiotensin system, (2) low-frequency component (LF, 0.04–0.15 Hz), with typical variations occurring at frequency 0.1 Hz, i.e. 6-times per minute. It represents predominantly sympathetic control with certain amount of vagal influence, (3) high-frequency component (HF, 0.15–0.4 Hz), with cycles fluctuating at average frequency 0.25 Hz, ie 15-times per minute. This power is related to respiratory activity and parasympathetic control14. While the VLF band is mediated primarily by sympathetic control and the HF by the parasympathetic one, the middle one, LF band, includes both, with predominance of the sympathetic branch of the autonomic control14,18,30. The main parameters of the analysis are the spectral power (area under the curve) in each of the individual bands and in the total frequency band, the centroid frequencies and the relative proportion of individual frequency bands contents in the total spectral power. We have shown previously that the cumulative numbers generated by summing up the individual frequency band spectral powers over both test positions increase the discrimination power/capability of respective parameters32.\n\nStatistical analysis was performed using standard statistical packages (SPSS, Statistical Package for the Social Sciences V10.0, SPSS Inc., Chicago, USA). Normality of data distribution was verified by Kolmogoroff-Smirnoff test. A two-tailed paired Student’s t-test was applied to estimate differences between groups in case of normal data distribution. Relations among variables were assessed using Pearson’s correlation analysis. Data are presented as means ± SD, unless indicated otherwise. The significance level was set a priori at p<0.05.\n\n\nResults\n\nDuring all four sessions, the average skin temperature values before, at 10, 20 and 30 minutes of treatment were 31.8±1.1, 39.8±0.7, 39.6±0.6 and 39.2±1.0°C respectively, while the delivered total average maximum energy was 158.5±13.0 W and the total average effective energy was 156.2±13.1 W. While starting the treatment session at 160 W energy level as suggested by manufacturer, in 25 out of 84 (29,8%) sessions the energy intensity could be increased -- in accordance with subject’s heat sensation -- to 170–200 W within the first 10 minutes of treatment, and in 13 out of 84 (15.5%) sessions the energy intensity had to be reduced to 100–150 W due to excess heat perception. The average effective emitted energy in each of four treatment session was therefore 156±14, 160±17, 160±19, and 153±16 W respectively. A significant correlation between the averaged skin temperature after 30 minutes of treatment and reduction in abdominal circumference was observed 1 month after the last treatment (r=-0.49, p=0.03).\n\nWhen compared with initial values, the average casual blood pressure was significantly lower after the 4th treatment session (134±12 vs. 127±10 mmHg, p=0.003) and raised to 129±9 mmHg (p=0.04 vs. initial value) 1 month after the treatment. The average heart rate has changed from 69±12 to 67±9/min (p=0.04) after 4th treatment session, and raised to 69±11/min (p=0.33, both p vs. initial value) after 1 month. No other significant changes have been observed in ECG.\n\nThe radiofrequency selective-field treatment lead to a significant reduction of abdominal circumference as measured at 3 different locations after the 4th session (umbilicus, 96.2±9.3 vs 93.7±9.0 cm, p<0.001 vs. initial value), and during follow-up after 1 month (92.6±9.6 cm, p<0.001) as well after 3 months (93.3±10.1 cm, p<0.02). Despite the significant drop in body weight at follow-up 1 month after the treatment (from 78.8±12.4 to 78.0±12.1 kg, p=0.001), no significant correlation has been found between the deltas in body weight and abdominal circumference values vs. their respective initial values at this time point (r<0.41, p>0.07). The weight increased to 78.4±12.0 kg after 3 months follow-up. No statistically significant change in body composition (in percentage of body fat and muscle mass) has been recorded during all three measurements vs. initial values.\n\nRegarding the immediate effects of the treatment on autonomic balance, a significant increase in low frequency (p=0.02) and reduction in high frequency (p=0.05) band cumulative spectral powers have been observed in HRV 20±14 minutes after the treatment. No sustained effects on autonomic balance, however, have been observed during the follow-up period after the treatment. Figure 1 and Figure 2 summarize the impact of the treatment on autonomic balance immediately after one treatment and 39±18 days (sustained effect) after the last treatment, respectively.\n\nFor description of spectral parameters see Figure 1.\n\nAt follow-up after 1 month, there was a significant correlation between the reduction of abdominal circumference and the initial very-low frequency band cumulative spectral power (r=-0.58, p=0.007, Figure 3). Moreover, in a subgroup comparison, subjects with a higher initial cumulative VLF power (6.4±0.4 LN ms2) demonstrated a significantly bigger drop in abdominal circumference after the 4th treatment (4.1±1.9 vs. 2.6±0.9 cm, p=0.045) than those with lower initial cumulative VLF spectral power (5,1±0.5 LN ms2).\n\nAs expected, a significant correlation between weight and insulin resistance index based on HOMA2 calculations has been observed before the treatment (p=-0.53, p=0.016). Change of body weight correlated significantly with the initial HOMA2 indices after the 4th treatment (r=-0.54, p=0.014 for HOMA2, and r=-0.47, p=0.036 for % beta function) and 1 month after the last treatment (p=-0.57, p=0.009 for % beta function). There was a significant correlation between the initial adiponectin values and deltas of total body fat percentage (r=-0.45, p=0.05) and body weight (r=-0.52, p=0.02) observed after the 4th treatment.\n\nThe immediate autonomic response to one treatment (Figure 4), as observed in the VLF band (r=0.60, p=0.005) and in the total spectral power (r=0.45, p=0.04) correlated significantly with the initial adiponectin values. Furthermore, a subgroup analysis based on initial adiponectin values (cut-off value 13.0 ng/ml) revealed a significantly stronger acute autonomic response to one treatment in those with higher initial adiponectin level (15.8±1.8 ng/ml) than with a lower one (10.3±1.8 ng/ml). Similarly, in respect to sustained effects, there was a significant correlation between delta of adiponectin values 1 month follow-up vs. initial values and delta of autonomic response in VLF band 1 month follow-up vs. initial values (r=0.48, p=0.03).\n\nOverall, two drop-outs have been recorded. In two cases the local skin irritation led to interruption of the protocol after the 2nd treatment. These subjects were not included in the analysis. One subject underwent elective surgery at 1 month of follow-up and did not attend the planned visit. Four subjects did not attend the last follow-up visit after 3 months.\n\nAfter first two treatments, one subject reported abdominal discomfort, and another one a hyperesthesia around umbilicus. These symptoms resolved within 1 week after the treatment session. No more adverse reactions have been observed after exchange of spacer used for proper positioning of the energy emitting panel at the 3rd treatment session.\n\n\nDiscussion\n\nThe principal findings of our study are threefold: the efficacy of selective-field radiofrequency treatment in terms of reduction of waist circumference up to 3 months after the treatment series was confirmed. The treatment is safe, as no clinically relevant side-effects were observed. The impact on autonomic cardiovascular balance is significant but transient, while being limited to an increased sympathetic response immediately after this energy-based treatment in abdominal area. No sustained effect of the intervention on autonomic balance has been observed 1 month after the last treatment. The treatment efficacy is inversely associated with insulin resistance and other features of metabolic syndrome and may be explained by the inhibitory effect of higher insulin levels on the (treatment-induced) lipolysis32. Whether the treatment efficacy could be better predictable using assessment of VLF spectral power and insulin resistance profile, and/or it could be further supported e.g. by pharmacological agents (such as metformin or insulin sensitisers33) or other means, this should be investigated in further prospective trials.\n\nThere is a pathogenic link among autonomic imbalance, insulin resistance and obesity. In addition to genetic background and lifestyle factors, autonomic imbalance could be a common root of obesity, hypertension and/or type 2 diabetes mellitus. At early stages of obesity/metabolic syndrome development, parasympathetic control is decreased while sympathetic overactivity usually occurs34. This dysfunction increases cardiovascular workload, hemodynamic stress and induces potentially significant cardiac pathology leading to serious arrhythmias. It remains open, however, whether elevated sympathetic tone is a primary feature that contributes to the development of obesity and metabolic syndrome or if it develops and/or changes secondary to the obese state.\n\nIt has been shown that sympathetic overactivity precedes the development of insulin resistance and type 2 diabetes mellitus35. Studies with genetically predisposed humans with insulin resistance have shown that early insulin resistance is already associated with increased sympathetic control, and it has been suggested that hyperinsulinemia is the initiating factor leading to increase of sympathetic neural activity36. Subsequently, adrenoceptor down-regulation and/or reduced sensitivity are likely to develop which situation results in a secondary reduction of sympathetic responsiveness. As adrenoceptors are involved in control of energy expenditure, their down-regulation leads further to impaired food-induced thermogenesis and post-prandial fat oxidation, promoting the accumulation of body fat. In this way, the development of obesity can be seen as a consequence of inappropriate/insufficient sympathetic control, energy dissipation, gaining weight and then insulin resistance37. This theory also confirms the earlier popular Bray’s MONALISA hypothesis, stating that “Most Obesities kNown Are Low In Sympathetic Activity”38. It is also consistent with findings from population studies, e.g. in observation of 7000 individuals without hypertension at baseline, low heart rate variability predicted greater risk of incident hypertension over 9 years of follow-up39. Similarly, in almost 2000 participants of Framingham Offspring Study, LF power and LF/HF ratio were lower in diabetic subjects than in those with normal fasting glucose. HRV was inversely associated with plasma glucose levels and was reduced in diabetic individuals as well as in subjects with impaired fasting glucose levels40.\n\nHeart rate variability measurement is an established tool for the assessment of impact of intervention on autonomic balance15–17,41,42. While the HRV LF and HF frequency bands have been sufficiently studied in short- and long-term ECG recordings, interpretation of the VLF band -- particularly in short-term recordings – is less explored. Along with influences coming from sympatho-thermoregulation, renin-angiotensin system and chemoreceptors, a clear VLF response to excessive temperature variations has been demonstrated19. Further on, significant impacts of a spicy food20 or capsaicin21 on VLF spectral power have been reported. These findings are consistent with our results where a significant correlation has been observed between the initial adiponectin level and the immediate VLF band autonomic response to a single treatment, as well as between the initial adiponectin and reduction of percentage of body fat after the treatment series (Figure 4). Additionally to fat percentage, the initial VLF spectral power significantly correlated with change in waist circumference seen after the treatment series (Figure 3). These observations raise a question whether individuals with higher VLF spectral power and higher adiponectinemia/lower insulin resistance might enjoy a better sympatho-thermogenic capability to “burn” the available energy while more readily inducing lipolysis processes, than those individuals with lower VLF tone. This hypothesis might have clinical implications in weight management programs and/or body contouring treatments for subcutaneous fat layers reduction.\n\nAt present, there are some new therapeutic targets and procedures taking into account autonomic imbalance in obesity as an independent and sensitive marker of health. Autonomic dysfunction is reversible with lifestyle changes such as hypocaloric nutrition or fasting15 and physical endurance training16. Recently, the approach of cold-induced, facultative thermogenesis aiming for sympathoadrenergically-mediated weight reduction by stimulation of brown fat tissue has been introduced43,44. A sympathetic stimulation of brown fat tissue leading to increased daily energy expenditure by 200–400 kcal45 has been suggested as the main mechanism in this successful model, and there is some hope for success of this approach on the reduction of obesity46. The conversion of white adipose tissue to the highly thermogenic beige adipose tissue has been shown to be influenced by acute sympathetic activation, as well47. Since the autonomic imbalance is a marker of adverse risk14,18, its improvement resulting from weight loss should be beneficial for the health of obese/diabetic individuals.\n\nOur relatively short and limited observation could not deliver sufficient evidence on whether subcutaneous abdominal fat reduction using selective-field RF treatment improves obesity-related cardiovascular risk. However, despite even only little changes in body weight, patients with significantly reduced waist circumference are reported to have an improved metabolic profile48. It has been shown that the waist circumference is a proven marker of higher total mortality risk49 as well as of a cardiovascular risk50. Therefore, reducing waist circumference and percentage of fat in body composition may represent a useful and clinically relevant target48. Moreover, the initial successful waist reduction may play a significant role in further stimulating the motivation of patients with metabolic syndrome for long-term adaptation and adherence to “healthier” lifestyle habits. As this is a proof-of-concept uncontrolled retrospective analysis, we cannot fully exclude external confounding factors that might have contributed to our observations. As a logical step, a randomized, controlled trial verifying the results in an appropriate patient sample would contribute to a better understanding of our findings.\n\nIn conclusion, our analysis provided a proof of concept for safety and efficacy of selective field RF treatment using the standard 4 × 30 minutes protocol for moderate reduction of subcutaneous fat tissue. Only transient and non-sustained effects on autonomic balance have been found during the follow-up after the treatment series. The efficacy of Vanquish RF treatment in terms of waist circumference reduction was shown and it was significantly related to initial VLF spectral power and adiponectin levels. This implicates that less insulin resistance may offer better conditions for lipolytic action of the treatment. This body contouring procedure was the most efficient in moderate abdominal overweight with lower insulin resistance, and as such can well complement other, systemic clinical measures for weight reduction based on lifestyle and nutritional changes. As the measurements of HRV and fat hormone status are easily performed, we suggest considering the inclusion of these parameters into the clinical prescreening armamentarium in order to enhance the outcomes of aesthetic body contouring methods.\n\n\nConsent\n\nWritten informed consent for publication of their anonymised clinical details was obtained from all patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of non-contact radiofrequency-induced reduction of abdominal fat HRV, 10.5256/f1000research.5708.d3830951", "appendix": "Author contributions\n\n\n\nJP and KH conceived the analysis. JP and KH designed the treatment protocol following the standard recommendation of the manufacturer. JP, ZK and KH carried out the research.\n\nES contributed to the analysis and provided expertise in cardiology. JP and KH prepared the first draft of the manuscript. ES contributed to the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nJP received speaking honorarium from BTL Industries. No competing interests were disclosed for KH, ZK and ES.\n\n\nGrant information\n\nThe author(s) declared that no financial grants were involved in supporting this work.\n\n\nReferences\n\nEuropean Guidelines on cardiovascular disease prevention in clinical practice (version 2012): The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). Atherosclerosis. 2012; 223(1): 1–68. 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Data Source" }	[ { "id": "7774", "date": "18 Mar 2015", "name": "Klaus Fritz", "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\nThe study shows data that were previously missing, on a therapy that increases temperature of the body and especially of the tissue treated. Data were missing on whether this might be a danger for cardiovascular diseases, circulation, or metabolism. These missing data are presented here for the first time, showing that the RF treatment described in the article is safe.", "responses": [] }, { "id": "8553", "date": "06 May 2015", "name": "Andreas Thomas", "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\nThis study shows the success of the radiofrequency-induced reduction of abdominal fat, while the correlation with the HRV changes (VLF spectral band). This is a very interesting study confirming the important link between obesity and cardiovascular risk parameters. It could discuss whether the HRV measurement is a good marker for the success of weight loss. It should be noted that the HRV measurement is highly selective, but not very specific. The only downside is that in the sense of a \"proof of concept\" study, the evidence is not high. The results should be confirmed with a randomized controlled trial.Congratulations to the authors!", "responses": [] } ]	1	https://f1000research.com/articles/4-49
https://f1000research.com/articles/3-288/v1	21 Nov 14	{ "type": "Research Article", "title": "UV-independent induction of beta defensin 3 in neonatal human skin explants", "authors": [ "Erin Wolf Horrell", "John D'Orazio", "Erin Wolf Horrell" ], "abstract": "In order to determine the effect of UV radiation on β-defensin 3 (BD3) expression in human skin, freshly-isolated UV-naïve skin was obtained from newborn male infants undergoing planned circumcision. Skin explants sustained ex vivo dermis side down on RPMI media were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explant through 72 hours after radiation. mRNA expression was measured by qRTPCR and normalized to TATA-binding protein. BD3 expression at each time point was compared with an untreated control taken at time 0 within each skin sample. Extensive variability in both the timing and magnitude of BD3 induction across individuals was noted and was not predicted by skin pigment phenotype, suggesting that BD3 induction was not influenced by epidermal melanization. However, a mock-irradiated time course demonstrated UV-independent BD3 mRNA increases across multiple donors which was not further augmented by treatment with UV radiation, suggesting that factors other than UV damage promoted increased BD3 expression in the skin explants. We conclude that BD3 expression is induced in a UV-independent manner in human skin explants processed and maintained in standard culture conditions, and that neonatal skin explants are an inappropriate model with which to study the effects of UV on BD3 induction in whole human skin.", "keywords": [ "The melanocortin 1 receptor (MC1R) is a Gs-protein-coupled receptor expressed on melanocytes that regulates several key aspects of cutaneous UV responses. When bound by agonistic ligands", "most notably α-melanocyte stimulating hormone (MSH)1", "MC1R initiates a cascade of UV-protective events mediated by activation of adenylyl cyclase and generation of cAMP that result in melanin production and enhanced genome stability via enhancement of DNA repair2. In addition to MSH", "MC1R signaling is regulated by other soluble ligands", "most notably agouti signaling protein (ASIP) which antagonizes MC1R signaling", "decreases cAMP levels", "and diminishes downstream melanocyte responses such as pigment induction3", "4. Recently", "it has become clear that β-defensin 3 (BD3)", "known for its role in innate antimicrobial immunity", "binds and influences MC1R signaling as a neutral MC1R agonist that blunts MSH-mediated MC1R activation as well as ASP-mediated MC1R antagonism5–8. Thus", "BD3 may be an important regulator of MC1R-dependent melanocyte responses in the skin." ], "content": "Introduction\n\nThe melanocortin 1 receptor (MC1R) is a Gs-protein-coupled receptor expressed on melanocytes that regulates several key aspects of cutaneous UV responses. When bound by agonistic ligands, most notably α-melanocyte stimulating hormone (MSH)1, MC1R initiates a cascade of UV-protective events mediated by activation of adenylyl cyclase and generation of cAMP that result in melanin production and enhanced genome stability via enhancement of DNA repair2. In addition to MSH, MC1R signaling is regulated by other soluble ligands, most notably agouti signaling protein (ASIP) which antagonizes MC1R signaling, decreases cAMP levels, and diminishes downstream melanocyte responses such as pigment induction3,4. Recently, it has become clear that β-defensin 3 (BD3), known for its role in innate antimicrobial immunity, binds and influences MC1R signaling as a neutral MC1R agonist that blunts MSH-mediated MC1R activation as well as ASP-mediated MC1R antagonism5–8. Thus, BD3 may be an important regulator of MC1R-dependent melanocyte responses in the skin.\n\nBecause UV radiation is a major causative agent for melanoma and other skin cancers and because MC1R signaling mediates critical UV-protective responses such as melanization of the skin and melanocytic resistance to UV mutagenesis, it is important to understand how UV affects expression of MC1R ligands in the skin. MSH levels increase in response to UV exposure of the skin. Cui and coworkers reported that UV promoted transcriptional increases in pro-opiomelanocortin (POMC), the protein precursor for MSH, in a cell damage and p53-dependent manner in epidermal keratinocytes9, supporting the hypothesis that melanocytic MC1R responses are modified by intracutaneous UV-regulated mechanisms. Similarly, recent studies reported that UVB radiation caused an increase BD3 mRNA and protein levels both in vivo and in vitro10, either in a cell-autonomous, damage-dependent manner or in response to inflammatory mediators such as interleukin-1 (IL-1β) and tumor necrosis factor (TNFα) known to promote its induction11,12. In an effort to understand the mechanisms of how BD3 production may be influenced by UV radiation, we determined its expression in freshly isolated human skin explants. Here we report that BD3 expression increases in a UV-independent manner in neonatal human skin explants in response to processing and culturing of tissues ex vivo.\n\n\nMethods\n\nNeonatal foreskin explants. Freshly-isolated, de-identified neonatal foreskins were collected from normal newborn infants undergoing planned circumcision from the University of Kentucky Birthing Center under an IRB-exempted protocol. Foreskins were collected only from patients who were consented prior to delivery. Samples were placed into 30 ml of Roswell Park Memorial Institute (RPMI) media (Life Technologies) and stored at room temperature for a maximum of four hours before processing. Samples were rinsed in phosphate buffered saline (PBS) + 1% penicillin streptomycin (Life Technologies), and dermal fat was manually removed by forceps to the point that explants would lie completely flat. Explants were placed in 3 cm cell culture dishes and floated dermal side down on 3 mL of RPMI media with 10% fetal bovine serum for 16–18 hours at 4°C until use.\n\nSkin color measurement. Skin reflective colorimetry was assessed with a CR-400 Colorimeter (Minolta Corporation, Japan) calibrated against a white background. Degree of melanization (darkness) was quantified as the colorimetric measurement on the L axis (white-black axis) of the CIE standard color axis13. The degree of pigmentation was determined by three independent measurements for each sample.\n\nUV exposure. Skin explants were exposed (epidermal side up) to an overhead double bank of UVB lamps (UV Products, Upland, CA) to receive 0.5 kJ/m2 UVB, a dose similar to that reported previously with respect to cutaneous BD3 induction in vivo10,14. UV emittance was measured with a Model IL1400A handheld flash measurement photometer (International Light, Newburyport, MA) equipped with separate UVB (measuring wavelengths from 265–332 nm; peak response at 290 nm) and UVA (measuring wavelengths from 315–390 nm; peak response at 355 nm) filters corresponding to International Light product numbers TD# 26532 and TD# 27108 respectively. Spectral output of the lamps was determined to be roughly 75% UV-B and 25% UV-A.\n\nmRNA isolation. Total RNA was harvested from skin using TRIzol (Invitrogen). 25 mg of sample were placed in 500 ul of TRIzol and ground to a fine consistency using a dounce homogenizer. RNA was isolated in the aqueous phase and dissolved in RNase free water. cDNA was reverse transcribed in a Mastercycler epgradient thermocycler (Eppendorf International) utilizing random hexamers and M-MLV reverse transcriptase (Promega).\n\nqPCR. Quantitative real-time PCR (qRTPCR) analysis was performed using an Applied Biosystems 7500 Real Time PCR System (Life Technologies) (10 ng cDNA/reaction) utilizing TATA-binding protein (TBP) as a reference gene. Primer sets for TBP were 5´-CAGCGTGACTGTGAGTTGCT (left) and 5´-TGGTTCATGGGGAAAAACAT (right) and for BD3 were 5´-TAGGGAGCTCTGCCTTACCA (left) and 5´-CACGCTGAGACTGGATGAAA (right) (Integrated DNA Technologies).\n\nStatistics and data analysis. Correlation and linear regression analysis were performed using GraphPad Prism 5.0 (GraphPad Software, CA). Data were considered statistically significant if p values were less than 0.05 from multiple independent experiments.\n\n\nResults\n\nTo understand the effects of UV radiation on BD3 expression in human skin, freshly-isolated foreskins were exposed to 0.5 kJ/m2 UVB. Fourteen de-identified samples were obtained from normal healthy male infants undergoing elective circumcision before discharge from the neonatal nursery. Skin pigmentation was measured for each sample by reflective colorimetry in order to estimate melanin content of the epidermis. The skin samples exhibited a range of melanization as determined by the L score which quantifies color on a black-white color axis (a lower L score is indicative of a blacker/darker color and correlates with epidermal eumelanin content15). The majority of the samples were derived from light-skinned infants, however at least 3 samples were darker in color (Figure 1). Skin explants were exposed to 0.5 kJ/m2 UVB, and biopsies were taken from the explants at 6, 12, 24, 48, and 72 hours following UV exposure. BD3 mRNA expression was measured by qRTPCR at 6, 12, 24, 48 and 72h after radiation, normalized to TBP, and compared to an unirradiated control taken at time 0. Due to the small size of the skin explants (roughly 1 cm2), it was not possible to have a time-matched mock-irradiated control at each time point, therefore values were normalized to unirradiated controls from each skin sample. We noted extensive variability in both the timing and magnitude of BD3 induction across individuals (Figure 2). Normalized BD3 fold induction ranged from 1.3-fold to 44.8-fold, and peak induction ranged from 6–72 hours depending on the sample (Figure 3). We tested whether the amount of BD3 expression correlated with skin pigmentation, hypothesizing that more melanin in the skin might inhibit UV penetration into the skin and therefore blunt UV effects on BD3 expression. In fact, BD3 expression did not appear to be influenced by pigment phenotype, as manifested by a positive trend between higher BD3 expression and darker skin samples (Figure 4A; r2 = 0.057, p = 0.41). Similarly, a negative trend between skin color and time of peak BD3 expression was observed, although this too did not reach statistical significance (Figure 4B; r2 = 0.234, p = 0.08).\n\nSkin color determination is shown for each sample. L Score is measured by reflective colorimetry and represents color of the skin on a black-white axis. Lower L score is indicative of a more darkly pigmented phenotype. Data represent the average L score ± SEM for three measurements per skin sample.\n\nFourteen independent human skin explants were treated ex vivo with 0.5 kJ/m2 UVB radiation. BD3 mRNA expression was determined at 6, 12, 24, 48, and 72 hours following UV treatment and compared to an untreated control. qRTPCR was performed in duplicate for each sample, and results are expressed as mean fold change over control ± SEM.\n\nPeak BD3 mRNA expression for human skin explants (n=14) is arranged by time of maximal induction for each individual donor. qRTPCR was performed in duplicate for each sample, and results are expressed as mean fold change over control ± SEM.\n\nA) L score versus peak BD3 mRNA induction. qRTPCR was performed in duplicate for each sample, and data represent mean BD3 induction for 14 human skin explants. There was no correlation between donor L score and amplitude of BD3 induction (r2 = 0.057, p = 0.41). B) L score versus time of peak BD3 mRNA induction. qRTPCR was performed in duplicate for each sample, and data represent mean BD3 induction for 14 human skin explants. Although a weak negative trend existed between donor L score and time of BD3 induction, the correlation was not statistically significant (r2 = 0.234, p = 0.08).\n\nFive additional human skin explants were treated ex vivo with 0.5 J/m2 UVB radiation. BD3 mRNA expression for the UV treated biopsy and an untreated time matched control was compared to an untreated control taken at the time of UV treatment. qRTPCR was performed in duplicate for each sample, and data represent the mean fold change over the untreated control taken at the time of UV treatment ± SEM. BD3 induction in the human skin samples was independent of UVB treatment.\n\nWe then considered the possibility that BD3 expression might be affected simply by time in culture and measured BD3 expression over time in samples exposed to 0 or 0.5 kJ/m2 UV exposure. Each of five explants were divided into three sections and sampled either at time 0 (no UV) or at 24h following exposure to either 0 or 0.5 kJ/m2 UVB. Similar to prior experiments, BD3 expression was measured by qRTPCR and normalized to TBP, however values could also be compared with mock-irradiated, time-matched conditions. We observed clear induction of BD3 expression in each of the mock-irradiated samples over time (Figure 5), and exposure to 0.5 kJ/m2 UV did not substantially alter BD3 mRNA expression when compared to individual mock-irradiated time-matched controls. These data suggest that either tissue removal or the process of culturing skin explants ex vivo is sufficient to enhance BD3 expression in whole human skin and that the addition of 0.5 kJ/m2 UV does not impact BD3 expression in this setting.\n\n\nConclusions/discussions\n\nIn an effort to develop a model in which to study UV induction of cutaneous BD3, we measured its expression over time in UV-naïve human skin explants. Although there was a high degree of variability in the magnitude and kinetics of BD3 induction between samples harvested from different donors, we observed BD3 up-regulation in each case. To control for the possibility that tissue processing and/or ex vivo culture conditions might impact BD3 expression in the explants, we compared BD3 mRNA levels between mock-irradiated versus UV-treated sections of skin samples harvested from the same donor. This experiment, which included samples from five donors, indicated that BD3 expression increased over time irrespective of UV exposure (at 0.5 kJ/m2), suggesting that BD3 expression is induced in human skin explants in a UV-independent manner.\n\nBD3 expression is reported to be up-regulated in wound healing processes16, therefore it might be plausible that its increase over time in skin explants may be related to normal wound physiologic processes activated by surgical excision of the skin and/or its processing after harvest. Indeed, the small size of the skin samples isolated from neonatal circumcision (on average 1–1.5 cm2) implies that the majority of the tissue in the explant will be in close proximity to at least one cut surface, raising the possibility of local trauma-induced factors contributing to BD3 expression in the samples. It may also be possible that harvesting skin biopsies from the sample over the experimental time course may have promoted wounding responses in the explants and fueled BD3 expression. Alternatively, it is possible that one or more factors involved in sustaining the skins in culture (media, temperature, oxygen tension, pH, etc.) may have promoted BD3 expression in the explants. We do not as yet understand the mechanisms underlying variability of BD3 induction amplitude or kinetics observed between samples, however it is possible that wounding or inflammatory responses induced by tissue removal may vary between normal individuals. We conclude that because of confounding variables involved in their generation and maintenance, neonatal foreskin explants may not be an appropriate model to isolate the effects of UV on BD3 expression in the skin, however other models may still be appropriate.\n\n\nConsent\n\nDe-identified neonatal foreskin samples were obtained from the University of Kentucky’s Chandler Medical Center Newborn Nursery without accompanying clinical information under an institutionally-reviewed IRB-exempted status.\n\n\nData availability\n\nF1000Research: Dataset 1. Colorimetry measurements from each donor, 10.5256/f1000research.5794.d3905717\n\nF1000Research: Dataset 2. Cycle threshold values for qRTPCR, 10.5256/f1000research.5794.d3905818", "appendix": "Author contributions\n\n\n\nEWH and JD conceived the study and designed the experiments. EWH carried out the research and data analysis. JD and EWH wrote the manuscript. All authors agree to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the National Cancer Institute (R01 CA131075) awarded to JD, as well as T32CA165990 which supported EWH.\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 wish to thank the staff of the University of Kentucky Obstetrics and Pediatric clinical services for their help in obtaining freshly-isolated neonatal foreskin samples and alerting us to their collection in a timely manner.\n\n\nReferences\n\nSuzuki I, Cone RD, Im S, et al.: Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology. 1996; 137(5): 1627–33. PubMed Abstract \| Publisher Full Text\n\nKadekaro AL, Kavanagh R, Kanto H, et al.: alpha-Melanocortin and endothelin-1 activate antiapoptotic pathways and reduce DNA damage in human melanocytes. Cancer Res. 2005; 65(10): 4292–9. PubMed Abstract \| Publisher Full Text\n\nMillar SE, Miller MW, Stevens ME, et al.: Expression and transgenic studies of the mouse agouti gene provide insight into the mechanisms by which mammalian coat color patterns are generated. Development. 1995; 121(10): 3223–32. PubMed Abstract\n\nSuzuki I, Tada A, Ollmann MM, et al.: Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin. J Invest Dermatol. 1997; 108(6): 838–42. PubMed Abstract \| Publisher Full Text\n\nCandille SI, Kaelin CB, Cattanach BM, et al.: A β-defensin mutation causes black coat color in domestic dogs. Science. 2007; 318(5855): 1418–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaelin CB, Candille SI, Yu B, et al.: New ligands for melanocortin receptors. Int J Obes (Lond). 2008; 32(Suppl 7): S19–27. PubMed Abstract \| Publisher Full Text\n\nBeaumont KA, Smit DJ, Liu YY, et al.: Melanocortin-1 receptor-mediated signalling pathways activated by NDP-MSH and HBD3 ligands. Pigment Cell Melanoma Res. 2012; 25(3): 370–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwope VB, Jameson JA, McFarland KL, et al.: Defining MC1R regulation in human melanocytes by its agonist alpha-melanocortin and antagonists agouti signaling protein and beta-defensin 3. J Invest Dermatol. 2012; 132(9): 2255–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui R, Widlund HR, Feige E, et al.: Central role of p53 in the suntan response and pathologic hyperpigmentation. Cell. 2007; 128(5): 853–64. PubMed Abstract \| Publisher Full Text\n\nGlaser R, Navid F, Schuller W, et al.: UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo. J Allergy Clin Immunol. 2009; 123(5): 1117–23. PubMed Abstract \| Publisher Full Text\n\nJia HP, Schutte BC, Schudy A, et al.: Discovery of new human beta-defensins using a genomics-based approach. Gene. 2001; 263(1–2): 211–8. PubMed Abstract \| Publisher Full Text\n\nHarder J, Bartels J, Christophers E, et al.: Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic. J Biol Chem. 2001; 276(8): 5707–13. PubMed Abstract \| Publisher Full Text\n\nWagner JK, Jovel C, Norton HL, et al.: Comparing quantitative measures of erythema, pigmentation and skin response using reflectometry. Pigment Cell Res. 2002; 15(5): 379–84. PubMed Abstract \| Publisher Full Text\n\nHong SP, Kim MJ, Jung MY, et al.: Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement. J Invest Dermatol. 2008; 128(12): 2880–7. PubMed Abstract \| Publisher Full Text\n\nD'Orazio JA, Nobuhisa T, Cui R, et al.: Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning. Nature. 2006; 443(7109): 340–4. PubMed Abstract \| Publisher Full Text\n\nKesting MR, Stoeckelhuber M, Hölzle F, et al.: Expression of antimicrobial peptides in cutaneous infections after skin surgery. Br J Dermatol. 2010; 163(1): 121–7. PubMed Abstract \| Publisher Full Text\n\nWolf Horrell EM, D’Orazio JA: Colorimetry measurements from each donor. F1000Research. 2014. Data Source\n\nWolf Horrell EM, D’Orazio JA: Cycle threshold values for qRTPCR. F1000Research. 2014. Data Source" }	[ { "id": "6759", "date": "04 Dec 2014", "name": "Zalfa Abdel-Malek", "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 study by Horell and D'Orazio aimed at addressing the production of BD3, a MC1R antagonist, in neonatal human foreskins, using skin explants, and its possible regulation by UV irradiation. The authors did not find a correlation between the pigmentary status of the skin and BD3 production, or a consistent effect of UV radiation. However, they noted altered production of BD3 over time in the skin explants, which they attributed to a wound repair-like reaction, or to inadequate culture conditions for the skin explants. They concluded that the use of foreskins in organotypic culture is not an appropriate model to assess BD3 production.It is not clear from the report exactly how the skin explants were maintained for the duration of the experiments. Were they maintained in a humidified incubator at 37°C? Was the culture medium changed daily? Obviously, the culture conditions can affect the viability of the explants. Did the authors check on the viability of the skin, especially at the end of the experiment (e.g. by examining the histology after H&E staining)? This is important, and the \"health\" of the skin can explain the erratic production of BD3. If the viability of the skin is compromised, this will have a generalized effect on its metabolic state.Since IL-1 is known to stimulate BD3 expression, did the authors check the levels of IL-1, particularly after UV exposure?If the change in BD3 production is due to a wound healing-like response, the authors might have to consider cutting the foreskins into equal parts at the beginning of the experiment, to avoid taking biopsies at different time points.It could very well be that the experimental conditions are responsible for the unexpected results that were obtained. So the conclusion that using foreskin explants is not an appropriate model might not be necessarily true.I suggest that the authors submit this report as a \"methodology\" report, after investigating the concerns raised above.", "responses": [ { "c_id": "1221", "date": "19 Feb 2015", "name": "John D'Orazio", "role": "Author Response", "response": "We sincerely thank you for your thorough reading of our manuscript and insightful comments. We have provided a point-by-point response to each of your concerns.Methods: It is unclear as to how the skin explants were maintained.We have expanded the methods to include these details. General Comment: Did the authors check the viability of skin samples?We have included hematoxylin and eosin stained histological slides for the skin explants at 0 and 24 hours following culture conditions to assess the viability of the samples and have determined the samples were still viable. Furthermore induction of TNF alpha suggests viability of the tissues over the time course of the experiment. General Comment: Did the authors check the level of IL-1B following UV radiation?We have determined the levels of TNF alpha which is also known to induce beta-defensin 3 expression. TNF induction appears to correlate with the beta-defensin 3 induction suggesting they are related. Methods: The authors may consider cutting the foreskins into equal parts at the beginning of the experiment to determine whether the change is due to a wound healing response.The samples were divided into equal parts at the beginning of the experiment prior to UV radiation. We apologize for the confusion in the methods and have updated the methods to include this information. General Comment: The conclusion that the use of human neonatal foreskins may not be an appropriate model to study BD3 induction may not be true and the results may be due to the experimental conditions. We thank the reviewer for this comment and have adjusted our discussion to accommodate it. General Comment: The authors should submit this report as a “methodology report.”Given the fact that our data raise important caveats about the use of neonatal human foreskins to study BD3 induction, we feel that our findings may be better reported as a research article rather than as a methodology report." } ] }, { "id": "6760", "date": "23 Dec 2014", "name": "Lidia Kos", "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 attempt to establish a skin explant model to investigate the effect of UV exposure on the expression levels of b-defensin 3, an antimicrobial peptide that also binds to Melanocortin 1 receptor and attenuates downstream signaling. They find extensive variability in the expression levels of b-defensin 3 in the UV-induced samples and cannot demonstrate that is in in fact up-regulated. They do show some minor increase in b-defensin 3 expression over time that is independent of UV irradiation and suggest that this may be due to a wound healing response to the skin excision process. The authors should have included in their analysis a few more genes known to be upregulated after UV exposure (for example, MC1R) both in keratinocytes and melanocytes. This way, they would have been able to define whether their culture system was improper to study b-defensin3 regulation specifically. In the mock irradiated experiment they should have also looked at levels of IL1 and TNFalpha to support their hypothesis that b-defensin 3 increases as a result of a wound healing response. In the paper by Glaser et al (2009) where b-defensin 3 was reported to be up-regulated after UV exposure, the authors used explants of adult skin. Is it possible that newborn and adult skin (perhaps based on the anatomy or level of cellular differentiation) respond differently to UV exposure that could explain the disparate results?", "responses": [ { "c_id": "1222", "date": "19 Feb 2015", "name": "John D'Orazio", "role": "Author Response", "response": "We sincerely thank you for your thorough reading and insightful comments. We have provided a point-by-point response to each of your concerns.General comment: The authors should have included a few more genes known to upregulated after UV exposure in both keratinocytes and melanocytes.We have included the induction of additional genes including tyrosinase in four human neonatal skin samples. We determined that tyrosinase gene expression was induced following UV radiation in half of the skin samples suggesting the model with our culture conditions may be appropriate to study other genes. General Comment: The authors should have looked at levels of IL1 and TNF alpha to support their hypothesis that BD3 increases as a result of wound healing.We have included the gene expression data for TNF alpha following UV radiation and determined that its expression correlates with BD3 expression. General Comment: Is it possible that the neonatal skin explants behave differently than the adult skin explants?Yes, this is a possibility, however as our study was limited to neonatal explants, we cannot directly address it with data. However, we thank the reviewer for this comment and have raised this caveat in our revised discussion." } ] }, { "id": "7161", "date": "20 Jan 2015", "name": "Pamela Cassidy", "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\nThe authors describe their studies of the effects of UV-irradiation on the expression of beta defensin on human neonatal foreskins treated and cultured in vivo. After finding variable responses between donors which did not correlate with skin phototype, they investigated the possibility that beta defensin expression was affected by culture conditions. They found that there were no differences between mock- and UV-irradiated samples. We have seen similar changes in genes of interest in the mock-treated tissues using ex vivo skin cultures. This study is valuable in that it highlights the need to include proper controls in this system. I have several additional minor comments to make about the manuscript and data:The protocol for mRNA isolation notes that tissue is ground in trizol and RNA is purified from the aqueous layer. There will not be two layers until chloroform is added and this should be included in the protocol. Figure 1 claims to represent degree of skin pigmentation +/- SEM but there are no error bars. The qPCR data shows Ct values as high as 32.9 for beta defensin, with almost all of the time=0 samples above 29. Many qPCR assays are not linear in this range. Did the authors do an examination of the performance of their primers in this range? Although these high Ct values make me a little skeptical about the absolute values of the fold change reported in figures 2 and 5, I am nevertheless persuaded that the differences between mock- and UV-treated samples is negligible as the authors conclude.", "responses": [ { "c_id": "1223", "date": "19 Feb 2015", "name": "John D'Orazio", "role": "Author Response", "response": "We sincerely thank you for your thorough reading and insightful comments. We have provided a point-by-point response to each of your concerns.Methods: The protocol for mRNA isolation via Trizol is not complete.We have expanded our methods to include these details. General Concern: Figure 1 claims to represent degrees of skin pigmentation +/- SEM but there are no error bars.We have expanded our methods to be more specific with how the measurements were determined. The degree of pigmentation for each skin sample was determined three times by colorimetry measurement. Neonatal skin samples were, as a rule, fairly uniform in their pigmentation. Their homogeneity between measurements therefore resulted in very small standard error of the mean values." } ] } ]	1	https://f1000research.com/articles/3-288
https://f1000research.com/articles/4-47/v1	19 Feb 15	{ "type": "Method Article", "title": "Procedure and datasets to compute links between genes and phenotypes defined by MeSH keywords", "authors": [ "Erinija Pranckeviciene" ], "abstract": "Algorithms mining relationships between genes and phenotypes can be classified into several overlapping categories based on how a phenotype is defined: by training genes known to be related to the phenotype; by keywords and algorithms designed to work with disease phenotypes. In this work an algorithm of linking phenotypes to Gene Ontology (GO) annotations is outlined, which does not require training genes and is based on algorithmic principles of Genes to Diseases (G2D) gene prioritization tool. In the outlined algorithm phenotypes are defined by terms of Medical Subject Headings (MeSH). GO annotations are linked to phenotypes through intermediate MeSH D terms of drugs and chemicals. This inference uses mathematical framework of fuzzy binary relationships based on fuzzy set theory. Strength of relationships between the terms is defined through frequency of co-occurrences of the pairs of terms in PubMed articles and a frequency of association between GO annotations and MeSH D terms in NCBI Gene gene2go and gene2pubmed datasets. Three plain tab-delimited datasets that are required by the algorithm are contributed to support computations. These datasets can be imported into a relational MySQL database. MySQL statements to create tables are provided. MySQL procedure implementing computations that are performed by outlined algorithm is listed. Plain tab-delimited format of contributed tables makes it easy to use this dataset in other applications.", "keywords": [ "ontology", "medical subject headings", "MySQL", "annotation", "phentypes" ], "content": "Introduction\n\nUnderstanding molecular mechanisms underlying both normal cellular processes and disease-causing gene perturbations has numerous applications in clinical diagnostics, personal genomics and engineering1–5. Most of the genomic studies address two major questions: (i) What genomic and molecular markers are associated with an observed phenotype? (ii) What molecular mechanisms lead to that phenotype in the studied organism? Answering these questions and uncovering gene-phenotype relationships mostly relies on experimental research that has already generated very large amounts of high-throughput data stored in public databases6–10. New knowledge about genes and their functions is acquired all the time based on a constant gathering of genomic data. To date there are more than 1500 databases hosting various types of genomic and molecular biology data11 acompanied by increasing number of research publications analyzing newly-generated data12. For this reason integrative algorithms to analyze high-throughput data by mining genomic databases and literature are in the focus of intensive research resulting in many publicly available bioinformatics tools for biologists and clinical researchers6,13–19.\n\nBiologists analyze lists of genes to dissect individual or collective gene involvement in the biological function that is being investigated, for example:\n\nfunctions of differentially expressed genes identified in a microarray or RNA-Seq experiment;\n\nrelationships between a biological process of interest and target genes regulated by a transcription factor identified by ChIP-Seq experiment;\n\ncausative relationships between functions of genes found in a chromosomal deletion or duplication identified in a patient and a clinical phenotype of the patient;\n\nidentifying candidate genes from gene lists in literature and databases.\n\nFinding meaningful relationships between genes in a large list and a phenotype by manually reviewing the literature and genomic databases is very laborious and time-consuming. Efforts to automate this process mostly have been directed towards the prioritization of human disease genes20,21 and less for model organisms and general phenotypes10. Gene prioritization tools, that can be used to infer relationships between genes and phenotypes, differ from each other with respect to computational algorithms and data sources used in prioritization21–23. In computations, a definition of a phenotype will determine the rules by which the algorithm will mine available data resources to retrieve gene-phenotype links.\n\nThe definition of a phenotype widely accepted in biology is “the observable trait or the collection of traits of an organism resulting from the interaction of the genetic makeup of the organism and the environment” meaning different things in different contexts24,25. In medicine the phenotype often refers to disease or abnormality26. In cellular contexts measurable cellular phenotypes are represented by features of cells such as the morphology (shape, size), the behavior (motility, growth), the developmental stage, the expression of specific genes and the rate of bio-chemical reactions8,27.\n\nSpecific vocabularies of phenotype terminology are implemented as ontologies containing concepts, the relationships between the concepts and the definitions of both28,29. Specialized phenotype vocabularies are available for model organisms30,31, life sciences32–36 and human diseases37,38. Phenotype can also be defined as a subset of genes known to be functionally related to the phenotype of interest, usually used in gene prioritization algorithms23,39. However, if the phenotype of interest hasn’t been well studied and does not have genes linked to it, then it is difficult or even impossible to use this approach.\n\nTerms of Medical Subject Headings (MeSH) vocabulary can serve as appropriate phenotypic descriptions38. MeSH terms are curated and are assigned to the articles in PubMed to adequately reflect the content of each article since they are meaningfully associated with the biological processes that they denote. Phenotypes in Mammalian Phenotype Ontology (MPO) used in Mouse Genome Informatics (MGI) database40 are also mapped to MeSH terms.\n\nGene prioritization tools have to establish links between genes and phenotypes by use of some algorithm. Several overlapping categories of tools can be distinguished based on how a phenotype is defined: by training genes known to be related to the phenotype; by keywords and tools designed to work with disease phenotypes. Table 1 lists maintained prioritization tools from these categories that are frequently cited in GoogleScholar. Algorithms defining phenotypes by training genes in prioritization evaluate similarity between training genes and candidate genes. Supervised machine learning (most often kernel methods) are used in this category of tools39,41. Algorithms describing phenotypes by keywords usually use frequencies of gene-associated documents that have keyword matches. Majority of algorithms are designed to prioritize genes with respect to disease phenotypes defined by either the keywords or the training genes or by both.\n\nEndeavour. In Endeavour the phenotype is defined by the training genes. It builds a phenotype model using different sources of genomic information derived from the training genes. Endeavour data sources consist of gene annotations, gene sequences, expressed sequence tags over multiple conditions, protein-protein interaction data and known transcription factor binding sites. The program works with the genes of human, mouse, rat, fly and worm organisms. It builds the model of the phenotype using information of the training genes in each of the genomic sources. It ranks the candidate genes according to how well they compare with the built model. Individual rankings in the Endeavour are combined by the order statistics42. In the table of the ranked genes the explanations are provided about the genes.\n\nToppGene. The candidate gene prioritization is one of the functions provided by the ToppGene tool. The user submits a set of training genes and a set of the test genes. The ToppGene first finds the significantly enriched annotations for the training genes in multiple data sources: GO annotations, literature, Interaction, Pathway, human and mouse phenotype data, TF binding sites, Cytobands, Co-expression Atlas, Drugs, microRNA and more. The candidate genes are ranked by the similarity of their functional annotations to the enriched annotations in the training genes. The similarity is computed as fuzzy-based measure54 or Pearson correlation coefficient. The user can examine the genes and the enriched terms of the the training set.\n\nGeneWanderer. In GeneWanderer the candidate genes are retrieved from the genomic region given the genomic coordinates. The phenotype is defined either by the disease keyword or by the list of the training genes known to be related to the phenotype. If the phenotype is defined by the keyword then the known genes associated with it are retrieved. The tool measures the distance between the candidate genes and the training genes in the protein-protein interaction network. The tool is specific to the human diseases.\n\nPolySearch. PolySearch allows queries in the form of: Given X find all Y. X and Y can be diseases, tissues, cell compartments, gene/protein names, SNPs, mutations, drugs and metabolites. If the phenotype is defined by keywords, then PolySearch retrieves the documents matching all keywords in the Pubmed, OMIM, DrugBank, Swiss-Prot, Human Mutation Database, Genetic Association Database and Human Protein Database. The ranked list of requested biomedical entities that are associated with the text of the query is returned. The score of the entity is proportional to the number of document matches in the databases. User can browse the results and examine the matching publications and sentences.\n\nPosMed. Positional PubMed is the semantic engine that ranks biomedical entities by the statistical significance of the associations with the provided keywords. The strength of the associations between biomedical entities and keywords is based on the number of the documents they share. The document categories comprise PubMed (PubMed titles, abstracts and MeSH terms), REACTOME (Pathway information from REACTOME), Protein-protein interaction (Protein-Protein Interactions in Human and Mouse from IntAct and Arabidopsis from AtPID), Gene ontology Human disease ontology Mammalian phenotype ontology Microarray based co-expression data for Arabidopsis. Given the keyword defining the phenotype and the type of biomedical entity to score (either gene or metabolite or drug) the PosMed returns list of the scored entities linked to the phenotype, sorted according to the strength of the connection between them. The PosMed supports human, mouse, rat, arabidopsis and rice organisms. The user can browse through all documents of the established links.\n\nG2D. In G2D the disease phenotype is defined by the OMIM identifier which is mapped to the associated MeSH terms of the diseases. The candidate genes are selected from the provided genomic region possibly containing a marker associated with the disease phenotype. G2D establishes a chain of evidence connecting the disease phenotype to the genes by forming the links between the terms in MeSH and GO annotations. The MeSH terms of the disease (category C) are linked to the MeSH terms of the chemicals and drugs (category D) which are linked to the Gene Ontology annotations. The connections between the terms are established by computing the normalized frequency of PubMed documents in which the MeSH terms (C and D) occur together. The connection between the protein GO annotations and the MeSH D terms are established by computing the normalized frequency of cooccurrence of GO and MeSH D term in papers supporting experimental evidence for the GO annotation. The GO annotation is weighted by a combined score which is used for ranking of the candidate genes.\n\nThis inference is illustrated in Figure 1 through the example of exploring candidate genes associated with cleft lip phenotype. Association between the cleft lip and the rs987525 variant from region 8q24.21 has been replicated independently in several different populations55 but no associated gene was found. G2D suggests the MYC gene as candidate. The link between this gene and the cleft lip disease phenotype is inferred through the relationship between the terms “Craniofacial abnormalities” and “Homeodomain proteins” and the relationship between the later term and the GO annotation a “Sequence specific DNA binding transcription factor activity” of the MYC gene. The MYC gene is regulated by the CTCF transcription factor56 which has a binding site at the genomic location of rs987525 leading to a possible hypothesis that this SNP marker might be linked to the cleft lip through a regulatory interaction with the MYC gene57. Another connection between the BMP4 gene and cleft lip OMIM phenotype is inferred through the relationship between the “Cleft lip” term and the term “Bone Morphogenetic Protein 4” which is related to the GO annotation “BMP signalling pathway”. The BMP4 gene harbors the rs1957860 marker variant which is known to be associated with cleft lip58.\n\nGene prioritization algorithms produce lists of the best candidates which are most strongly related to the phenotype of interest according to the criteria set by the algorithm. Rankings are based on evidence scores of relationships computed by the prioritization algorithm for each candidate gene. For generation of meaningful hypothesis it is important to know what factors led to the obtained rankings and links established between genes and phenotypes. In methods relying on phenotype definitions by training genes a detailed examination of such evidence is difficult. In multipurpose systems such as PolySearch and PosMed provided evidence lacks specificity. Most comprehensive in this respect is G2D in which OMIM phenotype is translated into adequate MeSH term of the disease. In this study an attempt is made to develop means to support computations linking genes and phenotypes defined by the MeSH terms extending beyond the diseases and building upon the algorithmic principles of G2D43,59.\n\n\nMethods\n\nIt was shown in applications of Arrowsmith algorithm that biomedical knowledge can be discovered through finding hidden links between concepts in scientific literature. The concepts, co-occurring at high frequency in two disparate sets of literature articles, indicated meaningful links60,61. The link suggested that fish oil can reduce Raynaud’s syndrome symptoms, later confirmed experimentally62. An inference leading to this result was “fish oil reduces blood viscosity, platelet aggregations and vascular re-activity which are increased in Raynaud’s syndrome”63. In similar way algorithms, based on linking the concepts or entities in the collections of data, relate genes to phenotypes by using concept co-occurrences in literature and controlled vocabularies43,64.\n\nLinks between phenotypes and gene GO annotations can be computed through intermediate links with chemicals as shown in G2D59. It is hypothesized that phenotype defined by the MeSH term can be meaningfully related to a subset of MeSH D terms denoting molecular entities of drugs and chemicals. Similarly, gene functions encoded by GO annotations can be meaningfully related to molecular entities denoted by MeSH D terms through related chemical processes affecting gene functions. Strengths of relationships can be derived from information in annotations of PubMed articles and NCBI datasets gene2go and gene2pubmed65. Figure 2 outlines the idea of the algorithm in which a phenotype and GO annotations are linked through chemicals.\n\nLet us denote MeSH D terms pertaining to chemicals by dj, j = 1, …, N. A relationship m(phenotype, chemical) between the phenotype defined by MeSH term g and the chemical defined by MeSH D term dj is denoted by m(g, dj). Let us denote a relationship m(chemical, GO annotation) between the MeSH D term dj of chemical and GO annotation goi, i = 1, …, M by m(dj, goi). Values of the m(g, dj) and m(dj, goi) relationships represent strengths of the connections between terms. The strengths of connections between the phenotype g and GO annotation goi passing through the chemicals dj, j = 1, …, N are computed as wgoi = maxj (m(g, dj) × m(dj, goi)). These computed weights express the strength of association between the functional annotation goi and the phenotype of interest. Table in a bottom panel of Figure 2 illustrates one possible way to order annotated genes by the magnitude of weights of their association to the phenotype of interest. Principles underlying the algorithm to compute strengths of relationships m(phenotype, chemical) and m(chemical, GO annotation) between phenotypes and functional gene annotations can be founded on fuzzy set theory (FST)43,66. Using mathematical framework of FST the relationships are defined as fuzzy binary relationships (FBRs) and can take a variety of forms67. A thorough explanation can be found in68 on pages 69–84.\n\nLet us denote phenotype MeSH terms as gj, j ∈ (1 … NG) in which j refers to a particular MeSH term. Similarly, let us denote MeSH D terms by dk, k ∈ (1 … ND). A subset of PubMed articles annotated by a specific gj term is denoted by Gj. Similarly, a subset articles annotated by a particular term dk is denoted by Dk. A fuzzy binary relation RGD between two MeSH terms (gj, dk) is defined as:\n\n{[(gj, dk), mgd (gj, dk)] \| (gj, dk) ∈ GNG × DND}, (1)\n\nwith membership function\n\n\n\nThe brackets \| · \| in Equation 2 denote the cardinality, a number of elements in a set, of an intersection \|Gj ∩ Dk\| of the two sets. The intersection represents a set of the articles annotated by both the gj term and the dk MeSH D term. FBR in Equation 1 is defined on all pairs of selected annotations in the universe of all articles annotated by those MeSH terms. The membership function in Equation 2 models a degree of inclusion of a narrower concept dk (chemical) into a broader concept gj (phenotype) dk ⊆ gj. The FBR of inclusion in a quantitative way defines a semantic relationship between meanings of the broader and narrower concepts69,70.\n\nInclusion relationship between GO annotations and MeSH D terms is defined using a universe of genes instead of articles. Let us denote GO annotations by goi, i ∈ (1 … NGO) in which i refers to a particular annotation. NGO is a total number of GO annotations of genes in gene2go. Let us denote by GOi a subset of genes in gene2go annotated by a particular goi. Let us denote by GDk a subset of genes in gene2pubmed associated with articles, annotated by the MeSH D term dk ∈ (1 … NGD), where NGD is total number of MeSH D terms associated with genes through articles. Fuzzy binary relation RDGO between these terms is defined as:\n\n{[(dk, goi), mdgo (dk, goi)] \| (goi, dk) ∈ GONGO × GDNGD}, (3)\n\nwith membership function\n\n\n\nThe degree of connection between GO annotation and MeSH D chemical in Equation 4 is determined by a number of genes sharing these two annotations over a number of genes annotated by that GO.\n\nA relationship between GO annotation and phenotype defining MeSH term is computed by applying maximum composition operation RGD ○ RDGO on fuzzy binary relations defined by Equation 1 and Equation 3 resulting in a following FBR:\n\n{[(gj, goi), max(mgd(gj, dk) * mdgo (dk, goi))]\n\n\| goi ∈ GONGO, dk ∈ (DND ∩ GDNGD), gj ∈ GNG}. (5)\n\nMySQL database and SQL procedures were created in order to experiment with and to support outlined inference70. The created datasets are limited to the annotated genes of human, mouse and fly organisms. The MeSH terms (mtree 2012) defining phenotypes are provided for the categories of Anatomy (A), Diseases (C), Drugs and chemicals (D) and Biological processes and phenomena (G). Information in the created datasets is as of September 2013.\n\n\nResults\n\nIn this section three contributed data sets are described. These datasets and presented MySQL procedure support computations of links between phenotype and GO annotations outlined in a previous section. Datasets were created by using NCBI E-utilities71 and custom scripts. The data sources (as of September 2013) used to create these datasets are described in Table 2. MeSH terms of category B are present but are not used to define phenotype in computation. The datasets are submitted in a format of tab delimited tables that can be imported into MySQL database or used as plain data. In this work a presented data management framework is based on MySQL.\n\nThe mesh_terms table stores associations between MeSH terms defining phenotype and MeSH D terms defining chemicals. Statements to create this table in MySQL database are presented in Table 3. Each row stores a pair of MeSH term (category A,C,D and G used to define phenotypes) and a MeSH D term defining a chemical and their relationship as defined by Equation 1 and Equation 2 with supporting information. Data in this table are based on annotated PubMed content as of September 2013. Meaning of columns in mesh_terms table is as follows:\n\nmid is unique identifier of a row;\n\nmterm is MeSH term in which spaces are replaced by underscores (for example Cell_Fusion);\n\ndterm is MeSH D term in which spaces are replaced by underscores (for example BMP4_Protein);\n\ndscore is a float number representing a strength of connection between mterm and dterm computed as in Equation 2;\n\nnm number of PubMed articles annotated by mterm;\n\nnd number of PubMed articles annotated by dterm corresponding to \|Ddterm\| in Equation 2;\n\ninters number of PubMed articles annotated by both mterm and dterm corresponding to \|Gmterm ∩ Ddterm\| in Equation 2;\n\nunio number of PubMed articles annotated by either mterm or dterm or both;\n\npmids comma separated list of PMID identifiers of PubMed articles that are the inters articles;\n\ndtid numerical key identifying dterm in another table dterm_go.\n\nThe dterm_go table stores associations between MeSH D terms defining chemicals and gene ontology annotations of genes. This table was created by custom scripts from NCBI gene2go and gene2pubmed datasets. These datasets can be found on NCBI ftp site ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/.\n\nThe gene2go dataset stores pairs of gene and its GO annotation. Annotations and genes of human, mouse and fly were retrieved. The gene2pubmed dataset stores pairs of genes and PMIDs of articles associated to them. From this datset pairs of genes and MeSH D annotations of their associated articles were retrieved. These intermediate datasets were used to create the dterm_go table. Statements to create this table in MySQL are presented in Table 4. Meaning of the columns in the dterm_go table is as follows:\n\ndterm is MeSH D term in which spaces are replaced by underscores (for example BMP4_Protein);\n\ngoterm is identifier of GO annotation in the Gene Ontology (for example GO:0000001 is identifier for annotation “mitochondrion inheritance”);\n\ngscore is a float number representing a strength of connection between goterm and dterm computed according to Equation 3 and Equation 4;\n\ngogenes number of genes (of mouse, human and fly) annotated by goterm as was recorded in gene2go dataset in NCBI ftp repository corresponding to \|GOgoterm\| in Equation 4;\n\ngenenum number of genes (of mouse, human and fly) sharing the goterm and dterm annotations corresponding to\| GDdterm ∩ GOgoterm\| in Equation 4;\n\ngenetot number of genes (of mouse, human and fly) associated with articles annotated by dterm recorder in gene2pubmed dataset in NCBI ftp repository;\n\ngenes comma separated list of Entrez Gene identifiers of genes that form genenum genes;\n\nid unique identifier of the row;\n\ndtid numerical key identifying dterm in table mesh_terms.\n\nTable go_terms stores description information of gene ontology annotations. Statements to create this table in MySQL are presented in Table 5. Meaning of the columns in the dterm_go table is as follows:\n\ngokey is a unique row identifier;\n\ngoterm is an identifier of GO annotation in the Gene Ontology (for example GO:0000001 is identifier for annotation “mitochondrion inheritance”);\n\ndescription is a description of GO annotation in the Gene Ontology in which spaces are replaced by underscores (for example mitochondrion_inheritance is description of GO:0000001 identifier);\n\ncategory is an indicator of a category of GO annotation and can take value of “Process”, “Function” or “Component”.\n\nFigure 2 and Equation 1, Equation 3, Equation 5 outline a possible way of establishing links between phenotypes defined by MeSH terms and GO annotations that pass through chemicals. These links can be computed by MySQL statements given that MySQL database tables were created as shown in Table 3, Table 4, Table 5. The suggested procedure is presented in Table 6. Three input parameters queryterm, dfrac, gofrac can be provided to the procedure mesh_to_go.sql. This MySQL procedure has a computation and an output part. The parameter gueryterm provides a MeSH term that defines phenotype. 16914 MeSH terms from 2012 MeSH edition72 can be queried in current implementation. These terms form pairs with 5908 MeSH D terms of chemicals. Textual fields of all MeSH terms and GO annotations have underscores instead of spaces between words that should be used in formulating queries.\n\nParameters dfrac and gofrac set thresholds on the corresponding dscore and gscore values. They can be used to filter terms in computation based on strengths of relationships between the phenotype and chemical and between the chemical and GO annotation (disallowing weaker relationships). Value of dfrac can vary in range of [0.0000041, 1] which is a range of dscore values. Value of gofrac can vary in interval of [0.0001, 1].\n\nIn computation presented in Table 6, a creation of t2 and t3 tables corresponds to performing a maximum composition operation defined by Equation 5. The table t2 contains all relationships between the phenotype in queryterm and GO annotations passing through all chemicals that have a connection to the queryterm phenotype. Fewer GO annotations with only maximum weight in relationship to the phenotype are selected into table t3. Statements in the output part create plain sectioned text file. Weighted GO annotations are listed in the first section. Second section identified by “list_of_all_links_go_dterms”, lists all connections in the table t2.\n\nFor example, a command line query for phenotype “Intellectual Disability” (user X and database Xdb) can be executed in a following way:\n\n\n\nThe first output file id_out section will have rows:\n\n\n\nThese GO annotations are weighted by strength of their relationship to the “Intellectual Disability” phenotype. These weighted GO annotations can be used to rank genes as in Figure 2. Second section of output details relationships between the phenotype and chemicals and between the chemicals and GO annotations, for example considering information on GO:0045362:\n\n\n\nA connection between “Intellectual Disability” phenotype and MeSH D term “Interleukin-1 Receptor” is quantified by dscore which equals to 0.0714286. Strength of connection between this chemical and “positive regulation of interleukin-1 biosynthetic process” GO term equals to 1. These two values determine the weight ms of this GO term in connection to “Intellectual Disability” (ID) phenotype. This computational procedure with respect to ID phenotype was previously explored73.\n\n\nUse case\n\nIn cancer genes accumulate a large number of mutations74 and next generation sequencing screening may produce a vast number of genetic variants and genes. If a gene harboring a variant was not previously reported, then outlined computation can be used to explore connections of that gene to specific cancer based on a current available knowledge.\n\nAmong highly mutated genes identified by the whole genome and exome sequencing of breast tumors are PIK3CA, TP53, GATA3, CDH1, RB1, MLL3, MAP3K1 and CDKN1, which were previously observed in clinical breast cancer tumors75. Genes not previously observed in those tumors were TBX3, RUNX1, LDLRAP1, STNM2, MYH9, AGTR2, STMN2, SF3B1. Both sets of genes were explored in relation to “Breast Neoplasms” by ranking genes in whole human genome. The genes were ranked by magnitudes of weights of their annotations with respect to relationship to “Breast Neoplasms” as depicted in Figure 2 employing the outlined computational principle. Top genes from those sets appearing within the Top 5% of the ranked human genome and closer to this interval are listed in Table 7.\n\nCancer genes are well characterized and widely studied in literature. The genes, not previously reported as carrying clinically important mutations in studies of breast cancer in75 had stronger links with cancer phenotype in question through their GO annotations. The genes from that study: RB1, GATA3, TP53 and CDH1 appeared in high ranking positions. Current exploration identifies “Tamoxifen” being strongly related to the breast cancer phenotype. Such link is logical because this chemical is used to treat hormone-sensitive tumors. Applied computational procedure through relationships between phenotypes and chemicals helps to explore contexts in which biological processes of interest take place. Unexpected links may be discovered that may help to formulate novel biological hypothesis.\n\n\nDiscussion\n\nAs of now, biologists still find it challenging to interpret large lists of poorly characterized genes with algorithms, which are somewhat limited in terms of how they define phenotypes. These lists may originate from a variety of sources, including microarray experiments, ChIP-Seq experiments identifying transcription factors’ target genes, and scientific literature. The algorithm described here is useful in formulating biological hypotheses in situations in which little is known about the phenotype and the genes in question. The algorithm begins by linking lists of gene GO annotations to phenotypes (non-disease and disease) described by meaningful keywords through the MeSH and PubMed databases. The algorithm then deduces which of the links between the genes and phenotypes are strongest and presents the results in an organized manner. This is different from most of existing algorithms in terms of the methods used to define phenotypes of interest and infer their relationships with genes. To better understand how the outlined algorithm is unique, the existing algorithms are parsed into three categories overlapping at some extent and examined.\n\nThe first category of existing algorithms only uses known phenotype-related genes, or training genes, while the second focuses solely on human disease phenotypes. The third category uses general keywords from literature to define phenotypes. All must deduce how genes and phenotypes are related by mining selected information sources, retrieving and integrating data from them, but there are some differences between them. Each will be discussed in turn.\n\nAlgorithms based on the use of training genes known to be related to the phenotype of interest, as in the Endeavour39 and ToppGene45 tools, make prioritizations on the basis of pattern classification76. Training genes extract phenotype-defining information from various data sources based on how similar the phenotype is to the training genes, and then build a model of a phenotype based on this extraction42. In other words, the model represents gene features that are most characteristic of that specific phenotype. The candidate genes are ranked by how similar their features are to the features of the model. For example, Endeavour relies on genomic data fusion from multiple information sources41. This tool may be very useful if the properties of the training genes clearly define the phenotype properties of interest in the organisms being investigated. Knowing these properties, one can characterize candidate genes by comparing them to the training genes. Genes that have similar characteristics to the training genes may also play an important role in previously unknown phenotype expressions. This principle of discovery is known as “guilt by association”77. Although very useful in detecting similarities between candidates and training genes, the integration of data from multiple sources has limitations. The main limitation is in existing schemes of combining the information from different sources to rank the candidates41. First, the prioritization algorithms using training genes generally differ with respect to the data sources they use23. Different information sources of training genes lead to different models and similarity metrics. Second, some data sources do not have complete information on some genes, so if the phenotype in question has not been sufficiently studied and there are no genes known to be associated with it, then the training genes approach is not effective. Third, the training genes might represent a heterogeneous group biasing phenotype definition in some way. For example, the data fusion scheme relies on the independence of information sources about gene properties, but in practice they are not entirely independent. Protein-protein interaction databases, the gene interaction databases and gene ontology refer to scientific publications as supporting evidence for the information they store, and might even be derived from the literature. While it should do so, the scheme does not always account for these possible interactions and overlaps between sources.\n\nMany tools specific to human diseases use phenotype definitions from databases47,59. Because human disease phenotypes have been extensively studied and are well represented by OMIM78, and because they contain structured information suited to uncovering meaningful links between human diseases and genes, it is relatively easy to associate genes with said phenotypes. However, phenotypes other than diseases and phenotypes in Mammalian Phenotype Ontology37 are not yet represented by well-structured and information-rich resources33,34.\n\nThe third category of algorithms, which use general keywords from literature to define phenotypes, are exemplified by tools such as PosMed, PolySearch, GeneProspector and CANDID48,51–53. These tools rely on finding matching documents in MEDLINE or locally-created databases, and then associating genes with the matching documents. General purpose discovery-oriented systems such as iHOP79, Anni2.080, Arrowsmith60,61 and PosMed52, use conceptual networks. Users can browse through the network and create textual profiles describing genes, proteins, or other biomedical concepts. However, once again, there will be genes and processes that are not well represented in literature and there is little information about them that can be retrieved.\n\nThus, while the obvious advantage offered by specialized gene information databases is that specific information can be extracted very quickly, complementing the literature with more information sources for gene prioritization is advantageous in allowing potential use of algorithms that offer novel interpretations of existing information. G2D43,59 is the only existing method which provides underlying ideas for the algorithmic approach outlined here, which prioritizes genes with respect to human disease phenotypes. However, the scope of the application of the developed algorithm to link genes with phenotypes70 outlined here is distinct from G2D by contributing the following:\n\nThe outlined algorithm establishes meaningful links between genes and phenotypes, and enables prioritization, beyond human disease phenotypes by using concepts of MeSH vocabulary from the categories A, D, and G.\n\nThe proposed algorithm can be applied beyond human organisms as the annotated genes of the entire genome for human (Homo sapiens), mouse (Mus musculus) and fly (Drosphila melanogaster) are used. In contrast, G2D focuses on human genes.\n\nThe data in the gene2go and gene2pubmed NCBI databases65 are used to link GO annotations to MeSH terms of Drugs and Chemicals describing the molecular entities. In contrast, G2D works with the RefSeq database for this purpose43.\n\nThe outlined algorithm similarly to G2D utilizes fuzzy binary relationships between concepts, based on mathematical operations of fuzzy set theory67,68, to infer gene-phenotype links. G2D uses a similarity relationship43 while this algorithm uses an inclusion relationship70.\n\nThe outlined algorithm is an attempt to remedy some of the challenges presented by information shortages and the way existing algorithms described above are configured to define phenotypes and determine relationships. It has important advantages compared to the other gene prioritization algorithms, in addition to G2D, reviewed extensively in Introduction section.\n\n\nConclusions\n\nThe approach to link genes and phenotypes outlined in this work represents one out of existing possible approaches. Contributed datasets opens possibility to experimentation and development of other applications. These datasets, although in need of updating comprise co-occurrences of selected categories of MeSH terms in PubMed and co-occurrences of MeSH D terms with GO annotations created from NCBI Gene datasets. Availability of such offline data saves time of a researcher who may want to explore and apply text and data mining algorithms to analyze relationships between concepts.\n\nExisting tools provide limited explanations for reasons for phenotype gene association. Using the outlined approach, evidence supporting the obtained strongest links can be easily examined. As a result of inference, the MeSH D terms which are most strongly related to both the candidate genes through their GO annotations and phenotype are identified. This is useful as it reveals the physical background domains related to the candidate genes gleaned from associated articles without reading their full text. The availability of this background information opens up the possibility of identifying and examining unique aspects of the functions of the studied genes.\n\nHowever, a single information source cannot account for all aspects of gene relations to phenotypes even if MeSH vocabulary contains information about the processes, phenomena and phenotypes studied in literature. And while functional gene annotations are also associated with scientific publications, there will be genes and processes that are not well represented in literature, as stated earlier. In this situation inferring links between genes and phenotypes might be more effective using other information sources, as genes can also be characterized by their interactions with other molecular entities, by their sequences and by the information about the protein domains of the products. These gene properties can be retrieved computationally from other specialized databases.\n\n\nData availability\n\nF1000Research: Dataset 1. Table mesh_terms, 10.5256/f1000research.6140.d4316782\n\nF1000Research: Dataset 2. Table dterm_go, 10.5256/f1000research.6140.d4316883\n\nF1000Research: Dataset 3. Table go_terms, 10.5256/f1000research.6140.d4317684", "appendix": "Competing interests\n\n\n\nAuthor declares no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nPart of this work was performed during completion of graduate training at department of Cellular and Molecular Medicine (CMM), University of Ottawa. Author gratefully acknowledges University of Ottawa for their admission scholarship and thanks Dr. C. Perez-Iratxeta from CMM and affiliated Ottawa Hospital Research Institute for her advise on G2D and related discussions. Author thanks Ms. Lindsey Li from Carleton University, Ottawa for her help in improving English expressions.\n\n\nReferences\n\nShendure J, Aiden EL: The expanding scope of DNA sequencing. 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Reference Source\n\nNCBI Medical Subject Headings. yesMesh browser. . 2012\n\nPranckeviciene E, Pranculis A, Preiksaitiene E, et al.: Computational pipeline to analyze genomic variants with respect to clinical phenotypes by mining literature. Study of genomic regions related to intellectual disability. European Journal of Human Genetics. 2014; 22(Supplement 1): P16.48–M,p314. Reference Source\n\nRoukos DH: Integrated clinical genomics: new horizon for diagnostic and biomarker discoveries in cancer. Expert Rev Mol Diagn. 2013; 13(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nEllis MJ, Ding L, Shen D, et al.: Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature. 2012; 486(7403): 353–360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Bie T, Tranchevent LC, van Oeffelen LM, et al.: Kernel-based data fusion for gene prioritization. Bioinformatics. 2007; 23(13): i125–132. PubMed Abstract \| Publisher Full Text\n\nWang PI, Marcotte EM: It’s the machine that matters: Predicting gene function and phenotype from protein networks. J Proteomics. 2010; 73(11): 2277–2289. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamosh A, Scott AF, Amberger JS, et al.: Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Res. 2005; 33(Database issue): D514–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernandez JM, Hoffmann R, Valencia A, et al.: iHOP web services. Nucleic Acids Res. 2007; 35(Web Server issue): W21–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJelier R, Schuemie MJ, Veldhoven A, et al.: Anni 2.0: a multipurpose textmining tool for the life sciences. Genome Biol. 2008; 9(6): R96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValentini G, Paccanaro A, Caniza H, et al.: An extensive analysis of disease-gene associations using network integration and fast kernel-based gene prioritization methods. Artif Intell Med. 2014; 61(2): 63–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPranckeviciene E: Dataset 1 in “Procedure and datasets to compute links between genes and phenotypes defined by MeSH keywods”. F1000Research. 2014. Data Source\n\nPranckeviciene E: Dataset 2 in “Procedure and datasets to compute links between genes and phenotypes defined by MeSH keywords”. F1000Research. 2014. Data Source\n\nPranckeviciene E: Dataset 3 in “Procedure and datasets to compute links between genes and phenotypes defined by MeSH keywords”. F1000Research. 2014. Data Source" }	[ { "id": "7736", "date": "03 Mar 2015", "name": "Jason E. McDermott", "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\nMajor concerns:The process of ranking individual genes by their relationships as depicted in Figure 2 and in the Use Case (page 9) was very confusing and it was not apparent how the process of associating functions with phenotype could be mapped to genes of interest to the researcher. Greater care needs to be taken to describe this process (which is a somewhat complicated one) since this seems to be the main application of the method described. The last five sentences in the Abstract provide detail that is not necessary here but should be included in the main text instead. It would be very helpful to have a comparison of the results given by the method described and another method (likely one of those described in the paper) in terms of functions identified for the use case and ranking of genes given. This would not be a performance evaluation (since it is difficult to tell what the 'right' answer would be in this case) but would provide a nice comparison with previous methods.Minor concerns:The word \"cancer\" is misspelled in the title for the Use Case section. In the use case it is unclear where the list of \"Genes not previously observed in these tumors\" comes from. I would assume that it was genes that were associated with cancer in the referenced work, but hadn't been previously associated? This needs to be made clear.", "responses": [] }, { "id": "7738", "date": "27 Apr 2015", "name": "Emidio Capriotti", "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 article addresses one important issue in the field but misses the following important aspects that should be discussed:Most of the available annotation database such as GO, MESH are biased toward specific terms. Thus, the accuracy of the method could biased toward specific concepts and terms. The author should analyze the distribution of the m scores and associate the statistical significance calculated with respect to a background distribution. For the definition of phenotypes the Human Phenotype Ontology (HPO) database is the reference. The author should cite this database in their paper and use it as benchmark set for the predictions.3) A comparison with other methods should be provided.Minor:The description of similar methods previously developed is too long.", "responses": [] } ]	1	https://f1000research.com/articles/4-47
https://f1000research.com/articles/3-254/v1	27 Oct 14	{ "type": "Systematic Review", "title": "Malignant pleural effusions and the role of talc poudrage and talc slurry: a systematic review and meta-analysis", "authors": [ "Srinivas Mummadi", "Anusha Kumbam", "Peter Y. Hahn", "Anusha Kumbam", "Peter Y. Hahn" ], "abstract": "Background: Malignant Pleural Effusion (MPE) is common with advanced malignancy. Palliative care with minimal adverse events is the cornerstone of management. Although talc pleurodesis plays an important role in treatment, the best modality of talc application remains controversial. Objective: To compare recurrence rates, rates of respiratory and non-respiratory complications between thoracoscopic talc insufflation/poudrage (TTI) and talc slurry (TS). Data sources and study selection: MEDLINE (PubMed, OVID), EBM Reviews (Cochrane database of Systematic Reviews, ACP Journal Club, DARE, Cochrane Central Register of Controlled Trials, Cochrane Methodology Register, Health Technology Assessment and NHS Economic Evaluation Database), EMBASE and Scopus. Randomized controlled trials published between 01/01/1980 - 10/1/2014 and comparing the two strategies were selected. Results: Twenty-eight potential studies were identified of which 24 studies were further excluded, leaving four studies. No statistically significant difference in the risk of recurrent pleural effusions was observed between TS and TTI groups (RR 0.72; 95 % CI 0.50-1.05; Q statistic, 3.58). There was a higher risk of post procedural respiratory complications in the TTI group compared to the TS group (RR 1.91, 95% CI= 1.24-2.93, Q statistic 3.15). No statistically significant difference in the incidence of non-respiratory complications between the TTI group and the TS group was observed (RR 0.88, 95% CI= 0.72-1.07, Q statistic 4.61).Conclusions: There is no difference in MPE recurrence based on patient centered outcomes between talc poudrage and talc slurry treatments. Respiratory complications are more common with talc poudrage via thoracoscopy.", "keywords": [ "malignant pleural effusion", "palliation", "pleurodesis" ], "content": "Introduction\n\nMalignant Pleural Effusion (MPE) is a well described event in the natural history of advanced malignancy. Malignant etiology accounts for 22% of the diagnosed pleural effusions1. Using data from the 2012 Nationwide Inpatient Sample (NIS), Healthcare Cost and Utilization Project (HCUP) and Agency for Healthcare Research and Quality, it is estimated that the aggregate charges (the “national bill”) were 722 million dollars in the USA2.\n\nPalliation with minimal adverse events remains the cornerstone of management3. Talc pleurodesis and Indwelling Pleural Catheters (IPC) are the two most commonly used palliative approaches.\n\nTalc pleurodesis can be achieved either by thoracoscopic instillation i.e.; talc insufflation/poudrage (TTI) or via a bedside chest tube i.e. talc slurry (TS). Existing systematic reviews concluded that thoracoscopic talc insufflation/poudrage was more efficacious in preventing recurrences when compared to bedside chest tube talc slurry4,5. New prospectively designed studies comparing TTI and TS have been published since then6–8. However the best initial approach for talc pleurodesis remains still unclear. To address the need for an update, a systematic review and meta-analysis of studies comparing thoracoscopic talc insufflation/poudrage and talc slurry in terms of patient centered outcomes was performed.\n\n\nMaterials and methods\n\nWe conducted a systematic review with meta-analysis of studies undertaken between 01/01/1980 and 10/1/2014 using MEDLINE (PubMed, OVID), EBM Reviews (Cochrane database of Systematic Reviews, ACP Journal Club, DARE, Cochrane Central Register of Controlled Trials, Cochrane Methodology Register, Health Technology Assessment and NHS Economic Evaluation Database), EMBASE and Scopus. Unpublished data sets such as conference abstracts and ClinicalTrials.gov were also included in the full review phase to reduce the effect of publication bias9.\n\nThe following keywords were used: chemical pleurodesis, pleurodesis, talc pleurodesis, bedside pleurodesis, surgical pleurodesis, medical pleurodesis, thoracoscopic pleurodesis, thoracoscopic talc pleurodesis, thoracoscopic poudrage, thoracoscopic talc poudrage, talc insufflation, thoracoscopic talc insufflation, pleuroscopy, medical thoracoscopy, talc poudrage, talc slurry, tube thoracostomy, chest tube talc slurry and malignant pleural effusion. Both keywords and medical subject headings were used in a Boolean search strategy. An example search strategy can be found in the Appendix 1.\n\nIn addition, a pearl growing strategy was employed using frequently cited reviews of malignant pleural effusion treatments. They were included to be analyzed in the full review phase of the study. Approval from the Institutional Review Board was unnecessary because this is a meta-analysis.\n\nInclusion and exclusion criteria were framed prior to the implementation of the search strategy. To evaluate outcomes in adult malignant pleural effusion patients (18 + years) undergoing talc pleurodesis, we included studies based on the following criteria:\n\n1) A randomized design was used in studying talc pleurodesis in patients with malignant pleural effusion between 01/01/1980 and 10/1/2014.\n\n2) Patients undergoing bedside TS were compared with patients undergoing thoracoscopic talc insufflation/poudrage (TTI) in the above fashion.\n\n3) Sufficient outcomes data were reported [Risk of recurrence of the pleural effusion, respiratory complications and non-respiratory complications].\n\nNon-English publications, case reports and series, pediatric studies, descriptive studies without a control group, retrospective studies and prospective controlled studies without randomization were excluded. Eligible articles were reviewed by two reviewers for inclusion; disagreements were resolved via discussion. An examination of the full-length articles was carried with the intent of eliminating duplicate studies or same patient cohorts.\n\nTwo reviewers independently extracted and rated the data from the selected full length articles using a standardized form. From each study, the data abstracted included study name/year, study design (prospective controlled, randomized controlled trial, retrospective etc.), cancer cell type, patient inclusion criteria, sample sizes for the bedside/surgical pleurodesis arms, technique employed in the bedside/surgical arms and the follow-up schedule.\n\nOutcomes data pertaining to the risk of recurrent pleural effusions, respiratory complications, and non-respiratory complications were also extracted.\n\nTalc pleurodesis for recurrent malignant pleural effusion is a palliative procedure and does not aim to have a mortality benefit. Therefore, measuring mortality outcomes was not the focus of the meta-analysis.\n\nVarious definitions for recurrent pleural effusions have been used in prior studies6,10. A clinically significant recurrent pleural effusion was defined a priori as accompanied by symptoms or a need for repeat pleural procedures. Where possible, asymptomatic radiological recurrences were not included in the meta-analysis.\n\nRespiratory complications were defined as occurrence of respiratory conditions such as pneumonia, Acute Respiratory Distress Syndrome (ARDS), acute respiratory failure, re-expansion pulmonary edema, bronchospasm, empyema, pulmonary embolism, prolonged air leak, bronchopleural fistula, atelectasis requiring bronchoscopy and subcutaneous emphysema.\n\nImmediate non-respiratory complications were tabulated from the complications mentioned in the full length articles. These included fever, wound infection, chest pain, tumor recurrence at site, myocardial infarction, need for blood transfusions, arrhythmias and immediate post procedural death.\n\nThe randomized controlled trials that met inclusion criteria were evaluated for quality using components of the modified Jadad scale11. The presence of the following features was appraised:\n\nA) A description of the study confirming the randomized nature.\n\nB) Method of allocation to the study arms described and whether adequate/inadequate.\n\nC) Description of withdrawals and dropouts.\n\nDue to the nature of the comparison (surgical vs bedside procedure), we felt that other features of the scale (description of a double blind nature) could not be appraised during our quality assessment. Two raters independently determined the quality of the studies included. Disagreements were resolved by discussions and final consensus.\n\nOutcomes data for recurrent pleural effusions, respiratory and non-respiratory complications were summarized using descriptive statistics (simple count, proportion of the study sample). They were visually presented in Forest plots. The Mantel-Haenszel method12 was used to combine data from individual studies and the results were reported as pooled relative risks (RR). Heterogeneity among the studies included was investigated by performing the I2 test13. Meta-analyses were conducted using the fixed effects model when heterogeneity between studies was low (I2 <40%) and the random effects model otherwise9.\n\nTo confirm the robust nature of the results, a sensitivity analysis was performed by removing one study at a time and determining the outcome.\n\nPublication bias was examined by visually examining the filled funnel plots using trim and fill method. Other methods (Begg’s correlation14 and Egger’s linear regression intercept)15 were additionally used.\n\nAll analyses were performed using a statistical software package (Comprehensive Meta-Analysis, version 2.2.064; Biostat, Englewood, NJ).\n\n\nResults\n\nBased on initial search, 137 articles were obtained and reviewed independently by two reviewers. Pearl growing strategy was employed to seek additional articles and resulted in five articles. A clinical trial registry (www.ClinicalTrials.gov) was also examined and resulted in one additional article. These 143 articles were reviewed and 115 articles were excluded based on title and abstract. A total of 28 potential studies were thus identified with our search strategy. Twenty-four studies were further excluded, leaving four studies6,10,16,17 for the final analysis. The sequence describing the above process can be found in Figure 1.\n\nNone of the studies restricted the study population to a single cancerous cell type.\n\nNone of the included studies employed thoracoscopic evacuation of the malignant pleural effusion prior to bedside TS insertion via a chest tube.\n\nFollow-up periods varied through the studies (Range = 30–425 days). Where available, recurrence data for the most distal available time point were selected for the meta-analysis.\n\nAll of the studies included in the analysis underwent quality assessment. The average Jadad score11 was 1.5 out of a maximum possible score of 4 (Ranges 1–2). Out of the possible seven ways to assess the quality11, only four questions could be answered due to the nature of the intervention. It was not possible reliably or ethically for the original investigators to have carried out efficient blinding in a surgical versus bedside clinical experiment.\n\nThe results of the pooled RR are shown in Figure 2. The four studies included in this analysis enrolled a total of 454 patients with malignant pleural effusion (Table 1). There was no statistically significant difference in the risk of recurrent pleural effusions between the bedside TS (recurrences/patients who underwent pleurodesis, n/N = 51/218 pts) and the TTI groups (recurrences/patients who underwent pleurodesis, n/N = 39/236 pts, pooled RR 0.72; 95% CI 0.50-1.05; Q statistic, 3.58; I2 statistic, 16.27%). There was no evidence of publication bias (P-value = 0.49 for the Begg’s test, P-value = 0.54 for the Egger’s regression intercept). After using the trim and fill methodology (Figure 3), these results did not change (RR- 0.78, 95% CI = 0.54-1.12, Q statistic, 6.8).\n\nRR, risk ratio, CI, confidence interval.\n\nTTI, Thoracoscopic talc insufflation, Also known as Thoracoscopic talc poudrage\n\nTS, Talc slurry applied via a bedside chest tube\n\nRCT, Randomized Controlled Trials\n\nq- Every\n\nThe definitions of a recurrent pleural effusion in the included studies varied. One study defined the recurrence based on radiological data alone6. The remainder of the studies clearly mentioned the number of patients who were symptomatic and required further pleural procedures once a recurrent pleural effusion was diagnosed10,16,17.\n\nA sensitivity analysis pooling data from studies reporting only clinically significant recurrences was performed leaving out one study6. This did not result in a different statistical outcome (pooled RR 0.49; 95% CI 0.11-2.22; Q statistic, 3.52; I2 statistic 43.22%).\n\nAs follow-up periods varied widely, a sensitivity analysis pooling the data from studies reporting 30 day outcomes was carried out and did not result in a different statistical outcome.\n\nThe results of the pooled RR are shown in Figure 4. The four studies included in this analysis reported outcomes on a total of 591 patients who underwent talc pleurodesis for palliation of malignant pleural effusion (Table 2).\n\nRR, risk ratio, CI, confidence interval.\n\nTTI, Thoracoscopic talc insufflation, also known as Thoracoscopic talc poudrage\n\nTS, Talc slurry via a bedside chest tube\n\nRCT, Randomized Controlled Trials\n\nN/A, Not available\n\nU.K, United Kingdom\n\nIV, Intravenous\n\nBP fistula, Bronchopleural fistula\n\nPE, Pulmonary Embolism\n\nThere was a statistically significant higher risk of post procedural respiratory complications in the TTI group (Incidence of respiratory complications/Pts who underwent pleurodesis, n/N = 59/307 pts) compared to the TS group (Incidence of respiratory complications/Pts who underwent pleurodesis, n/N = 28/284 pts, pooled RR 1.91, 95% CI = 1.24-2.93, Q statistic 3.15, I2 statistic 4.79%). There was no evidence of publication bias (P-value = 1.0 for the Begg’s test, P-value = 0.24 for the Egger’s regression intercept). After using the trim and fill methodology (Figure 5), these results did not change (RR- 1.99, 95% CI = 1.30-3.04, Q statistic, 4.32).\n\nA sensitivity analysis pooling data from studies with ≥ 2 score on the Modified Jadad scale was performed leaving out one study with a score of 117. This did not result in a different statistical outcome (pooled RR 1.99, 95% CI = 1.29-3.08, Q statistic 2.05, I2 statistic 2.49).\n\nThe results of the pooled RR are shown in Figure 6. The four studies included in this analysis reported outcomes on a total of 591 patients who underwent talc pleurodesis for palliation of malignant pleural effusion (Table 3).\n\nRR, risk ratio, CI, confidence interval.\n\nTTI, Thoracoscopic talc insufflation, Also known as Thoracoscopic talc poudrage\n\nTS, Talc slurry applied via a bedside chest tube\n\nRCT, Randomized Controlled Trials\n\nRBC- Red Blood Cell\n\nMI- Myocardial Infarction\n\nDVT- Deep Venous Thrombosis\n\nThere was no statistically significant difference in the incidence of non-respiratory complications between the TTI group (Incidence of non-respiratory complications/Pts who underwent pleurodesis, n/N = 110/307 pts) and the TS group (Incidence of non-respiratory complications/Pts who underwent pleurodesis, n/N = 116/284 pts, pooled RR 0.88, 95% CI = 0.72-1.07, Q statistic 4.61, I2 statistic 34.96%). There was no evidence of publication bias (P-value = 1.0 for the Begg’s test, P-value = 0.48 for the Egger’s regression intercept). After using the trim and fill methodology (Figure 7), these results did not change (RR- 0.93, 95% CI = 0.76-1.12, Q statistic, 9.8).\n\nA sensitivity analysis pooling data from studies with ≥2 score on the Modified Jadad scale was performed leaving out one study with a score of 117. This did not result in a different statistical outcome (pooled RR 0.93, 95% CI = 0.76-1.14, Q statistic 0.06, I2 statistic 0.0).\n\n\nDiscussion\n\nMany experts believe that serial thoracentesis is not an ideal choice for treating the recurrent malignant pleural effusion18,19.\n\nTalc pleurodesis was first performed in 193520 and is still commonly employed in the treatment of malignant pleural effusions. Although studies have shown talc to be the best chemical agent in terms of pleurodesis success and risk of recurrence21,22, the best method of applying talc remains controversial. Our meta-analysis demonstrates that both talc poudrage (TTI) and talc slurry (TS) offer similar protection against clinically significant recurrences. There was no difference in the risk of clinically significant recurrence (i.e., further need for pleural procedures or symptoms). TTI did have a greater risk of respiratory complications. There was, however, no difference in the rate of non-respiratory complications such as fever and need for blood transfusions.\n\nOur results are in contrast to those of previous meta-analyses5, including the recently withdrawn Cochrane analysis which suggested improved success rates of talc pleurodesis utilizing TTI. The conclusion of these analyses was that thoracoscopic pleurodesis with talc was the optimal method for pleurodesis in patients with malignant pleural effusions. However, several newer prospective studies have been published since6,7 and have been incorporated into the present analysis.\n\nArguments in favor of TTI include the observation that there is more complete lung expansion after the procedure18. This is certainly understandable given that take-down of adhesions is typically performed during the procedure itself as opposed to TS. Interestingly, Terra et al. using CT scanning post-TTI and TS to assess degree of post procedure lung expansion did not find a correlation between clinical outcomes and initial degree of lung expansion7. These authors postulated that factors other than the degree of visceral and parietal pleura apposition were important in determining the success of pleurodesis. Similarly, Mager et al. using 99m Tc-labeled talc showed that rotation protocols did not affect the overall dispersion of talc suspensions after TS23. The degree of dispersion also did not affect pleurodesis success23.\n\nIn comparing TTI and TS, several difficulties arise. Pleurodesis success rates vary in the literature, due to the inconsistent definition of pleurodesis success and failure used in different studies. Failure or recurrence has been defined radiologically in some studies6 but it has been argued that patient centered outcomes such as new symptoms and need for further pleural procedures are more pertinent outcomes7. In our meta-analysis, we determined recurrence a priori as the development of symptoms or the further need for pleural procedures and disregarded asymptomatic radiological recurrences where possible.\n\nThe technique of both TS and TTI vary significantly between centers and this is evident in the included studies. TS varied in regard to length of chest tube clamping, rotating or not-rotating the patient, size of chest tube, and timing of chest tube removal. With regard to TTI, in three of the four studies TTI was performed under general anesthesia and the ability to tolerate general anesthesia was in fact an entry criteria. Overall, 88% of patients in our analysis underwent general anesthesia for TTI. One could argue that the increased respiratory complications observed with TTI may be related to general anesthesia and single lung ventilation. Despite the concerns of ARDS with the use of ungraded talc, the studies included in our meta-analysis did not report specific cases of ARDS. Non-specific respiratory failure was, however, reported in patients in the study by Dresler6 and Yim et al. reported a case of acute respiratory failure in the TS group16.\n\nWith the increasing numbers of interventional pulmonologists performing pleuroscopy (medical thoracoscopy)24 under local and/or moderate sedation, the question of which procedure is the most optimal for talc pleurodesis is increasingly relevant. Whether talc poudrage performed during pleuroscopy with local or moderate sedation and dual lung ventilation is equivalent to surgical thoracoscopy (VATS) in terms of pleurodesis success and complications is unknown. Further studies are needed to compare talc poudrage performed with pleuroscopy versus TS.\n\nOne may wonder whether the question of TTI versus TS is still relevant in the era of indwelling pleural catheters (IPCs). Certainly, in the patient with trapped lung, both TS and TTI would likely be ineffective and indeed all of the studies in this meta-analysis excluded patients with possible trapped lung physiology. In the patient with malignant effusion without trapped lung, however, clear superiority of IPCs has not been demonstrated3. In fact, talc pleurodesis may be more economical compared to IPC in patients with good performance status and projected life expectancy of > 6 weeks25,26. The issue of cost is especially relevant in the era of health care reform and accountable care organizations. With the advent of newer molecular/hormonal therapies especially in breast cancer, malignant pleural effusion is increasingly recognized as a non-terminal event27. Perhaps most importantly, patient preference is paramount28 and no study has clearly demonstrated the superiority of IPCs compared to talc pleurodesis.\n\nOur study has several limitations. The results of meta-analyses are dependent on the quality of the studies included. The inclusion of only randomized controlled trials was necessary due to significant bias inherent in non-randomized prospective studies. Despite the lack of heterogeneity between studies, the individual studies varied substantially (technique of talc pleurodesis, varying definitions of recurrence and follow-up schedule). Sensitivity analyses performed leaving out one study at a time did not impact the results, suggesting robust data. Publication bias is an inherent limitation of meta-analyses. It is reassuring to see that accounting for it did not result in a statistically significant departure from the original point estimates.\n\nIn conclusion, our meta-analysis demonstrates that there is no difference in malignant pleural effusion recurrence based on patient centered outcomes between talc poudrage and talc slurry. Respiratory complications are more common with talc poudrage via thoracoscopy. Further studies are needed, however, to look at the role of talc pleurodesis via pleuroscopy. The decision of which procedure to perform needs to take into account also the patient preferences.", "appendix": "Author contributions\n\n\n\nSM co-conceived the study, participated in the design of the study, search strategy execution, performance of the statistical analysis, and writing of the manuscript.\n\nAK participated in the search strategy execution, performance of the statistical analysis and writing of the manuscript.\n\nPH co-conceived the study, participated in the design of the study, performance of the statistical analysis, and writing of the manuscript.\n\nAll authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMarel M, Zrustova M, Stasny B, et al.: The incidence of pleural effusion in a well-defined region. Epidemiologic study in central Bohemia. Chest. 1993; 104(5): 1486–1489. PubMed Abstract \| Publisher Full Text\n\nHCUP national inpatient sample (NIS). The Healthcare cost and utilization project (HCUP). Agency for healthcare research and quality, Rockville, MD. 2012. Updated 2014. Reference Source\n\nDavies HE, Mishra EK, Kahan BC, et al.: Effect of an indwelling pleural catheter vs chest tube and talc pleurodesis for relieving dyspnea in patients with malignant pleural effusion: the TIME2 randomized controlled trial. JAMA. 2012; 307(22): 2383–2389. PubMed Abstract \| Publisher Full Text\n\nShaw P, Agarwal R: Pleurodesis for malignant pleural effusions. Cochrane Database Syst Rev. 2004; (1): CD002916. PubMed Abstract \| Publisher Full Text\n\nTan C, Sedrakyan A, Browne J, et al.: The evidence on the effectiveness of management for malignant pleural effusion: a systematic review. Eur J Cardiothorac Surg. 2006; 29(5): 829–838. PubMed Abstract \| Publisher Full Text\n\nDresler CM, Olak J, Herndon JE 2nd, et al.: Phase III intergroup study of talc poudrage vs talc slurry sclerosis for malignant pleural effusion. Chest. 2005; 127(3): 909–915. PubMed Abstract \| Publisher Full Text\n\nTerra RM, Junqueira JJ, Teixeira LR, et al.: Is full postpleurodesis lung expansion a determinant of a successful outcome after talc pleurodesis? Chest. 2009; 136(2): 361–368. PubMed Abstract \| Publisher Full Text\n\nStefani A, Natali P, Casali C, et al.: Talc poudrage versus talc slurry in the treatment of malignant pleural effusion. A prospective comparative study. Eur J Cardiothorac Surg. 2006; 30(6): 827–832. PubMed Abstract \| Publisher Full Text\n\nCrowther M, Lim W, Crowther MA: Systematic review and meta-analysis methodology. Blood. 2010; 116(17): 3140–3146. PubMed Abstract \| Publisher Full Text\n\nTerra RM, Junqueira JJ, Teixeira LR, et al.: Is full postpleurodesis lung expansion a determinant of a successful outcome after talc pleurodesis? Chest. 2009; 136(2): 361–368. PubMed Abstract \| Publisher Full Text\n\nJadad AR, Moore RA, Carroll D, et al.: Assessing the quality of reports of randomized clinical trials: is blinding necessary? Control Clin Trials. 1996; 17(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nMantel N, Haenszel W: Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst. 1959; 22(4): 719–748. PubMed Abstract\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBegg CB, Mazumdar M: Operating characteristics of a rank correlation test for publication bias. Biometrics. 1994; 50(4): 1088–1101. PubMed Abstract \| Publisher Full Text\n\nEgger M, Davey Smith G, Schneider M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997; 315(7109): 629–634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYim AP, Chan AT, Lee TW, et al.: Thoracoscopic talc insufflation versus talc slurry for symptomatic malignant pleural effusion. Ann Thorac Surg. 1996; 62(6): 1655–1658. PubMed Abstract \| Publisher Full Text\n\nManes M, Rodriguez-Panadero F, Bravo JL, et al.: Talc pleurodesis prospective and randomized study.clinical follow up. Chest. 2000; 118(4): 131S.\n\nLee P: Point: Should thoracoscopic talc pleurodesis be the first choice management for malignant effusion? Yes. Chest. 2012; 142(1): 15–7; discussion 20–1. PubMed Abstract \| Publisher Full Text\n\nLight RW: Counterpoint: should thoracoscopic talc pleurodesis be the first choice management for malignant pleural effusion? No. Chest. 2012; 142(1): 17–9; discussion 19–20. PubMed Abstract \| Publisher Full Text\n\nBethune N: Pleural poudrage: New technique for the deliberate production of pleural adhesion as preliminary to lobectomy. J Thorac Surg. 1935; 4: 251.\n\nDiacon AH, Wyser C, Bolliger CT, et al.: Prospective randomized comparison of thoracoscopic talc poudrage under local anesthesia versus bleomycin instillation for pleurodesis in malignant pleural effusions. Am J Respir Crit Care Med. 2000; 162(4 Pt 1): 1445–1449. PubMed Abstract \| Publisher Full Text\n\nXia H, Wang XJ, Zhou Q, et al.: Efficacy and safety of talc pleurodesis for malignant pleural effusion: a meta-analysis. PLoS One. 2014; 9(1): e87060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMager HJ, Maesen B, Verzijlbergen F, et al.: Distribution of talc suspension during treatment of malignant pleural effusion with talc pleurodesis. Lung Cancer. 2002; 36(1): 77–81. PubMed Abstract \| Publisher Full Text\n\nMori PA, Casalini AG: Therapeutic medical thoracoscopy. Monaldi Arch Chest Dis. 2011; 75(1): 89–94. PubMed Abstract\n\nOlden AM, Holloway R: Treatment of malignant pleural effusion: PleuRx catheter or talc pleurodesis? A cost-effectiveness analysis. J Palliat Med. 2010; 13(1): 59–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuri V, Pyrdeck TL, Crabtree TD, et al.: Treatment of malignant pleural effusion: a cost-effectiveness analysis. Ann Thorac Surg. 2012; 94(2): 374–379. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohsen TA, Zeid AA, Meshref M, et al.: Local iodine pleurodesis versus thoracoscopic talc insufflation in recurrent malignant pleural effusion: a prospective randomized control trial. Eur J Cardiothorac Surg. 2011; 40(2): 282–286. PubMed Abstract \| Publisher Full Text\n\nMaskell NA: Treatment options for malignant pleural effusions: patient preference does matter. JAMA. 2012; 307(22): 2432–2433. PubMed Abstract \| Publisher Full Text\n\n\nSupplementary materials\n\nFrom: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(6): e1000097. doi:10.1371/journal.pmed1000097" }	[ { "id": "6535", "date": "31 Oct 2014", "name": "Gary Lee", "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\nOverview:The topic is important and clinically relevant. Talc pleurodesis is commonly practiced. The debate on the superiority of thoracoscopic poudrage (TTI) versus talc slurry (TS) has been ongoing for decades. A meta-analysis incorporating the most recent data is useful.The methodology appears sound. The four selected articles are well described and acknowledged in most review articles as the only randomized studies directly addressing this topic.The interpretation of the data is fair and balanced. The endpoints chosen are clinically relevant and important. Most other reviews or book chapters on the issue have only focused on the success/failure rate of the pleurodesis. The authors rightly highlighted the increased complication rates of TTI. The figures and graphs are clear and easy to understand.The discussion is based on the analyses and the postulations for the observed results are sound and reasonable. Overall this is an excellent paper and I would support its indexing.There are several few points that the authors may wish to consider. I believe that this article may be improved if the authors can address the following:The study by Manes et al. (2000)The authors quite rightly included not only peer reviewed papers but also all published abstracts. This approach is according to standard meta-analysis practice, in order to avoid publication bias such that ‘negative’ studies (often not published in full) are not excluded. However, including data from published abstracts preclude scrutiny of the detailed methods, analyses and thus raises questions on the quality of the results. The situation in the meta-analysis of TTI vs TS highlights the pros vs cons of including data that were not peer reviewed.Three of the four selected studies were published in respected journals and were subjected to peer-reviewing processes. The study by Manes however was published only as an abstract 14 years ago, and to date had not been published as a full paper. This by itself raises great concerns. The results of this particular study deviated significantly from all the other three. The authors have identified clear methodological concerns (especially ‘recycling’ of patients into randomization after failing pleurodesis). Unless the authors have obtained details from the primary research group, it is doubtful that a short abstract could provide adequate details for proper critique of the methods and results. Including this study without qualifying its many limitations may distort the interpretation of the readers.I suggest that the authors should:Highlight the point that the Manes study was never published in full in the text/legends;Perform and show a separate analysis excluding the Manes study;Discuss the rationale of including/excluding this single study in the Discussion section.I believe the above measures are justified as the Manes study was not a ‘negative’ study and would never have been biased against if ever submitted for full publication.An alternate way of presenting the dataAlthough it would not change the actual conclusion or the raw data, presenting the results as ‘success’ rather than ‘failure’ rates would quite significantly change the ‘visual effects’ of the graphs. Take for example the Dresler study. The RR for failure is 0.74 but if expressed as ratio of success rates it would become 1.11, the Yim study 1.04 and the Terres study 0.96. This probably presents a more useful interpretation for clinicians and patients – that the magnitude of superiority of TTI in any of the studies is at best 1.11 times over TS (excluding Manes et al.). DiscussionIt would be useful to include that there are no scientific grounds why insufflation should be more advantageous than slurry. Talc does not work as a glue (otherwise we would have had major problems when talc was still included in baby nappy powders). Even distribution of talc over the pleural surface is not therefore critical. Radio-active isotope studies have shown that talc, even when applied as a slurry, can distribute around the pleural cavity via respiratory motions.", "responses": [ { "c_id": "1233", "date": "18 Feb 2015", "name": "Srinivas Mummadi", "role": "Reader Comment", "response": "Thank you very much your comments and a detailed critique of our manuscript. Response to “The study by Manes et al. (2000)” We agree with your assessment of the pros and cons of including the study by Manes et al. (2000) published only as an abstract. As our inclusion criteria were defined a priori, we included the study in our analysis.We agree with your comments that it suffers from potential quality concerns. In addition to our stated concerns regarding the inappropriate allocation of randomization, we have made efforts to highlight additional quality concerns outlined below.Footnotes to the tabular data have been added, highlighting the existing form of publication as an abstract. We also discussed in detail the lack of change in point estimates for all the predefined outcomes after removing the study as a part of the sensitivity analysis.Due to its unique conclusion (TTI is superior to TS in terms of pleurodesis success rates) and a relatively large treatment effect, we believe that the inclusion of this study with the clear mentioning of its inherent limitations gives us a unique opportunity to present both sides of the talc poudrage versus talc slurry debate in a systematic review.Inclusion of this study in the analysis allows meta-analysis to play the role of an adjudicator.As mentioned earlier, removal of this study in the sensitivity analysis did not influence the results.For the sake of clarity, we would like to report the results for all the studied outcomes after removing the above mentioned study in this reply.1) Pooled relative risks (RRs) of success rates post talc pleurodesis (TTI vs TS)Point estimate (RR) = 1.04, 95% CI (0.97-1.1), P-value – 0.24, Q statistic -1.282) Pooled relative risks (RRs) for respiratory complications post talc pleurodesis (TTI vs TS)Point estimate (RR) = 1.99, 95% CI (1.29-3.08), P-value – 0.002, Q statistic -2.053) Pooled relative risks (RRs) for non-respiratory complications post talc pleurodesis (TTI vs TS)Point estimate (RR) = 0.93, 95% CI (0.76-1.14), P-value – 0.5, Q statistic - 0.06 An alternate way of presenting the dataWe agree with your suggestion that the clarity of the take-home message would be improved by presenting the data as rates of success rather than rates of failure. We have therefore renamed the outcome as “successful pleurodesis” [defined as no need for further pleural procedures despite asymptomatic radiological recurrence in a few cases]. Measuring patient centered outcomes was the predefined objective of the study, therefore asymptomatic radiological recurrences where clearly defined were counted towards success (3 patients)Relevant changes have been made in the body of the manuscript, figures, and tables. Reassuringly, measuring the outcome as either relative rates of success or relative risks of recurrence did not influence the existing conclusion. DiscussionWe agree that there are no quality data to substantiate the “intuition” that thoracoscopic talc insufflation is superior to talc slurry in terms of talc dispersion in the pleural space. We would like to draw your attention to our mentioning of these points in the existing discussion section (Mager et al.). We have fine-tuned the write up to buttress the above point to increase the visibility to the reader." } ] }, { "id": "7143", "date": "31 Dec 2014", "name": "Pyng Lee", "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\nMalignant pleural effusion is also one of the leading causes of exudative effusion; studies have demonstrated that 42 to 77% of exudative effusions are secondary to malignancy (Marel et al.,1993; Valdés et al.,1996) Need statistician review.", "responses": [] } ]	1	https://f1000research.com/articles/3-254
https://f1000research.com/articles/4-46/v1	17 Feb 15	{ "type": "Opinion Article", "title": "Why do we bother? Exploring biologists' motivations to share the details of their teaching practice", "authors": [ "Graham Scott" ], "abstract": "There exists in the UK (and across the global HE sector) a community of practitioners who define themselves as biologists but who are more than that. They are reflective educators involving themselves in the Scholarship of Teaching and Learning (SoTL). In this paper I explore the motivations of these individuals to disseminate the detail of their teaching practice. I reflect upon my own experience and my observations of the experiences of others and in doing so I explore common enablers/disablers to engagement with SoTL. I discuss the prime importance of a supportive disciplinary SoTL community and of inspirational individuals (peers and managers alike). I reflect upon the tensions that exist between teaching and research focused career paths and I consider the possibility that this tension is of variable significance. I conclude that the barriers to individual engagement with SoTL can be overcome and that the individual drive to do so is a powerful one.", "keywords": [ "teaching", "practice", "biologist", "scholarship of teaching and learning" ], "content": "Introduction\n\nDuring one of the early planning sessions for the Society of Experimental Biology Symposium Teaching and Communicating Science in the Digital Age (15th–17th December 2014, Charles Darwin House, London, United Kingdom) it occurred to me that as we discussed what digital communication might mean, how it might be utilised to increase our personal profiles as practicing biologists and how it might be used to change the ways in which we disseminate our science and teach our students, we seemed to routinely miss one area of our day to day activities namely the dissemination of teaching practice itself. Not what we teach, but rather how we teach and how we evaluate and disseminate our teaching practice.\n\nHere I am not primarily interested in the mechanisms by which we disseminate our teaching practice – we illustrate symposium presentations using digital media, we disseminate through social media, we blog and we submit papers to online journals for example. Instead my primary focus is a desire to better understand our motivations to share our practice. In so doing I hope to gain some insight into our experiences as professionals who are often be viewed as being at the intersection of two areas of academic practice, teaching and research, and at the boundary that exists between disciplinary areas (in this case the biosciences, the social sciences and the educational sciences).\n\nThrough discussion and reflection it became apparent to me that the motivation to disseminate ones teaching practice is bound up with the development of an individuals scholarship, the key enablers/disablers we each experience, and the audience to whom one “speaks”. These three areas are therefore the secondary foci of this paper.\n\nThis is in large part a personal reflection, an attempt that I have made to make sense of my own motivations and the professional pathway that I have undertaken and continue to follow. I acknowledge therefore that this is a very UK-centric viewpoint and that the breadth of my experiences limits it. I have tried to reflect upon my practice in the context of the following:\n\n1. My reflections upon my own pedagogical practice and dissemination activities and my observations of those of my peers.\n\n2. My discussions with colleagues who are new to teaching (as a mentor to newly appointed lecturers in my own institution over more than 10 years; and, as a facilitator of several new to teaching CPD events organised by both the Higher Education Academy and the Society of Biology).\n\n3. My discussions with the authors of papers about the teaching of biology in my role as an associate editor and editor of journals.\n\n4. My readings of the relevant literature (and in particular for example the work of Healey, 2000; Lave & Wenger, 1991; Trigwell et al., 2000; Wenger, 1998).\n\n5. Interviews with seven academic colleagues based in the UK (see below).\n\n\nThe interviews\n\nI carried out seven interviews with UK-based Higher Education (HE) bioscience educators to provide myself with a sounding board against which to test my own reflections. The interviews were conducted by telephone and interviewees were selected because I was aware, or I was made aware, that they actively engaged in the dissemination of their teaching practice. All but one of the interviewees were, or had until recently been, employed on traditional teaching and research (T&R) contracts. Of these, three I considered to be established (two Chairs – both awarded on the basis of teaching/engagement rather than biological research, and a Senior Lecturer; one female, two male), and three I considered to be new to teaching (having less than four years formal experience) (one female and two male). The final (female) interviewee had not held a T&R contract but had more than 15 years of formal teaching practice in mainstream UK HE Biology departments and a wealth of experience in both the Scholarship of Teaching and Learning (SoTL) and the professional development of teaching faculty within the biosciences. The interviewees represented mainstream bioscience departments at UK HEIs from the Russell Group, the post 1992 group and other institutions from three of the four UK home nations and so were drawn from across the sector. (I accept however that they could in no way be described as a fully representative sample). The relevant ethics committee of the University of Hull approved this data collection.\n\nInterviews were conducted by telephone and lasted between 30 and 40 minutes each. The discussion was recorded and transcribed and interviewees were provided with an opportunity to comment upon their own transcript. The discussion was semi-structured focusing upon four broad areas each phrased as questions that were sent to the interviewees in advance:\n\n1. Can you describe to me the ways in which you have communicated your teaching practice to colleagues. How has this changed over time?\n\n2. Who do you consider to be the audience when you disseminate your teaching practice?\n\n3. At what stage in your career did you first feel the need to tell colleagues formally about your teaching practice? What motivated you to do so?\n\n4. Have there been personal costs/benefits associated with the efforts that you have gone to share your teaching practice with others? To what extent have you been encouraged/discouraged in your efforts (and by whom)?\n\n This question was not sent to interviewees in advance. It arose during the first interview and was subsequently added to the other discussions at an appropriate juncture.\n\nThese questions were asked/discussed in the context of a wider discussion about SoTL and about the career trajectory of the individual concerned.\n\n\nDisseminating our teaching practice\n\nThe dissemination or communication of ones teaching practice is a key dimension of Trigwell et al. (2000) four dimension model of the Scholarship of Teaching and Learning (Table 1). As the model illustrates some teaching faculty (those at level 1), who see teaching in a teacher-centric way, do not communicate their practice and demonstrate limited awareness of the scholarship of others. On the other hand, some members of the faculty move far beyond this and see teaching as a student-focused activity. These colleagues publish the outcomes of their scholarship in the international peer-reviewed literature and in doing so demonstrate a capacity to conduct action research and an advanced knowledge of the pedagogic literature. The transition from level 1 to level 4 is not a pre-requisite of teaching, nor is it a necessary consequence of teaching activity. Rather it represents the potential ontogenetic pathway of a reflective practitioner as they negotiate Threshold Concepts (see Meyer & Land, 2003) and overcome the barriers imposed by Troublesome Knowledge (A. Tierney, pers. com.).\n\nOver recent years, I have observed the practice of many of my peers as it has developed from level 2 through level 3 and into level 4 of the Trigwell et al. model; a journey that I have shared with them. Three of my four more established interviewees described their dissemination activities and the materials presented as having evolved over time. Their initial disseminations were spoken rather than written papers and they tended to be quite descriptive. Their more recent work has been more likely to be underpinned by pedagogic theory and robust data, but interestingly none of them saw themselves as having made a transition from being biologists to researchers in education. One down-played their grasp and use of the literature to inform their work (comments that I took to indicate a level 2 Informed dimension and a level 3 Communication dimension from Table 1). Another explained that they still felt less comfortable with an audience of what they described as social scientists, by which I understood them to mean non-biologists interested in SoTL in comparison to an audience of biologists with an interest in SoTL. This was because they felt that they lacked the grounding in SoTL that a PhD and Post-Doctoral Research Associates (PDRA) track had provided for them in biology. This is a common perception and one that has been discussed by Kelly et al. (2012), who suggest that this is an issue that goes beyond disciplinary research methodology differences and is intimately bound up with the disciplinary identities of the individuals involved. My interviews with new to teaching faculty provide an indication that a subtle shift might be taking place as a result of changes in the way in which UK HEIs develop new academics. Although two of them described spoken presentations in which they explained to colleagues what they had done and suggested that this was a sharing of good practice rather than a presentation of research (level 2 communication dimension in Table 1), the third (on a standard T&R contract) described the fact that as part of a compulsory Postgraduate Certificate (PGCert) in HE practice they were required to undertake a piece of action research involving the re-development of a module. As a minimum this project had to be disseminated as a presentation to their peer group, but in this case it resulted in a publication in the journal Bioscience Education. As a journal editor handling papers on biology education I believe that this is becoming more commonplace. It is possible therefore that an emphasis upon SoTL from the outset as part of the routine practice of an academic may provide an opportunity for some individuals to enter the Trigwell model at a higher level and perhaps therefore subsequently progress at an accelerated rate (this is a possibility that I will return to later in this paper).\n\n\nThe audience\n\nFrom the preceding section it is clear that the primary audience for the dissemination of SoTL is one’s peers. Commonly we start by discussing our ideas with a mentor or other key colleague (see below) and from there move on to present posters and oral presentations at events organised within our institution. Our audience grows as we develop as SoTL practitioners and we begin to present our work at national and international meetings, through publications and via social media. But it would be wrong to think that this has always been an easy journey to make. Consider the following quotation taken from an interview with an established academic:\n\n“Being keen on learning and teaching was, certainly when I started out, it was almost you know, it was something you kept under your hat.” Established interviewee.\n\nThis statement typifies an experience common to many of us who started on our academic careers 15–20 years ago. At that time (in a UK context) opportunities to formally present our teaching practice were significantly less common than they are today, particularly in the pre-digital world when outlets such as blogs and social media were not available to us. Two of the more established interviewees discussed the critical importance of their disciplinary learned societies in this regard, both having had the opportunity to present their pedagogy in education themed sessions at disciplinary research conferences. Both talked about the importance of these sessions as places to both present ones own work but also to hear about the good practice of others. These meetings were seen as a way to avoid the redundancy of “reinventing the wheel”. On the face of it this is a positive state of affairs but the same interviewees also recollected that at that time (15 to 20 years ago) these sessions were seen as “a bit of a side show to the main [disciplinary research] event”. Common to the experience of all of my interviewees was the perceived importance of an audience of peers beyond their own institution (be that the readership of a journal or blog, the users of social media, or the participants of a symposium or conference). All of them described the importance of being part of a supportive community. One explained:\n\n“One [benefit of making the effort to disseminate ones teaching practice] is becoming involved with the really good community of like-minded people who are very supportive and good to be with and good to discuss with. You’ll always have a good debate but it’s very collegial and I think one of the real differences that I’ve noticed between presenting at research conferences, certainly in my discipline, and learning and teaching ones is that collegiality. People aren’t trying to catch you out and prove how clever they are. They’re genuinely interested in what you have to say.” Established interviewee.\n\nHowever, all of the established interviewees expressed some concern that in the UK context at least, the perceived shift in focus of the Higher Education Academy away from disciplinary workshops and conferences towards more generic ones may stifle debate within the biosciences and limit opportunities for dissemination. There was even a suggestion that a golden age of UK SoTL may have passed. This is not a view that I necessarily share. Whilst I agree that the HEA Bioscience Subject Centre and the HEA STEM group were key to the development of a broader Bioscience SoTL community in the UK it is evident that that community did exist in nascent form prior to that period and that through its own efforts can continue to thrive. We have seen that the learned societies have played (and continue to play) a role in this by providing opportunities for SoTL and the dissemination of SoTL alongside disciplinary research. As long as SoTL does not remain/become a “side-show” at their meetings there is a potential for continued activity. As one of my interviewees explained, it is essential that as a community of practitioners we do our part to maintain the development of a culture in which SoTL is seen as being on a par with research across the HE sector. This is perhaps particularly important given that it was recently estimated that as many as 25% of UK academics are described as “teaching-only” (Times Higher Education, 2008) many of whom are contractually obliged to engage in “scholarship” (Cashmore, 2009).\n\nOne outcome of my interviews, although not a surprise, was something of a disappointment. With the exception of the established interviewee who is currently primarily involved in the teaching of pedagogy and SoTL to in-post academics and one who was new to teaching, none of the interviewees mentioned their students as an audience for their SoTL outputs. When pressed all explained that they regularly told their students how learning would take place (i.e. they described the various learning strategies employed) and that their own students were often involved (as participants) in their SoTL activity, but only the two of them reported using the outcomes of SoTL activity (their own or that of others) to demonstrate to their students the benefits of the pedagogy that they employed. My wider experience of discussing this with colleagues suggests that not including ones students as part of the SoTL audience is the norm rather than the exception. However, my personal experience is that doing so can be a useful exercise. It helps one to reflect upon the pedagogies employed and upon ones own scholarship. It can also provide students with a useful insight into their own learning experience (pers obs).\n\n\nMotivations\n\nSo what makes a biologist want to talk about how to teach biology rather than about biology per se? By this I don’t mean why do we have the coffee room or water cooler type discussions that are commonplace in any educational setting about how a session has gone or might be improved, or how a course could be changed to take into account a development in the field or a new administrative requirement. I mean why do a minority of biology faculty (and in most institutions it is a minority) take that extra step and start to formally share their practice in the same way that they might be expected to share the details of their disciplinary research.\n\nFor many of us (myself included) an inspirational colleague or mentor set us on our path. In some cases they modelled good practice themselves; they were innovators who took the time to explain to us why they were changing the way they taught. Their passion for innovation and willingness to share the details of both their success and their failures provided us with a model to follow. In other cases supportive mentors and senior colleagues provided us with a space in which to work – perhaps they gave us free rein to attempt a new way of teaching or perhaps they encouraged us to undertake CPD that exposed us to the possibilities of teaching in a different way. These key motivational figures were common to both the established and new to teaching interviewees.\n\nAll of the interviewees and many of the new to teaching academics that I have discussed this with share a common desire to improve their own teaching and to enhance the student experience. But whilst this clearly underpins the reflective development of teaching and the motivation to share good practice, it is not in itself a sufficient explanation of the drive to disseminate – after all, it would be perfectly possible for a lecturer to strive to do both without ever presenting their practice to colleagues. Several interviewees described the presentation of their own practice almost as an obligation, a way of making a contribution to the community from which they themselves benefit:\n\n“I wanted to keep attending those sort of events … and it seemed only fair to also contribute if you’re going to listen. It’s pretty rare that I would attend any event without offering to speak at it.” New to teaching interviewee.\n\nThere was also a sense that people wanted to present a realistic account of what worked or did not work. To do more than showcase their outputs by giving an honest “warts and all” account of the difficulties encountered. This once again emphasises the importance of a feeling of collegiality and belonging to an active community. Being part of the group is in itself a clear motivation for many to continue to disseminate their work.\n\nAll of the interviewees gave a very positive account of their role within an established or growing community of scholars and demonstrated a strong commitment to SoTL at whatever level of engagement they had achieved. I have already identified the importance of the community to established biology faculty with a history of SoTL type activity but I suspect that it is also very important to the new generation of academics in the UK for who SoTL is a requirement of a Teaching and Scholarship type contract. None of the established interviewees thought that it would have been possible to be awarded a Chair on the basis of Teaching and Scholarship in their own institution until relatively recently and all saw the formal recognition of excellence in teaching that has become more prevalent in the UK HE sector in recent years as a positive development. Clearly then career development (promotion) has become a new motivation for engagement with SoTL and the dissemination of teaching practice in recent times – particularly in the case of the 25% of UK academics are described as “teaching-only” (Times Higher Education, 2008) and those on teaching and scholarship contracts who are contractually obliged to engage in “scholarship” (Cashmore, 2009) previously mentioned. These colleagues have the potential to become a mainstream SoTL community within the biology discipline and are likely to continue to be motivated to undertake SoTL activity throughout their careers.\n\nHowever, this may not be a sufficient motivation for a continued involvement in SoTL for those colleagues who remain on traditional teaching and research contracts. As I have previously mentioned, I have recently observed the development of a distinct sub-community of academics who discuss the preparation of a presentation or a paper related to their teaching practice as a key hurdle to be jumped on the road to promotion via a teaching and research route. This is particularly the case amongst many new to teaching academics on T&R contracts who have undertaken what could be described as a SoTL activity as part of a PGCert (or similar qualification/training course). It is possible, as I suggested previously, that these colleagues are motivated to engage in a meaningful way with SoTL and effectively enter the Trigwell et al. (2000) model at a higher level. But it may also be possible that as a result of their potentially narrow range of motivation to disseminate they do not fully establish themselves across the breadth of the dimensions of the model.\n\n\nEnablers/Disablers\n\nFor those employed on a traditional teaching and research contract the tensions existing between research and teaching (and in particular the lack of time to do both) are a key barrier to SoTL. There is for these colleagues a significant pressure to win disciplinary grant funding, to write high impact research papers and to teach well. For many the extra efforts involved in the dissemination of teaching practice may simply be beyond them. Some rare individuals do manage to maintain both a disciplinary research profile and become very active within the bioscience education community, but for many a choice is made along the way to specialise in one area or the other. For some the choice is less problematic than for others. Those on a teaching and research contract are contractually obliged to carry out research and it is very unlikely that SoTL activities would be seen as research in that context. This does not mean that the journey from biologist to scholar of biology teaching is an easy one for those who are contractually empowered to make it. Some of the colleagues I have discussed this with describe a sense of guilt that they are turning their backs on aspects of their disciplinary background. Rowland (2012) has expanded upon this problem in her discussion of the fact that some academics do not see those who specialise in teaching rather than in research as being sufficiently current to deliver advanced classes and supervise students undertaking disciplinary research projects. Others explain that they feel like imposters in a new field. Self-doubt may therefore be a key barrier that prevents some colleagues from achieving their full potential.\n\nAs has been previously mentioned, particular people are often a key enabling factor in the development and maintenance of an individuals motivation to engage in SoTL and to disseminate ones teaching practice. I have already indicated the importance of inspiring mentorship and of participation in a supportive community of practice. However, other inter-personal relationships are important too. Several interviewees discussed the significance of strong leadership at the institutional level. One established interviewee commented that it “took a brave Vice Chancellor to establish those routes” (teaching and scholarship/engagement promotion pathways to the Professorial level) and added that it then took “people who really wanted to develop their careers in teaching to actually then take it forward”. A new to teaching interviewee described how their Vice Chancellor had instigated a series of teaching awards that were celebrated alongside the achievements of graduating students. This very public recognition of teaching excellence was described as bringing value to the efforts that academics make. However, whilst reward and recognition in the form of awards was seen as a general positive and as a validation of the value of individual practice, the view was also expressed that institutional notoriety could be short-lived. One established interviewee, themselves the recipient of numerous institutional and international awards to recognise excellence in teaching, expressed the view that in their personal experience there remained a disconnect between this type of recognition and rewards linked to institutional promotion criteria. Middle-managers and line-managers were also described as being very important enablers and/or disablers. Several interviewees (and many of the colleagues I meet through my facilitation of workshops) describe the support that a good line manager provides as being essential to their engagement with SoTL and key to their ability to disseminate their practice. Often this support is very practical, taking the form, for example, of the financial support needed to undertake SoTL activities and to attend dissemination events. Sometimes it is simply being given the time and space in which to develop this facet of an individuals practice. On the other-hand an unsupportive line-manger is often cited as being a complete barrier to SoTL. In the case of reward of SoTL through promotions processes one established interviewee expressed the view that although new criteria had been established to recognise excellence in teaching they were likely to be used by panels of academics who had themselves been promoted on the basis of research and may not understand how to apply them. Whilst such a situation would undoubtedly be problematic robust promotional systems and criteria should mitigate against this.\n\n\nConclusion\n\nI titled this paper “Why do we bother?” a question that at the time I thought was a neutrally phrased one. Early in the writing of the paper however I was challenged by an interviewee who assumed that I had intended it to be read in a negative fashion. As a result I have realised that in fact my preconception was that my reflections on the topic would reveal to me a very simple picture – we bother because we enjoy it, because the work we do has value, and quite simply because we can’t imagine not doing it. I think that on the whole I have found evidence to support my view but I have also revealed to myself that the picture is not cut and dried.\n\nThere exists in the UK (and across the HE sector) a community of practitioners who define themselves as biologists but who are more than that. We are reflective educators involving ourselves in the Scholarship of Learning and Teaching. Ours is a self-sustaining community and one that at the moment is enjoying the benefit of a period of supported growth (recently primarily supported in the UK context by the HEA Bioscience Subject Centre). It is in a position to continue to grow as students and student driven market forces apply pressure upon institutions but we should not be complacent. I would add to all of the motivations that have been discussed above the part that disseminating our practice can play in show-casing our work and our worth to the senior managers and policy makers who have the future of our community partly in their hands.\n\nAs a closing thought, lack of support has been seen as a key barrier to individual engagement with SoTL, but lack of support can be overcome if the motivation to do so is sufficient. As one established interviewee commented, “somebody said to me what would happen if your line manager said you couldn’t do this [SoTL] anymore? And I said, well I’d do it anyway.”", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nI am grateful to the seven interviewees who took part in this study. I am also grateful to Anne Tierney with whom I discussed this work and who provided constructive feedback on the manuscript during its development.\n\n\nReferences\n\nCashmore A: Reward and Recognition of Teaching in Higher Education: Institutional Policies and their Implementation (No. 2). The Higher Education Academy and GENIE Centre for Excellence in Teaching and Learning. University of Leicester. 2009. Reference Source\n\nHealey M: Developing the Scholarship of Teaching in Higher Education: A discipline-based approach. Higher Education Research & Development. 2000; 19(2): 169–189. Publisher Full Text\n\nKelly N, Nesbit S, Oliver C: A Difficult Journey: Transitioning from STEM to SoTL. International Journal for the Scholarship of Teaching and Learning. 2012; 6(1): Article 18. Reference Source\n\nLave J, Wenger E: Situated Learning: Legitimate Peripheral Participation. Cambridge University Press. 1991. Reference Source\n\nMeyer J, Land R: Threshold concepts and troublesome knowledge: linkages to ways of thinking and practising within the disciplines. In Improving student learning: Improving student learning theory and practice - Ten Years On. (Rust, C. (Ed).). Oxford: Oxford Centre for Staff and Learning Development. 2003. Reference Source\n\nRowland S: Teaching-focused science academics supervising research students in science education: what’s the problem? Higher Education Research and Development. 2012; 31(5): 741–743. Publisher Full Text\n\nTrigwell K, Martin E, Benjamin J, et al.: Scholarship of Teaching: A model. Higher Education Research & Development. 2000; 19(2): 155–168. Publisher Full Text\n\nWenger E: Communities of Practice: Learning, Meaning and Identity. Cambridge University Press. 1998. Reference Source" }	[ { "id": "8674", "date": "22 May 2015", "name": "Catherine J. Hack", "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\nThis is an interesting and timely piece, given the current drivers to enhance teaching quality across the UK Higher Education sector, and the growth in discipline specific pedagogic research over the last decade.This paper is based on a small qualitative study, which used semi-structure interviews with seven participants. Although a small cohort, it was purposefully recruited to include an appropriate range of experience, age and gender balance. The methodology was appropriate for the objective of the study which was to explore the motivations of academics, specifically those involved in research and teaching in the biosciences, to evaluate their teaching and share their practice. The limitations of the research were identified. The analysis was informed by Trigwell’s multi-dimensional model of scholarship of teaching and was situated within the authors’ extensive experience of publishing pedagogic research and scholarship in the life sciences. I would be interested to hear whether the author agrees with others (e.g. Stierer, 2007,and Tight, 2013) that there has been significant change in the quality of scholarship and pedagogic research conducted by discipline specialists (i.e. not educationalists) and whether discipline-specific researchers are conducting more robust studies which are informed by the relevant pedagogy- i.e. moving beyond Trigwell's model.The author comments on his choice of title, and I wonder whether a change to ‘why we bother’ may be more reflective of the article content?", "responses": [] }, { "id": "8753", "date": "26 May 2015", "name": "Vivien Rolfe", "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\nTitle and content:A paper of interest to the ‘bioscience’ community exploring why we share our teaching practices with others? This is very topical in light of changes to the HE sector and the decline in support and project funding for education research. So I am hoping the paper will enlighten me on not just the ‘why’ but ‘how do we bother’ to become excellent practitioners and share to support our subject communities?Methodology:A small number of interviews were conducted reflecting a range of institutions. It wasn’t clear how the author got from interview transcripts to aligning to Trigwell’s model of scholarship of teaching? How was the data analysed and was any particular software used? The findings are discussed in accordance with the interview lines of enquiry.Discussions and conclusions:I find it a little strange that none of the interviewees or reflections by the author mention ‘open education’ as a motivational factor and enabler for sharing teaching practices? The advent of open licenses and easy technologies has made it simple for people to share news and practices to global audiences and reach out to learners beyond their institution. I think the global open education movement is a significant driver and possibly worth a sentence in the discussion that it was not mentioned by any of the interviewees.Under motivations also I’m surprised that seeking professional fellowships (e.g. HEA / SEDA UKPSF or HEA National Teaching Fellowships) are not mentioned as a driver, as teaching excellence is a National key performance indicator and many institutions have set staff targets for these. The need for teaching staff in all disciplines to reflect and enhance their practice has grown rapidly, so again, it might be worth a comment noting that this was not mentioned during the interviews.A final area I’m surprised was not raised is the lack of funding for education projects in the UK. Changes in direction for organizations including the HEA and Jisc have had a major impact on financial support for teaching innovation and the ability to build communities around projects as used to take place.Overall, a well conducted study addressing a very topical question that will be of interest to the biology and bioscience community.", "responses": [] } ]	1	https://f1000research.com/articles/4-46
https://f1000research.com/articles/3-234/v1	03 Oct 14	{ "type": "Clinical Practice Article", "title": "Breaking a Guinness World Record on Hand Sanitizing Relay, initiating a call for vital research in overcoming campaign fatigue for hand hygiene", "authors": [ "Wing Hong Seto", "Kwok-Hung Li", "Christina Woon Yee Cheung", "Patricia Tai Yin Ching", "Benjamin J. Cowling", "Wing Hong Seto", "Kwok-Hung Li", "Christina Woon Yee Cheung", "Patricia Tai Yin Ching" ], "abstract": "Hand hygiene has been shown to be effective in significantly reducing hospital acquired infections for many years. However it is difficult to maintain and enhance compliance with hand hygiene guidelines. In Hong Kong, we previously reported a strategy to counter campaign fatigue from 50%-55% in 2009-11 to 83% in 2012. Here we report a creative activity that we used to sustain and enhance hand hygiene compliance. In May 2014 we broke the first Guinness World Record for a Hand Sanitizing Relay. A total of 277 participants performed hand hygiene before two official and approved witnesses. Following this team-directed strategy, an increase in hand hygiene compliance was identified in two clinical areas with previously poor compliance. The longer term impact of this strategy remains to be determined. More broadly, further research is urgently needed on meeting the challenge of campaign fatigue, and maintaining and enhancing compliance with hand hygiene guidelines.", "keywords": [ "Hand hygiene (HH) has been shown to be effective in significantly reducing hospital acquired infections for many years1. To ensure that this is practiced all over the world", "the World Health Organization (WHO) launched the First Patient Safety Challenge in 20052. This is highly successful and as of 24 September 2014 a total of 134 member states of WHO have pledged their support3. The WHO has also provided guidelines on HH with full details on the proper procedures and also guidance on the proper implementation strategy4." ], "content": "Introduction\n\nHand hygiene (HH) has been shown to be effective in significantly reducing hospital acquired infections for many years1. To ensure that this is practiced all over the world, the World Health Organization (WHO) launched the First Patient Safety Challenge in 20052. This is highly successful and as of 24 September 2014 a total of 134 member states of WHO have pledged their support3. The WHO has also provided guidelines on HH with full details on the proper procedures and also guidance on the proper implementation strategy4.\n\nAlthough the effects of HH are firmly established, it is not easy to achieve compliance to the HH guideline. This is because all staff in the hospital must practice it consistently before, during and after contact with patients and their immediate surroundings. Fully recognizing that compliance is difficult, the WHO guideline has laid out a clear strategy for implementation which includes five essential steps:\n\nStep 1: Facility preparedness including resources and formulating a plan\n\nStep 2: Baseline evaluation – establishing the current situation\n\nStep 3: Implementation – introducing the improvement activities\n\nStep 4: Follow-up evaluation – evaluating the implementation impact\n\nStep 5: Further planning and review cycle.\n\nIt should be noted that the WHO also recommends that a plan be developed for the review cycle that is ongoing for a minimum of five years.\n\nThe WHO has formulated a Multimodal Hand Hygiene Improvement Strategy (MHHIS) for promoting the HH practice. This strategy is not limited to a single promotional component such a posters and banners. Rather the WHO endorses a myriad of coordinated actions including system change to ensure that alcohol-based handrub and handwashing facilities are in place, continuous training and education programs, HH audits with feedback, reminders in the workplace, and fostering institutional safety climate in the hospital.\n\n\nSystematic reviews on efficacy of HH campaigns\n\nThree reviews on HH compliance have been reported in the literature. One review was conducted before 2010 which included all studies in English from a market economy with proper description of results5. A total of 96 studies were included and the results were rather variable but the authors concluded that noncompliance with hand hygiene guidelines is a universal problem and that this can be present even when all the basic requirements for implementation as recommended above were in place. It is stressed that to develop successful interventions, more research on behavioral determinants is needed. The second is a Cochrane review with updates searches up till November 2009 and was published in 20116. The standards for selection were much more stringent and only studies with controls or interrupted time series with explicit entry and quality criteria were considered and finally only four studies were included. Two studies had data on HH compliance and only one showed improvement from the campaign. The other two reported improvement based in increase in usage of hand disinfectants. The authors of the review concluded that soundly designed studies are still required with at least 12 months of follow up. This is because the short duration of the included studies did not permit assessment of the long term impact of the campaigns.\n\nAnother review by Huis et al. reported in 2012 was conducted but with a focus on classifying the improvement activities based on their determinants of behavior change7. It was stated in the review that the criteria for inclusion were not as stringent as Gould et al.6 but rather the standards of Anderson and Sharpe were used8. Nevertheless the papers included must have clearly described their intervention methods for these to be evaluated and finally 41 studies were included. The review concluded that the strategies are rather limited and “we should be more creative in the application of alternative activities”. The authors encouraged further research particularly on group-directed and team-directed strategies in addition to strategies focusing on the individual or organization.\n\n\nRecent studies on the long term impact of HH campaigns\n\nAs stated in the review by Erasmus et al.5, there is a lack of data showing the long term impact of HH campaigns. There are however two studies published in 2014 with data on the impact beyond two years. The first by Myer et al.9 reported a significant increase of HH compliance from 22%–40% in 2000 to 65%–81% in 2003–2006. Data for compliance of the different categories of healthcare workers from 2003 to 2006 were also provided and there were no increases recorded during these three years. The compliance data in 2003 and 2006 were the following: physicians (63%; 60%), Nurses (72%; 75%) and other ancillary (65%; 63%). In another study, Biswal et al. reported an increase in HH compliance from 28% to 43% after six months10. However an audit conducted two years later shows that compliance had fallen to 36%. These two studies indicate that it is not easy to further enhance compliance in the long term.\n\n\nMeeting the challenge of campaign fatigue\n\nThe WHO guidelines can be extremely helpful but with the passage of time, maintaining compliance can indeed be problematic. We were the first to report a paper on campaign fatigue in HH, in a hospital in which all components of the WHO MHHIS were utilized11. The compliance to HH successfully increased from 41% to 58% in 2008. However in spite of adding the various components of the MHHIS as recommended, compliance remained at 53% in 2009, 55% in 2010 and 50% in 2011. Compliance that remains static despite active promotional activities is evidence of campaign fatigue. This is similar to advertisement fatigue, defined as the lack of effects on users resulting from frequent and repeated exposures12.\n\nWe reported a technique to overcome campaign fatigue utilizing the help of frontline link nurses11. This was successful in bringing the HH compliance rate from 50% to 83% in 2012. The improvement was noted in all the 19 clinical areas except for two where together there was a decline in compliance from 42% to 10%.\n\nAs recommended by Huis et al.7 it is important to explore creative alternative activities to promote HH especially for a program that is ongoing for an extended period. Breaking a world record is an exciting endeavor and perhaps this can be one way to meet the challenge but it must be formatted in a manner that will enhance compliance with HH. Breaking the world record for HH had been done before but the record was to be the largest group of participants to do hand washing. It was first started in Bangladesh in 2008, and the latest record was organized by WHO Pan American Health Organization with a total of 740,870 participants in 201113. In this world record, the emphasis was on the total number of participants and not on the proper hand hygiene technique and thus there was no attempt to demonstrate any increase in compliance.\n\nIn the recent breaking of the first Hand Sanitizing Relay in May 2014, the concept is different. Every individual participant must perform HH before two official and approved witnesses who timed each person using a stopwatch, and recorded by video as required (Figure 1). It is done in a relay and the participant must perform HH correctly before passing on the alcohol hand rub to the next person (Figure 2). The entire process was validated by Guinness World Records. In the breaking of this record, a total of 277 participants were involved in the relay (Figure 3). What is pertinent is that the compliance of the two clinical areas showing a decline in compliance mentioned above increased to 95% in June 2014. It seems that not all health care workers will respond similarly to a promotional campaign and other high impact activities are needed to enthuse the relatively slow movers. Furthermore, Huis et al.7 also stressed the need for team-directed strategies and the relay format in this record fits this criterion precisely.\n\nIn conclusion, the essential point to stress in this report is that for health promotional campaigns that are ongoing, it is important to have further research on meeting the challenge of campaign fatigue. This is especially relevant because the HH campaign of WHO is approaching its ten year anniversary and campaign fatigue must certainly be emerging all over the world. Presently the data on this vital subject is meager and indeed more work is needed. The hospital is recognized as one of the most complex organizations in existence. Understanding campaign fatigue is essential not only for HH but also for in the promotion of appropriate healthcare in general.", "appendix": "Author contributions\n\n\n\nAll authors declare that they have participated in the conception, design, analysis and writing of the manuscript and have read and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPittet D, Hugonnet S, Harbarth S, et al.: Effectiveness of a hospital-wide programme to improve compliance with hand hygiene. Infection Control Programme. Lancet. 2000; 356(9238): 1307–1312. PubMed Abstract \| Publisher Full Text\n\nPittet D, Donaldson L: Clean Care is Safer Care: a worldwide priority. Lancet. 2005; 366(9493): 1246–1247. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization. Clean care is safer care. Reference Source\n\nWorld Health Organization. WHO guidelines on hand hygiene in health care. Geneva: Switzerland, 2009. Reference Source\n\nErasmus V, Daha TJ, Brug H, et al.: Systematic review of studies on compliance with hand hygiene guidelines in hospital care. Infect Control Hosp Epidemiol. 2010; 31(3): 283–294. PubMed Abstract \| Publisher Full Text\n\nGould DJ, Moralejo D, Drey N, et al.: Interventions to improve hand hygiene compliance in patient care. Cochrane Database Syst Rev. 2010; (9): Art. No.: CD005186. PubMed Abstract \| Publisher Full Text\n\nHuis A, Van Achterberg T, de Bruin M, et al.: A systematic review of hand hygiene improvement strategies: a behavioural approach. Implement Sci. 2012; 7: 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson LA, Sharpe PA: Improving patient and provider communication: A synthesis and review of communication interventions. Patient Educ Couns. 1991; 17(2): 99–134. Publisher Full Text\n\nMayer J, Mooney B, Gundlapalli A, et al.: Dissemination and sustainability of a hospital-wide hand hygiene program emphasizing positive reinforcement. Infect Control Hosp Epidemiol. 2011; 32(1): 59–66. PubMed Abstract \| Publisher Full Text\n\nBiswal M, Rajpoot S, Dhaliwal N, et al.: Evaluation of the short-term and long-term effect of a short series of hand hygiene campaigns on improving adherence in a tertiary care hospital in India. Am J Infect Control. 2014; 42(9): 1009–10. PubMed Abstract \| Publisher Full Text\n\nSeto WH, Yuen SW, Cheung CW, et al.: Hand hygiene promotion and the participation of infection control link nurse: an effective innovation to overcome campaign fatigue. Am J Infect Control. 2013; 41(12): 1281–1283. PubMed Abstract \| Publisher Full Text\n\nAbrams Z, Vee E: Personalized Ad Delivery when Ads Fatigue: An Approximation Algorithm. Proceeding, WINE’07 Proceedings of the 3rd international conference on Internet and network economics. Springer-Verlag Berlin, Heidelberg. 2007; 4858: 535–540. Publisher Full Text\n\nPeru, Mexico, and Argentina Help the Region of the Americas Beat the Guinness World Record for Simultaneous Handwashing. Reference Source" }	[ { "id": "6323", "date": "06 Nov 2014", "name": "Mary-Louise McLaws", "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\nThis manuscript reports on a unique project aimed at refreshing the support for a WHO global hand hygiene compliance program in healthcare workers from a tertiary teaching hospital in Hong Kong. The manuscript is well written and has succinctly reported the literature to highlight the lack of research into methods to amelioration fatigue. The only comment I have to improve on this excellent manuscript is to include a reference about the need for behavioural research (Page 2, paragraph 6), after “....behavioural determinants is needed” e.g. by Whitby M et al. (2007).", "responses": [ { "c_id": "1217", "date": "11 Feb 2015", "name": "Ben Cowling", "role": "Author Response", "response": "Thank you for these comments which we appreciate. We have incorporated the reference suggested by the reviewer in our revised manuscript." } ] }, { "id": "7084", "date": "19 Jan 2015", "name": "Vicki Erasmus", "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 interesting intervention study with a good twist on keeping hand hygiene interesting for healthcare professionals. The intervention can also inspire other hospitals to undertake quality improvement which is excellent! In general the paper is well written with a good literature base, and great effort were undertaken to ensure reliability during the record breaking attempt, including two witnesses. I have two reservations though, and hope the authors could add something on these points.The authors report an increased compliance from 50->83%, although it in unclear how or where this was measured. Was this data collected after the intervention (how long after?) in hospital wards? More details on this could help place the results in perspective and help other professi9onal undertake similar efforts. I miss a discussion of the project. By listing some of their limitations others can be helped in further improving similar activities.", "responses": [ { "c_id": "1216", "date": "11 Feb 2015", "name": "Ben Cowling", "role": "Author Response", "response": "Thank you for these comments which we greatly appreciate. We have made some minor revisions to our article to address the points made by the reviewer. Specifically, we have now clarified the dates of the Guinness World Record activity and the dates of subsequent assessment of HH compliance, in May 2014 and June 2014 respectively. We have also added a brief discussion of the project and the limitations, which will help others planning similar activities." } ] }, { "id": "7083", "date": "10 Feb 2015", "name": "Alison Holmes", "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\nThe authors of this study should be praised for approaching the matter of behavioural fatigue from such a refreshing angle. As such, their experience was an innovative activity.Technically, the paper is well written, although I feel that perhaps the initial sections focusing on evidence regarding hand hygiene campaigns was too long, considering the overall extension of the paper. Likewise, the authors may have been able to benefit related from including some existing evidence from other fields related to overcoming behavioural fatigue.Whilst the authors indicate that their intervention managed to increase compliance in 2 areas from 10% in 2012 (mentioned in \"...improvement was noted in all the 19 clinical areas except for two where together there was a decline in compliance from 42% to 10%\") to 95% in June 2014, it would have been extremely useful to see the decay rate of such boost (I accept that the authors highlight as a limitation of their paper the need to see the longer term impact of their intervention, but I feel that as the problem they tried to solve was related to compliance fatigue, we would have benefited from examining how well their initiative fared).But these suggestions should not impact on the merit of the paper, which is showcasing an innovative approach focused on maintaining engagement with desired hand hygiene behaviours.", "responses": [ { "c_id": "1215", "date": "11 Feb 2015", "name": "Ben Cowling", "role": "Author Response", "response": "Thank you for these comments which we greatly appreciate. We will look into the literature on overcoming campaign fatigue in other fields. We agree that examining the medium to longer term compliance is important and we are continuing to monitor HH compliance in this hospital." } ] } ]	1	https://f1000research.com/articles/3-234
https://f1000research.com/articles/4-34/v1	02 Feb 15	{ "type": "Research Article", "title": "Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection", "authors": [ "Veljko Veljkovic", "Philippe M. Loiseau", "Bruno Figadere", "Sanja Glisic", "Nevena Veljkovic", "Vladimir R. Perovic", "David P. Cavanaugh", "Donald R. Branch", "Philippe M. Loiseau", "Bruno Figadere", "Sanja Glisic", "Nevena Veljkovic", "Vladimir R. Perovic", "David P. Cavanaugh", "Donald R. Branch" ], "abstract": "The ongoing Ebola virus epidemic has presented numerous challenges with respect to control and treatment because there are no approved drugs or vaccines for the Ebola virus disease (EVD). Herein is proposed simple theoretical criterion for fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. We performed a repurposing screen of 6438 drugs from DrugBank using this criterion and selected 267 approved and 382 experimental drugs as candidates for treatment of EVD including 15 anti-malarial drugs and 32 antibiotics. An open source Web server allowing screening of molecular libraries for candidate drugs for treatment of EVD was also established.", "keywords": [ "Ebola virus", "drug candidates", "entry inhibitors", "virtual screening" ], "content": "Introduction\n\nThe current Ebola virus outbreak is one of the largest outbreaks of its kind in history and the first in West Africa. By January 14, 2015, a total of 21296 probable and confirmed cases, including 8429 deaths from Ebola virus disease (EVD), had been reported from five countries in West Africa - Guinea, Liberia, Nigeria, Senegal, and Sierra Leone (http://apps.who.int/iris/bitstream/10665/148237/2/roadmapsitrep_14Jan2015_eng.pdf?ua=1). EVD with a high case-fatality rate of 40% and with currently no approved vaccine or therapy, represents a major public health threat.\n\nIn response to the current Ebola virus outbreak, the international community has urged for accelerated development of drugs against EVD but also has endorsed the clinical use of unregistered treatments for Ebola1. Conventional time and the money consuming approach of drug development (> 10 years; > 2 billions $) does not meet the current urgent need for anti-Ebola drugs. Repurposing or repositioning of existing drugs could overcome some of these obstacles and help in the rapid discovery and development of therapeutics for EVD, although this approach does not negate the need for some preclinical studies and clinical trials for validation of the proposed indications. Recently, results of two large repurposing screenings of Food and Drug Administration (FDA)-approved drugs have been reported. In the first study, Madrid and co-workers performed in vitro and in vivo (in mice) screening of 1012 FDA-approved drugs and selected 24 candidate entry inhibitors for Ebola virus2. In the second study, 53 inhibitors of Ebola virus infection with IC50 < 10 µM and selectivity index SI > 10-fold have been identified by in vitro screening of 2816 FDA-approved drugs3. In the same study, an additional 95 drugs which are active against Ebola virus infection with IC50 > 10 µM and SI <10-fold were also reported.\n\nAlthough in vitro and in vivo screening for repurposing/repositioning of existing drugs could significantly accelerate discovery of new drugs these approaches are time-consuming and costly for screening of large drug libraries. Recently, we proposed a novel approach for in silico screening of molecular libraries for drug candidates4–8. This approach, which uses the average quasi valence number (AQVN) and the electron-ion interaction potential (EIIP), parameters determining long-range interaction between biological molecules, might hold a key to overcoming some of these obstacles in experimental screening by significantly reducing the number of compounds which should be in vitro and in vivo tested9.\n\nHerein, 267 approved and 382 experimental drugs, selected by the EIIP/AQVN-based virtual screening of DrugBank (http://www.drugbank.ca), have been proposed as candidate drugs for treatment of EVD. An open access portal allowing screening of molecular libraries for candidate drugs for treatment of EVD was established.\n\n\nMaterial and methods\n\nFor screening of drugs for repurposing to select candidates for Ebola virus entry inhibitors, 1463 approved and 4975 experimental drugs from DrugBank (http://www.drugbank.ca) were screened. For development of the predictive criterion used in this analysis, the learning set (Dataset 1) encompassing 152 drugs which are selected as inhibitors of Ebola virus infection by in vitro and in vivo screening of 3828 FDA-approved drugs2,3, was established. As control data sets 45,010,644 compounds from PubChem (http://www.ncbi.nlm.nih.gov/pccompound) and 49 Ebola virus entry inhibitors collected by data mining of literature and patents, were used. For screening of literature data the NCBI literature database PubMed (http://www.ncbi.nlm.nih.gov/pubmed) was used. For search of patents and patent applications we used the Free Patent Online browser (http://www.freepatentsonline.com).\n\nSpecific recognition and targeting between interacting biological molecules at distances > 5Å are determined by the average AQVN and the EIIP10, which are derived from the general model pseudopotential11,12. These parameters for organic molecules are determined by the following simple equations10:\n\n\n\nWhere Z* is the average quasi-valence number (AQVN) determined by\n\n\n\nwhere Zi is the valence number of the i-th atomic component, ni is the number of atoms of the i-th component, m is the number of atomic components in the molecule, and N is the total number of atoms. EIIP values calculated according to equation 1 and equation 2 are expressed in Rydberg units (Ry).\n\nAmong 3300 currently used molecular descriptors, AQVN and EIIP represent the unique physical properties which characterize the long-range interactions between biological molecules10. Small molecules with similar AQVN and EIIP values interact with the common therapeutic target, which allow establish criterions for virtual screening of molecular libraries for compounds with similar therapeutic properties4–9. Here we develop the EIIP/AQVN-based criterion for virtual screening of molecular libraries for candidate drugs against Ebola virus infection.\n\n\nResults and discussion\n\nPreviously, analyses of the EIIP/AQVN distribution of 45,010,644 compounds from the PubChem database (http://www.ncbi.nlm.nih.gov/pccompound) revealed that 92.5% of presented compounds are homogenously distributed within EIIP and AQVN intervals (0.00 – 0.11 Ry) and (2.4 – 3.3), respectively). This domain of the EIIP/AQVN space, encompassing the majority of known chemical compounds, is referred to as the “basic EIIP/AQVN chemical space” (BCS)6. Analysis of the molecular training set (Dataset 1), encompassing 152 small molecule inhibitors of Ebola virus infection selected by in vitro screening of 3828 FDA approved drugs2,3, show that 79% of these compounds are placed within AQVN and EIIP region (2.3 – 2.7) and (0.0829 – 0.0954 Ry), respectively (“Ebola Virus Infection Inhibitors Space”, EVIIS). The AQVN region (2.36 – 2.54) and the EIIP region (0.0912 – 0.0924 Ry) form the part of EVIIS which encompasses 55.5% of all drugs from the learning set (core EVIIS, cEVIS). Literature data mining reveals 49 compounds with experimentally proved activity against Ebola virus infection (Table 1)13–29. Most of these compounds 47 (95.9%) are placed within EVIIS (Table 1). Of note is that EVIIS and cEVIIS domains contain only 14.6% and 6.5% of compounds from PubChem, respectively. This confirms high specificity of clustering of Ebola virus infection inhibitors within the EIIP/AQVN space. Comparison of distributions of Ebola virus infection inhibitors and compounds from PubMed is given in Figure 1.\n\n*Experimental drug applied for treatment of Ebola patients in Liberia (http://www.ox.ac.uk/news/2014-11-13-oxford-lead-trial-experimental-drug-ebola-patients)\n\n(A) 45010644 compounds from the PubChem database (http://www.ncbi.nlm.nih.gov/pccompound); (B) FDA-approved drugs which are active against Ebola virus infection (Dataset 1)2,3; (C) Entry inhibitors of Ebola virus (Table 1).\n\nIt was shown that Ebola virus glycoprotein (GP)-mediated entry and infection is subordinated with a membrane-trafficking event that translocates a GP binding partner to the cell surface, which depends on microtubules30,31. Consistently, microtubule inhibitors which block this trafficking process could decrease infection without interfering with the direct binding and translocation of the Ebola virus into cells. AQVN and EIIP values of microtubule modulators and transcription inhibitors with reported anti-Ebola virus activity are given in Table 2. As can be seen, all these compounds, which do not directly affect binding and internalization of Ebola virus, are located outside of EVIIS. This additionally confirms the specificity of the EVIS domain.\n\nIn further analysis we used EVIIS as a filter for virtual screening for candidate Ebola virus infection inhibitors. In Dataset 2 622 approved and 1089 experimental drugs in Dataset 3 selected by EVIIS screening of 6532 drugs from DrugBank are reported. Using cEVIIS, we located 267 approved and 382 experimental drugs. This small molecular library represents a source of candidate drugs for treatment of Ebola virus disease (EVD), which can be further experimentally tested.\n\nMadrid and co-workers selected 24 drugs by in vitro screening of 1012 FDA-approved drugs, which are effective against Ebola virus infection2. They also showed that among these compounds, four antimalarial drugs (chloroquine, hydroxychloroquine, amodiaquine and aminoquinoline-13) also are effective against Ebola virus infection in vivo2. Among 53 compounds which effectively inhibit Ebola virus infection in vitro, which Kouznetsova and co-workers selected from 2816 approved drugs, are also three anti-malarial drugs (mefloquione, chloroquine, amodiaquine)3. It was also suggested that application of chloroquine for prevention of virus transmission should be considered because this compound significantly inhibits Ebola virus infection13. Our analysis showed that 15 of 22 approved ant-malarial drugs (http://en.wikipedia.org/wiki/Antimalarial_medication) are located in EVIIS (Table 3). Six 2-alkylquinolines have been also included in this study. This chemical series is promising as some derivatives exhibited antiviral activity such as 2PQ, and 2QQ32,33 antimalarial activity such as 2PQ and 2PentQ234, antileishmanial activity such as 2PQ35,36 and neurotrophin-like activity on dopaminergic neurons such as 2QI1537. These compounds exhibit some advantages in regard to their chemical synthesis with few steps and good yields as well as their chemical stability in tropical conditions of storage. Their combined effects against virus and Leishmania parasites suggested they could be an advantage for the treatment of Leishmania/HIV co-infections and they were considered as attractive enough to enter the pipeline of DNDi on 2010.\n\nAll these data strongly suggest that this class of drugs should be further investigated as a promising source of therapeutics for treatment of EVD. Anti-malarial drugs with dual activity should be of special interest because malaria represents the highest health-related disease in African countries with EVD.\n\nAmong 3828 FDA-approved drugs screened for anti-Ebola activity were six antibiotics which inhibit Ebola virus infection (azthromycin, erythromycin, spiramycin, dirithromycin, maduramicin, charitromycin)2,3. All these antibiotics are within EVIIS and four of them are in cEVIIS. Analysis of 184 approved antibiotics (Dataset 4) showed that only 32 (17.4%) have AQVN and EIIP values in EVIIS, and that 11 of them are located within cEVIIS. Previously we reported domains of AQVN and EIIP which characterize different classes of antibiotics (Table 4)6. According to these data, among antibiotics some macrolides, pleuromutilins and aminoglycosides have the highest chance for inhibition of Ebola virus infection. Of note is that five of six antibiotics with experimentally proved activity against Ebola virus infection (azthromycin, erythromycin, spiramycin, dirithromycin, charitromycin) are macrolides. Antibiotics representing candidate Ebola virus infection inhibitors selected by EIIP/AQVN criterion are given in Table 5.\n\nPrevious, we determined AQVN and EIIP domains characterizing different classes of anti-HIV drugs4–9. As can be seen in Table 6, the EIIP/AQVN domain of CCR5 HIV entry inhibitors is within EVIIS, and domains of CXCR4 HIV entry inhibitors and HIV protease inhibitors partially overlaps EVIIS. The EIIP/AQVN domains of other classes of anti-HIV agents are located outside EVIIS. This indicates that some HIV entry inhibitors and HIV protease inhibitors could also be effective drugs against Ebola virus infection.\n\nIn conclusion, the presented results show that the EIIP/AQVN criterion can be used as an efficient filter in virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. Approved (Dataset 2) and experimental drugs (Dataset 3), anti-malarial drugs (Table 3) and antibiotics (Table 5) selected by this criterion represents a valuable source of candidate therapeutics for treatment of EVD, some of which are already approved by FDA for treatment of other diseases which can be repurposed for use in EVD. We hope that these data, obtained by an in silico drug repurposing screen, will accelerate discovery of drugs for treatment of EVD, which are necessary in this ongoing emergency situation caused by the current unprecedented Ebola virus outbreak. To enable other researchers working on online EIIP/AQVN-based screening of different sources of small molecules for candidate Ebola drugs, we established an open web server (http://www.biomedconsulting.info/ebola_screen.php).\n\n\nData availability\n\nThe virtual screen for candidate inhibitors of EBOLA virus infection web tool is available at: http://www.biomedconsulting.info/tools/ebolascreen.php. An archived version can be accessed at: http://www.webcitation.org/6Vxtuojgx38\n\nF1000Research: Dataset 1. FDA-approved drugs which are active against Ebola virus infection2,3, 10.5256/f1000research.6110.d4287639\n\nF1000Research: Dataset 2. Approved and experimental drugs selected as candidate for treatment of EVD, 10.5256/f1000research.6110.d4287740\n\nF1000Research: Dataset 3. Experimental drugs selected as candidate for treatment of EVD, 10.5256/f1000research.6110.d4287841\n\nF1000Research: Dataset 4. Approved antibiotics screened for candidate anti-Ebola drugs, 10.5256/f1000research.6110.d4287942", "appendix": "Author contributions\n\n\n\nConceived and designed the study: VV SG NV. Developed the analysis tools: VP. Analyzed the data: VV SG NV DRB PPML BF DPC. Wrote the paper: VV DRB PPML.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant no. 173001).\n\n\nAcknowledgments\n\nThe authors would like to gratefully acknowledge networking support by the COST Action CM1307.\n\n\nReferences\n\nEnserink M: Infectious diseases. Debate erupts on ‘repurposed’ drugs for Ebola. Science. 2014; 345(6198): 718–9. PubMed Abstract \| Publisher Full Text\n\nMadrid PB, Chopra S, Manger ID, et al.: A systematic screen of FDA-approved drugs for inhibitors of biological threat agents. PLoS One. 2013; 8(4): e60579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKouznetsova J, Sun W, Martínez-Romero C, et al.: Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerg Microb Infect. 2014; 3: e84. Publisher Full Text\n\nVeljkovic V, Veljkovic N, Este J, et al.: Application of the EIIP/ISM bioinformatics concept in development of new drugs. Curr Med Chem. 2007; 14(4): 441–53. PubMed Abstract \| Publisher Full Text\n\nVeljkovic V, Mouscadet JF, Veljkovic N, et al.: Simple criterion for selection of flavonoid compounds with anti-HIV activity. Bioorg Medic Chem Lett. 2007; 17(5): 1226–32. PubMed Abstract \| Publisher Full Text\n\nVeljkovic N, Glisic S, Perovic V, et al.: The role of long-range intermolecular interactions in discovery of new drugs. Exp Opin Drug Disc. 2011; 6(12): 1263–70. 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Reference Source\n\nPanchal RG, Reid SP, Tran JP, et al.: Identification of an antioxidant small-molecule with broad-spectrum antiviral activity. Antiviral Res. 2012; 93(1): 23–9. PubMed Abstract \| Publisher Full Text\n\nDube D, Schornberg KL, Shoemaker CJ, et al.: Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry. Proc Natl Acad Sci USA. 2010; 107(38): 16637–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYonezawa A, Cavrois M, Greene WC: Studies of Ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha. J Virol. 2005; 79(2): 918–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFakhfakh MA, Fournet A, Prina E, et al.: Synthesis and biological evaluation of substituted quinolines: potential treatment of protozoal and retroviral co-infections. Bioorg Med Chem. 2003; 11(23): 5013–23. PubMed Abstract \| Publisher Full Text\n\nFournet A, Mahieux R, Fakhfakh MA, et al.: Substituted quinolines induce inhibition of proliferation of HTLV-1 infected cells. Bioorg Med Chem Lett. 2003; 13(5): 891–4. PubMed Abstract \| Publisher Full Text\n\nGantier JC, Fournet A, Munos MH, et al.: The effect of some 2-substituted quinolines isolated from Galipea longiflora on Plasmodium vinckei petteri infected mice. Planta Med. 1996; 62(3): 285–6. PubMed Abstract \| Publisher Full Text\n\nCampos Vieira N, Vacus J, Fournet A, et al.: Antileishmanial activity of a formulation of 2-n-propylquinoline by oral route in mice model. Parasite. 2011; 18(4): 333–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakayama H, Loiseau PM, Bories C, et al.: Efficacy of orally administered 2-substituted quinolines in experimental murine cutaneous and visceral leishmaniases. Antimicrob Agents Chemother. 2005; 49(12): 4950–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt F, Champy P, Séon-Méniel B, et al.: Chemicals possessing a neurotrophin-like activity on dopaminergic neurons in primary culture. PLoS One. 2009; 4(7): e6215. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerovic VR: Virtual screen for candidate inhibitors of EBOLA virus infection. F1000Res. 2015. Reference Source\n\nVeljkovic V, Loiseau PM, Figadère B, et al.: Dataset 1 in: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Research. 2015. Data Source\n\nVeljkovic V, Loiseau PM, Figadère B, et al.: Dataset 2 in: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Research. 2015. Data Source\n\nVeljkovic V, Loiseau PM, Figadère B, et al.: Dataset 3 in: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Research. 2015. Data Source\n\nVeljkovic V, Loiseau PM, Figadère B, et al.: Dataset 4 in: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Research. 2015. Data Source" }	[ { "id": "7552", "date": "11 Feb 2015", "name": "Patrick Butaye", "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\nThis manuscript deals with the in silico analysis of molecules for their activity against Ebola Virus (EBV). They started from a reference library of compounds who have previously demonstrated in vitro and/or in vivo activity against EBV and analyzed these compounds by the determination of their EIIP and AQVN. With these data, they scanned a larger collection of compounds with unknown activity against EBV and selected possible candidates to test for their activity in vitro and/or in vivo. This is a straight forward manuscript however it may be better structured. The part of the M&M dealing with the EIIP and AQVN is more appropriate to go into the introduction since this is background information of the calculation. For clarity of the manuscript is also better to separate the results section as it is difficult to follow now. Start with the analysis of the compounds with known activity, the two datasets, and then proceed with results from the unknown dataset. Then in the discussion, the different products of interest may be evaluated. This will largely increase the readability. Upon the antibiotics, it would be good to elaborate a bit on how they work on EBV, since common sense tells that antibiotics do not work on viruses. A better explanation on how these products may interact and inhibit/kill EBV would also be good.", "responses": [ { "c_id": "1224", "date": "13 Feb 2015", "name": "Veljko Veljkovic", "role": "Author Response", "response": "The antibiotics presented in this article are not selected because of their antibiotic activity but they are proposed as candidate entry inhibitors of Ebola virus (drug repurposing)." } ] }, { "id": "7633", "date": "11 Feb 2015", "name": "Bruno Botta", "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\nTo identify drug candidates against Ebola virus infections is surely an urgent need, especially in light of recent virus outbreaks registered mostly in Africa. In this respect, Velijkovic's article is presented in a timely manner and offers a fast and reliable opportunity to screen among large databases to reposition old drugs against Ebola.The experimental design relies on a consolidated methodology, developed by some of the authors and successfully applied in multiple projects. Overall, the manuscript is clear and few minor editing would be necessary, in my personal opinion, to improve its consistency.In Materials and Methods, a dataset of 152 drugs that counteract Ebola virus in vitro and in vivo has been selected as training set. However, it seems that an inconsistency does exist within this number. As authors have reported, these anti-Ebola drugs have been identified by Madrid (24 molecules) and Kouznetsova (53 molecules and 95 weaker drugs). Accordingly, the total number of FDA-approved drugs identified in these studies is 172. Why authors used a smaller set of 152? Is there any structural redundancy? A clarification of this discrepancy would improve the reproducibility of the work.Finally, if I understood properly authors have selected more than 500 molecules (including FDA-approved and experimental drugs) as anti-Ebola candidates by means of in silico screening and suggest that further in vitro/in vivo tests should be performed on these molecules. In my opinion, this number is still too large for enabling efficient and fast in vitro and/or in vivo assays. Experimental testing of this set would require significant efforts. Just for comparison, the number of candidates selected in silico by authors is about half of those selected by Madrid by means of HTS (ref 2). Is there a way to prioritize small molecules by using the EIIP/AQVN-based approach, and to provide a lower number of compounds to be submitted to experimental evaluation? Authors should comment on this point, because the advantage of using the EIIP/AQVN-based screening in silico appears to be limited in the current version of the manuscript.", "responses": [ { "c_id": "1225", "date": "13 Feb 2015", "name": "Veljko Veljkovic", "role": "Author Response", "response": "The aim of the work was not only to reduce the number of candidate drugs for EVD but to select all approved drugs which will efficiently target GP or its receptor. Further filtering of these candidate anti-Ebola drugs by other structural tools (pharmacophoric modeling, docking studies, etc.) will reduce the number of compounds for experimental testing." } ] } ]	1	https://f1000research.com/articles/4-34
https://f1000research.com/articles/3-174/v1	28 Jul 14	{ "type": "Research Article", "title": "Strategies of the honeybee Apis mellifera during visual search for vertical targets presented at various heights: a role for spatial attention?", "authors": [ "Linde Morawetz", "Lars Chittka", "Johannes Spaethe" ], "abstract": "When honeybees are presented with a colour discrimination task, they tend to choose swiftly and accurately when objects are presented in the ventral part of their frontal visual field. In contrast, poor performance is observed when objects appear in the dorsal part. Here we investigate if this asymmetry is caused by fixed search patterns or if bees can use alternative search mechanisms such as spatial attention, which allows flexible focusing on different areas of the visual field. We asked individual honeybees to choose an orange rewarded target among blue distractors. Target and distractors were presented in the ventral visual field, the dorsal field or both. Bees presented with targets in the ventral visual field consistently had the highest search efficiency, with rapid decisions, high accuracy and direct flight paths. In contrast, search performance for dorsally located targets was inaccurate and slow at the beginning of the test phase, but bees increased their search performance significantly after a few learning trials: they found the target faster, made fewer errors and flew in a straight line towards the target. However, bees needed thrice as long to improve the search for a dorsally located target when the target’s position changed randomly between the ventral and the dorsal visual field. We propose that honeybees form expectations of the location of the target’s appearance and adapt their search strategy accordingly. Different possible mechanisms of this behavioural adaptation are discussed.", "keywords": [ "Attention", "learning", "vision", "honey bees" ], "content": "Introduction\n\nWhen honeybees search for targets presented on a vertical plane, they show a distinct spatial asymmetry in colour and pattern learning between the ventral and the dorsal half of their frontal visual field. They easily learn a target when its defining features are perceived by the ventro-frontal area of the eye, but are less accurate when the crucial features of the target appear in the dorso-frontal area (Baumgärtner, 1928; Chittka et al., 1988; Giurfa et al., 1999; Lehrer, 1999; Morawetz & Spaethe, 2012; Wehner, 1972). This behavioural asymmetry might in theory be explained by specialization in eye morphology. This is found in the eye of the honeybee drone, where the dorsal area is adapted to queen detection by increasing visual acuity and sensitivity, which is partly achieved by enlarged facet diameters and a reduction of the interommatidial angles (Menzel et al., 1991; Seidl, 1982; Streinzer et al., 2013; van Praagh et al., 1980). However, such regional specialisation in eye optics is not found in the worker honeybee, where the interommatidial angles and facet diameters are similar in the frontal visual field 30° below and above the horizontal plane (Seidl, 1982).\n\nIt is also possible that regional specialisation of the visual system occurs at the neuronal level. Indeed, ascending neurons of the medulla show differences in arborisation patterns between the ventral and dorsal area (Ehmer & Gronenberg, 2002), indicating that both areas of the visual field are to some extent processed separately. Furthermore, the output of these two areas becomes segregated in the anterior optic tubercle and in the collar of the calyxes of the mushroom bodies (Ehmer & Gronenberg, 2002; Mota et al., 2011). The data hint at differences in neuronal processing between these two eye regions and correspond with the behavioural evidence of a dorso-ventral differentiation (Baumgärtner, 1928; Chittka et al., 1988; Giurfa et al., 1999; Lehrer, 1999; Morawetz & Spaethe, 2012; Wehner, 1972).\n\nAn alternative explanation for the dorso-ventral asymmetry is the usage of attentional mechanisms, which focuses the visual processing capacity of the brain flexibly to the currently most important area in space (spatial attention; Carrasco & McElree, 2001; Druker & Anderson, 2010; Geng & Behrmann, 2002; Posner, 1980; Yantis & Jonides, 1984). Attention here can be thought of as a kind of ‘inner’ eye, focusing on a spatial subset of the information that is available from the visual sensory periphery. The existence of spatial attention is well known in vertebrates, but has only recently been described for the first time in insects, the fruit fly Drosophila melanogaster (Sareen et al., 2011) and the honeybee Apis mellifera (Paulk et al., 2014).\n\nSpatial attention optimizes search processes in detection tasks, where the subject has an expectation of the appearance of the object, using external cueing or own experience (Posner, 1980). Search efforts can then be directed to this region which leads to faster and more accurate decisions (Carrasco & McElree, 2001). Hence, spatial attention would be a useful tool for foraging bees helping to adapt to various spatial settings of flower distribution. The dorso-ventral asymmetry observed in visual discrimination tasks could be explained by attention being focused on the ventral part of the visual field by default, but the attentional focus could be moved to other parts of the visual field, if necessary. To test if bees can employ spatial attention during foraging, we confronted honeybees with three search scenarios differing in the positioning of the target in the visual field. This approach allows to analyse the changes in search performance and flight behaviour over time and therefore to identify possible adaptations of the search behaviour to the particular target presentation.\n\n\nMaterial and methods\n\nThe experiments were conducted between July and September in 2011 on the terraces of the Biozentrum, University of Vienna, where several hives of Apis mellifera were located. Bees were trained to an experimental box and marked individually.\n\nA wooden box (30 × 54 × 40 cm) served as experimental arena (Figure 1A; see also Morawetz & Spaethe, 2012). The bees could enter the box through a Plexiglas tube on the front and shutters in the tube allowed to control access to the box. Two video cameras (DCR-SR55, Sony, Minato, Tokyo, Japan) were placed above and at one side of the box to record the flights through a small-meshed net and a Plexiglas wall, respectively.\n\nA. Side view of the experimental arena. Bees enter the arena through a Plexiglas tube, view the objects on the back wall and fly towards one of them. The bee’s decision is counted as correct when the bee crosses the decision line (5 cm in front of the back wall) at the position of the target. The bee’s flight can be observed and filmed through a small-meshed net from above and through a Plexiglas cover from the side. The number gives the angle of the target subtended on the bee’s eye when viewed from the entrance of the box. B. Reflectance curves of the back wall cover (grey BG - background), the orange target (HKS 7N) and the blue distractor (HKS 49N). C. Colour hexagon showing the colour loci of the target and the distractor (colour distance of HKS 7N to background: 0.23 hexagon units; HKS 49N to background: 0.25 hexagon units; distance between the two colours: 0.27 hexagon units); calculations after Chittka (1992).\n\nThe back wall was divided into nine fields (3 rows by 3 columns) of which only the top and the bottom row were used for training and testing. In the centre of each field a platform provided reward for a correct choice (1 M sucrose solution ad libitum) and punishment for an incorrect choice (0.1% quinine solution w/w; Chittka et al., 2003), respectively. Stimulus discs (K + E, Stuttgart-Feuerbach, Germany) of 9 cm diameter were cut from coloured paper; the target discs were orange (HKS 7N) and distractor discs were blue (HKS 49N, for colour details see Figure 1B,C). The stimuli subtended a visual angle of >15° on the eye of the bee when sitting at the box entrance, which enabled the bee to perceive chromatic information from the beginning of the search (Dyer et al., 2008; Giurfa et al., 1996). The stimuli were presented on the back wall of the arena subtending a visual field of 82° in the horizontal and 60° in the vertical on the eye of a bee (Figure 1A). Stimuli located in the dorsal row were thus perceived by the dorsal part of the eye, while objects in the ventral row were perceived by the ventral part of the eye, when the bee entered the arena (Figure 1A).\n\nBees were trained to enter the experimental arena and to search for the orange target on the back wall. During this pre-training, special care was taken in presenting the target at different heights and positions in the experimental arena to avoid any position learning. After the first successful visit of the target, a training phase of 20 visits followed. The position of the target was changed in a pseudo-random pattern: on average, in 50% of the visits the target was presented in the top row, and in the other 50% in the bottom row. The target discs and feeders were exchanged with clean discs/feeders after every third visit to avoid odour contamination.\n\nThe training phase was followed by an experimental phase, which consisted of 30 flights (five blocks, six flights per block). Bees were divided in three experimental groups. Bees from all groups had to search for one orange target among two blue distractors, but the groups differed in the placement of the objects (see insets of Figure 2). In the ‘dorsal group’, all objects were always placed in the top row; in the ‘ventral group’, they were only presented in the bottom row. In the ‘mixed group’, target and distractors could appear in the top row as well as in the bottom row. The target position was changed in a pseudo-random order assuring that the target was presented 50% of the trials in the top row and 50% of the trials in the bottom row. The distractor positions were changed randomly between the remaining five positions of the two rows. Therefore, bees of the ‘dorsal’ and ‘ventral group’ needed to search only in a subarea of the search field (three possible positions), while bees of the ‘mixed group’ had to search the target within the entire search area (six possible positions).\n\nTreatments differed in the positioning of the rewarding target and distractors which were associated with a punishment (insets: grey circle = target, black circle = distractor). In the ‘dorsal group’ (straight grey line, grey circle) the target was always presented in the top row, in the ‘ventral group’ (straight black line, black circle) it was always located in the bottom row, and in the ‘mixed group’ the position of the target alternated randomly between both rows (top row: dashed grey line, grey triangle; bottom row: dashed black line, black triangle). A, B. Comparison between the one row condition and the two row condition separated in trials where the target appears in the bottom row (A) and trials with the target in the top row (B). C. Comparison of the first 12 decisions of the dorsal group and the ‘ventral group’. Statistics: general linear model with binomial distribution: n.s. P>0.10, * P<0.05, P<0.01, P<0.001; data points: mean ± s.e.m., N=9 individuals per group.\n\nSearch efficiency was measured using two parameters – error rate and decision time. This allowed to check for possible speed-accuracy trade-offs (Burns & Dyer, 2008; Chittka et al., 2003). We counted a bee’s choice when it crossed a decision line, which was indicated by a grid of white twine 5 cm in front of the back wall of the experimental box (Figure 1; Morawetz & Spaethe, 2012). We counted an error when bees crossed this decision line at a location without a target. The decision time was defined as the time the bees needed from entering the box until they crossed the decision line.\n\nWe analysed the video recording using The Observer XT Version 7 (Noldus, Wageningen, The Netherlands) and reconstructed the flight paths of the bees using SkillSpector 1.3.0. (Video4coach, Svendborg, Denmark). Statistics was calculated using the statistical package R version 2.14.1 (R Development Core Team, 2011). We used mixed linear models to test the effect of treatment group, target location and learning on the error rate and on the decision time, respectively. Furthermore, the identity of the individual bees was implemented into the model as a random factor. For analysing the error data, a binomial distribution was applied using the function lmer of the package ‘lme4’ (Bates et al., 2011), while for the decision time, a normal distribution was applied using the same function. Significance levels were determined by calculating a type-III ANOVA using the function Anova (package ‘car’, Fox & Weisberg, 2011). To check if the bees showed a general tendency to fly upwards or downwards during the different flight stages, the flight angles were tested against 0° (horizontal flight) with a Wilcoxon test. Figures were created using Sigma Plot 11.0 (Systat Software Inc., San Jose, USA) and Corel Draw X3 (Corel Corporation, Ottawa, Canada).\n\n\nResults\n\nDuring the training phase, when only the target was present, bees of all treatment groups showed similar accuracy (χ2(2)=1.37, P=0.502). At the end of the training, the bees made four times fewer mistakes when searching for ventrally located targets compared to targets located in the dorsal row (bottom row: 15% ± 25% s.d.; top row: 65% ± 33%; χ2(1)=34.32, P<0.001; see also dataset 1). During the test phase, the overall error rate was similar in all three treatment groups (χ2(2)=2.79, P=0.248; see also dataset 2), but the speed of improvement differed between the three groups (interaction treatment group x learning block: χ2(8)=19.58, P=0.012).\n\nBees of the group presented with targets in the ventral visual field showed a constant performance during the test with a mean error rate of 17% ± 14 s.d. (χ2(4)=2.72, P=0.606; Figure 2A, straight line). Conversely, when bees were presented with targets in the dorsal visual field, they made about twice as much mistakes at the beginning of the test than at the end (χ2(4)=12.84, P=0.012; Figure 2B, straight line). However, the overall error rate did not differ from the ‘ventral group’ (χ2(1)=0.78, P=0.377; Figure 2A). The main change in learning performance in the ‘dorsal group’ took place between the first and second learning block; thus a possible difference between both treatments at the beginning of the test phase may have been masked. We then analysed the first 12 decisions in more detail, subdividing the 12 visits into blocks of three (Figure 2C). During the first three flights, bees of the ‘dorsal group’ made four times more mistakes than bees of the ‘ventral group’ (z = 2.77, P=0.006; Figure 2C). After this initial phase, both groups achieved similar levels of accuracy (all further learning blocks: P>0.05; Figure 2C).\n\nThe ‘mixed group’ was the only group in which the target and distractors were presented in both the ventral and the dorsal part of the visual field. Similar to the ‘dorsal group’, bees improved their accuracy during the test (χ2(4)=9.54, P=0.049). Bees of the ‘mixed group’ made fewer mistakes when the target was presented in the ventral row compared to the situation when it was presented in the dorsal row (χ2(1)=5.27, P=0.022; Figure 2A,B dashed lines). Next, we analysed both data sets separately and compared them to the correspondent single row treatment group (Figure 2A: flights of the ‘mixed group’ when the target was ventrally presented compared to the ‘ventral group’; Figure 2B: flights of the ‘mixed group’ when target was dorsally presented compared to the ‘dorsal group’). The performance of bees from the ‘mixed group’, when searching for a ventral target, was similar to the performance of the ‘ventral group’ where the target was always presented ventrally (χ2(1)=0.35, P=0.556; Figure 2A). Furthermore, bees of the ‘mixed group’, when searching for ventral targets, showed a constant performance during the experiment (χ2(4)=3.90, P=0.420). However, they made significantly more mistakes than bees of the ‘dorsal group’ in finding a dorsally positioned target (χ2(1)=7.69, P=0.006; Figure 2B). Nonetheless, the ‘mixed group’ improved their performance for dorsal targets (χ2(4)=14.05, P=0.007), and in the fourth learning block, bees managed to achieve a similarly low error rate as the bees of the ‘dorsal group’ (Figure 2B).\n\nAll three experimental groups became faster during the experiment (χ2(4)=16.83, P=0.002; Figure 3), but the decision time did not differ among treatment groups (χ2(2)=1.04, P=0.595) or in the interaction of both factors (χ2(8)=10.00, P=0.265). However, bees of the ‘mixed group’ were slower when the target was presented in the top row than when it was presented in the bottom row (χ2(1)=28.6, P<0.001). Bees of the ‘dorsal’ and ‘ventral group’, on the other hand, showed similar decision times during most of the time (χ2(1)=0.24, P=0.624). However, during the first three flights, bees of the ‘dorsal group’ were significantly slower than bees of the ‘ventral group’ (t(13.1)=2.21, P=0.045; Figure 3C).\n\nDecision time of the three experimental groups: ‘ventral group’ (black straight line, black circle), ‘dorsal group’ (grey straight line, grey circle) and ‘mixed group’ (ventral half: black dashed line, black triangle; dorsal half: grey dashed line, grey triangle). A. When searching for the target in the ventral half of the visual field, the bees of the ‘mixed group’ made their decision at a similar speed as the ‘ventral group’ (χ2(1)=0.00, P=0.979). They did not improve their performance (χ2(4)=2.25, P=0.689). B. Comparing the ‘dorsal group’ with the dorsal flights of the ‘mixed group’, the bees showed no significant difference in their decision time (χ2(1)=3.02, P=0.082). All bees became faster in the course of the experiment (χ2(4)=22.84, P=0.001). C. Comparison of the ‘dorsal’ and the ‘ventral group’. Only during the first three decisions the ‘dorsal group’ was significantly slower than the ‘ventral group’ (t(13.10)=2.211, P=0.045), afterwards bees of both groups had similar decision times (all other data points P>0.05). Statistics: t test: n.s. P>0.10, x P<0.10, P<0.05; data points: mean ± s.e.m., N=9 per group.\n\nWhen analysing the flight structure of the first flights in detail, it became evident that bees of the ‘dorsal group’ increased the steepness of their departure angle (flight angle between 0 and 5 cm distance from the entrance) significantly from the first to the third flight (Figure 4; Wilcoxon test between first and third flight: P<0.05). During their first flight, they started horizontally into the box (median flight angle 5.9°, Figure 4), but from the third flight on they flew upwards when taking off (median flight angle third flight: 12.1° upwards; fifth flight: 10.0° upwards). This change in flight structure indicates that the bees decided earlier to fly upwards in their third flight compared to their first flight. All other experimental groups showed median departure angles between 7° downwards and 5° upwards – no significant trend in flying up- or downwards during the departure was found.\n\nMedian flight angles of different phases in the bee’s flights crossing the experimental arena from the entrance (0 cm) to the decision line (25 cm). For all four search situations the first (black arrows), the third (blue arrows) and the fifth flight (green arrows) are shown (N=7). Please note that in the mixed group the flight number refers to number of trials absolved with the particular target situation and not to the total number of trials the bee has absolved. Arrows: median flight angles; grey area: first to third quartile; statistics: one-sample Wilcoxon test against 0° (flight without significant trend of changing flight height).\n\nWhen the target was located in the bottom tier, the bees’ flight was composed of downward angles during most of their flight path, suggesting a continuous downward flight (Figure 4, Figure 5). Bees searching for a dorsally located target, however, differed in their flight structure between treatment groups: bees of the ‘mixed group’ flew in a steep angle downwards during the first flight period (maximum flight angle 40° downwards) and showed a significant upwards direction between 20 and 25 cm distance from the entrance (median flight angle between 33° and 58° upwards, Figure 4). During the fifth flight of the experiment with the target in the top row, these bees lost their tendency to fly downwards at the beginning of their flight and flew straight forward for the first 20 cm and ascended steeply afterwards. Bees of the ‘dorsal group’ flew horizontally between the first 5 to 20 cm, with some animals already beginning to ascend. Between 20 and 25 cm they ascended in a steep angle of around 25° similar to the animals of the ‘mixed group’. Therefore, all bees searching for a top row target approached the target from below. Flight paths of individual bees demonstrate the described differences of the three different groups (Figure 5).\n\nThe flight paths describe the differences in flight behaviour among the three experimental groups. The 16 squares represent cross sections of the flight box with the bee entering the box from the left side searching for the target (presented on the right side; orange rectangle). The blue line 25 cm away from the entrance marks the decision line. For each experimental group three individual bees are shown (straight black line, dashed black line, dashed grey line).\n\nIn conclusion, the accuracy, decision time and flight path of bees searching for a vertically presented target was partly dependent on the target’s location and partly on the bees’ possibility to predict the target’s location. Searching for ventrally located targets was an easy task – all bees showed high accuracy, short decision times and a direct flight path. The predictability became important in situations in which bees had to detect the target with the dorsal part of their eyes. When the target was always in the top row (‘dorsal group’), bees adapted their search strategy rapidly to the situation –after three search flights they detected the target swiftly, decided accurately and approached the target directly. When the target was positioned randomly in the top or the bottom row (‘mixed group’), the bees showed a more downward directed flight pattern when being confronted with dorsally located targets. As a consequence, these bees took longer in making their decisions and made more mistakes.\n\n\n\n\nDiscussion\n\nWe simulated three foraging situations that a bee might encounter in her natural environment: approaching flowers from above (flowers perceived by the ventral part of the eye), foraging within a 3D floral environment, either in a tree or a meadow with flowers at various heights (flowers may appear in the dorsal and ventral part of the visual field) and approaching a flower from below when foraging, for example, upwards on vertical inflorescences (flowers perceived by the dorsal area of the eye). We demonstrated that bees were able to adapt their detection strategy to all three foraging situations, although the course of performance improvement differs among the experimental conditions. In the following paragraphs, we discuss the possible proximate and ultimate mechanisms of this behavioural flexibility.\n\nBees of this group were presented with one target and two distractors in the bottom row. They decided accurately and swiftly and had a downwards directed flight vector. It is therefore possible that bees, by default, focus on the ventral part of the frontal visual field when being confronted with vertically presented objects, a behaviour which might explain the dorso-ventral asymmetries in visual discrimination observed by several independent studies (Baumgärtner, 1928; Chittka et al., 1988; Giurfa et al., 1999; Lehrer, 1999; Morawetz & Spaethe, 2012; Wehner, 1972).\n\nIn the ‘dorsal group’, both target and distractors were presented in the top row. During the initial flights, bees’ search was inaccurate and slow compared to the ‘ventral group’. The bees flew straight ahead for the first 20 cm in the flight arena and then turned upwards in a steep angle during the final approach. After the first three flights, bees showed two major changes in their behaviour: (1) the error rate dropped from 40 to 20% (Figure 2C) and (2) they changed their departure angle from horizontal to, on average, 11° upwards (Figure 4), a behaviour which was exclusively shown by the ‘dorsal group’.\n\nThe reason for the bees’ improvement might be explained by two independent but not exclusive mechanisms. Firstly, a top-down attentional mechanism could have accelerated information processing from the dorsal part of the frontal visual field by shifting the attentional focus to this area. In this case, bees might have detected the target stimulus just after entering the experimental arena and started an upward flight towards the top row. Alternatively, bees could have learned to adjust their flight path to the area where they expected the target: they learned to initiate an upward flight immediately after entering the arena before detecting the target. By flying upwards they were able to analyse the top row with the ventral part of their eye and therefore increase their accuracy. Evidence for both mechanisms comes from earlier studies: bees can learn to perform directional changes and changing flight routes to optimize foraging success (Cheng & Wignall, 2006; Lihoreau et al., 2010; Perry & Barron, 2013), as well as use attentional processes to selectively react to visual stimuli (Giurfa et al., 1999; Paulk et al., 2014; Spaethe et al., 2006). However, our results do not allow discriminating between both mechanisms.\n\nBees of the mixed group were confronted with one target and two distractors, which were randomly distributed between the bottom and top row. During most trials, objects were located in both the bottom and the top row. Hence, the search strategy of the ‘mixed group’ was fundamentally different from the two other groups. Bees were not simply able to fly towards a group of objects, but had to decide which group of objects was relevant for them, i.e. contained the target. At the beginning of the experiment, bees flew downwards regardless of the target position – probably executing their ‘default’ behavioural pattern and flying towards the lowermost objects. This behaviour resulted in a low error rate when the target showed up in the bottom row, but in a high error rate and long decision times when the target appeared in the top row. Furthermore, our data indicate that bees detected ventrally located stimuli first, but analysed the chromatic features of the stimuli only later during flight.\n\nBees of the mixed group had to change their flight pattern to improve the search for targets located in the top row. A downwards orientated flight route can hamper the detection of a dorsally located target due to an unfavourable angle of view for objects in the top row. Additionally, it makes complex course corrections necessary (demonstrated by the flight curves in Figure 5). Indeed, bees of the ‘mixed group’ stopped flying downwards in the first part of their flight after their tenth trial (Figure 4) and instead flew straight before deciding for an upwards or downwards movement.\n\nAdditionally, bees of the mixed group may have also applied mechanisms of visual attention to increase the detection performance of targets located in the top row. For example, bees could have increased the area of their attentional focus to a size large enough to cover both rows. This would have allowed the bees to process all presented objects simultaneously (Eriksen & St. James, 1986; Pashler, 1987). In humans, an increase in the size of processing focus is normally accompanied by a decrease of processing quality (Castiello & Umiltà, 1990; Eriksen & St. James, 1986). We did not find a decrease of performance level in the search for ventrally located target in the ‘mixed group’, which makes this explanation unlikely. An alternative search mechanism might be that bees learn to move their attentional focus from the ventral part of the visual field to the dorsal part after they had not detected a target in the ventral part. This explanation fits with the constant high search performance for targets in the bottom row and with longer decision times and late upward directed flight vectors, when searching for a target in the top row. The high error rate in the first part of the experiment probably owes to the learning process, in which the bees (1) learned to avoid flying downwards immediately after entering the arena and (2) learned to continue searching the top row when the target could not be detected in the bottom row.\n\nWhen searching for a flower patch, bees often approach the meadow or tree from above (Kevan, 1990; Lehrer, 1999) – a situation represented by the conditions of the ‘ventral group’. Bees seem especially capable to deal with this type of search, as they detect ventrally positioned targets more easily (Figure 2A; Lehrer, 1999; Morawetz & Spaethe, 2012; Skorupski et al., 2006). However, when bees are foraging in a meadow with flowers at various heights, flowers can also appear in the upper part of the visual field and information received from the dorsal part of the eyes become important. For example, bees which visit raceme inflorescences tend to begin collecting nectar at the bottom of the inflorescence and ascend vertically step by step (Fisogni et al., 2011; Ishii et al., 2008; Pyke, 1978; Valtueña et al., 2013; Waddington & Heinrich, 1979). In this situation the flowers below the just probed ones, which are perceived by the ventral visual field, typically contain no nectar, because the bee had just visited them (Heinrich, 1975; Ishii et al., 2008). To optimize foraging efficiency, bees must focus visual processing on the dorsal part of the visual field or adopt an upward directed motor pattern. We demonstrated that bees can adapt to the described situation, improving their detection ability of dorsally located targets within three visits only (Figure 2C).\n\nWhen foraging in a blooming tree, a bee can expect a rewarding flower appearing in the ventral and the dorsal visual field with a similar probability. Flowers are densely distributed in a tree and bees normally see several flowers simultaneously. Interestingly, large bees as Bombus and Xylocopa reveal a complex pattern of upwards and downward movements inside a tree: when moving between neighbouring flowers, they move upwards, similar to the movement pattern on a vertical inflorescence (Kevan, 1990). However, when moving to an inflorescence at distance larger than 20 cm they tended to fly downwards (Kevan, 1990). This behaviour matches our observation that bees tend to fly to the lowermost object when approaching from a distance. However, we also demonstrated that bees can overcome this motor pattern within 10 learning trials, when it hinders the bees’ foraging success. Likewise, Kevan (1990) described that movement patterns of bees differed between tree species with different flower distributions and suggested that bees optimize their foraging strategy to the particular resource distribution.\n\nIn conclusion, we showed that bees flexibly adapt to a given foraging situation by focusing their detection and discrimination effort to the appearance of the object within their visual field. They probably use both attentional mechanisms and behavioural strategies to optimize their foraging success, although more data are needed to clearly separate between these mechanisms. This flexibility provides the ability to choose among different search strategies and to quickly adapt to various foraging environments.\n\n\nData availability\n\nfigshare: Datasets for strategies of Apis mellifera during visual search for vertical targets, doi: http://dx.doi.org/10.6084/m9.figshare.1104387 (Morawetz et al., 2014).", "appendix": "Author contributions\n\n\n\nLM and JS conceived the study and designed the experiments. LM carried out the research and performed the data analysis. LM, LC and JS were involved in data interpretation and writing the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nL.M. was recipient of a DOC-fFORTE fellowship of the Austrian Academy of Science at the Department of Integrative Zoology, University of Vienna.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Martin Streinzer for performing the colour measurements.\n\n\nReferences\n\nBates D, Maechler M, Bolker B: lme4: Linear mixed-effect models using S4 classes: R package version 0.999375-42. 2011. Reference Source\n\nBaumgärtner H: Der Formensinn und die Sehschärfe der Bienen. Z Vergl Physiol. 1928; 7(1): 56–143. Publisher Full Text\n\nBurns JG, Dyer AG: Diversity of speed-accuracy strategies benefits social insects. Curr Biol. 2008; 18(20): R953–R954. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeidl R: Die Sehfelder und Ommatidien-Divergenzwinkel von Arbeiterin, Königin und Drohn der Honigbiene (Apis mellifica), Dissertation. Darmstadt: Techn. Hochsch. 1982.\n\nSkorupski P, Spaethe J, Chittka L: Visual search and decision making in bees: time, speed, and accuracy. Int J Comp Psychol. 2006; 19(3): 342–357. Reference Source\n\nSpaethe J, Tautz J, Chittka L: Do honeybees detect colour targets using serial or parallel visual search? J Expl Biol. 2006; 209(6): 987–993. PubMed Abstract \| Publisher Full Text\n\nStreinzer M, Brockmann A, Nagaraja N, et al.: Sex and caste-specific variation in compound eye morphology of five honeybee species. PLoS One. 2013; 8(2): e57702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValtueña FJ, Ortega-Olivencia A, Rodríguez-Riaño T, et al.: Behaviour of pollinator insects within inflorescences of Scrophularia species from Iberian Peninsula. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "5787", "date": "26 Aug 2014", "name": "Bruno van Swinderen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study Morawetz, Chittka, and Spaethe present results from a behavioral paradigm for honeybees where they examine spatial attention. Bees were trained to select a color, using classical conditioning with sugar versus quinine. Trained bees were then presented with various scenarios whereby the targets were presented together with distracters, either in the upper or lower visual field. The authors find that targets in the ventral field led to a more efficient search strategy. Importantly, it appears that honeybees form expectations about where their target will be, in repeated flights. These are interesting results, which help consolidate the view that insects display a selective attention. In particular, the data supporting the view that there is an expectation already formed early in the flight angle is very intriguing. It is nice to see a paper where the behavior itself is examined closely, rather than only the proportion of choice outcomes. This helps to better show how attention might be operating in these simple animals. The paper is close to indexation quality. There are a couple questions that might need to be attended to, to further improve the quality of the work. First, it is quite unclear how the position of the “decision line” was arrived at. While it is evidently depending on previously published work, one concern is that varying the position of this decision line might substantially change the results and conclusions. The decision line is currently 5cm away from the end wall, and both metrics (error rate and decision time) depend on this distance. Clearly, a bee flying downwards is exploiting gravity to get to the decision line, while a bee flying upwards is fighting gravity. This simple factor could account for the different flight dynamics seen for either situation (as is evident in the informative single examples shown in Figure 5). Can the authors exclude a non-trivial explanation here for the effects of gravity? With regard to the error rate for example, it is not clear whether a “mistake” is meaningful here. Maybe bees fly more erratically upwards (they do). More generally, it would be good to have a bit more of a rationale for why this 5cm point was chosen. How different would the results have been at 7cm or 10cm? Second, the error rate metric does not seem to account for proximity. Thus, a bee crossing the decision line close to the target is as equally wrong as a bee crossing a whole square away? This seems strange. Should the error rate be weighted in some way? How might that change the results? Minor: Sareen et al (2011) showed that fruit fly attention is mostly ventral. This might be discussed a bit more, in connection to the current findings in the bee.", "responses": [ { "c_id": "1228", "date": "16 Feb 2015", "name": "Lars Chittka", "role": "Author Response", "response": "First of all, we thank the referee for the kind consideration of our manuscript and the suggestions, which helped to improve our manuscript. First, it is quite unclear how the position of the “decision line” was arrived at. While it is evidently depending on previously published work, one concern is that varying the position of this decision line might substantially change the results and conclusions. The decision line is currently 5cm away from the end wall, and both metrics (error rate and decision time) depend on this distance.In response to this comment and those by the other referees, we have now first explored the bees’ flight structure and error rates at different distances in more detail, providing a justification why it is useful to focus on an analysis of error rates at 5cm distance from the target. Clearly, a bee flying downwards is exploiting gravity to get to the decision line, while a bee flying upwards is fighting gravity. This simple factor could account for the different flight dynamics seen for either situation (as is evident in the informative single examples shown in Figure 5). Can the authors exclude a non-trivial explanation here for the effects of gravity?This is an interesting point. We would expect gravity to affect all bees who fly towards a dorsally located target in the same way. However, bees of the 'dorsal group' and bees of the 'mixed group' searching for a dorsally located target differed significantly in their error rate (Fig. 2), error locations (Fig. 7), flight structure (Fig. 5, 6) and decision time (Fig.4). We therefore suspect that gravity cannot provide a good explanation for the dorso-ventral asymmetry observed in this experiment. With regard to the error rate for example, it is not clear whether a “mistake” is meaningful here. Maybe bees fly more erratically upwards (they do). More generally, it would be good to have a bit more of a rationale for why this 5cm point was chosen. How different would the results have been at 7cm or 10cm?To answer this question, we included a new video analysis into the manuscript describing the error rate in distances of 10 cm, 7.5 cm and 0 cm (fig. 3). For the first learning block it shows that 5 cm distance allows a good differentiation between direct flights (as in the ventral group) and less directed flights (as in the dorsal group). This provides a justification for our choice to analyse error rate at this distance. However, it is necessary to state, that there is no 'ideal' distance for error analysis as flight structure may change over time. This is demonstrated by the analysis of the last learning block, where differences between groups are most pronounced at 7.5 cm distance. Second, the error rate metric does not seem to account for proximity. Thus, a bee crossing the decision line close to the target is as equally wrong as a bee crossing a whole square away? This seems strange. Should the error rate be weighted in some way? How might that change the results?This is a good point. In response, we decided to depict the different error locations in a graph (fig. 7). The graph shows that groups searching for targets at different heights differed in their way in making errors. The results corresponded well with data from flight structure analysis. For example, bees of the dorsal groups gradually developed a trend to fly upwards. At the same time, they made most errors while being located in the top row - demonstrating the distinct upwards movement of these bees during their flights. Sareen et al. (2011) showed that fruit fly attention is mostly ventral. This might be discussed a bit more, in connection to the current findings in the bee.We thank the referee for the suggestion and included the findings of Sareen et al. into the introduction." } ] }, { "id": "5610", "date": "27 Aug 2014", "name": "Martin Egelhaaf", "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\nGeneral remarksThe amount of information that is perceived by the peripheral visual system is thought to exceed the amount of information that can be processed by subsequent stages of the brain. Based on a host of experimental data mainly obtained with humans, both overt and covert strategies were concluded to facilitate the extraction of the behaviourally relevant information and to cope with the problem that the entire visual information cannot be processed at once. In previous studies by the same authors (or a subgroup of them) it was convincingly shown that honeybees (in contrast to bumblebees) employ mainly an overt serial strategy to search for previously learnt coloured target objects, if these are embedded between distracting cues. In analogy to similar results obtained with human observers, these conclusions were interpreted as being based on a kind of serial shift of the animals’ focus of attention. The analyses were based on the search performance (i.e. measuring both error rate and search time) while detecting a target object embedded in a variable number of distracting differently coloured objects.The present study is based on a similar experimental design as employed in these previous studies and asks the highly interesting question of whether the visual system of honeybees is functionally regionalised with regard to a pattern detection/discrimination task. Honeybees were trained to detect a coloured target either in the upper or bottom row of a 3x3 matrix of possible object positions. In the test phase distracting objects were added in three different constellations to the stimulus matrix. Both target and distractor objects were placed either in the top row (‘dorsal group’) or only in the bottom row (‘ventral group’); in a ‘mixed group’, target and distractors could appear in the top row as well as in the bottom row. From the way the search performance improved under the different stimulus constellations, the authors concluded that honeybees flexibly adapt to a given foraging situation by focusing their search strategy to the location of the object within their visual field. The accuracy, decision time and flight paths of honeybees were concluded to depend on both the target’s location in the visual field and on the bees’ possibility to predict the target location. It is discussed whether honeybees use attentional mechanisms to increase their success rate in the detection task.Although I am sympathetic with the overall goal of this project and agree with the authors’ overall conclusion, I have to admit that I am somewhat confused by parts of the experimental design of the study. Some further explanation of the rationale behind the analysis is likely to largely raise the potential of the study. Major issues\n\nAfter the training phase the bees could not have any a priori expectation with regard to target position, since target position was pseudo-randomized during the training procedure. Interestingly, a clear dorso-ventral asymmetry of the error rate of target detection was found even at the end of the training period. For the test phase the bees were subdivided into the three experimental groups that were tested with just one of the stimulus constellations. The rationale behind this design is not well explained and not immediately obvious. At least at first sight, one might have expected that the different constellations were tested again with all bees in a pseudo-random fashion.One reason that this has not been done might have been the extreme dorso-ventral performance gradient after the training phase. Even without any distractor the coloured target could be detected with only a very high median error rate of 65% when presented in the top row. Surprisingly, no strong point is made that the detection performance is not much above chance level (this issue should have been tested statistically!), when the target was presented in the top row. Theoretically, this poor performance should not increase, if in the test phase – i.e. after adding two distractor objects - the target were also presented randomly in the top and bottom row of the stimulus arrangement. Hence, it might have been scientifically meaningful to ask whether the detection performance in the dorsal part of the visual field might increase, if the bee had the chance to build up an expectation that the target is not located ventrally. This might be a rationale for subdividing the bees into the three groups. Irrespective of whether or not this supposition is correct, an explanation for the experimental design should be given.In any case, it can be concluded from the development of error rates, decision times, but also the trajectory data that the performance of the ‘dorsal group’ improves over time and that this finding is consistent with the interpretation that also dorsally located targets can be readily detected, if the bee has a priori knowledge that the target is likely to be found in an otherwise ‘suboptimal’ part of the visual field. Again, it might be helpful if this kind of conclusion were drawn explicitly in the paper. Whether this finding is just a consequence of a learning process or might be also linked to attentional mechanisms can then be discussed. However, it might be hard to conceptualise how attentional mechanisms can be pinpointed experimentally in the type of experiments that were performed in this study.The hardest issue that remains to be explained are the data obtained with the ‘mixed group’ of bees. Although here no expectation can develop with regard of the location of the target, the performance improves over stimulus presentations to values that are much better than those obtained at the end of the training phase – even without any distractors (!). This highly surprising issue definitely needs to be discussed. This somehow apparent contradiction raises also the question whether – given a tremendous variability between bees but also between different flights of individual bees – the data base is sufficiently large to allow for drawing strong conclusions.\n\nMinor issuesTitle of the paper:Since the paper does not focus on the topic of attention, I would omit in the title of the paper the addendum “: a role for spatial attention?” Attention is just one possibility, in addition to several others, to explain the experimental results. Putting too much emphasis on attention already in the title may be somewhat misleading.Abstract:“Here we investigate if this asymmetry is caused by fixed search patterns or if bees can use alternative search mechanisms such as spatial attention, which allows flexible focusing on different areas of the visual field.” I do not see that the investigation of this alternative has been the focus of the experimental analysis of the study. At least the results do not allow answering this question in an unambiguous way.Introduction:1st paragraph: It appears to be surprising that the paper starts by considering whether the distinct spatial asymmetry in colour and pattern learning of honeybees as observed in previous studies might be a consequence of specialisations in eye morphology, as has been proposed for detecting the queen by the drone bee eye. It should be noted that this sort of detection task is conceptually very different: in the drone bee case we are dealing with detection thresholds where - for obvious reasons - spatial acuity and sensitivity, i.e. anatomical and physiological retinal parameters, are the relevant factors, because they determine at which distance a small target (i.e. in this case the queen) can be seen. In the learning experiments the targets were usually sufficiently large, and no spatial resolution or sensitivity problem existed for them to be detected.Methods:What was the frame rate and resolution of the camera system?“The stimuli subtended a visual angle of …” (and following sentence): The authors probably mean the individual discs. “stimuli” not specific enough.It should be explained why a virtual decision line was chosen. It might have been more intuitive to monitor the landing on the different targets. Would the error rate change, if the landings were recorded rather than the crossing of a virtual decision line?What happened to the bees after the individual training and test flights? Do they return to the entrance hole of the flight arena or were they caught and ‘manually’ bought back to their hive?Were the individual bees labelled and could be identified? This might have been a precondition to be able to monitor changes in detection performance.What about the orientation of body and head in 3D space? How well can these parameters be resolved? This is important information, especially if the retinal position of the objects needs to be assessed. The term ‘flight angle’ should be explained.“We used mixed linear models to test the effect of treatment group, target location and learning on the error rate and on the decision time, respectively. Furthermore, the identity of the individual bees was implemented into the model as a random factor.” It is not entirely clear what these sentences really mean. ‘Mixed linear models’ and ‘implementing individual bees into a model as a random factor’ should be explained.Results:What does ‘overall error rate’ mean?Figure 3: If I understand the explanation of the figure correctly, the data point ‘6’ ‘N° of flights’ shown in A and B should be the mean of the corresponding data points ‘3’ and ‘6’ ‘N° of flights’ in C. Since in C both ‘dorsal group’ data points have a larger value than the corresponding data point in B, this interpretation cannot be correct – or something went wrong with the data. Please check.Legend of figure 4: “Please note that in the mixed group the flight number refers to number of trials absolved with the particular target situation and not to the total number of trials the bee has absolved”. This statement is somewhat cryptic. Please explain.Discussion:Most issues concerning the Discussion have already been mentioned above. Here just some minor, though relevant details:During the flights towards the target bees change their height. How is this done? Do they change the pitch angle of their body and, in particular, of the head when changing height as compared to the pitch angle during horizontal flights? This is important, since it pertains to the retinal area with which the target is seen. Drosophila, for instance, changes the pitch angle of its body when changing height, whereas it keeps the head orientation in space relatively constant (by changing the head angle relative to the body).“… bees could have increased the area of their attentional focus to a size large enough to cover both rows”. Obviously, by assuming such an attentional process almost every change in detection performance can be explained. However, from an epistemological point of view such a hypothesis makes only sense, if an experiment could be designed by which this hypothesis can be tested.", "responses": [ { "c_id": "1227", "date": "13 Feb 2015", "name": "Lars Chittka", "role": "Author Response", "response": "We thank the reviewer for the careful reading, the extensive suggestions and the help in closing gaps in the logic of the manuscript.After the training phase the bees could not have any a priori expectation with regard to target position, since target position was pseudo-randomized during the training procedure. Interestingly, a clear dorso-ventral asymmetry of the error rate of target detection was found even at the end of the training period. For the test phase the bees were subdivided into the three experimental groups that were tested with just one of the stimulus constellations. The rationale behind this design is not well explained and not immediately obvious. At least at first sight, one might have expected that the different constellations were tested again with all bees in a pseudo-random fashion.One reason that this has not been done might have been the extreme dorso-ventral performance gradient after the training phase. Even without any distractor the coloured target could be detected with only a very high median error rate of 65% when presented in the top row. Surprisingly, no strong point is made that the detection performance is not much above chance level (this issue should have been tested statistically!), when the target was presented in the top row.Thank you for this useful suggestion. We included this point in the analysis. Theoretically, this poor performance should not increase, if in the test phase – i.e. after adding two distractor objects - the target were also presented randomly in the top and bottom row of the stimulus arrangement. Hence, it might have been scientifically meaningful to ask whether the detection performance in the dorsal part of the visual field might increase, if the bee had the chance to build up an expectation that the target is not located ventrally. This might be a rationale for subdividing the bees into the three groups. Irrespective of whether or not this supposition is correct, an explanation for the experimental design should be given.Yes, your explanation is correct. We apologize for the poorly crafted description of our rationale and now include a more careful explanation. In any case, it can be concluded from the development of error rates, decision times, but also the trajectory data that the performance of the ‘dorsal group’ improves over time and that this finding is consistent with the interpretation that also dorsally located targets can be readily detected, if the bee has a priori knowledge that the target is likely to be found in an otherwise ‘suboptimal’ part of the visual field. Again, it might be helpful if this kind of conclusion were drawn explicitly in the paper. Whether this finding is just a consequence of a learning process or might be also linked to attentional mechanisms can then be discussed. However, it might be hard to conceptualise how attentional mechanisms can be pinpointed experimentally in the type of experiments that were performed in this study.Your conclusion is in accordance with our explanation, although it seems that we did not explain it sufficiently in the earlier version. In the new version, we made our point more clear by describing the rationale in the Material & Methods. Furthermore, we addressed this point in the Discussion section more extensively. The hardest issue that remains to be explained are the data obtained with the ‘mixed group’ of bees. Although here no expectation can develop with regard of the location of the target, the performance improves over stimulus presentations to values that are much better than those obtained at the end of the training phase – even without any distractors (!). This highly surprising issue definitely needs to be discussed. This somehow apparent contradiction raises also the question whether – given a tremendous variability between bees but also between different flights of individual bees – the data base is sufficiently large to allow for drawing strong conclusions.We now discuss this issue carefully in the Discussion, suggesting a possible mechanism to cope with the search situation: we demonstrated that even in this group bees adapted their flight vectors over time, which may help them to detect the target faster and avoid difficult flight manoeuvres. It is difficult to draw conclusions from the comparison of the pre-training phase with the end of the training phase (=experimental phase) as bees had more than twice as much experience with the task at the end of the training phase than at the end of the pre-training phase and may cope more easily with the search task for this reason alone. Title of the paper: Since the paper does not focus on the topic of attention, I would omit in the title of the paper the addendum “: a role for spatial attention?” Attention is just one possibility, in addition to several others, to explain the experimental results. Putting too much emphasis on attention already in the title may be somewhat misleading.The title has been changed to address this. Abstract: “Here we investigate if this asymmetry is caused by fixed search patterns or if bees can use alternative search mechanisms such as spatial attention, which allows flexible focusing on different areas of the visual field.” I do not see that the investigation of this alternative has been the focus of the experimental analysis of the study. At least the results do not allow answering this question in an unambiguous way.We changed that sentence into \"Here we investigate if this asymmetry is caused by fixed search patterns or if bees can increase their detection ability of objects in the dorsal part of the visual field in search scenarios, where the target appears often or always in this area of the visual field.\" Introduction, 1st paragraph: It appears to be surprising that the paper starts by considering whether the distinct spatial asymmetry in colour and pattern learning of honeybees as observed in previous studies might be a consequence of specialisations in eye morphology, as has been proposed for detecting the queen by the drone bee eye. It should be noted that this sort of detection task is conceptually very different: in the drone bee case we are dealing with detection thresholds where - for obvious reasons - spatial acuity and sensitivity, i.e. anatomical and physiological retinal parameters, are the relevant factors, because they determine at which distance a small target (i.e. in this case the queen) can be seen. In the learning experiments the targets were usually sufficiently large, and no spatial resolution or sensitivity problem existed for them to be detected.We understand your point of view. However, we wanted to point to the fact that a dorso-ventral asymmetry in visual pattern recognition can at least in theory be explained by eye optics. The honeybee drone is a relevant example of where such an asymmetry occurs. Even if such optical asymmetries do not explain the behavioural asymmetry in our study, this is at least a theoretical explanation for unequal performance in various parts of the visual field, so we can’t disregard this possibility. Methods: What was the frame rate and resolution of the camera system?We now include the frame rate. “The stimuli subtended a visual angle of …” (and following sentence): The authors probably mean the individual discs. “stimuli” not specific enough.We have changed the term into 'stimulus discs' It should be explained why a virtual decision line was chosen. It might have been more intuitive to monitor the landing on the different targets. Would the error rate change, if the landings were recorded rather than the crossing of a virtual decision line?We now evaluate the flight behaviour in much more detail, including locations at various distances from the screen as well as errors upon landing. We find that the distance of 5 cm from the search screen allows for best differentiation between the different experimental group, and therefore focus some aspects of the evaluation on this distance. What happened to the bees after the individual training and test flights? Do they return to the entrance hole of the flight arena or were they caught and ‘manually’ bought back to their hive?We have now included the following sentence into material & methods: 'After drinking to satiation, the bee left the arena to fly back to the colony.' Were the individual bees labelled and could be identified? This might have been a precondition to be able to monitor changes in detection performance.We had stated that bees were marked individually. However, for clearer understanding we extended the sentence which now says: ' Bees were trained to an experimental box and marked individually by applying differently coloured paint markings on the thorax.' What about the orientation of body and head in 3D space? How well can these parameters be resolved? This is important information, especially if the retinal position of the objects needs to be assessed. The term ‘flight angle’ should be explained.Unfortunately the video quality does not allow for analysis of orientation of body and head. We included the following sentence into material & method: “To analyse the flight directions (vertical flight angles) of the bees while crossing the arena, we measured the vertical position of the bee at different distances to the search screen and calculated the angle between the flight direction and the horizontal.” “We used mixed linear models to test the effect of treatment group, target location and learning on the error rate and on the decision time, respectively. Furthermore, the identity of the individual bees was implemented into the model as a random factor.” It is not entirely clear what these sentences really mean. ‘Mixed linear models’ and ‘implementing individual bees into a model as a random factor’ should be explained.We now state: 'We used general linear models to test the effect of treatment group, target location and learning on the error rate and on the decision time, respectively. As the behaviour of each individual bee was measured several times during experimental course (repeated measurement design), the identity of the individual bees was embedded into the model as random effect.' What does ‘overall error rate’ mean?This term has now been removed. Figure 3: If I understand the explanation of the figure correctly, the data point ‘6’ ‘N° of flights’ shown in A and B should be the mean of the corresponding data points ‘3’ and ‘6’ ‘N° of flights’ in C. Since in C both ‘dorsal group’ data points have a larger value than the corresponding data point in B, this interpretation cannot be correct – or something went wrong with the data. Please check.We used not the mean but the median of the decision time to attenuate effects of rare outlier trials. This is now clarified in Material & Methods. Legend of figure 4: “Please note that in the mixed group the flight number refers to number of trials absolved with the particular target situation and not to the total number of trials the bee has absolved”. This statement is somewhat cryptic. Please explain.The sentence was changed into 'In the mixed group the flight number refers to number of trials absolved with the particular target location (top row or bottom row) and not to the total number of trials the bee has absolved in total (all trials regardless of the target location).' Discussion: Most issues concerning the Discussion have already been mentioned above. Here just some minor, though relevant details:During the flights towards the target bees change their height. How is this done? Do they change the pitch angle of their body and, in particular, of the head when changing height as compared to the pitch angle during horizontal flights? This is important, since it pertains to the retinal area with which the target is seen. Drosophila, for instance, changes the pitch angle of its body when changing height, whereas it keeps the head orientation in space relatively constant (by changing the head angle relative to the body).As mentioned, we have no information about the head angle during the approach flight, nor about the pitch of the body axis. The limitations of this lack of information are now discussed. “… bees could have increased the area of their attentional focus to a size large enough to cover both rows”. Obviously, by assuming such an attentional process almost every change in detection performance can be explained. However, from an epistemological point of view such a hypothesis makes only sense, if an experiment could be designed by which this hypothesis can be tested.It is possible to test for such a hypothesis – behavioural experiments in human psychology have been testing various parameters of attentional focus (see citations in the manuscript). Theoretically these could be adapted to fit a behavioural experiment for insects." } ] }, { "id": "5608", "date": "09 Sep 2014", "name": "Martin Giurfa", "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\nThis paper analyzes visual target recognition in bees and focuses on previously reported asymmetries between the dorsal and the ventral visual fields in terms of color/pattern recognition and discrimination. The authors studied whether a pattern detection/discrimination task varies when patterns are displayed either in the ventral or in the dorsal visual fields and discuss different possible mechanisms accounting for this variation. Specifically, bees were trained to choose a rewarded orange target which was presented among blue distracters either in the ventral visual field ('ventral' group' of bees), the dorsal field ('dorsal' group') or in both fields ('mixed group'), and choice errors, decision time, and flight pathways upon entering into the experimental arena were recorded.The authors show in a convincing way that bees presented with targets in the ventral visual field were most efficient in detecting the rewarded target as they made rapid decisions with high accuracy and direct flight paths. In contrast, bees perceiving the patterns in the dorsal visual field made more errors and took more time to detect the pattern. Yet these 'dorsal bees' improved significantly their performance after a few learning trials as they decreased both the number of errors and decision time and flew in a straight line towards the target. Bees of the 'mixed group' needed considerably more time to improve the search of the target whose position changed randomly between the ventral and the dorsal visual field.The conclusions drawn by the authors focus on the notion of expectation as it is said that \"bees form expectations of the location of the target’s appearance and adapt their search strategy accordingly\". Furthermore, the authors insist on the notion of attention (more specifically, spatial attention), which is certainly attractive in the light of the results obtained, but as the authors themselves acknowledge, is probably not the unique explanation for their data.As a consequence, a more suitable title for the paper, given the results presented, would besomething like: \"Honey bees exhibit flexible (plastic) visual search strategies for targets presented vertically at various heights\". It seems to me that the main finding of the paper is precisely the flexibility and adaptability of the bees' visual search strategies to the various experimental situations rather than the proof of attentional processes guiding these strategies.A main concern raised by this paper relates to the criteria (unclear to me) used to define the position of the so-called \"decision line\" in the setup. Clearly such a line was very close (5 cm) to the targets so that decision counting may have under/over estimated real decisions performed (and perhaps) modified at further distances from the targets. It would be therefore interesting to contrast the decisions counted and established following the authors' method with a different one, which, for instance, may be based on quantifying angular deviations of trajectories along the flight path; one can establish a criterion for a decision based on an angular deviation from the entrance vector and quantify decision which may occur before reaching the decision line defined by the authors.A minor concern relates to incomplete citations in the paper. In particular, given that the focus is on ventral/dorsal asymmetry of visual performances on bees, it would be worth considering and mentioning the work on ventral vs. dorsal target detection in bees (Giurfa et al., 1999) . Additionally, the statement on the relative recency of studies on attention in insects is questionable. Although it is not explicitly said - probably because of constraints imposed by reviewers - the work by van Swinderen and Greenspan (2003) constitutes a study on visual attention. Also, if one focuses on bees, the use of this concept of attention dates to 2004 (Giurfa, 2004) when it was argued that discrimination between perceptually closer stimuli was possible after differential conditioning but not after absolute conditioning due to differences in attention inculcated by these two training procedures. The idea was further developed in later papers, so that referring the use of this concept in insects to 2011 seems a bit unfair to prior contributions which also focused on the same idea.All in all, the manuscript is sound and well-written. The results are quite convincing (but see remark on the appropriateness of the decision line criterion) and constitute an important contribution to field of insect vision.", "responses": [ { "c_id": "1229", "date": "16 Feb 2015", "name": "Lars Chittka", "role": "Author Response", "response": "The conclusions drawn by the authors focus on the notion of expectation as it is said that \"bees form expectations of the location of the target’s appearance and adapt their search strategy accordingly\". Furthermore, the authors insist on the notion of attention (more specifically, spatial attention), which is certainly attractive in the light of the results obtained, but as the authors themselves acknowledge, is probably not the unique explanation for their data.As a consequence, a more suitable title for the paper, given the results presented, would be something like: \"Honey bees exhibit flexible (plastic) visual search strategies for targets presented vertically at various heights\". It seems to me that the main finding of the paper is precisely the flexibility and adaptability of the bees' visual search strategies to the various experimental situations rather than the proof of attentional processes guiding these strategies.We thank the referee for the title suggestion; the title now reads ' Honeybees (Apis mellifera) exhibit flexible visual search strategies for vertical targets presented at various heights' A main concern raised by this paper relates to the criteria (unclear to me) used to define the position of the so-called \"decision line\" in the setup. Clearly such a line was very close (5 cm) to the targets so that decision counting may have under/over estimated real decisions performed (and perhaps) modified at further distances from the targets. It would be therefore interesting to contrast the decisions counted and established following the authors' method with a different one, which, for instance, may be based on quantifying angular deviations of trajectories along the flight path; one can establish a criterion for a decision based on an angular deviation from the entrance vector and quantify decision which may occur before reaching the decision line defined by the authors.This mirrors the other referees’ concern and we have now added a much more detailed analysis at various distances from the target screen. A minor concern relates to incomplete citations in the paper. In particular, given that the focus is on ventral/dorsal asymmetry of visual performances on bees, it would be worth considering and mentioning the work on ventral vs. dorsal target detection in bees (Giurfa et al., 1999) . Additionally, the statement on the relative recency of studies on attention in insects is questionable. Although it is not explicitly said - probably because of constraints imposed by reviewers - the work by van Swinderen and Greenspan (2003) constitutes a study on visual attention. Also, if one focuses on bees, the use of this concept of attention dates to 2004 (Giurfa, 2004) when it was argued that discrimination between perceptually closer stimuli was possible after differential conditioning but not after absolute conditioning due to differences in attention inculcated by these two training procedures. The idea was further developed in later papers, so that referring the use of this concept in insects to 2011 seems a bit unfair to prior contributions which also focused on the same idea.We have now added these references as suggested." } ] } ]	1	https://f1000research.com/articles/3-174
https://f1000research.com/articles/4-43/v1	12 Feb 15	{ "type": "Method Article", "title": "Guiding Ebola patients to suitable health facilities: an SMS-based approach", "authors": [ "Mohamad-Ali Trad", "Raja Jurdak", "Rajib Rana" ], "abstract": "Access to appropriate health services is a fundamental problem in developing countries, where patients do not have access to information and to the nearest health service facility. We propose building a recommendation system based on simple SMS text messaging to help Ebola patients readily find the closest health service with available and appropriate resources. The system will map people’s reported symptoms to likely Ebola case definitions and suitable health service locations. In addition to providing a valuable individual service to people with curable diseases, the proposed system will also predict population-level disease spread risk for infectious diseases using crowd-sourced symptoms from the population. Health workers will be able to better plan and anticipate responses to the current Ebola outbreak in West Africa. Patients will have improved access to appropriate health care. This system could also be applied in other resource poor or rich settings.", "keywords": [ "ebola", "SMS", "text message", "health service" ], "content": "Background\n\nThe current Ebola virus outbreak in West Africa that originated in Guinea in December 2013, and now has spread to two neighbouring countries, is still uncontained and remains the largest known Ebola virus disease outbreak to date. Some of the challenges identified include the geographical spread of cases, people’s movement between affected countries, the transport and burial of bodies by relatives, the weak healthcare systems, severe human resource constraints, lack of proven therapeutics, and lack of community engagement and cooperation1,2.\n\nThere have been numerous reports where probable Ebola-infected patients had to be driven away from health care facilities due to lack of bed availability. Furthermore, mobility of a positive case imposes further public health risks1. The current Centre for Disease Control and Prevention (CDC) recommendations to stop the spread of Ebola includes: case finding and diagnosis, case isolation and contact tracing, and safe burial practices (http://www.cdc.gov/vhf/ebola/resources/index.html). Difficult access due to remoteness and lack of transportation3 further widen the gap between the patient and the healthcare provider. Access to information that guides patients to the nearest facility with appropriate resources is another challenge. There is currently no known system that can improve this access.\n\nWe propose building a recommendation system based on simple SMS text messaging to help Ebola patients readily find the closest health service with available and appropriate resources. The system will map people’s reported symptoms to likely Ebola case definitions and suitable health service locations. In addition to providing a valuable individual service to people with curable diseases, the proposed system will also predict population-level disease spread risk for infectious diseases automatically using the crowd-sourced symptoms from the population.\n\nThis will be extremely valuable for health workers to better plan and anticipate responses to the current Ebola Outbreak in West Africa and hopefully prevent many new cases. An Ebola tracking service has been developed by Cedric Moro (See Figure 1), who has been collecting data using the Whatsapp platform to produce maps showing the spread of infection. The key limitations of using Cedric Moro’s approach is that Whatsapp requires an advanced Smartphone, so it can capture a biased sample of the population only. In addition, it only tracks Ebola and does not predict the spread trajectory.\n\nIn contrast, simpler and more affordable mobile phones are highly utilized in affected West African countries. For example, in Guinea mobile phone penetration is as high as 71 percent (http://www.budde.com.au/Research/Guinea-Telecoms-Mobile-and-Broadband-Market-Insights-and-Statistics.html). Therefore, mobile phones can potentially be an inexpensive yet effective way of communication in overcrowded urban or remote settings. For example, the Somali experience with this technology has been encouraging in controlling cholera outbreaks. (http://blogs.oxfam.org/en/blogs/12-11-13-mobile-phones-help-tackle-cholera-somalia).\n\n\nGeneral approach\n\nThe workflow of the proposed end-to-end system is shown in Figure 2. The use case for the patients will be straightforward. Using any basic mobile phone they can send a text message about their symptoms based on CDC case definition (http://www.cdc.gov/vhf/ebola/resources/index.html) to a designated toll-free number. The message once received will be analysed with Natural Language Processing Algorithms to extract the symptoms. Patient location will be approximated by the available cell-tower information or provided post-code or village name. Symptoms will then be fed to the classification module to determine whether it is a suspected Ebola case. The classification module maps the reported symptoms to the CDC provided clinical symptoms of Ebola. If suspected, health service(s) within a chosen radius (configurable) with sufficient resources will be recommended using a return text message. This message will contain name, location and phone number of the health service(s).\n\nThe symptoms, which occur in Ebola, are very similar to those, which occur in many other diseases in tropical Africa (https://web.stanford.edu/group/virus/filo/humandiseases.html). Therefore, our classification module will take into account available meta information, such as the spatial extent of the current outbreak while determining the suspected case. If the symptoms only partially matches the case definition of Ebola, but the patient is based in an Ebola affected zone, he or she will be recommended to visit the health service for full check-up. The patients would then be texted a few hours later asking whether they went to the recommended hospital and if so to provide feedback.\n\nTo obtain information about currently available facilities in a health service, we will adopt a phased approach. The knowledge base will be initially constructed using the location and contact number of the health services extracted from web portals using an automated web crawler; later on, we will use both positive and negative patient feedback to infer the current facilities at the service. For example, if patient feedback shows the recommendation was useful, we will assume that the health service has capacity to admit more patients. Otherwise, we will assume that the health service is out of capacity and keep the status for some time (which would need to be determined). However, there will be a provision for the health services to update their information anytime regarding resource, location (e.g. NGO field clinics) in the knowledge base using text messages. At the server end the text messages indicating information update will undergo Natural Language Processing algorithms to extract the key words (e.g., how many beds available) and the knowledge base will be updated accordingly. Once the information is updated, the health services/patients will be notified using a reply text message.\n\nThe text messages received from the population of end-users will be continuously analysed at the back-end to predict the outbreak risk into the future and its spread pathways. The locations of users extracted from the text messages will be used to estimate their orbits of movement. These locations will be coupled with the inferred patient conditions to predict the geographic extent of disease-spread risk.\n\n\nDiscussion\n\nWe use inference (from positive or negative feedback) to predict the capacity of a health service to accommodate more patients. Such a method could be useful if the service cannot update their service status. In order to significantly improve the performance of our system we would recommend appointing a communication officer in every service, who will regularly update the service (number of beds, doctors, medicine etc.) availability in the system. Update interval can be hourly or even 4-hourly - a time unit that would be variable and dependent on the facility’s capacity.\n\nIn some cases the system may not be able to recommend health services within the nominated radius. In those unsuccessful cases the system will store the patient information and as soon as any availability is spotted, a follow-up text message will be sent to the patient.\n\nWhen Ebola outbreaks take place simultaneously across bordering countries, such as in Liberia and Sierra Leone, our recommendation system can be adapted to recommend health services across borders, which could potentially help managing patient load.\n\n\nConclusion\n\nThe current Ebola outbreak is shaping up to be a large-scale problem, not only in the three affected West African countries, but also with cases starting to appear in Western countries such as Spain and the USA. The CDC recommendations for controlling the spread further are at the source, i.e. in West Africa. Given the large spatial scale of the disease, any assistive technology should be equally prevalent and widely used in the target countries. It is particularly for this reason that we believe and advocate for the use of simple SMS messaging as a mechanism to guide Ebola patients to suitable health facilities.\n\n\nProject stage\n\nCurrently we are actively seeking funding to commence the project. We are approaching the Gates Foundation in addition to mobile phone operators in West Africa. We are also looking for international and local partner organisations operating in the affected areas.", "appendix": "Author contributions\n\n\n\nThe idea was conceptualized by MT and RJ. Literature review was conducted by all authors, in addition to the article write-up. RJ and RR constructed and developed the technical aspects of the project.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThis article was previously posted on arXiv (http://arxiv.org/abs/1410.3576) and has been accepted as a poster for ASID-Auckland 2015. It was also quoted by TIME magazine (November 13, 2014 issue; http://time.com/3583191/social-network-scientists-builder), and reported in the media (http://www.wired.co.uk/news/archive/2014-10/14/sms-ebola).\n\n\nReferences\n\nEbola in west Africa: gaining community trust and confidence. The Lancet. 2014; 383(9933): 1946. PubMed Abstract \| Publisher Full Text\n\nTrad M, Fisher DA, Tambyah PA: Ebola in west Africa. The Lancet Infectious Diseases. 2014. Publisher Full Text\n\nSheik-Mohamed A, Velema JP: Where health care has no access: the nomadic populations of sub-Saharan Africa. Trop Med Int Health. 1999; 4(10): 695–707. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7657", "date": "26 Feb 2015", "name": "Corinna Schmitt", "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\nThe authors present a good idea how toestablish a SMS-based approach to guide Ebola patients to health facilities. The idea is great and the strategy sound reasonable, but the following issues must be clarified in more detail:How do the authors guarantee that the patient will get the information of the most nearest facility? What is with the support of poor regions with low phone access? Which information is stored in the \"knowledge base\" you are talking about? What do the authors mean by the term \"snd-to-end system\"?I suggest the author's solution could be further influenced by existing emergency solutions. Thus, look on the drafts of the IETF working group ecrit. They are looking for standards in such research fields.", "responses": [] }, { "id": "7655", "date": "10 Mar 2015", "name": "Patrick Charles", "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\nThe manuscript describes a proposed SMS-based system to use in locations where Ebola virus disease (EVD) is occurring to help direct possible cases to the nearest treatment centre that has beds available at the time.This is an interesting and useful idea and would work well in Africa where mobile phone technology is widely available.The manuscript is well written and the idea is presented well.", "responses": [] } ]	1	https://f1000research.com/articles/4-43
https://f1000research.com/articles/3-202/v1	26 Aug 14	{ "type": "Opinion Article", "title": "Animal-oriented virtual environments: illusion, dilation, and discovery", "authors": [ "Bradly Alicea" ], "abstract": "As a research tool, virtual environments (VEs) hold immense promise for brain scientists. Yet to fully realize this potential in non-human systems, theoretical and conceptual perspectives must be developed. When selectively coupled to nervous systems, virtual environments can help us better understand the functional architecture of animals’ brains during naturalistic behaviors. While this will no doubt allow us to further our understanding of the neural basis of behavior, there is also an opportunity to uncover the diversity inherent in brain activity and behavior. This is due to two properties of virtual environments: the ability to create sensory illusions, and the ability to dilate space and/or time. These and other potential manipulations will be characterized as the effects of virtuality. In addition, the systems-level outcomes of virtual environment enhanced perception will be discussed in the context of the uncanny valley and other expected relationships between emotional valence, cognition, and training. These effects and their usefulness for brain science will be understood in the context of three types of neurobehavioral phenomena: sensorimotor integration, spatial navigation, and interactivity. For each of these behaviors, a combination of illusory and space/time dilation examples will be reviewed. Once these examples are presented, the implications for improving upon virtual models for more directly inducing the mental phenomena of illusion and space/time dilation will be considered. To conclude, future directions for integrating the use of VEs into a strategy of broader biological inquiry will be presented.", "keywords": [ "Simulation", "Behavioral Neuroscience", "Cognitive Neuroscience", "Virtual Environments" ], "content": "Introduction\n\nVirtual Environments (VEs) are increasingly being used to uncover the fundamental features of cognition. Areas of investigation include spatial cognition, sensorimotor control, and emotional processing (Bohil et al., 2011). While VEs are an up-and-coming method for studying human cognition, they are increasingly also being used in the study of animal cognition. VE systems usually consist of a sensory or experiential analogue. This allows us not only to faithfully replicate naturalistic conditions for behaviors in the lab, but also explore the limits of the underlying neural systems.\n\nOne popular aim in the brain science community is to understand the basis of cognitive functions or disorders (Mar, 2011; Menzel, 2012). By using virtual environments, we wish to control as many environmental variables as possible. Tight control of environmental conditions in an immersive environment should allow us to isolate the biological sources of behavioral variation. This should apply to both human and non-human animals, although the design of visual arrays and other forms of sensory manipulation must conform to a specific animal’s sensory abilities and specializations. Removing the environmental vagaries of a behavior may also allow us to induce mental phenomena that can only be simulated in a virtual environment. While the role of mental phenomenology is a controversial topic when talking about non-human animals, VE systems should allow us to better investigate the possible existence of mental worlds in animals. These include sensory illusions and the dilation of space and/or time.\n\nGiven that these concepts are not immediately intuitive, how do we formally and operationally define sensory illusion and space/time dilation? The working definition of sensory illusion is focused on a virtual stimulus which can be confused as a real stimulus. The key property of sensory illusion is perceptual ambiguity, where the virtual stimulus looks nearly real, but is nevertheless a simulation. This has the potential to introduce ambiguities in constructing a unified percept of the object, particularly in the context of multisensory integration. By contrast, the working definition of space/time dilation involves a virtual stimulus that speeds up or slows down action in a visual reference frame (or sensory event) relative to the natural motion of an object. The definition of natural motion is either intuitive or innate. Intuitive natural motion can be defined as physical objects evaluated by the observer in terms of naive physics (Povinelli, 2003). Innate natural motion can be defined as biological motion, or the movement patterns of organismal bodies as sensed by an observer (Grossman & Blake, 2001). Both of these can be violated through the use of virtual environments, and the neural response can mimic that of sensory illusion.\n\nThese phenomena have been demonstrated in a number of contemporary papers that look at cognitive behaviors such as sensorimotor integration, spatial navigation, and interactivity. The papers reviewed here represent the state-of-the-art application of VEs to the naturalistic study of brain activity and behavior in animals. Aside from serving the needs of neuroethologists, who can now study behavior in a controlled setting, animal models also allow us to better understand the neural correlates of behavior. This is due to the relative ease of conducting direct recordings of neuronal populations and circuits. They also serve as important clues to more subjective issues that warrant further investigation.\n\n\nVirtual environments meet cognitive neuroethology\n\nIn the past few decades, a number of pop-culture references and technological developments have turned virtuality into a relevant, shared human experience. Virtuality itself can be defined as the collective effects of a virtual world stimulus on perception, behavior, and social interactions. While there are many dimensions to this experience, two of the most fundamental are perceptual illusion and space/time dilation. Because VE models are immersive, the technology that simulates perceptual cues creates the illusion of being in a sensory cocoon. Inside of this cocoon, the participant can transcend perceptual limits whilst maintaining a highly-faithful representation of the physical world. Yet VE models are also engaging, and when this level of engagement is high, the potential exists for other forms of sensory distortion. Space/time dilation exists when perception and action can be sped up or slowed down, creating different time-scales. Reality itself can also be dilated in space. In this case, dilation involves expanding and contracting the scope of attentional resources. Both of these effects result directly from the technological environment.\n\nVE models provide an alternate environment which has a high degree of representational similarity but varying degrees of experiential similarity. Yet it also provides us with a means to explore cognitive neuroethology, or the cognitive dynamics of naturalistic animal behavior (Giurfa, 2003). As VEs provide a means to explore behavioral effects beyond trial-by-trial presentations, it also requires us to account for unique emotional and cognitive responses. While the effects of virtuality might seem to be obscure, it is actually a common theme in movies such as “The Matrix” and “Inception”. VEs allow for exploration of these fictive aspects of the real world represented as cognitive processes. In applications to animals, this can be extended further into the world of neuroethology. In fact, analogies based on these movies have been made between fictive mental responses and manipulations of hippocampal-dependent memories (see Spiers & Bendor, 2014). In this paper, these types of effects will be applied to animal models, and shown to exist for three types of behavior.\n\nWhy would this be interesting to the study of non-human brain and behavior? With VE systems, we can provide high-fidelity reconstructions of the real world and environments in which typical sensory cues are either dilated in space/time, temporally distorted, or combinations of both. In this paper, we will explore how virtual environments allow us to uncover the cognitive and neural processing behind illusion and space/time dilation in animals. These effects, seen in a number of contexts and neural systems, can be collectively referred to as the effects of virtuality. By using a model from the human-robot interaction literature (e.g. uncanny valley), we can better generalize the effects of virtuality to cross-species behaviors and neural mechanisms.\n\nThere is evidence that these factors are most relevant to animal behavior research, for which naturalistic settings are of primary importance (Zupanc, 2010). But how much of the environment must be replicated in order for an animal to recognize it as “just like the real thing”? One way this can be characterized is through the uncanny valley phenomenon. The uncanny valley characterizes the subjectivity inherent in how observers perceive and act upon virtual environment avatars and robots that embody various degrees of realism (see Figure 1). The uncanny valley is based on an emotional response occurring in the very early stages of perceptual processing, which can be elicited for any object that generates an emotional response or involves recognition mechanisms. Both emotional response and recognition result from experience, which emerges in development and occurs in non-human contexts (Lewkowicz & Ghazanfar, 2012). Experience also conditions the classification of stimuli as being real or a facsimile in terms of recognition. Whenever a real object clearly has the attributes of such, the early emotional response resolves the ambiguity of classification as real or virtual (Steckenfinger & Ghazanfar, 2009). It is when this ambiguity cannot be resolved that the problem lies.\n\nRealism (x-axis) represents the fidelity and/or resolution of this representation. Emotional valence (y-axis) represents the positive or negative emotional valence associated with a given representation of familiar objects or conspecifics. A: the first phase of the response curve, showing an initial rise in emotional valence with moderate degrees of realism. B: the second phase of the response curve, showing a dip and rebound in emotional valence at very high levels of realism. Shaded region represents hypothetical individual variation exhibited in the response. Figure adapted from the uncanny valley principle as originally proposed by Mori (1970).\n\nFor purposes of this paper, let us map recognition to classification using the uncanny valley curve as a referent. Initially (see Figure 1A), the more “real” an object becomes, the more it is associated with its real-world analogue. This phase of the curve is associated with gains in sensory fidelity. The second phase of the curve (see Figure 1B), which consists of two inflection points, is associated with a drop-off in the feeling of realness just before a fully “real” emotional response occurs. At this point in the response curve, there is a predicted perceptual decoupling between the highly-realistic representation and the recognition that a robot is human or an object is real. This is an ongoing challenge in the world of human-robot interaction and VE design. However, this technical challenge might also be used to facilitate the effects of virtuality mentioned previously.\n\nThere are a few caveats to the arguments and ideas presented herein. In animals, the uncanny valley has been observed only in primates (Penn & Povinelli, 2007; Steckenfinger & Ghazanfar, 2009). However, the strategic use of VEs to provide stimuli could reveal a similar neural response in other animals. In addition, the effects of virtuality are expected to exhibit a variable effect size depending on the species chosen. Species that have high levels of what is traditionally considered animal intelligence (Matzel & Kolata, 2010) should exhibit these effects most strongly. Effects such as illusion can also be very strong in organisms with highly-specialized sensory systems, particularly given that the VE manipulation is highly specific. I propose that the key component that relates the hyper-realism of VE to the uncanny valley effect is not a set of higher-cognitive mechanisms, but rather the information held in perceptual ambiguities. It is these ambiguities and the uncanny valley effect in general that can actually be leveraged to produce illusory or space/time dilation effects.\n\n\nPotential means of measurement\n\nLet us now turn to potential ways to measure the effects of virtuality and the predicted patterns of these measures for each type of effect (illusion, space/time dilation). There are four general types of measurement for which the neural substrate will vary across taxa: emotional valence, perceptual ambiguity and coherence, adaptation and motion perception, and spatial memory. A summary of these measurement types can be found in Table 1.\n\nThe first effect of virtuality involves the production of illusory effects. In terms of emotional valence, it is predicted that when stimuli are either completely or not at all illusory, there is little emotional response. It is when stimuli are slightly illusory that we expect to see the greatest emotional response. A similar situation is expected to exist for perceptual ambiguity and coherence, and not surprisingly is linked to emotional valence. As was just discussed, it is predicted that the slight degrees of illusion elicit the greatest amount of emotional valence. As a consequence, slight degrees of illusion can correspondingly degrade perceptual performance. In this case, perceptual performance can be measured in the form of response times, object recognition, and kinematic patterns.\n\nIllusion can also be measured by looking at the correlates of adaptation and motion perception. In general, illusory effects should utilize existing capacity for adaptation and result in phenomena such as visual aftereffects. Correlates of spatial memory can also provide potential measurement of illusory effects, as such effects should produce new episodic but not associative memories. Applying VEs to animals can also produce space/time dilation effects which can be measured in a number of ways. As with illusion, there are four general types of measurement for which the neural substrate will also vary across taxa: emotional valence, perceptual ambiguity and coherence, adaptation and motion perception, and spatial memory.\n\nAs in the case of illusory effects, emotional valence should be highest when the effects of virtuality are slight. In the case of space/time dilation, the greatest amount of emotional valence occurs when stimuli are slightly disjoint in space/time. By contrast, stimuli that are either entirely integrated or entirely disjoint in space/time should elicit little emotional response. Also as with illusion, perceptual ambiguity and coherence are linked to emotional valence. In this case, moderate amounts of space/time dilation are expected to elicit the greatest amount of emotional valence. Much like in the case of illusion, these conditions contribute to the degradation of perceptual performance.\n\nSpace/time dilation should also be apparent in measurements of response times, object recognition, and kinematic patterns. However, space/time dilation is particularly effective at systematically warping the reference frames of perception and action. This should be similar to the phenomena of rotational and gravitational reference frame manipulation (Leone, 1998; Lipshits et al., 2005) and plasticity of the multimodal gravitational reference frame (Luyat et al., 2005) that have been observed in humans. Due to the wider-ranging nature of this effect, the effects of space/time dilation on adaptation and motion perception should facilitate new adaptations and a generalized neuroplastic response. Spatial memory should also be affected by space/time dilation, as modification of mental representations such as the gravitational reference frames should produce new associative memories.\n\nIn cases where there is ambiguity in the stimulus (e.g. agents that look real but do not exhibit all of the cues of a real individual), a distinctive neural response related to the mismatch between appearance and motion can be elicited (Saygin et al., 2012). Part of this response involves physiological adaptation to motion (Celebrini & Newsome, 1994) as expected of real-world objects. The response to mismatch also involves the associated function of visual motion and theory of mind (ToM) mechanisms (Gerrans, 2002). This principle of associated function may also allow for perceptual ambiguities to influence a more general set of neural mechanisms (Changizi, 2011). For example, in humans the ambiguous nature of some virtual stimuli (e.g. agents or complex objects) elicits activity in the bilateral anterior intraparietal sulcus. While this is usually related to prediction error, it can also affect the global state of the action-perception system (Saygin et al., 2012). Thus, simple ambiguities may be intentionally introduced using virtual environments to trigger controlled departures from the context of reality.\n\nTo resolve the issue of equivalent responses to real and virtual environments in non-human animal species, it is worth noting that what individuals generally consider to be reality is based on personal experience and perceptual coherence (Engert, 2013). If this premise holds true for the neural basis of sensation and perception (for an example from primate vision, see Andersen et al., 2014), then we should be able to discover the limits of this illusory capacity by manipulating the environment and rousing the organism from this illusion. It is important to remember that in this context, illusory responses are not dependent on the animal reaching some sort of philosophical epiphany. Rather, the illusory effect is a metaphor that encapsulates an immersive versus non-immersive experience. Depending on the level of immersion, it may be possible to control not only the sensory cues experienced by the non-human animal, but the entirety of the experience itself. In the case of human experience, reality is defined as perceptual and cognitive norms which permeate the context of everyday living. The effects of this context are limited to current (e.g. non life-history dependent) experience. However, it also serves as a contrast to perception and action outside of the VE. In many cases, non-human animals should respond to both rudimentary sensory cues (illusion) and dilated perceptual representations and sensory cues (space/time dilation). In these cases, then the application of VEs to the study of animal cognition and behavior will have much predictive and comparative value.\n\n\nCurrent examples\n\nTo outline the potential of VE systems for animal research, I will focus on three areas of contemporary investigation: sensorimotor integration, spatial navigation, and interactivity (see Table 2). All three of these areas have been studied extensively in humans. Furthermore, the first two areas have also been studied extensively in animals, but until recently have not leveraged the advantages of VE technology. These examples utilize a range of experimental apparatus, from simple illusory stimuli and tracking systems to extensive mimicry of sensory cues. The simulation of any one set of environmental stimuli results in the activation of multiple neural circuits and may involve multiple cognitive systems. Yet this diversity of approaches has roughly the same effect: to enable control over the environment and to extend the range of experimentally-observable behaviors. Newly-observed behaviors and neural responses include: semi-realistic neural coding at the cellular level, transferring experience between spatial scales (e.g. beaming), and dynamic changes in distributed population codes. These and other unique findings also allow us to gain an appreciation for the spectrum of neural responses associated with these behaviors in an analytically tractable manner.\n\nTo better appreciate these examples, recall that the efficacy of VE systems is based on more than the ability to generate a series of high-fidelity visual images or tactile stimulations. Part of this unexplained variance has to do with the emotional state and cognitive response (Seyama & Nagayama, 2007) to specific stimuli. The other component involves the form of virtual intervention. Would it simply be enough to show an animal a familiar visual scene, or can experimental outcomes of large effect be elicited by reducing the environment to key features of an experience? The uncanny valley effect suggests that the former is just as important as the latter, and both interact with emotional responses. While the main effect of using VE to generate the effects of virtuality might seem to depend upon selectively manipulating the fidelity of a simulation, perceptual information that triggers an emotional response might be just as important.\n\nFor further clarity, we can turn to two examples of how robotic models have been utilized to study animal behavior. Robotic conspecifics can be used to mimic key mating signals. In this case (Patricelli & Krakauer, 2010), it is not the fidelity of the robot that is important, but rather the quality of the mimicked signal. Robotic approximations of conspecifics can be used to replicate commonly-observed, species-specific behaviors such as ant trail building and rat pup behavior (Akst, 2013). As with the simulation of mating rituals, it is not the details of the behavior and how it is represented in the brain that are important. Experiments replicating social learning and conspecific interactions using biomimetic robots demonstrate that full replication of sensory cues is not necessary to elicit a response (Krause et al., 2011). These findings suggest that successful simulation and the elicitation of desired behaviors can be reduced to a few key features depending on the cognitive or technological domain.\n\nAn experimental apparatus that is both capable of tightly reproducing the original environment (maintaining integration) and selectively distorting it (disrupting integration) is highly useful for understanding the effects of movement disorders. Being able to conduct experiments with this level of environmental control in non-human animals allows for single cell-level contributions to behavioral variation.\n\nAhrens et al. (2012) have developed an innovative virtual environment for zebrafish that is customized for fish cognition and swimming behavior. Visual scenes are projected onto a screen located underneath the fish’s location (Petri dish), and consist of square gratings that move along the fish’s body from snout to tail. Importantly, the speed of visual cue presentation can be adaptively adjusted relative to swim speed. Immersion in such a context is sufficient for initiating short-term forms of motor learning (Gray, 2012). The neural populations responsible for motor learning are distributed across the brain, including the inferior olive and cerebellum. This is the expected location for motor learning consolidation, which is conserved from fishes to humans.\n\nZebrafish VE also allows for flexibility in the experimental setup which in turn provides a means to dissect components of the sensorimotor loop in a systematic manner. Engert (2013) has proposed two alternate interaction modes (e.g. experimental preparations) for creating illusory stimuli related to zebrafish swimming behavior. In this case, possible illusory stimuli include (but may not be limited to) oscillating visual gratings and animations that are inconsistent with an organism's perception of self-motion (Lappe et al., 1999). These type of illusions presented in an experimental setup allows for direct measurement of movement and the recording of neural responses to active behavior. The other involves immobilizing the fish and recording the neural activity associated with intended (or fictive) locomotion. In both cases, the contributions of visual stimuli, motion, and the corresponding neural response can be decoupled through an inconsistency between an organism’s self-motion and the surrounding environment.\n\nWhile this effect might be explained as an experimental artifact, robot-fish interaction studies might help us further appreciate the role of conspecific-like self-motion cues in regulating how perception and action are coupled and decoupled. In the work of Marras & Porfiri (2012), biological fish were attracted to the locomotion of a robotic fish. Rather than actively decoupling sensory cues, the robot-fish interaction involves replicating the hydrodynamic and other mechanical cues of conspecific swimming behavior. While the coupling or decoupling of self-motion and behavior may be context-dependent in nature, VE and robotic studies have shown (in an almost accidental fashion) how true to context stimuli must be to elicit the proper neural responses. As we will see in the case of interactivity, neural activity associated with intentional behavior can be both a useful and important indicator of dynamic cognitive responses.\n\nIn another set of experiments in insects, virtual environments are used to dilate visual stimuli with regard to motor control. Gray et al. (2002) use the walls of a flight arena to present visual cues that mimic depth and motion to an immobilized insect. This was done in a specialized arena which is shown and discussed in Gray et al. (2002) and Seelig et al. (2010). In Seelig et al. (2010), a head-fixation task is replicated by having a fly walk on an air-supported ball concurrent with the presentation of visual stimuli. Using this type of VE design, an integrated response was found in horizontal system neurons. Using systems such as these, adaptive behaviors can be initiated in a highly-controlled environment. This not only allows for a range of behavioral regimes to be explored, but multiscale (e.g. cellular and behavioral dynamics) experimental investigations as well.\n\nSpatial navigation is perhaps the best understood of the three featured behaviors due to our extensive knowledge of neural mechanisms at both the structural (hippocampus) and single-cell (place and grid cell) levels. Indeed, virtual environments enable the development and confirmation of sophisticated theoretical models of spatial navigation. This is exactly what was done in Holscher et al. (2005) and Harvey et al. (2009). In the Harvey et al. (2009) approach, a mouse is situated atop an air supported-spherical treadmill, and its head is fixed for purposes of in vivo measurement. The virtual environment consists of a projection-based visual display. The first-person display features a fisheye-view of a linear track with a reward at the end of the track. This experimental setup resulted in semi-realistic firing patterns for place cells, which encode locations in virtual space. The authors also found three distinct sub-threshold signatures for place fields, which in turn may allow us to confirm theoretical models of neuronal coding (Ekstrom et al., 2003).\n\nWhile traditional spatial navigation experiments require very few illusory or space/time dilation-related manipulations, there is the potential to do experiments in animals where spatial relationships (and perhaps even mental representations of space-time) are warped. The work of Gershow et al. (2012) demonstrates how gradients of airborne cues can be delivered to organisms in a controlled manner using a series of microcontrollers. Some invertebrate species such as moths engage in a form of spatial navigation behavior called plume tracking. Plumes of odorants or other chemicals do not diffuse through their environmental media (e.g. air or water) in a linear fashion, and the information embedded in a plume is made highly nonlinear due to turbulent conditions. By delivering these gradients as highly laminar flows, the diversity and complexity of motor responses associated with plume tracking can be made tractable.\n\nInteractivity can be defined as the ability to manipulate and adaptively respond to a wider range of objects and behaviors than would found in a non-virtual context. This is a term I am presenting here for purposes of describing a series of experiments that feature animals interacting with VE systems. This could include computer-generated stimuli or avatars. Depending on the application, this can provide either the experience of enveloping interactivity or an experience of dilating the temporal or spatial scale of perception and action.\n\nNormand et al. (2012) use an ingenious experimental design to study interactivity between rats and humans using a technique called “beaming”. In this approach, a rat interacts with a robotic human analogue (ePuck). Humans interact with a telerobotic virtual environment system that maps behavior to ePuck that size-wise is similar to the rat’s body. To provide closed-loop feedback, the rat’s movements are then tracked and mapped to a human-like avatar in the virtual environment. The beaming approach allows for human interactions to take place at the rat’s size scale and vice versa. This also enables inter-species interactions such as the neuroanthropological studies of human-animal interaction featured in Keil & Downey (2012). Using beaming in this context might more directly address the existence of ToM within and between species.\n\nInteractivity can also be explored using brain-machine interfaces (BMIs). BMIs share many attributes with virtual environments, and allow us to better contextualize the potential interactions between brain, behavior, and environment observed during virtual world immersion. We can look to the application of BMIs in understanding the neural mechanisms underlying grasping in non-human primates as a relevant example. In O’Doherty et al. (2011), his group introduces the brain-machine-brain interface, which uses electrophysiological signals from the motor cortex (motion planning) as input to a virtual arm that grasps virtual objects. The additional (e.g. feedback to the brain) component involves stimulation of the sensorimotor cortex that serves as haptic (e.g. touch) feedback. This set of experiments has applications to brain-controlled prosthetic devices. This brain-machine-brain interface is currently being realized in application form as the Walk Again project, which aims to enable prosthesis-wearers to engage in activities such as soccer (Yong, 2011). This includes robotic limbs that require close coordination with intentional behaviors, or even devices which record behaviorally-relevant electrical signals in one animal and uses that signal to stimulate the brain of another animal (Pais-Vieira et al., 2013).\n\nDespite these examples from specific cognitive domains, it is not clear what the effects of VE actually are. As the neural response is characterized as semi-realistic by the authors, this suggests VE may not be perceived by the animals as a real world (the virtual representation falling partially into the uncanny valley featured in Figure 1). But how does the uncanny valley-like effects become manifest in sensorimotor integration, spatial cognition, and interactivity? These are not clearly emotional behaviors, but also involve making distinctions between the real and the artificial. In the case of sensorimotor integration, the uncanny valley might involve slightly unnatural movement patterns. This could involve a detectable discontinuity in the integration of vision and touch. Such an outcome could be registered as an emotional ambiguity (e.g. what is this object?), which could in turn disrupt how the animal treats its environment. A similar outcome might be seen for spatial cognition in terms of disruptions of the spatial reference frame. Like sensorimotor integration, there is a reliance of multisensory integration as a seamless process. When this consistency is violated in terms of an animal’s locational self-awareness (e.g. where am I?), an emotional response is triggered. However, in terms of interactivity, an uncanny valley-like emotional response is more straightforward. Interactivity involves interpersonal interactions with objects and agents, and so an uncanny valley-like response occurs in much the same way as predicted by the original theory.\n\nAlternatively, the possibility exists that virtual worlds simply expose the diversity of responses to highly similar environmental phenomena. This is not only due to cross-talk between different cognitive processes, but also involves individual variation in learning abilities and attentional capacity. In human experiments that focus on the effects of training, subjects can be switched back and forth between virtual and real-world tasks (Rose et al., 2000). Ideally, the virtual condition should provide gains in expertise that are transferrable to the real world analogue task. A similar experimental approach might be used for disentangling the effects of a virtual environment (such as sub-threshold neuronal activations) on an animal. While it is impossible to know which interpretation is correct at this point, future experiments specifically focused on perceptual realism in animals might provide us with a clearer picture.\n\n\nIllusion, space/time dilation, and virtual models\n\nThere may be other ways to understand the phenomena of illusion and space/time dilation independently of the three previous examples. Virtual models rely on two assumptions about the generalized animal response to virtuality supported by the previous experiments just reviewed. One assumption is that these responses are rooted in symbolic and adaptable representations of the sensory world. While there is scant evidence of higher-level representation in non-human animals, basic representational systems such as the ability to identify quantities and specific groupings of objects (numerosity) have been observed in animals ranging from fish (Agrillo et al., 2011) to macaques (Roitman et al., 2007).\n\nAnother assumption is that these representations may be subject to fictive conditioning. Fictive conditioning, which could be considered a form of associative learning, involves the acquisition of a learned response due to a stimulus via one sense that compensates for a lack of stimulus in another sense. One example of this is the supernumerary hand illusion in humans (Guterstam et al., 2011). In this phenomenon, information from one sense (vision) compensates for the lack of information from another sense (touch) to establish a stable (but fictive) association between the body and a third (prosthetic) arm. Yet despite such assumptions, there is an opportunity for systems neuroscientists to better understand the nuances of function for various pathways and processes. This is particularly true when comparing brain function between an animal subject to the effects of virtuality and a control animal behaving in the absence of virtual manipulation.\n\nReturning to the issue of realism in VE, it is worth noting that whether or not non-human animals possess a bona-fide ToM is controversial. While behavioral tests have shown a propensity of reflective behavioral responses in certain species, the neural mechanisms of this are unclear. In addition, while the neural correlates for ToM in humans are fairly well-established (see Saxe, 2009), the neural correlates for mental behaviors in non-human taxa are not as well characterized. Despite these caveats and limitations, eliciting species-specific responses to virtual stimuli consistent with the uncanny valley effect should be quite possible. To explain how this might occur, we can turn to the work of Maravita & Iriki (2004). In this study, experimenters trained a monkey to use a physical rake to retrieve objects from the environment. Electrophysiological and behavioral evidence post-training suggests that the rake had become incorporated into the animal’s body schema (Macaluso & Maravita, 2010), as the tool becomes an extension of the arm.\n\nIn extending the Uncanny Valley model to virtual environments, it is generally true that objects become more real as their fidelity increases. However, as they are incorporated into the body schema, they become less emotionally salient as real objects. This dropoff is not observed for physical objects (Carlson et al., 2010), but is predicted to occur for virtual objects even of high fidelity. Finally, once the individual is fully immersed in the VE and becomes acclimated to the use of the virtual object, the virtual object then becomes fully consistent with the body’s self-representation and sensory representation of the surrounding environment. In this sense, the virtual becomes real, and in some cases serves as a link between affect and cognition (Lewis & Lloyd, 2010). The extent to which this is true will partially determine the future potential of using VE in animal contexts.\n\nA virtual representation for illusion follows three sets of observations. The first involves the sensory systems that are engaged by the environment. Due to the immersive and flexible aspects of VEs, behaving animals can engage the environment in a naturalistic fashion. This includes engaging an environmental stimulus in a way analogous to behaviors such as foraging, free navigation, and mating. Therefore, considering the connections between higher-level cognition (e.g. attention) and psychophysiological phenomena (e.g. microsaccades) might be useful in selectively manipulating the input (Otero-Millan et al., 2012). In immersive contexts, the selective decoupling of vision from touch/proprioception and even audition is very important.\n\nThe use of VE systems also results in neural correlates that are distinct from real world analogues in humans, in concrete forms such as comparisons between static images and animated video (Han et al., 2005), or 2-D versus 3-D images of hand movements (Perani et al., 2001). While the sensory systems are engaged during interactions with virtual environments, areas related to multisensory integration and memory consolidation are also engaged. This is particularly true for long-lived illusions that are more than the by-product of visual after effects. As a result of this neural and sensory engagement, we should expect certain behavioral dynamics that correspond with those exhibited in the natural world. This is a consequence of behaviors being engaged in context. Ideally, an animal should produce a behavioral response to the illusion that is similar or identical to the same stimulus in the natural world. More likely (and more common with less immersive stimuli) is a behavioral shift that does not mimic the real world. This can be due to a lack of realism in the virtual stimuli, but may also be due to a lack of contextual cues.\n\nThis expected result is based on the idea that once a virtual environment reaches a certain level of realness, the brain can no longer distinguish between real and virtual stimuli. In the case of highly immersive environments, there may be an augmented effect on cognitive processes such as attention and memory (Ragan et al., 2010). Yet much like in the case of the uncanny valley, there is a regime where the brain treats virtual stimuli very differently from their physical world counterparts. Therefore, we can use informed speculation to better characterize the theoretical relationship between a continuous measure of immersion and task performance. The general variable called performance indicates a potential measure of goal-oriented behavior (e.g. swimming orientation, target accuracy) relative to a real-world control.\n\nIn the cases of space/time dilation and illusion, we can make an educated guess as to what the consequences on performance should look like. For example, the predictions for space/time dilation should show a roughly linear relationship between the degree of immersion and performance. In this case, immersion can be operationalized as the degree of exposure an organism has to a VE system. Generally, the degree of immersion increases with the level of performance. On the other hand, previous experience with a specific set of perceptual cues might change this response in certain individuals. Other types of responses might also be possible. A secondary prediction is that there should be a tendency for a flattening out of the response curve at very high and very low levels of immersion, as immediate distinctions between the real and virtual worlds become impossible.\n\nBy contrast, the predictions for illusion might involve an inverted U-shaped relationship between performance and environmental realism. As the amount of environmental realism increases from very low resolution simulation of the environment, performance should increase. Yet for very high resolution simulations, where multiple sensory modalities are simulated at very-high fidelity, performance should drop off. However, any such response would likely be expertise-dependent (C. Bohil, pers. comm.), and might be very different when the stimuli are significantly different from what is normally experienced by the organism. The Uncanny Valley effect and inverted U-shaped relationship is expected to be most prominent in cases where stimuli are unexpected with respect to experience. This can in turn interfere with higher-level mechanisms involved in perception and action.\n\nSimilar questions to those that define illusory experiences in animals can also be asked in the context of space/time dilation. Depending on the degree of immersion, there are a range of sensory systems that could be engaged during space/time dilation. In mammals, this might include the visual and vestibular systems working in concert to register the location and position of the organism’s body in the environment (Fetsch et al., 2012). Unlike illusion, multisensory integration must not be disrupted over long periods of time.\n\nThe neural substrates of space/time dilation involve structures related to learning and memory, spatial cognition, and time-keeping. In mammals, these include the hippocampus (Jacobs et al., 1990) and frontostriatal-cerebellar connections (Stevens et al., 2007). In cases where space/time dilation is successfully achieved, we should expect enhanced activity in these regions. Space/Time dilation should lead to unique behavioral dynamics, very different from those expected from illusion. Highly-immersive environments should produce sped-up or slowed-down responses that are consistent with the type of space/time dilation employed. The outcome of space/time dilation is a learning effect that may reconstitute neural synchrony (Axmacher et al., 2006).\n\n\nChallenges and future directions\n\nThere are a number of hurdles for eliciting the effects of virtuality (illusion and space/time dilation) in animals. Of course, these hurdles are not unique to non-human animals, as VE systems applied to humans are often far from an immersive experience. But animal models provide additional constraints in that systems reliant upon symbolic representations and fictive conditioning may not have much of an effect on the individual. While these are key and often complex features of human cognition, depending on the species they may be absent altogether in animal cognition. Taking this into consideration, the best strategy would be to tailor VE system content to specific animal species. In fishes, symbolism is likely absent and fictive conditioning must be done at a highly abstract level. In other animal species such as birds or social insects, symbolism might be used as a means to mediate the encoding of memories.\n\nAnother consideration is the interaction between cognitive mechanisms such as attention, memory, and psychophysiological phenomena (e.g. arousal). These connections between neurocognitive mechanisms and cross-talk have been shown to be important in mediating human-VE interactions (Parsons & Courtney, 2011). In non-human animals, the interaction of these mechanisms provides an opportunity to make a stronger link between affect and the effects of virtuality. This also provides a means to understand the traditionally affect-driven Uncanny Valley effect in the context of illusion and space/time dilation, which in their totality are products of higher-level cognition.\n\nEven more interesting is the effect of decoupling affect or other psychophysiological responses from their cognitive context. A simple example might be a virtual version of the nictitating membrane response. This form of conditioned learning can lead to an effect called overexpectation (Rescorla, 2006), which can affect memory formation across taxa for both fear conditioning and perhaps even other forms of acquisition (Kehoe & White, 2004). Coupling simple mechanisms with VE systems might open up new avenues for manipulating and exploring higher-cognitive processes.\n\nWhile there are many unknowns in terms of how animals respond to their environment, not to mention the diversity inherent in animal brains and sensory systems, we can nevertheless selectively manipulate these variables using virtual environments. In the broader scheme of animal cognition, parallels with human cognition can be drawn in to illustrate potential neural mechanisms that might be involved in producing behavioral effects observed across a range of experiments. While these effects constitute a relatively unexplored component of animal behavior, they may lead to new discoveries in animal cognition and perhaps in the genetic substrates of conserved animal behaviors (Figure 2).\n\nElicitation of these behavioral effects is dependent on the configuration of the virtual environment itself. Unlike natural environments, virtual environments are highly stereotyped and do not include much of the noise associated with biological realism (Dennett, 2013). Nevertheless, environmental realism can be high, and findings in human experiments suggest that this is not an epiphenomenon (Blascovich & Bailenson, 2011). In addition, virtual environments are highly flexible and provide an experimental test bed for exploring the potential richness of animal perceptual, cognitive, and social behavior (Bohil et al., 2011). Since there are a range of possible design configurations for animal research-oriented VE systems, many of which can be tailored to a scientific question and organism of interest, the possibilities for further application and future research are potentially endless. Furthermore, costs can be minimized through clever design features.\n\nTailoring the virtual world to the perceptual specializations of a given organism would help in this regard. One example is the high critical fusion frequency (CFF) of the housefly (Healy et al., 2013). Tightly-controlled environments can be constructed by using the fly’s natural visual sampling rate as a baseline. The rate of presentation can then be systematically varied. Another example is the electrosensory and mechanosensory capabilities of sharks, rays, and certain bony fishes (Coombs et al., 2002). A VE system that models fluids and fluid dynamics in the sensory environment could enable the creation of perceptual ambiguities, which could then allow for the power of sensory illusion to be leveraged. These type of examples ultimately provides the experimentalist with a highly-controllable, selectively enriched (Nithianantharajah & Hannan, 2006), and customizable environment.\n\nThe benefit of this might be also considered in terms of gene-environment interactions (Figure 2). One way in which virtual environments might be able to assist in uncovering gene-environment interactions is by using a logic similar to that which twin studies rests upon. In twin studies, the genetic similarities of identical twins are used to control for unknown genetic variation (van Dongen et al., 2012). In a similar manner, virtual environments might be employed to control for unknown environmental noise. For experimental purposes, a random sample exposed to the same highly-controlled environment is predicted to exhibit minimal environmental variation. This should allow for the effects of the genetic background to be magnified, enabling stronger associations between genes and behavior to be made.\n\nWith the rapid adoption and increasing affordability of next-generation sequencing technologies, it is now possible to target assays of a genome in combination with genome-wide association (GWAS) studies to uncover the genetic components of a trait. What is still a mystery are the interactions between genes, behavior, and environment. Gene sequencing combined with robust environmental control can elucidate some of these interactions, while also providing insights into the ultimate processing limits of functionally-distinct neural systems.\n\nWhile the link between genotype and controllable environment is more speculative, the promise of VEs for the study of animal behavior and cognition is real and the returns can be immediate. I have shown how different forms of VE have been used to elucidate and perhaps even augment animal behavior. In fact, VE might be particularly useful in understanding particularly difficult-to-define problems such as neural coding (Kumar et al., 2010) and human-animal interaction (Wilson & Barker, 2003). Overall, however, VE systems provide a flexible mode of investigation for both general and specific mechanisms that govern brain and behavior. In addition, two specific types of manipulation (illusion and space/time dilation) can be used to produce novel experimental outcomes. These effects of virtuality provide an opportunity to advance the naturalistic study of animal brain and behavior.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI would like to thank Corey Bohil and Frank Biocca for their insights and collaboration during my time in the MIND Laboratory. I would also like to thank Corey Bohil for his editorial assistance during the writing of this manuscript.\n\n\nReferences\n\nAgrillo C, Piffer L, Bisazza A: Number versus continuous quantity in numerosity judgments by fish. Cognition. 2011; 119(2): 281–287. PubMed Abstract \| Publisher Full Text\n\nAhrens MB, Li JM, Orger MB, et al.: Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature. 2012; 485(7399): 471–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkst J: Send in the Bots. 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Publisher Full Text" }	[ { "id": "5934", "date": "02 Oct 2014", "name": "Alan Dorin", "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\nOverall I think a survey/position paper like the one presented here is relevant and helpful. The survey explores the application of technology for generating and manipulating virtual environments to the study of animal behaviour and cognition. Some important potential issues and benefits the practice raises (or might raise) have been explored in this initial version of the article. I still have some major reservations about the article as it stands and would recommend a few changes prior to its finalisation.The article is very broad and, in many places highly speculative. The many claims phrased \"should\", \"may\", \"potential\" and \"expected\" (e.g. see para 1 of Introduction) would be more convincing to me if supported with strong arguments based on published or presented evidence. Since the paper is so broad in its scope this might require a very long paper indeed, perhaps an entire book! As an alternative, I would suggest narrowing the scope considerably (e.g. to consider experiments with one species or other experimental category such as Primates, insects, or something still more specific such as Zebrafish or Hymenoptera, or anything for which there is already a lot of evidence gathered in the literature) and focusing on how the arguments presented here might apply, and be supported, in that case. A second reason for suggesting a reduction in scope relates to the differences in perceptual and cognitive systems between animals. For instance avian colour vision and hymenopteran colour vision are very different, as are the learning mechanisms these organisms seem to implement. Hence, discoveries in one case are not necessarily transferable between species. This makes claims about animal perception in general difficult to state with authority unless they are so broad as to be meaningless or at least unhelpful. I suspect many readers familiar with these issues would be skeptical about the utility of making generalisations at all. This contributes to my perception that the article is biting off a little more than can be chewed in a single publication. The introduction of the concept of the uncanny valley to this context is interesting! As the author points out we need to be very careful about applying it beyond humans/primates. It would indeed be fascinating to test for the effect in other animals. The author needs to be careful though that their hypothesis that it might apply beyond primates and in specific cases, doesn't get confused even loosely with the \"truth\" of this, and hence its relevance. E.g. see para. 2 of \"Current examples\" where you indicate that the \"effect suggests...\". Indeed in one fun collaboration, a researcher/artist team have tested for something related to the uncanny valley (although this wasn't their motivation) in honeybee attraction to artists' renditions of flowers (https://www.artlink.com.au/articles/3722/insects-as-art-lovers-bees-for-van-gogh/). Bees are not fussy and can be trained to visit coloured squares of paper for rewards so I guess they fall at the \"no uncanny valley\" end of the spectrum. But I am not sure if any measure of initial confusion, discomfort, or any unusual neural activity, might have been recorded experimentally to ascertain the presence of the valley. A group of researchers at Graz, Austria are working on robots, (not VEs), that are intended to influence the behaviour of animals/insects. This might well be research that could investigate the effect the author is considering (http://zool33.uni-graz.at/artlife/node/208). I hear it took the researchers some patient work to encourage bees to accept their robot hive-mates and I am unsure of the degree of their success! Additionally, this indicates another avenue besides complete virtuality for interacting with organisms as the author notes in para. 3 of \"Current examples\" and later under \"Sensorimotor integration\" in the context of fish studies. A note on the valley diagram: individual variation may spread horizontally too - i.e. the \"pinch points\" as the curves cross the x-axis in the graph might be better rendered as broad \"fords\" across the axis.Lastly, even if the author chooses to remain broad in your discussion of different animals being tested within virtual environments, please consider editing the article, and individual sentences, much more tightly. Perhaps delete references to robot work, noting it is being done, and focus on screen-based work. Or vice versa. Or discuss only the relevance of the uncanny valley. At the moment, as a reader I find the paper is a little too broad. A tighter focus would (for me) make it more enjoyable to read and more informative.", "responses": [] }, { "id": "6813", "date": "26 Nov 2014", "name": "Jesse D. Cushman", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides an interesting attempt to synthesize theories and empirical findings regarding the potential utility of virtual reality in animal research. I found many of the referenced papers to be quite interesting from my fairly localized perspective on this subject. Overall, however, I find the article to be excessively speculative, insufficiently referenced and often repetitive. The speculations are quite often so vague that they are essentially meaningless. Below are some specific concerns: Application of the uncanny valley concept: It is a huge leap to assume there is an uncanny valley in all aspects of perception, the original idea is quite specific to “humanness” and there is very good reason to think that the “close but not quite” uncanny valley does not generalize beyond this. Why would such a non-linearity be expected to be present in virtual reality? This argues that a poorly instantiated VR would actually be better than a very good, but not quite perfect one. Secondly, the emotional valence that is plotted to produce the uncanny valley comes from the immediate reaction of a human observer to a robot. This should not be confused with the emotional valence that is acquired by learning in a virtual environment based on the specific task contingencies and rewards or punishments that may be presented. A huge omission in this article is the concept of immersion vs. presence. The author refers to immersion in several places but fails to incorporate the critical concept of presence, which is defined as the psychological experience being “present” in the virtual environment. This concept is referred to variously as “engagement” and “performance.” The absence of this important concept in the paper is perhaps the most serious underlying problem. “Engagement” and “good performance” do not necessarily require much immersion and should not be considered evidence of presence: an animal or human can be quite engaged and perform well in very rudimentary “virtual environments” as long as proper feedback is given (rewards, punishment, etc.), without being present in the virtual environment. Fundamentally, immersion vs. presence gets at the distinction between sensation and perception. Illusion, a term used frequently in the article, operates at the perceptual level and is therefore somewhat akin to presence, but it’s definition as “confusing” a virtual stimulus for a real one does not fully capture the complexity of being present in a virtual environment. Virtuality is another frequently used term in the article, but its precise definition and how it relates to the immersion vs. presence distinction is entirely unclear. Several recent papers that are highly relevant to the paper should also be cited and discussed: Ravassard et al. (2012) - Multisensory control of hippocampal spatiotemporal selectivity. – This paper directly compares “place cells” in real world and virtual environments in rats. Cushman et al. (2013) - Multisensory control of multimodal behavior: do the legs know what the tongue is doing? – This paper shows spatial navigation in virtual reality in rats and utilizes isolation of the visual and auditory modalities combined with the simultaneous measure of two behavioral outputs. Aronov and Tank (2014) - Engagement of Neural Circuits Underlying 2D Spatial Navigation in a Rodent Virtual Reality System. – This paper extensively characterizes place cells and grid cells in virtual reality.Aghajan et al. (2014) - Impaired spatial selectivity and intact phase precession in two-dimensional virtual reality. - This paper compares place cells in 2-D virtual and real world environments", "responses": [] } ]	1	https://f1000research.com/articles/3-202
https://f1000research.com/articles/4-30/v2	10 Feb 15	{ "type": "Research Note", "title": "Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry", "authors": [ "Jason Long", "Edward Wright", "Eleonora Molesti", "Nigel Temperton", "Wendy Barclay", "Jason Long", "Edward Wright", "Eleonora Molesti", "Nigel Temperton" ], "abstract": "Emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. The former was the only available intervention when the current unprecedented Ebolavirus (EBOV) outbreak in West Africa began. Prior to this, the development of EBOV vaccines and anti-viral therapies required time and resources that were not available. Therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of EBOV and could be used immediately. Here we test three such medicines and measure their ability to inhibit pseudotype viruses (PVs) of two EBOV species, Marburg virus (MARV) and avian influenza H5 (FLU-H5). We confirm the ability of chloroquine (CQ) to inhibit viral entry in a pH specific manner. The commonly used proton pump inhibitors, Omeprazole and Esomeprazole were also able to inhibit entry of all PVs tested but at higher drug concentrations than may be achieved in vivo. We propose CQ as a priority candidate to consider for treatment of EBOV.", "keywords": [ "ebola", "emerging viral disease", "avian influenze", "H5N1", "Marburg", "chloroquine", "omemprazole", "esomeprazole" ], "content": "Introduction\n\nEmerging pathogens such as Ebolaviruses (EBOV), Avian Influenza viruses, Severe Acute Respiratory Syndrome (SARS) virus, Middle-East coronavirus (MERS), Chikungunya virus (CHIKV) and Dengue virus pose public health challenges that demand researchers and governments work together to assess their pandemic potential and plan mitigating strategies. Of the five species of EBOV belonging to the Filoviridae (including Zaire ebolavirus (EBOV-Z), Bundibugyo ebolavirus (EBOV-B), Reston ebolavirus, Sudan ebolavirus (EBOV-S) and Tai Forest ebolavirus1), EBOV-Z and EBOV-S are responsible for the majority of outbreaks of highly pathogenic haemorrhagic fevers causing high fatality rates2. Past outbreaks have been of limited size affecting a local population, however a strain of EBOV-Z is the causative agent of the current outbreak that began in late 2013 and has since become an unprecedented and devastating epidemic3,4, resulting in over 20,000 suspected cases, of which those confirmed had a case fatality rate of around 60% in the afflicted West African countries (http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary-20150107?lang=en and http://www.who.int/csr/disease/ebola/situation-reports/en/). Towards the end of 2014 the trend in case numbers reversed in Liberia and the epidemic slowed in Sierra Leone and Guinea, but the virus continues to transit in new geographical areas5. This epidemic has triggered a significant global health response relying on primary hygiene and other containment measures that have proved successful in limiting the spread of the virus in previous outbreaks. Given the scale of this outbreak and the fear that traditional containment measures may fail to prevent global spread, several vaccines have been fast-tracked into phase I clinical trials6–8 although even if proved efficacious, the limited supply of sufficient quantities of vaccine will hinder their use in the current situation. For disease treatment, patients suffering a haemorrhagic fever have relied on the clinical management of symptoms (http://www.cdc.gov/vhf/ebola/treatment/), with a handful of patients in this outbreak receiving experimental therapies such as ZMapp, TKM-Ebola, brincidofovir and favipiravir (http://www.nature.com/news/ebola-trials-to-start-in-december-1.16342)9–12. Alternatively antibody treatment by transfusion therapy using blood or plasma from Ebola virus survivors has been approved11,13–16; although issues with safety and lack of resources for this method limit its suitability in West Africa today. Having no approved or widely available therapeutics for EBOV, as with many other emerging viral diseases, focus has turned to possible re-purposing of drugs already licensed for other uses by the EMA and FDA. Several clinically approved drugs have been identified by researchers17–20, including amiodarone, one of the several cationic amphiphiles found to inhibit filovirus entry which is currently being trialled in Sierra Leone21. However reservations have been expressed about the complications that could be caused by side effects of the drug in EBOV patients. The anti-malarial drug chloroquine (CQ) has also been shown to inhibit EBOV entry and protected mice from EBOV infection18,22 and has been previously highlighted as a possible drug to treat EBOV infection11.\n\nThe possible difficulties that may arise with use of re-purposed drugs include unforeseen interactions between virus/drug and host causing exacerbation of disease. Therefore it is important to try and understand the mechanism of virus inhibition by such drugs. To this end we re-examined the anti-viral properties of CQ, and show here that it inhibited the pH-dependent endosomal entry of a pseudotyped virus (PV) bearing EBOV glycoproteins, in the same way as did the potent and specific vacuolar-ATPase (vATPase) inhibitor bafilyomycin A1 (BafA1) (a non-medical laboratory compound). We also show that licensed and widely used proton pump inhibitors (PPIs) for treatment of gastric acid reflux, omeprazole (OM) and esomeprazole (ESOM), inhibited PV EBOV entry, likely by their off-target inhibitory activity on endosomal vATPase.\n\n\nMethods\n\nHuman embryonic kidney (293T/17) (ATCC) and Human lung adenocarcinoma epithelial cells (A549) (ATTC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS) (Biosera) and 1% Penicillin-streptomycin (PS) (Invitrogen). The cell lines were maintained at 37°C in a 5% CO2 atmosphere.\n\nChloroquine diphosphate salt (CQ), bafilomycin A1 from Streptomyces griseus (BafA1), omeprazole (OM) and esomeprazole magnesium hydrate (ESOM) (Sigma) were resuspended as per manufacturer’s instructions and aliquots stored at -20°C: CQ was prepared in sterile dH2O; BafA1, OM and ESOM were prepared in sterile DMSO (Sigma).\n\nThe Bundibugyo ebolavirus (EBOV-B) envelope glycoprotein (GP) (FJ217161) coding sequence was synthesised (Bio Basic Inc.) and the HA glycoprotein of avian influenza A/turkey/England/50-92/91(H5N1) (FLU-H5) was amplified from the HA plasmid of the H5N1 reverse genetics system23. Both were sub-cloned into the pCAGGS expression vector. Expression vectors containing the envelope glycoproteins of Zaire Ebolavirus (Mayinga) (EBOV_Z), Marburg virus (Lake Victoria isolate; MARV) and Gibbon Ape Leukemia Virus (GALV) (modified to contain the trans-membrane domain of amphotropic murine leukemia virus (A-MLV) envelope glycoprotein) are described previously24,25. The Renilla luciferase gene was sub-cloned into pCAGGS expressing vector from a minigenome reporter described previously26.\n\nThe generation of all lentiviral pseudotype viruses was based on the methods detailed previously27–29. Briefly, 293T/17 cells were seeded into 10cm3 tissue culture plates (Nunc™ Thermo Scientific). The HIV gag-pol plasmid, pCMV-Δ8.91 and the firefly luciferase reporter construct, pCSFLW, were transfected together with either influenza HA, GALV, EBOV or Marburg GP expression constructs at a ratio of 1:1.5:1 (core:reporter:envelope) using Fugene6 transfection reagent (Promega). At 24 h post-transfection, cells were washed and fresh media applied. For the generation of H5 PVs, 1U exogenous recombinant neuraminidase from Clostridium perfringens (Sigma-Aldrich) was also added 24 h after transfection to effect egress from the producer cells. PV supernatants were harvested at 48 and 72 h post-transfection and passed through a 0.45m pore filter. EBOV PVs were aliquoted and stored at 4°C; the remaining PVs were stored at -80°C.\n\n293T cells in 10cm3 plates were transfected with 15ug of Renilla luciferase expressing plasmid using Lipofectamine 2000 according to manufacturer’s instructions (Life Technologies™). CQ, BafA1, OM and ESOM were serially diluted in 96-well white-bottomed plates (Nunc™ Thermo Scientific) to give the final described concentrations. After 20h the transfected cells were trypsinised and 1×104 cells were added to each well. After 30min cells were transduced with no more than 1×105 RLU of PV per well (estimated from raw RLU values of previously infected 293T cells), and to an equal volume per well. 48 h later supernatant was removed and cells were lysed with 30µl of passive lysis buffer (Promega), and firefly/Renilla luciferase activity measured using a FLUOstar Omega plate reader (BMG Labtech) and the Dual luciferase assay system (Promega).\n\nA549 cells were pre-treated with drug 1 h before 75nM of the pH sensitive Lysotracker® Red DND-99 (Life Technologies™) was added to the media of each well30. After 30minutes in growth conditions, cells were analyzed for fluorescence using an Axiovert 40 confocal laser (CFL) microscope and an AxioCam MRc camera (Carl Zeiss).\n\nPV transduction RLUs were normalised to the Renilla value in the corresponding wells. Percent infection of each drug dilution was calculated compared to untreated cells. Two-way ANOVA with Bonferroni’s multiple comparisons test between untreated and treated mean values (α-0.05) was performed to measure statistically significant differences. IC50 values were calculated using non-linear regression analysis (log[inhibitor] vs normalised response). All manipulation of data was performed on GraphPad Prism 6 (GraphPad software).\n\n\nResults\n\nThe envelope glycoproteins of several emerging viruses with high pathogenicity and pandemic potential were used to create lentiviral based pseudotype particles as previously described29. PVs were generated bearing the envelope glycoproteins from Zaire ebolavirus (Mayinga strain) (EBOV-Z), Bundibugyo ebolavirus (EBOV-B), Marburg (Lake Victoria isolate) virus (MARV), H5 HA from a highly pathogenic avian influenza virus A/turkey/England/50-92/91(H5N1) (FLU-H5), and Gibbon Ape Leukaemia virus (GALV). GALV PVs were included because GALV is a virus that does not require acidification of endosomes for its entry into cells. All the PVs generated were shown to transduce 293T cells and firefly luciferase expression from the packaged reporter gene was measured above mock infected cells (non-transduced cells) (Dataset 1).\n\nIn order to assess the ability of CQ, BafA1, OM and ESOM to inhibit PV entry, drugs were serially diluted in triplicate in white bottomed 96-well plates. Next, 293T cells transfected 24 hours previously with a Renilla luciferase expression plasmid to allow monitoring of cell viability, were added to each well. Appropriately diluted PVs were then added to each dilution, including a no-drug control. After 48 hours incubation, the supernatant was removed and firefly and Renilla luciferase RLUs were recorded using the Dual Luciferase Assay System (Promega).\n\nPV RLUs were normalised to the corresponding Renilla values, which reduced the edge effect observed in the 96-well plates, and controlled for toxicity of the drugs. Only BafA1 appeared to reduce expression of Renilla at the highest concentrations, suggesting cellular toxicity, (Dataset 1) and visible cytopathic effect was not observed in cells treated by CQ, OM and ESOM at the concentrations used in Figure 1.\n\n293T cells previously transfected with a Renilla expression plasmid were treated with differing concentrations of drug before being transduced with PV (carried out in triplicate). Data are the percent of infection compared to untreated cells. EBOV-Z, EBOV-B, MARV, FLU-H5 and GALV inhibition was measured for each drug compound. Cells were harvested and firefly and Renilla activity measured after 48 h incubation. A. Cells were treated with 10, 3.33 and 1.11nM of BafA1. B. Cells were treated with 30, 10, 3.33 and 1.11µM of CQ. C and D. Cells were treated with 100, 50 and 25µM of OM and ESOM, respectively. Statistical analysis of these data are shown in Table 1.\n\na IC50 values were calculated using non-linear regression analysis (log[inhibitor] vs normalised response)\n\nb Two-way ANOVA with Bonferroni’s multiple comparisons test between untreated and treated mean values (α-0.05)\n\nns P>0.05, * P≤0.05, P≤0.01, * P≤0.001, **** P≤0.0001\n\nBoth BafA1 and CQ reduced EBOV-Z, EBOV-B, MARV and FLU-H5 entry in a dose dependent manner (Figure 1A and B). The IC50 value of BafA1 was in the nM range for EBOV-Z, EBOV-B, FLU-H5 and MARV and inhibition of entry was statistically significant at the 10nM concentration compared to the untreated control (Table 1). CQ inhibited EBOV-Z, EBOV-B, MARV and FLU-H5 with IC50 of 3.319, 3.585, 3.192 and 10.44µM respectively, and inhibition was statistically significant (Table 1). In contrast, GALV entry was augmented by both BafA1 and CQ above that of the untreated cells to a maximum of 143.83% (3.33nM) and 180.38% (3.33µM) respectively. Both OM and ESOM reduced entry of all PVs tested at 100µM but GALV PV was the least affected (Figure 1C and D). Inhibition of entry for EBOV-Z, EBOV-B, MARV and FLU-H5 PVs by ESOM was significant at 50µM, and GALV PV was not significantly inhibited at this dose (Figure 1D and Table 1).\n\nBafA1 and CQ are known endosomal acidification inhibitors (BafA1 being a potent and specific vATPase inhibitor and CQ a licensed lysotropic agent)31. The effects of OM and ESOM on endosomal acidification have also been previously reported32,33. To confirm that endosomal pH was being affected at doses used here, A549 cells were treated with drug for 1 hour before applying LysoTracker® Red DND-99 (LifeTechnologies). A549 cells were chosen here because 293T cells are poorly imaged due to their morphology. The lysotracker probe specifically fluoresces in acidic organelles. Fluorescence was decreased in cells treated with BafA1 and CQ in a dose dependant manner, but was unaffected in cells treated with vehicle alone (Figure 2). OM and ESOM appeared to decrease fluorescence, and therefore increase endosomal pH, only at a concentration of 200µM, higher than that required to inhibit PV entry. Moreover cellular toxicity was observed at this concentration after 24 hours.\n\nA549 cells were treated with drug for 1 h before 75nM Lysotracker® Red DND-99 was added to each well. DMSO (drug vehicle) only was diluted at 30mM, 3mM, 0.3mM and 0.03mM. (A–D). CQ was diluted 30, 10, 3.33 and 1.11µM (E–H), BafA1 was diluted to 10, 3.33 and 1.11nM (I–L) and OM and ESOM were diluted to 100, 50 and 25µM (M–P) and (Q–T) respectively. The level of fluorescence was imaged by confocal microscopy (x50 magnification).\n\n\n\n\nConclusions and discussion\n\nAfter attachment to cells, viruses require a mechanism of fusion to deliver the viral genome. Preventing this action by fusion inhibitors has been successful approach for HIV antiviral therapy34. Unlike HIV, EBOV and many other viruses are dependent on the naturally low pH of acidic endosomes to activate and trigger fusion by their envelope glycoproteins. In this instance, a ‘fusion inhibitor’ could target the host cell machinery preventing acidification of the endosome, working to inhibit virus entry of several different viruses. Here we have reiterated that cell entry by PVs representing EBOV, FLU-H5 and MARV can be inhibited by increasing the endosome pH using BafA1 and CQ (Figure 1), and this correlates with their ability to prevent the acidification of intracellular organelles (Figure 2).\n\nCQ has shown antiviral activity against several viruses in vitro, including EBOV, influenza, Nipah, Hendra, Dengue and CHIKV35–37. Disappointingly, this antiviral activity has not always translated into efficacy in vivo models or clinical trials, although CQ was effective in a mouse model against EBOV18,35,38–42. The variability in in vivo results may depend on study design and strains of virus used. In one study BafA1 treated mice were not protected from influenza infection but treatment with a related compound, SaliPhe, was protective, even though both drugs were potent in vitro43. Inhibition of endosome acidification as a target for inhibiting EBOV can be justified by the knowledge that the filoviruses depend on the low pH for two separate steps of their entry pathway. Not only is the fusion by G protein triggered by low pH, but its cleavage into a fusogenic form is carried out by endosomal enzymes cathepsins B and L whose activation is also pH dependent44. Some have argued that G protein cleavage by cathepsin is less essential than previously thought45,46 and that EBOV species other than Zaire together with closely related MARV do not require cathepsin cleavage for entry47,48. Nonetheless, entry of MARV PVs was still inhibited in our assays suggesting that inhibiting fusion alone is sufficient.\n\nRecently, using computational modelling, Ekins et al. suggested the anti-EBOV mechanism of CQ may be by binding the VP35 protein of EBOV49. If this drug had activity on several steps of the replication cycle it may not only be more effective in vivo but it may be even less likely that the virus could mutate to escape inhibition.\n\nAt first we were surprised that CQ actually increased entry of GALV PV (Figure 1). However this effect has been noted before for other retroviruses, including A-MLV and HIV-1, and is accounted for by the inhibitory effect of CQ on the autophagy pathway. CQ prevents degradation of phagosomes that contain virus particles and prevents them from otherwise being degraded50–52.\n\nCQ has been used for many years as an anti-malarial drug, although it is now only effective in parts of central America and the Caribbean due to accumulation of drug resistance by the plasmodium parasite53. Interestingly, compounds belonging to the omeprazole family have also been described as having anti-malarial properties in vitro, possibly via their reported ability to target vATPase in the plasma membrane of Plasmodium parasite54. Soon after its discovery OM was found to also inhibit intracellular vATPase at µM concentrations as opposed to its licensed target of gastric H+/K+-ATPase against which it is effective at much lower concentrations32,33. Indeed there are a plethora of publications indicating use of OM and ESOM in cancer therapy, as a means to inhibit the characteristic acidic intracellular environment, and thus permit sensitivity to cytotoxic therapies55–59. A role of OM and ESOM has also been noted in the suppression of bone resorption, another physiological process dependent on pH60–62. Given the volume of research suggesting these off target effects depend on an ability to affect intracellular pH, we hypothesised that these drugs would, like CQ and BafA1, inhibit EBOV, MARV and influenza virus pH dependent entry. We used GALV as a control again since its entry is reportedly independent of pH. Indeed, EBOV, FLU-H5 and MARV were inhibited by lower doses of OM or ESOM than GALV (Figure 1 and Table 1). GALV entry was also inhibited at the highest concentration, but we cannot exclude that this was due to a toxic effect that was not measured by the Renilla control we employed here. We did not observe as close a correlation between drug doses that mediated the inhibition of EBOV or influenza PV entry and increase in pH of intracellular vesicles for OM and ESOM as for CQ and BafA, (Figure 1 and Figure 2). More recently, it has been reported that OM and ESOM altered the localisation of vATPase in the cell as well as the pH of intracellular vesicles46 and this may explain their ability to inhibit PV entry more potently than the pH changes we observed would suggest.\n\nInhibition of influenza virus entry to cells by means of inhibiting acidification of endosomes has been known for decades63, although no current antivirals for influenza have been licensed on this basis. Some epidemiological evidence from population studies suggests that OM could exert a protective effect against influenza-like-illness64, but our studies suggest that doses required for potent inhibition might be difficult to achieve without significant toxicity. Despite these drugs being readily available, even without prescription in some countries, the licensed dosing would generate a plasma concentration reportedly 1.59–9.61µM for ESOM that falls short of the IC50 calculated in this study, although higher doses have been used clinically65. Therefore it seems unlikely that OM and ESOM would be a suitable therapy for ebolavirus infection, but more specifically designed vATPase inhibitors may have potential as broad acting antivirals against several emerging viruses in the future. With regard to CQ, the evidence suggests a more promising position for use against ebolavirus. Standard adult dosing (25mg/kg) achieves plasma concentration of 2µM, close to our IC50 value against EBOV PV entry. Protection in the mouse model was previously shown with a 90mg/kg dosage18,66.\n\nUsing re-purposed drugs to treat outbreaks of emerging diseases must surely be approached with caution. In Ebola patients with severe life-threatening disease it would be important to ensure that any side effects of a therapy did not enhance disease progression, particularly if higher doses of re-purposed drugs, as suggested here, were considered. On the other hand, CQ has been taken prophylactically in a tropical setting for many years to prevent malaria and we suggest that, with little additional need for scale up of production of a new agent, this might represent a useful adjunct to the current antiviral strategies being trialled in West Africa. We envisage that in contacts of EBOV cases, CQ might decrease the viral load that establishes in the early days after virus transmission. Further work in in vivo models including guinea pig and primates should inform about doses and administration regimens.\n\n\nData availability\n\nFigshare: Inhibition of pseudotype virus entry by existing FDA-approved drugs. doi: http://dx.doi.org/10.6084/m9.figshare.129480167", "appendix": "Author contributions\n\n\n\nDr Jason Long, Dr Edward Wright and Dr Eleonora Molesti generated the PVs. Jason Long performed the drug entry assay and pH assay. This work was planned by Prof Wendy Barclay, Dr Nigel Temperton and Dr Jason Long. All authors were involved in preparing and revising the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the grants FLUPIG EU FP7 258084 and BBSRC BB/K002465/1.\n\n\nAcknowledgments\n\nThe authors wish to thank Caroline Goujon for the kind provision of the GALV construct and Olivier Moncorgé for generating the Renilla expression plasmid.\n\n\nReferences\n\nLi YH, Chen SP: Evolutionary history of Ebola virus. Epidemiol Infect. 2014; 142(6): 1138–45. PubMed Abstract \| Publisher Full Text\n\nFeldmann H, Geisbert TW: Ebola haemorrhagic fever. Lancet. 2011; 377(9768): 849–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaize S, Pannetier D, Oestereich L, et al.: Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med. 2014; 371(15): 1418–25. PubMed Abstract \| Publisher Full Text\n\nGatherer D: The 2014 Ebola virus disease outbreak in West Africa. 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Calcif Tissue Int. 1986; 38(2): 123–5. PubMed Abstract \| Publisher Full Text\n\nSheraly AR, Lickorish D, Sarraf F, et al.: Use of gastrointestinal proton pump inhibitors to regulate osteoclast-mediated resorption of calcium phosphate cements in vivo. Curr Drug Deliv. 2009; 6(2): 192–8. PubMed Abstract \| Publisher Full Text\n\nYoshimura A, Kuroda K, Kawasaki K, et al.: Infectious cell entry mechanism of influenza virus. J Virol. 1982; 43(1): 284–93. PubMed Abstract \| Free Full Text\n\nGasparini R, Lai PL, Casabona F, et al.: Do the omeprazole family compounds exert a protective effect against influenza-like illness? BMC Infect Dis. 2014; 14: 297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLind T, Rydberg L, Kylebäck A, et al.: Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease. Aliment Pharmacol Ther. 2000; 14(7): 861–7. PubMed Abstract \| Publisher Full Text\n\nMaitland K, Williams TN, Kotecka BM, et al.: Plasma chloroquine concentrations in young and older malaria patients treated with chloroquine. Acta Trop. 1997; 66(3): 155–61. PubMed Abstract \| Publisher Full Text\n\nLong JS, Wright E, Molesti E, et al.: Inhibition of pseudotype virus entry by existing FDA-approved drugs. Figshare. 2014. Data Source" }	[ { "id": "8424", "date": "27 Apr 2015", "name": "Sean Ekins", "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\nThe work was of particular interest especially in light of several viruses shown to be taken up in this rather non specific way. I would have perhaps also like some discussion of receptor and channel mediated virus uptake - there are several publications in this space. The interplay between such different mechanisms may point to multiple targets or need for combined approaches to block them.The authors describe amiodarone, but there are many molecules that have been found as ebola replication or pseudoviral entry inhibitors, had they looked at more molecules to see if the pH mechanism was common across them?I would likely suggest adding repurposing in the title of the article.The conclusion might benefit from comparison of the chloroquine data with that previously published (higher EC50), potential ocular toxicity etc.Some discussion as to whether the pH effect is an in vitro specific effect or something of in vivo relevance - would also be worth mention.This study confirms the previous work on chloroquine and suggestions by others as to its potential utility. This begs the question why it is not used clinically. What other data would be needed to show that chloroquine could be clinically useful?The study is well designed and reported and adds to the growing literature on chloroquine and its potential as an antiviral.", "responses": [] }, { "id": "8175", "date": "29 Apr 2015", "name": "Robin A. Weiss", "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\nThis is an elegant study employing envelope pseudotypes of highly pathogenic viruses which demonstrates that certain inhibitors of low endosomal pH can inhibit viral entry. Because some of these molecules such as Chloroquine have been in clinical use for decades, and are inexpensive, they might tip the balance between survival and death during human infection.I have no criticism of the experimental work. However, I have been told by a reliable physician who has recently cared for patients with Ebola infection that treatment with Chloroquine offered no clinical benefit. Thus it is possible that an in vitro observation may not translate into a useful treatment in vivo. So one should be wary about the conclusions.", "responses": [] } ]	2	https://f1000research.com/articles/4-30
https://f1000research.com/articles/4-38/v1	09 Feb 15	{ "type": "Review", "title": "Small molecules with antiviral activity against the Ebola virus", "authors": [ "Nadia Litterman", "Christopher Lipinski", "Sean Ekins", "Nadia Litterman", "Christopher Lipinski" ], "abstract": "The recent outbreak of the Ebola virus in West Africa has highlighted the clear shortage of broad-spectrum antiviral drugs for emerging viruses. There are numerous FDA approved drugs and other small molecules described in the literature that could be further evaluated for their potential as antiviral compounds. These molecules are in addition to the few new antivirals that have been tested in Ebola patients but were not originally developed against the Ebola virus, and may play an important role as we await an effective vaccine. The balance between using FDA approved drugs versus novel antivirals with minimal safety and no efficacy data in humans should be considered. We have evaluated 55 molecules from the perspective of an experienced medicinal chemist as well as using simple molecular properties and have highlighted 16 compounds that have desirable qualities as well as those that may be less desirable. In addition we propose that a collaborative database for sharing such published and novel information on small molecules is needed for the research community studying the Ebola virus.", "keywords": [ "Ebola Virus", "FDA approved drugs", "Medicinal chemistry" ], "content": "Introduction\n\nViruses remain a constant threat to global health, with new infections from Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) killing more than 3 million people annually1,2. The flavivirus that causes dengue fever infects up to 100 million people each year, leading to death in 2.5% of cases3,4. Other viral outbreaks including severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory coronavirus (MERS-CoV), affect far fewer people but have high mortality rates and the potential to spread to epidemic size1,5,6. Thus, even with the development of vaccines and other treatments, viruses lead to a large burden on human health.\n\nMore than 30 small molecule drugs have been developed that have activity against individual viruses, including HIV, influenza, HBV, and more recently HCV7,8. However, a large number of virus types remain without any effective therapeutics, and there are few broad-spectrum anti-virals available. Thus, when viruses emerge that cause life-threatening infections, such as the recent Ebola virus epidemic in West Africa, there are no treatment options. Since it is likely that these and other types of infectious agents will emerge in the future, an important goal is to identify inhibitors to be available to contain such outbreaks.\n\nA large array of drug discovery efforts have proven that despite their small size, viral genomes represent suitable targets for drugs. Direct-acting antivirals, which target viral proteins rather than the host’s, have been the subject of extensive investigation3. Targets in this class fall into multiple categories: virus adsorption inhibitors, inhibitors of viral DNA or RNA synthesis, viral protease inhibitors required for virus maturation, and viral neuraminidase inhibitors required for virus elution7. In addition, cellular targets exist that are required for viral replication, including inosine monophosphate (IMP) dehydrogenase, which is required to supply the pool of guanosine triphosphate (GTP) that serves as a substrate for RNA and DNA, and S-adenosylhomocysteine (SAH) hydrolase, which is required for the methylation and hence maturation of viral DNA. While finding specific inhibitors to target each viral threat individually may be ideal, the cost-savings and feasibility of finding drugs that act as broad-spectrum antivirals, or those that target specific viral genus or family, is an important goal given the expense associated with developing any one drug for one disease3. There are important individual aspects of each virus, and viral family to consider, but nonetheless, many mechanisms of the viral life cycle are mirrored across families, and thus represent opportunities to learn from the many experimental studies that have already been performed.\n\nLike many around the world, we have been watching the devastating effects of the Ebola virus in West Africa. We have been impressed by the important contributions of the health organizations and the personal sacrifices the medical workers are making to serve patients and hamper the spread of disease. And yet we began to wonder, why has there been relatively little focus on small molecules apart from a recent review of Ebola virus therapeutic strategies in general9? Small molecules have several advantages over other therapeutic approaches including the ability to be produced at a large scale and stability necessary for broad distribution. We felt it was time to therefore focus more on small molecules while we await a vaccine. We have found that indeed there is much prior knowledge regarding small molecules that have been shown to be active against the Ebola virus in vitro or in animal models10–13, including a number of FDA-approved drugs14–16. A thorough literature search of PubMed, and CAS SciFinderTM (CAS, Columbus OH) using terms including “Ebola” identified 55 molecules suggested to have activity against Ebola virus in vitro and/or in vivo (Supplemental Table 1).\n\nRecently, a pharmacophore17 was generated from four FDA approved compounds for other diseases (non-antivirals) resulting from two high throughput screens against the Ebola virus14,15 and closely matched the receptor-ligand pharmacophores for the Ebola Viral protein 35 (VP35)10. Follow-up docking studies suggested that these compounds may have favorable inhibitory interactions with this receptor. VP35 is a cofactor in the RNA polymerase transcription complex, and helps the virus evade the immune response by blocking activation of the interferon regulatory factor 3, which is required for the induction of interferons alpha and beta. Thus, blocking VP35 should allow for an enhancement of the host immune response to the Ebola virus. It is proposed that similar compounds may be acting via a closely related mechanism, though there has been no experimental evidence to directly prove this yet.\n\nAnother recent study16 has highlighted the ability of three clinically approved ion channel blockers to inhibit the Ebola virus cellular entry. The drugs amiodarone, dronedarone, and verapamil, were given at concentrations that are possible in human serum, and were effective against a number of filoviruses. The authors hypothesized that these drugs may act by disrupting late endosomal processing or by disrupting calcium signaling that is required for viral entry.\n\nOf course, none of these aforementioned FDA approved drugs were designed to target the Ebola virus. Amodiaquine and chloroquine are antimalarials, clomiphene and toremifene are selective estrogen receptor modulators. Amiodarone, dronedarone, and verapamil are anti-arrhythmics. Interestingly, all of these compounds have a common tertiary amine feature, which may suggest they could act through similar mechanism18,19. However, they are all orally bioavailable and generally safe for humans. Thus these repurposed drugs may represent a fast track to potential evaluation and approval as a feasible option for preventing the spread and mortality associated with the Ebola virus in a large population.\n\nSeveral small molecules have actually been tested in very small numbers of humans for activity against the Ebola virus. For example there has been some press on favipiravir, which is undergoing phase 3 clinical trials in the US for influenza and is approved in Japan, as it has shown promising efficacy against the Ebola virus in mice20. Faviparavir is thought to act by inhibiting the viral RNA-dependent RNA polymerase selectively and has demonstrated activity against a number of other viruses. At least one Ebola patient, who has since recovered, was given favipiravir21, and Japan offered to supply it to the World Health Organization.\n\nA second experimental drug, brincidofovir22, in phase 3 clinical trials for treatment of cytomegalovirus and other DNA viruses has shown efficacy against the Ebola virus in vitro and animal studies are ongoing23. Brincidofovir is thought to mimic cytidine, a building block of DNA, and thereby inhibit viral DNA polymerases, and its mechanism of action against the Ebola virus, an RNA virus, is yet unknown. Brincidofovir, which has demonstrated safety in humans, has been given to at least two Ebola virus patients, one in Dallas and one in Nebraska. While unfortunately the Dallas patient died, the Nebraska patient survived24. It is of course too early to know the effect of this molecule on the progression of the disease. This compound is a pro-drug that is converted into the active antiviral, cidofovir diphosphate. Brincidofovir has higher oral bioavailability, intracellular concentrations of drug and increased antiviral potency22. This compound only appeared in the literature in 2014 and there is very little published information.\n\nBeyond these early stage drugs, there are a number of other compounds that have been identified as active against the Ebola virus as summarized by Erik De Clercq9. While many are not ready for in human use, they may present an attractive starting point to be refined in a future drug discovery effort. For example, a novel nucleoside analog, BCX4430 demonstrated efficacy in mice and non-human primates against the Ebola virus25. This compound targets viral RNA polymerase activity by inducing early termination of transcription and thus blocking replication. BCX4430 is not only active against Ebola virus, but also targets other members of the Filovirus family as well as 8 other RNA virus families. Because of the potent and efficacious effects, BCX4430 is being fast-tracked for clinical trials in humans.\n\nWe have recently described an expert’s medicinal chemistry26 analysis of the over 320 NIH probe compounds using public and commercial sources of chemical structures and the issues related to doing this type of analysis27. The likely chemistry quality of these probes was scored based on a number of criteria including literature related to the probe and potential chemical reactivity. Through a series of machine learning models, we also computationally predicted the scores which were being identified through a painstaking manual process26. External validation and comparison with other measures of drug-likeness and filtering rules suggested a comparable level of accuracy26.\n\nWe have now carefully analyzed in a similar manner the 55 small molecules with activity against the Ebola virus identified from our literature search. The chemist’s (C.A.L.) decisions on compound quality are summarized in Supplemental Table 1. In contrast to the previous work in scoring compounds as chemical probes, the current aim is very specific - to treat a very serious viral disease for which there is a phenotypic readout. All the known drugs in clinical use were rejected as the chemist looked for compounds that looked more interesting in his opinion. Only 16 out of 55 were selected as desirable in this analysis with no potential problems based on medicinal chemistry experience. In addition to this manual approach, we applied computational filters such as Pan Assay INterference compoundS (PAINS) to identify potentially problematic compounds from structures28,29 (Supplemental Table 1). PAINS analysis was enabled using the MMDS mobile app30. Based on this approach we have identified several molecules that appear problematic and agreed with the medicinal chemistry analysis. For example 4 molecules appear to fail the PAINS filters, including the rhodanine compound LJ-001 which was found to be active against numerous enveloped viruses and was found through a screen of inhibitors of Nipah virus entry (IC50 1μM)31. In vivo a steady state plasma concentration could not be maintained at a therapeutic level. Rhodanines are known to be problematic PAINS compounds28,29. Interestingly amodiaquine was not scored favorably by PAINS or the medicinal chemist, yet this is a successful antimalarial drug.\n\nIn addition we analyzed the simple chemical properties (calculated in the Collaborative Drug Discovery (CDD) Vault (Collaborative Drug Discovery, Inc) using the Chemaxon toolkit (Chemaxon, Budapest, Hungary)) of the molecules and compared these to their medicinal chemistry classification (Table 1). The mean calculated molecular property values for compounds were compared using the t-test and ANOVA with JMP v. 8.0.1 (SAS Institute, Cary, NC). Significant differences were noted in LogP and Lipinski Rule of 5 violations between desirable and undesirable compounds although one molecule SARA-133 skewed the data with a molecular weight over 3000 (Table 1). Removal of this compound leads to significant differences for molecular weight, number of hydrogen bond donors, heavy atom count and polar surface area, in addition to logP and Lipinski Rule of 5 violations for desirable compounds versus undesirable compounds.\n\n* statistically significant p < 0.05 using the t-test and ANOVA. ** statistically significant p < 0.0001 using the t-test and ANOVA. Note data are skewed by SARA-133. When this molecule is removed the mean values and significance data are shown in parentheses.\n\nThe research described for small molecule inhibitors against the Ebola virus has occurred in a disconnected manner and there have been few efforts to summarize the total medicinal chemistry efforts to date. We think this research can learn from other areas in which there are currently efforts to improve collaboration and screening. In the area of Tuberculosis research, funding from the Bill and Melinda Gates Foundation and The European Commission have enabled the TB Drug Accelerator and the More Medicines For Tuberculosis, as large-scale collaborations between academia, research institutes and industry. These collaborations have promoted the selective sharing of related data in a secure environment between collaborators using the CDD Vault32 (Figure 1). The centralized availability of publicly available data on compounds screened for activity against Mycobacterium tuberculosis enables researchers to leverage the existing literature alongside their private data. This knowledge can be used for building of validated computational models that can help in selecting additional compounds or lead optimization33,34, saving time and effort. By organizing the data on small molecules tested against the Ebola virus similarly in a central database and using machine learning models based on public data may help identify additional compounds for testing. Such an effort may also prevent duplication of efforts as we have seen with the screening of multiple libraries of FDA approved compounds against the Ebola Virus14–16. In order to catalyze this we have made the 55 compounds (Supplemental Table 1) freely available as a dataset in CDD Public (https://app.collaborativedrug.com/register). The compound representation in CDD Vault can be accessed via the text compound descriptors (as in the Supplemental Table 1) or in a batch connection table format as an MDL format structure data file (*.sdf) that is universally read by all chemistry aware software. Being able to easily retrieve the chemical structures in machine retrievable form has considerable value. Identifying compounds by name or company code number does not per se allow direct access to chemical structure and often fails entirely to link compound identifier with the chemistry structure of the compound. InChIKey has the great advantage of being searchable on the web (e.g. via Google) but requires a lookup table (e.g. EMBL’s Unichem) to get back to the chemical structure. SMILES and InChi representations allow direct access to chemical structure and are compatible with structure searching in most public chemistry databases as well as the proprietary chemistry ACS CAS SciFinder database. The IUPAC name is universally used in naming chemical compounds in patents and software exists for going from IUPAC name to chemical structure.\n\nThis illustrates how a comprehensive database could be created and used for collaborations. 55 molecules with predicted properties and scores from an experienced medicinal chemist and filters and shown alongside the publication link and target information.\n\n\nConclusions\n\nThe compounds which are FDA approved drugs for other diseases14–16 but with activity against Ebola virus in vitro or in vivo may represent useful starting points with the advantage that much is known regarding their ADME and Tox properties. However they may not be ideal in the opinion of a medicinal chemist. By bringing together all 55 compounds described in the literature with activity against the Ebola virus, this body of evidence may be more convincing than each study in isolation. In addition it allows us to consider structural features and molecular properties, which may provide insights into the target or mechanism of action. It is unclear whether any of the 55 compounds might also have activity when used as combination therapy as is the current standard of care for HIV, in order to overcome drug resistance, and this needs experimental evaluation. Clearly, we are a considerable distance from having an FDA approved drug for the Ebola virus, but this analysis illustrates that there are already drugs on the shelf that can be potentially repurposed in a shorter time period and at a lower cost and yet they do not appear to have been tested in patients with the Ebola virus. In addition there are at least 16 molecules which in the opinion of an experienced medicinal chemist are possibilities for further optimization. While novel compounds are likely more commercially viable they also will require considerable effort to assess safety. Resources will need to be allocated to this effort and administrators and scientists should perhaps consider some of the medicinal chemistry insights we have provided as well as using a collaborative database to share molecules that are active amongst all scientists. It is hoped these efforts could inspire further drug discovery efforts around small molecules.\n\nTo illustrate the level of interest in repurposing efforts for the Ebola virus, the following studies were identified upon submission that describe additional compounds as well as those already described herein35–37.\n\n\nSupplementary material\n\nSupplemental Table 1. 55 compounds identified from the literature as in vitro or in vivo active against the Ebola virus. Molecule structures in various 2D formats are provided along with simple molecular descriptors, information relating to the target if known, indication, publication reference as well as medicinal chemistry insights and PAINS filter failures.\n\nhttp://dx.doi.org/10.5256/f1000research.6120.s42952", "appendix": "Author contributions\n\n\n\nAll authors contributed to the collaborative writing of this project.\n\n\nCompeting interests\n\n\n\nN.K.L. works for Collaborative Drug Discovery Inc. S.E. works for Collaborations in Chemistry, and S.E. and C.A.L. consults for Collaborative Drug Discovery Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nSE acknowledges several discussions with Dr. Megan Coffee, Dr. Joel S. Freundlich, Dr. Nancy Connell and Dr. Peter Madrid.\n\n\nReferences\n\nMartinez JP, Sasse F, Brönstrup M, et al.: Antiviral drug discovery: broad-spectrum drugs from nature. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeljkovic V, Loiseau PM, Figadere B, et al.: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection. F1000Res. 2015; 4: 34. Publisher Full Text\n\nKouznetsova J, Sun W, Martínez-Romero C, et al.: Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerging Microbes Infect. 2014; 3: e84. Publisher Full Text\n\nLong J, Wright E, Molesti E, et al.: Antiviral therapies against Ebola and other emerging viral diseases using existing medicines that block virus entry. F1000Res. 2015; 4: 30. Reference Source" }	[ { "id": "7611", "date": "11 Feb 2015", "name": "John A. Lowe III", "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\nThe authors have searched a collection of compounds for a common pharmacophore directed against Ebola proteins, and detected 55 hits. These were filtered by an experienced medicinal chemist using well-established techniques and intuition to provide 16 legitimate compounds that may serve the Ebola research community as a publicly available resource. This is an excellent example of the power of shared, collaborative research databases providing valuable resources to the research community.", "responses": [ { "c_id": "1249", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "Thank you for your review and comments!" } ] }, { "id": "7713", "date": "19 Feb 2015", "name": "Paul S Anderson", "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\nThe authors have collected 55 small molecules reported to have activity against the ebola virus from the literature and organized them into a dataset that is easily searchable for those who may be interested in pursuing further research on the design of inhibitors of this virus.", "responses": [ { "c_id": "1250", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "Thank you for your positive review comments." } ] } ]	1	https://f1000research.com/articles/4-38
https://f1000research.com/articles/4-37/v1	04 Feb 15	{ "type": "Review", "title": "Emerging strategies to prevent heart failure after myocardial infarction", "authors": [ "Thomas R. Cimato" ], "abstract": "Congestive heart failure (CHF) remains a significant cause of death and disability in industrialized countries. Projections show that the prevalence of CHF will increase 46% from 2012 to 2030, resulting in over eight million adults with CHF in the United States. While substantial advances have been achieved in the treatment of CHF over the past two decades, CHF rivals cancer as a cause of mortality. Strategies focused on prevention of CHF should be emphasized to meaningfully impact the projected increase in CHF. Irrespective of the type of CHF, either systolic or diastolic, coronary artery disease has supplanted hypertension as the most prevalent cause for congestive heart failure, with a high rate of mortality and future hospitalizations. Since coronary artery disease plays a central role in the development of CHF, approaches to treat coronary artery disease and identification of patients at risk for recurrent myocardial infarction (RMI) are approaches to prevent development of CHF.\n\nSubjects who sustain recurrent MI represent a particularly high-risk group for development of CHF. Despite the evolution of therapy for MI from thrombolytic therapy to primary percutaneous coronary intervention (PCI), RMI occurs in ~ 10% of patients in the first year after first MI, and 3 years after their first MI. In this review I explore emerging approaches to prevent RMI including the rationale for recent trials of complete revascularization at the time of MI, newly emerging biomarkers that have additive predictive value for identifying patients with high risk of CHF and death when using existing biomarkers. Finally, the paradigm of hematopoietic stem cell mobilization in MI leading to monocyte expansion and acceleration of atherosclerosis is discussed as an emerging approach to identify patients at high risk of RMI, CHF, and death after MI.", "keywords": [ "Coronary", "artery", "disease", "congestive", "heart", "failure", "atherosclerosis", "myocardial", "infarction", "biomarkers", "hematopoietic", "stem", "cells" ], "content": "Introduction\n\nCongestive heart failure (CHF) remains a significant cause of death and disability in industrialized countries1. It is estimated that 5.1 million Americans over the age of 20 have CHF2. Projections show that the prevalence of CHF will increase 46% from 2012 to 2030, resulting in over eight million adults in the United States with CHF2. While substantial advances have been achieved in the treatment of CHF over the past two decades, CHF mortality remains as high as cancer mortality3,4. The number of deaths attributable at least in part to CHF was as high in 1995 (287,000 deaths) and in 2010 (279,000 deaths) indicating that while therapies to treat heart failure have improved individual survival, the prevalence of CHF continues to rise2. Strategies focused on prevention of CHF should be emphasized to meaningfully impact the projected increase in CHF. Irrespective of the type of CHF5, either systolic6 or diastolic7–9, coronary artery disease has supplanted hypertension as the most prevalent cause for congestive heart failure, with a high rate of mortality and future hospitalizations. For these reasons, prevention of coronary artery disease events represents an important approach for prevention of CHF in subjects with both reduced and preserved systolic function. While dramatic improvements in treatment for acute coronary syndromes (ACS) has improved survival, studies of new agents to treat ACS should also include CHF as an outcome, as these data are limited relative to standard major adverse cardiac events (MACE) end-points. CHF end-points in clinical trials are more challenging to achieve, as they require prolonged follow-up periods, but would go a long way to begin to understand their impact in prevention of CHF. Since coronary artery disease plays a central role in the development of CHF, approaches under investigation to prevent CHF after MI by reducing recurrent ACS events are discussed below.\n\nA first myocardial infarction (MI) often represents the initial entry to the health care system, and represents an important opportunity for intervention to prevent subsequent cardiovascular disease events and progression to CHF. Patients who suffer MI are at risk for death and illness from several causes including CHF, arrhythmias, sudden death, and recurrent myocardial infarction (RMI). With modern revascularization therapies for MI, often the majority of patients receive prompt revascularization of the infarct related artery, recover to near normal systolic function, and often have minimal symptoms of CHF consistent with what is defined as the American College of Cardiology/American Heart Association (AHA/ACC) Stage B CHF10. The development of CHF after MI depends on multiple factors. These include the size of the infarct, its location, and if it results in papillary muscle dysfunction causing mitral regurgitation11. Quantitatively, few studies are available reporting the incidence of heart failure after first MI. In Olmstead County Minnesota, 41% of subjects who suffered a prior MI but did not have heart failure at the time of MI developed new onset heart failure 6 years after MI12. Objective criteria to identify subjects likely to develop CHF 90 days after MI include decreased left ventricular (LV) systolic function, ejection fraction (EF) < 30% after percutaneous revascularization, presentation with Kilip class > I, and Q waves on post-revascularization electrocardiogram (ECG)13. Evidence based therapies to improve LV systolic function and prolong life are noted in the ACC/AHA Guideline for the Management of Heart Failure14. To avoid redundancy and re-iteration of these widely utilized therapies for post-MI heart failure I will briefly summarize the currently accepted medical therapies. Subjects with ST-segment elevation MI treated with primary angioplasty of the infarct-related artery (IRA) with normal ejection fraction should be treated with aspirin 81 mg daily, high intensity statin therapy, and a beta-blocker for 1-year post-MI15. A P2Y12 inhibitor should be used for a minimum of 1 year. ACE inhibitors are added if there is post-MI LV systolic dysfunction, and an angiotensin receptor blocker may be exchanged if the ACE inhibitor is not tolerated. Aldosterone antagonists (spironolactone or eplerinone) are indicated with post-MI LV systolic dysfunction14. The sections that follow outline the emerging approaches to prevent progression of atherosclerosis, RMI, and heart failure from MI.\n\nSubjects who sustain RMI represent a particularly high-risk group for development of CHF. RMI is particularly worrisome as it is associated with a high mortality rate, and high likelihood of CHF in those who survive. Despite the evolution of therapy for MI from thrombolytic therapy to primary percutaneous primary angioplasty (PCI), RMI occurs in ~10% of patients in the first year after first MI, and 3 years after their first MI16. Of those who sustain a RMI, 40% die within 1 year16,17. Age, diabetes mellitus, unstable angina17–19, congestive heart failure on admission with MI20 and underlying left ventricular systolic dysfunction21 are the strongest clinical predictors of RMI. Amongst medical therapies, ongoing treatment with NSAIDs, and in particular rofecoxib, celecoxib and diclofenac are associated with death and RMI22. Given the high risk of death associated with RMI, and the role of RMI in development of CHF, strategies to prevent its occurrence are an important focus. However, what is remarkably clear throughout the evolution of therapy for MI, patients with CHF and/or left ventricular systolic dysfunction, are at high risk for RMI. Below I will discuss the emerging strategies to prevent RMI with an underlying goal of preventing CHF.\n\nPCI is the preferred treatment strategy for restoring myocardial perfusion in patients with ST-segment elevation MI23. Guidelines from both the European Society of Cardiology (ESC) and the ACC/AHA discourage PCI of non-infarct- non-IRA at the time of primary or rescue PCI in stable patients with ST-segment elevation MI (class III recommendation ACC/AHA)23. This recommendation is based on observation studies and clinical trials with limited power to answer the question of whether there is any benefit in complete revascularization of all coronary lesions > 70% at the time of ST-segment elevation MI. However, multi-vessel coronary artery disease occurs in 40–65% of subjects undergoing PCI for ST-segment elevation MI24–27. While reperfusion of the IRA is routinely performed, the presence of critical stenosis in the other coronary vessels can result in ischemia in the non-infarct related territory. Additionally, ST-segment elevation MI is associated with an inflammatory surge that results in plaque rupture in other coronary vessels resulting in RMI28. Rupture of an unstable coronary-artery plaque that appears to be a single lesion on angiography is commonly deemed the culprit lesion in ST-segment elevation MI. However, patients often harbor multiple complex coronary plaques29. ST-segment elevation MI may represent a pan-coronary process of thin-cap fibroatheromas in non-IRAs30. Plaques in the non-IRA can be responsible for RMI. Therefore, treatment of these other coronary lesions by complete revascularization represents a potentially important approach to prevent progressive myocardial ischemia, RMI, and future CHF.\n\nThe concept of complete revascularization in patients with ST-segment elevation MI has been tested in several clinical trials recently, both completed and ongoing. The PRAMI Trial (Controlled Trial number ISRCTN73028481) tested whether performing preventative PCI as part of the procedure to treat the IRA would reduce the combined incidence of death from cardiac causes, non-fatal myocardial infarction, or refractory angina31. The PRAMI trial found that the complete revascularization approach reduced death from cardiac causes (hazard ratio versus culprit artery PCI, 0.34), non-fatal myocardial infarction (hazard ration 0.32), and refractory angina (hazard ratio 0.35) over the standard approach of IRA only PCI31. Importantly, the design of PRAMI did not allow for staged PCI of the non-IRA coronary lesions. Heart failure after MI was not an end-point reported by the study. PRAMI importantly did show that complete revascularization at the time of ST-segment elevation MI was feasible and safe. Criticisms of PRAMI were raised regarding the relatively small sample size of the study, raising questions regarding generalizability.\n\nSince the presentation and publication of the PRAMI trial, several other studies have emerged in support of the complete revascularization approach in treatment of ST-segment elevation MI. A similar trial called CVLPRIT (Complete Versus Culprit-Lesion only Primary PCI Trial; ISRCTN2166248) is a randomized prospective trial of 300 subjects32. The study tested if complete revascularization using a staged PCI approach for non-IRA coronary lesions within 45 days after MI versus the IRA only approach reduces all-cause mortality, RMI, heart failure, and need for revascularization on year after enrollment. Secondary end-points included safety end-points including stroke, intracranial hemorrhage, major non-intracranial bleeding, and vascular complications32. The trial results were presented in September 2014 at the European Society of Cardiology meeting and showed a significant reduction in major adverse cardiac events (hazard ratio 0.45, p < 0.009). All-cause mortality, RMI and repeat revascularization also showed similar reductions with the staged PCI approach versus infarct artery only approach, but did not reach statistical significance. There was a substantial reduction in heart failure with the staged PCI approach as well (hazard ratio 0.43, p = 0.14) but did not reach statistical significance likely due to the small sample patient cohort in the study33. While the results of the study are encouraging regarding the use of staged PCI for complete revascularization following ST-segment elevation MI, the small study cohort makes the generalizability of the findings questionable.\n\nA recent meta-analysis comparing the benefits and risks of routine IRA-only PCI vs. multi-vessel PCI in ST-segment elevation MI revealed that multi-vessel PCI during the intervention for ST-segment elevation MI resulted in increased mortality (odds ratio 1.35; p < 0.001), but when performed as a staged procedure, hospital mortality was lower (odds ratio 0.35, p < 0.001), as well as reduced long-term mortality, and need for repeat PCI with staged, multi-vessel PCI after MI34. The findings support the current view of the ACC/AHA guidelines indicating non-IRA intervention at the time of primary angioplasty for ST-segment elevation MI may be harmful, but also suggest that treatment of severe coronary artery disease without functional testing or assessment of recurrent angina symptoms with staged PCI of the non-IRA lesions may be beneficial. No heart failure related end-points were assessed in the study however.\n\nThe COMPLETE Trial (Complete vs Culprit-only Revascularization to Treat Multi-vessel Disease after Primary PCI for STEMI; NCT01740479) is designed to answer the question of complete versus IRA-only revascularization after PCI for ST-segment elevation MI35. The study will recruit 3900 subjects to determine whether opening all suitable narrowings or blockages (> 70% stenosis or > 50% stenosis with fractional flow reserve < 0.8) using a staged PCI approach with drug eluting stents of all suitable non-IRA lesions is superior to an IRA-only approach in reducing the composite outcome of death due to cardiovascular causes or RMI. The secondary end-point will be death due to cardiovascular causes, RMI, ischemia-driven revascularization, unstable angina, or heart failure over a 4-year follow-up period.\n\nCompletion of this study and others will help determine the efficacy of complete revascularization immediately after MI improves survival. However, the completed and proposed studies of complete revascularization after MI likely do not have an adequate follow-up period to determine if complete revascularization has any impact on heart failure. It would be most useful for a longer follow-up period to be considered in these trials to assess whether the frequency of heart failure is impacted by complete revascularization. Further insights may be gained if more precise heart failure classification is also determined, specifically treatment or admission to hospital for heart failure in subjects with both preserved and reduced systolic function, as both are likely heart failure specific end-points in subjects with prior MI. Findings from such a longitudinal study would potentially have significant impact in the management of patients with MI, and prevent progression to heart failure with either preserved or reduced systolic function over the longer term.\n\nComplete revascularization of critical coronary disease may prevent RMI and CHF. However these studies do not address coronary disease that would not normally be treated, lesions with < 70% occlusive stenosis of the coronary artery. These non-critical lesions can progress, leading to plaque rupture and RMI. Multiple angiographic studies have documented progression of coronary atherosclerosis after an initial acute coronary syndrome36–39. Importantly, both IRA and non-IRA lesions appear to progress over time. In a very insightful angiographic and intravascular ultrasound based study, the PROSPECT Investigators prospectively evaluated coronary artery disease lesions in 697 patients with acute coronary syndromes (ACS-Unstable angina, Non-ST segment elevation MI, and ST-segment elevation MI). Subjects were followed for 3–4 years for recurrent coronary disease events including death from cardiac causes, cardiac arrest, myocardial infarction, or unstable angina40. Adverse coronary disease events occurred in 20.4% of patients. Remarkably, these subjects had a relatively equal number of culprit lesions in the prior IRA as in the non-IRA. In the non-IRA lesions that caused the coronary event later, the lesions were described as mild, with a mean stenosis of 32.3 ± 20.6% by conventional angiography on the initial study. However by intravascular ultrasound the same lesions that caused a new event had > 70% plaque burden, a small luminal area, and were characterized as thin-cap fibroatheromas. This study suggests that after an initial acute coronary syndrome event, significant progression of atherosclerosis occurs in 20% of subjects from angiographically mild appearing lesions, and is as likely to occur in the prior IRA as in non-IRA lesions that would not have been treated during the index catheterization.\n\nIn the context of prevention of heart failure and death from recurrent coronary disease events, identification of the small subset of patients who will have recurrent events from recurrent disease in the IRA or non-IRA vessels is critical. In a related study, the PROSPECT investigators evaluated the utility of C-reactive protein (CRP) levels to predict which subjects had a recurrent coronary disease event41. Subjects with CRP levels that were elevated (3–10 mg/L) or very elevated (> 10 mg/L) 6 months after their index ACS event had greater rates of total and non-IRA coronary events after 3 years than the subjects with normal CRP levels (< 3 mg/L). Elevated CRP levels 6 months after ACS weakly predicted future coronary disease events with a hazard ratio of 1.02. While CRP is a widely utilized measure of inflammation, its use is not widely accepted. Often the results, if elevated, are attributed to non-specific sources such as a wound infection rather than as an index of inflammation that may be tied to coronary artery disease. However, this study highlights the importance of ongoing inflammation in the progression of coronary artery disease, and the principle of using a measure of inflammation to potentially identify subjects at risk for RMI. More specific markers of inflammation that are mechanistically linked to progression of atherosclerosis may be useful in identifying patients at high risk for RMI and CHF.\n\nPrediction of CHF and death after MI can be based on standard clinical parameters including using the TIMI risk score42 or the GRACE score43. However, substantial additional information can be obtained through the use of biomarkers. The value of troponin I, CRP, and brain-derived natriuretic peptide (BNP), in post-ACS risk stratification are well documented44. More recently, characterized biomarkers that are predictive of heart failure and death post-MI include growth differentiation factor-15 (GDF-15). GDF-15 is a member of the transforming growth factor-β cytokine superfamily, is induced and secreted from cardiac myocytes during ischemia and stress, and has anti-apoptotic actions45. The value of GDF-15 in prediction of death or CHF was tested in a prospective study of 1142 subjects with both non-ST segment elevation and ST-segment elevation MI with a follow-up period of 505 days46. GDF-15 levels were roughly equivalent to N-terminal BNP for prediction of death or CHF after 1 year. However, combining GDF-15 and N-terminal BNP improved the predictive power of either marker alone yielding an AUC C-statistic of 0.8146. The value of GDF-15 levels in subjects with ACS was further independently tested by the TIMI group using the PROVE IT-TIMI 22 cohort47. They found that GDF-15 levels predicted risk of death, MI, and CHF independent of cardiovascular risk factors, BNP, or CRP levels. Interestingly there was also no significant interaction between GDF-15 levels and intensive statin therapy for risk of death or MI, suggesting statins have minimal effects on GDF-15 levels47.\n\nAnother recently characterized biomarker of death and CHF after MI is C-terminal provasopressin (copeptin), the C-terminal portion of provasopressin. Arginine vasopressin (antidiuretic hormone) is released from the neurohypophysis to promote water resorption. Copeptin is secreted in equimolar quantities with arginine vasopressin, but is stable in the blood stream, while arginine vasopressin is unstable and rapidly cleared48. In the Leicester Acute Myocardial Infarction Peptide (LAMP) Study, the predictive value of copeptin levels to predict death and heart failure in 980 subjects with non-ST and ST-segment elevation MI, and followed for events for 342 days. Copeptin levels were roughly equivalent to N-terminal BNP levels, and prediction of death and CHF were improved when copeptin and N-terminal BNP were combined in the model48. The value of copeptin to predict death, RMI, stroke, or resuscitated cardiac arrest was independently tested in the OPTIMAAL cohort in subjects with MI and either clinical CHF or reduced LV systolic function (EF < 35%)49. In contrast to the LAMP Study, copeptin was a stronger predictor of mortality compared with either BNP or N-terminal BNP. Collectively, the findings from these two studies highlight additional biomarkers that may be used additively with BNP to identify subjects at risk for death, and for CHF after MI.\n\nThe biological mechanisms underlying RMI remained unclear until recently. A recent study in mice demonstrated that experimental MI or stroke results in enlargement of atherosclerotic lesions with more advanced morphology over weeks after total occlusion of the left coronary artery50. Mechanistically, myocardial infarction in mice causes stress and pain, resulting in a surge in release of norepinephrine. Nerves within the bone marrow regulate the release catecholamines51, which in turn reduce the levels of SDF-1 (CXCL12). Hematopoietic stem (HSC) and progenitor cells (HSPCs) are then released from the bone marrow. In the setting of myocardial infarction HSC and HSPC release dramatically increases rising 2-fold after 6 hours to over 20-fold higher than baseline 3 days into a myocardial infarction50. Coinciding with the release of HSCs and HSPCs, monocytes, macrophages, and neutrophils expand within atherosclerotic lesions in the aorta, and are replenished from the blood stream and spleen for up to 3 months after MI in mice. HSPC mobilization requires adrenergic signaling at least initially as HSC and HSPC mobilization was reduced in half by a β3 adrenergic receptor inhibitor, indicating an immediate effect of the sympathetic nervous system on HSPC mobilization. Similar findings were noted in a small animal model of chronic stress, resulting in mobilization of HSCs and HSPCs and progression of atherosclerosis52. The findings highlight an important new paradigm to verify in human subjects to begin to understand if activation of the HSC/HSPC/Monocyte axis underlies the mechanism of RMI and acceleration of atherosclerosis.\n\nKey differences underlie animal models of MI and humans that seek treatment for MI. In animal models, the left coronary artery, supplying the largest part of the heart, is tied off resulting in necrosis of the anterior wall of the heart. In contrast, human subjects who seek medical attention for MI undergo coronary angiography and revascularization within 90 minutes of admission to hospital. The size and duration of stimulus to mobilize HSPCs in human subjects may be substantially lower than that observed in mice with a completed infarct. However, it is also possible that patients who present late with ST-segment elevation MI may have the most substantial release of HSCs and HSPCs after MI. Human subjects are frequently treated with β1/β2-adrenergic inhibitors after MI, but those with heart failure may be treated with catecholamines (norepinephrine, epinephrine, dopamine, or dobutamine) for blood pressure support if there is heart failure or cardiogenic shock. This may result in more extensive HSC and HSPC mobilization in subjects with CHF after MI, making them at higher risk for RMI. This is only a speculation at this point but will be the subject of further studies.\n\nDespite these important differences between human subjects and animal models of MI, evidence of acceleration of atherosclerosis is still observed after MI in patients. A recent clinical observational study evaluated 449 patients 1 year after STEMI treated by primary angioplasty and β-blockers. One year after MI, 45 patients developed > 70% stenosis in non-IRA vessels53. Most remarkably, the patients that developed critical stenosis in non-IRA arteries had significantly higher levels of epinephrine, norepinephrine and CRP levels 1 year after their index MI53. In a similar prospective observational study, 449 subjects in China with chronic stable angina, non-ST segment elevation and ST-segment elevation MI underwent follow-up coronary angiography 1 year later54. One hundred Thirty-four of 449 patients, or 29.8% had some evidence of non-IRA progression of atherosclerosis of varying severity. Over the follow-up period, 52.9% of subjects with ST-segment elevation MI developed progression of atherosclerosis in the non-IRA while 19.6% of subjects with chronic stable angina and non-ST segment elevation MI had evidence of progression of atherosclerosis. Blood was collected 7 days after intervention for retrospective correlation after follow-up angiography. Remarkably, subjects who developed progression of atherosclerosis in the non-IRA had significantly higher levels of monocytes in their blood stream 7 days after their initial procedure. Collectively, the findings of these two studies provide a preliminary indication that the milleu of ST-segment elevation MI 1) elevates catecholamine levels, and 2) monocyte levels that correspond to progression of atherosclerosis 1 year later. While the paradigm of increased adrenergic tone associated with MI mobilizing HSC/HSPCs, monocyte expansion in the spleen and bone marrow, and acceleration of atherosclerosis remains to be validated in human subjects, these studies support this concept.\n\nFurther evidence to support this paradigm came from imaging studies using positron emission tomography. Activated inflammatory cells express high levels of glucose transporters and 18F-deoxyglucose is avidly absorbed, facilitating imaging of inflammation55. Several recently published studies56–58 have made use of this technique to show that ST-segment elevation MI but not chronic stable angina results in a dramatic increase in bone marrow, splenic and vascular inflammatory activity. The findings are supportive of the paradigm of HSC, HSPC and monocyte inflammatory activation by the post-MI milleu. However, it remains unclear if 18F-deoxyglucose imaging of inflammation can be used to identify subjects at high risk of RMI, or predict the development of CHF post-MI.\n\nThe paradigm of HSC and HSPC mobilization, monocyte expansion, and acceleration of atherosclerosis in ST-segment elevation MI remains to be verified in human subjects. While HSC and HSPC mobilization and monocyte expansion clearly occurs in animal models of completed, unrevascularized MI, the extent of HSC and HSPC mobilization in human subjects that undergo revascularization and treatment with medical therapy is unclear and requires validation. However, the paradigm and the mediators of HSC and HSPC mobilization and differentiation to monocytes, leading to acceleration of atherosclerosis represents a potential focus to identify patients at risk for RMI, and potentially reduce CHF and death from MI. Therapies directed at HSC and HSPC mobilization and monocyte differentiation may also be a promising approach to reduce acceleration of atherosclerosis after MI, but may also be challenging to administer as these same cell types are also involved in repair of MI.\n\n\nConclusions\n\nMI often leads to CHF, as longitudinal studies show that 40% of subjects with prior MI develop CHF 6 years later.\n\nRMI occurs in ~10% of subjects after an index ST-segment elevation MI and carries a high mortality rate of 40%. RMI raises the likelihood of developing CHF.\n\nComplete revascularization of coronary artery disease at the time of ST-segment elevation MI is an emerging approach to prevent RMI.\n\nAtherosclerosis progresses in 20% of patients after an initial acute coronary syndrome in non-IRA lesions, suggesting improved approaches to identify patients at high risk for progression of coronary disease may prevent RMI and CHF.\n\nIn small animal model experiments, non-revascularized MI results in mobilization of hematopoietic stem and progenitor cells, and expansion of monocytes in the spleen, that contributes to acceleration of atherosclerosis.\n\nPreliminary studies in human subjects with prior MI also have progression of non-IRA lesions, and subjects who show most substantial lesion progression have elevated levels of catecholamines and higher blood monocyte levels.\n\nHematopoietic stem, progenitor cell mobilization, and monocyte expansion in humans with MI have not been studied, but may represent biomarkers for RMI, and potential therapeutic targets to prevent RMI and acceleration of atherosclerosis in non-IRAs.", "appendix": "Author contributions\n\n\n\nTRC prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBraunwald E: Heart Failure. 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PubMed Abstract \| Publisher Full Text\n\nKornowski R, Goldbourt U, Zion M, et al.: Predictors and long-term prognostic significance of recurrent infarction in the year after a first myocardial infarction. SPRINT Study Group. Am J Cardiol. 1993; 72(12): 883–8. PubMed Abstract \| Publisher Full Text\n\nOrn S, Cleland JG, Romo M, et al.: Recurrent infarction causes the most deaths following myocardial infarction with left ventricular dysfunction. Am J Med. 2005; 118(7): 752–758. PubMed Abstract \| Publisher Full Text\n\nLamberts M, Fosbøl EL, Olsen AM, et al.: Ongoing treatment with non-steroidal anti-inflammatory drugs at time of admission is associated with poorer prognosis in patients with first-time acute myocardial infarction. Int J Cardiol. 168(2): 832–7. PubMed Abstract \| Publisher Full Text\n\nO’Gara PT, Kushner FG, Ascheim DD, et al.: 2013 ACCF/AHA guideline for the management of ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2013; 127(4): e362–425. PubMed Abstract \| Publisher Full Text\n\nJaski BE, Cohen JD, Trausch J, et al.: Outcome of urgent percutaneous transluminal coronary angioplasty in acute myocardial infarction: comparison of single-vessel versus multivessel coronary artery disease. Am Heart J. 1992; 124(6): 1427–33. PubMed Abstract \| Publisher Full Text\n\nKahn JK, Rutherford BD, McConahay DR, et al.: Results of primary angioplasty for acute myocardial infarction in patients with multivessel coronary artery disease. J Am Coll Cardiol. 1990; 16(5): 1089–96. PubMed Abstract \| Publisher Full Text\n\nMoreno R, García E, Elízaga J, et al.: [Results of primary angioplasty in patients with multivessel disease]. Rev Esp Cardiol. 1998; 51(7): 547–55. PubMed Abstract\n\nMuller DW, Topol EJ, Ellis SG, et al.: Multivessel coronary artery disease: a key predictor of short-term prognosis after reperfusion therapy for acute myocardial infarction. Thombosis and Angioplasty in Myocardial Infarction (TAMI) Study Group. Am Heart J. 1991; 121(4 Pt 1): 1042–9. PubMed Abstract \| Publisher Full Text\n\nTanaka A, Shimada K, Sano T, et al.: Multiple plaque rupture and C-reactive protein in acute myocardial infarction. J Am Coll Cardiol. 2005; 45(10): 1594–9. PubMed Abstract \| Publisher Full Text\n\nGoldstein JA, Demetriou D, Grines CL, et al.: Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med. 2000; 343(13): 915–922. PubMed Abstract \| Publisher Full Text\n\nKubo T, Imanishi T, Kashiwagi M, et al.: Multiple coronary lesion instability in patients with acute myocardial infarction as determined by optical coherence tomography. Am J Cardiol. 2010; 105(3): 318–22. PubMed Abstract \| Publisher Full Text\n\nWald DS, Morris JK, Wald NJ, et al.: Randomized trial of preventive angioplasty in myocardial infarction. N Engl J Med. 2013; 369(12): 1115–23. PubMed Abstract \| Publisher Full Text\n\nKelly DJ, McCann GP, Blackman D, et al.: Complete Versus culprit-Lesion only Primary PCI Trial (CVLPRIT): a multicenter trial testing management strategies when multivessel disease is detected at the time of primary PCI: rationale and design. EuroIntervention. 2013; 8(10): 1190–8. PubMed Abstract \| Publisher Full Text\n\nEuropean Society of Cardiology 2014 Congress. Press release. Reference Source\n\nBainey KR, Mehta SR, Lai T, et al.: Complete vs culprit-only revascularization for patients with multivessel disease undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: A systematic review and meta-analysis. Am Heart J. 2014; 167(1): 1–14.e2. PubMed Abstract \| Publisher Full Text\n\nComplete vs Culprit-only Revascularization to Treat Multi-vessel Disease After Primary PCI for STEMI (COMPLETE). Reference Source\n\nWaters D, Craven TE, Lespérance J: Prognostic significance of progression of coronary atherosclerosis. Circulation. 1993; 87(4): 1067–75. PubMed Abstract \| Publisher Full Text\n\nKaski JC, Chester MR, Chen L, et al.: Rapid angiographic progression of coronary artery disease in patients with angina pectoris. The role of complex stenosis morphology. Circulation. 1995; 92(8): 2058–65. PubMed Abstract \| Publisher Full Text\n\nAzen SP, Mack WJ, Cashin-Hemphill L, et al.: Progression of coronary artery disease predicts clinical coronary events. Long-term follow-up from the Cholesterol Lowering Atherosclerosis Study. Circulation. 1996; 93(1): 34–41. PubMed Abstract \| Publisher Full Text\n\nKaski JC, Chen L, Chester M: Rapid angiographic progression of “Target” and “Nontarget” stenoses in patients awaiting coronary angioplasty. J Am Coll Cardiol. 1995; 26(2): 416–21. PubMed Abstract \| Publisher Full Text\n\nStone GW, Maehara A, Lansky AJ, et al.: A prospective natural-history study of coronary atherosclerosis. N Engl J Med. 2011; 364(3): 226–35. PubMed Abstract \| Publisher Full Text\n\nKelly CR, Weisz G, Maehara A, et al.: Relation of C-reactive protein levels to instability of untreated vulnerable coronary plaques (from the PROSPECT Study). Am J Cardiol. 2014; 114(3): 376–83. PubMed Abstract \| Publisher Full Text\n\nMorrow DA, Antman EM, Charlesworth A, et al.: TIMI risk score for ST-elevation myocardial infarction: A convenient, bedside, clinical score for risk assessment at presentation: An intravenous nPA for treatment of infracting myocardium early II trial substudy. Circulation. 2000; 102(17): 2031–7. PubMed Abstract \| Publisher Full Text\n\nde Araújo Gonçalves P, Ferreira J, Aguiar C, et al.: TIMI, PURSUIT, and GRACE risk scores: sustained prognostic value and interaction with revascularization in NSTE-ACS. Eur Heart J. 2005; 26(9): 865–72. PubMed Abstract \| Publisher Full Text\n\nSabatine MS, Morrow DA, de Lemos JA, et al.: Multimarker approach to risk stratification in non-ST elevation acute coronary syndromes: simultaneous assessment of troponin I, C-reactive protein, and B-type natriuretic peptide. Circulation. 2002; 105(15): 1760–3. PubMed Abstract \| Publisher Full Text\n\nKhan SQ, Ng K, Dhillon O, et al.: Growth differentiation factor-15 as a prognostic marker in patients with acute myocardial infarction. Eur Heart J. 2009; 30(9): 1057–65. PubMed Abstract \| Publisher Full Text\n\nKempf T, Eden M, Strelau J, et al.: The transforming growth factor-beta superfamily member growth-differentiation factor-15 protects the heart from ischemia/reperfusion injury. Circ Res. 2006; 98(3): 351–60. PubMed Abstract \| Publisher Full Text\n\nBonaca MP, Morrow DA, Braunwald E, et al.: Growth differentiation factor-15 and risk of recurrent events in patients stabilized after acute coronary syndrome: observations from PROVE IT-TIMI 22. Arterioscler Thromb Vasc Biol. 2011; 31(1): 203–10. PubMed Abstract \| Publisher Full Text\n\nKhan SQ, Dhillon OS, O’Brien RJ, et al.: C-terminal provasopressin (copeptin) as a novel prognostic marker in acute myocardial infarction: Leicester Acute Myocardial Infarction Peptide (LAMP) study. Circulation. 2007; 115(16): 2103–10. PubMed Abstract \| Publisher Full Text\n\nVoors AA, von Haehling S, Anker SD, et al.: C-terminal provasopressin (copeptin) is a strong prognostic marker in patients with heart failure after an acute myocardial infarction: results from the OPTIMAAL study. Eur Heart J. 2009; 30(10): 187–94. PubMed Abstract \| Publisher Full Text\n\nDutta P, Courties G, Wei Y, et al.: Myocardial infarction accelerates atherosclerosis. Nature. 2012; 487(7407): 325–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatayama Y, Battista M, Kao WM, et al.: Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell. 2006; 124(2): 407–21. PubMed Abstract \| Publisher Full Text\n\nHeidt T, Sager HB, Courties G, et al.: Chronic variable stress activates hematopoietic stem cells. Nat Med. 2014; 20(7): 754–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Liu JH, Zhu XL, et al.: Nonculprit Lesion Progression in Patients with ST Elevation Myocardial Infarction After Primary Percutaneous Coronary Intervention. Int Heart J. 2014; 55(1): 48–52. PubMed Abstract \| Publisher Full Text\n\nHan Y, Jing J, Tu S, et al.: ST elevation acute myocardial infarction accelerates non-culprit coronary lesion atherosclerosis. Int J Cardiovasc Imaging. 2014; 30(2): 253–61. PubMed Abstract \| Publisher Full Text\n\nMulder WJM, Jaffer FA, Fayad ZA, et al.: Imaging and nanomedicine in inflammatory atherosclerosis. Sci Transl Med. 2014; 6(239): 239sr1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim EJ, Kim S, Kang DO, et al.: Metabolic activity of the spleen and bone marrow in patients with acute myocardial infarction evaluated by 18F-fluorodeoxyglucose positron emission tomographic imaging. Circ Cardiovasc Imaging. 2014; 7(3): 454–60. PubMed Abstract \| Publisher Full Text\n\nWollenweber T, Roentgen P, Schaefer A, et al.: Characterizing the inflammatory tissue response to acute myocardial infarction by clinical multimodality noninvasive imaging. Circ Cardiovasc Imaging. 2014; 7(5): 811–8. PubMed Abstract \| Publisher Full Text\n\nEmami H, Singh P, MacNabb M, et al.: Splenic Metabolic Activity Predicts Risk of Future Cardiovascular Events: Demonstration of a Cardiosplenic Axis in Humans. JACC Cardiovasc Imaging. 2014. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12448", "date": "01 Mar 2016", "name": "Arvind Bhimaraj", "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\nThomas Cimato addresses a key article in identifying the continuum of myocardial infarction leading to heart failure. The odds of a risk factor leading to heart failure is still the highest for myocardial infarction and this is hence a very relevant topic. There are some really minor changes:The statement in introduction “,coronary artery disease has supplanted hypertension as the most prevalent cause for congestive heart failure, with a high rate of mortality and future hospitalization” need an appropriate reference for fact check.To my knowledge based on recent statistics of CHF, CAD and MI have a higher association but by prevalence, HTN is a bigger contributor. There are couple of places where the author states “ I will briefly summarize” (5th line, page 2, left column) and “Below I will discuss the emerging strategies……” (Last line of the paragraph under subheading Recurrent MI and heart failure”. If possible, a reference to self will sound best avoided and a rephrasing to “is summarized” will have a better flow. Otherwise, it’s a well elaborated article reflecting the need for attention in such perfectives post MI.", "responses": [] }, { "id": "12447", "date": "08 Mar 2016", "name": "Steven Hollenberg", "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 2015 ACC/AHA/SCAI Focused Update on Primary Percutaneous Coronary Intervention for Patients With ST-Elevation Myocardial Infarction1 changed the recommendation concerning PCI of a non-culprit artery at the time of primary PCI, removing the Class III: Harm designation and replacing it with a Class IIb recommendation (may be considered). Uncertainty remains regarding timing, since, as noted in the article, in some trials multivessel PCI was performed at the time of the initial procedure, and in other trials it was done in a staged fashion prior to hospital discharge. It should be stressed that this recommendation applies to patients selected by trial entry criteria and was not intended to advocate routine multivessel PCI in all cases. As such, selection of patients at high risk for recurrent MI, as outlined in the article, remains important.As a minor point, the CVLPRIT trial cited as a presentation in the article has been published.2 The article at points seems to imply that studies that report major adverse cardiac events commonly do not include CHF as an outcome; this is not strictly correct in most cases. While the primary endpoint may include only death, myocardial infarction, and stroke in some trials, hospitalization for CHF is almost always recorded as a secondary endpoint. One challenge is that this latter endpoint is somewhat softer than others since it relies on the decision to admit.With respect to the proposed studies of complete revascularization after MI, the contention that studies “likely do not have an adequate follow-up period to determine if complete revascularization has any impact on heart failure” is not quite correct. The ongoing COMPLETE trial of 3900 patients cited in the paper has a composite of CV death and new MI as a primary endpoint and a composite of CV death, MI and CHF hospitalization over a 4-year follow-up period as the secondary endpoint. See clinicaltrials.gov for study design. As noted in the article, identification of patients with residual inflammation after MI may well be important. It may be worth mentioning two ongoing trials of anti-inflammatory therapy in patients with CAD. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) is testing a monoclonal antibody the blocks the inflammatory cytokine interleukin-1 in patients with acute MI and elevated CRP. The Cardiovascular Inflammation Reduction Trial (CIRT) is testing low-dose methotrexate in patients with stable CAD and either diabetes or metabolic syndrome. See clinicaltrials.gov for details. Identification of HSC and HSPC mobilization after myocardial infarction could identify a high-risk subgroup, but it is less clear that these cells are the primary actors in acceleration of atherosclerosis. Perhaps they are simply markers of larger infarctions or more severe underlying atherosclerosis. As the article mentions, they may be involved in the healing process, and one would want to be cautious before embarking on an intervention that might potentially interfere with cardiac repair.", "responses": [] } ]	1	https://f1000research.com/articles/4-37
https://f1000research.com/articles/3-206/v1	29 Aug 14	{ "type": "Software Tool Article", "title": "PAGAL - Properties and corresponding graphics of alpha helical structures in proteins", "authors": [ "Sandeep Chakraborty", "Basuthkar J. Rao", "Abhaya M. Dandekar", "Basuthkar J. Rao", "Abhaya M. Dandekar" ], "abstract": "Alpha helices (AH) are peptide fragments characterized by regular patterns of hydrogen bonding between the carbonyl oxygen and amino nitrogen of residues regularly spaced in sequence, resulting in spiral conformations. Their preponderance in protein structures underlines their importance. Interestingly, they are invariably present in all anti-microbial peptides. For example, the cecropin component of the chimeric anti-microbial protein designed previously by our group comprises of two AHs linked by a short stretch of random coil. These anti-microbial peptides are often amphipathic (quantified by a hydrophobic moment), aligning hydrophobic residues on one surface and charged residues on the others. In the current work, we reproduce previously described computational methods to compute the hydrophobic moment of AHs - and provide open access to the source code (PAGAL). We simultaneously generated input files for TikZ (a package for creating high resolution graphics programmatically) to obtain the Edmundson wheel and showing the direction and magnitude of the hydrophobic moment, and Pymol scripts to generate color coded protein surfaces. Additionally, we have observed an empirical structural property of AHs: the distance between the Cα atoms of the ith and (i+4)th residue is equal to the distance between the carbonyl oxygens of the ith and (i+4)th residue. We validated this using 100 non-homologous high resolution structures from the PISCES database. The source code and manual is available at http://github.com/sanchak/pagal and on http://dx.doi.org/10.5281/zenodo.11136.", "keywords": [ "A protein structure is formed by well ordered local segments defined by the hydrogen-bonding pattern of the peptide backbone (secondary structures)", "and conformations that lack any regular arrangement (random coils). The most prevalent secondary structures are alpha helices (AH) and β sheets", "while other conformations like π-helix occur rarely in natural proteins1. AHs are right-handed spiral conformations which have a hydrogen bond between the carbonyl oxygen (C=O) of every residue and the alpha-amino nitrogen (N-H) of the fourth residue away from the N-terminal." ], "content": "Introduction\n\nA protein structure is formed by well ordered local segments defined by the hydrogen-bonding pattern of the peptide backbone (secondary structures), and conformations that lack any regular arrangement (random coils). The most prevalent secondary structures are alpha helices (AH) and β sheets, while other conformations like π-helix occur rarely in natural proteins1. AHs are right-handed spiral conformations which have a hydrogen bond between the carbonyl oxygen (C=O) of every residue and the alpha-amino nitrogen (N-H) of the fourth residue away from the N-terminal.\n\nDSSP is the official program used to assign secondary structure to a protein when the atomic coordinates are known2,3. Several methods can also predict an AH from the sequence4,5. Essentially, any structure prediction tool can be used to predict an AH from the sequence by first predicting the structure and then applying DSSP to the predicted structure6–8.\n\nThe niche of AHs in protein structures is widespread. AHs are the functionally significant element in several motifs (DNA binding motifs)9, and the key components of any protein that permeates biological membranes10. AHs are also almost invariably present in anti-microbial peptides (AMP)11. For example, cecropin B, a component of a chimeric protein with anti-microbial properties that provides grapevines with enhanced resistance against the Gram-negative pathogen Xylella fastidiosa12, is composed of two AHs connected by a small random coil13. Other AMPs comprise only a single AH14,15. These peptides are characterized by a strong hydrophobic surface (defined by a hydrophobic moment16), and often have charged residues, either anionic or cationic, aligned on the opposite surface16. Previously, Jones et al. have implemented computational methods to extract the characteristics of AHs17.\n\nIn the current work, we first observe and propose an empirical structural property of AHs: that the distance between the Cα atoms of the ith and (i+4)th residue is equal to the distance between the carbonyl oxygens of the ith and (i+4)th residue. This hypothesis is validated on a set of high resolution non-homologous 100 proteins (775 AHs) taken from the PISCES database18. Next, we implement the methodologies described previously17 to compute the hydrophobic moments for AHs using the hydrophobicity scale used in19: PAGAL - Properties and corresponding graphics of alpha helical structures in proteins. There are other programs available online to do similar processing (http://rzlab.ucr.edu/scripts/wheel/ for example). We also specify a metric associated with each helix - the ratio of the positive to the negative residues (RPNR) in the AH - which helps identify AHs with a particular kind of charge distribution on their surface. The results are outputted as the input to a graphical program TikZ (for the Edmundson wheel20 and hydrophobic moment), and Pymol scripts (for showing the peptide surface). The source code and manual available at http://github.com/sanchak/pagal and on http://dx.doi.org/10.5281/zenodo.11136.\n\n\nMaterials and methods\n\nWe first outline the method to obtain the coordinates of each residue in the Edmundson wheel, and the computation of the hydrophobic moment (Algorithm 1). The input to the function is an alpha helix - either as a PDB structure or as a fasta sequence. The center of the wheel is taken as (0,0) and the radius as 5. The first residue has coordinates (0,5). Each subsequent residue is advanced by 100 degrees on the circle, as 3.6 turns of the helix makes one full circle.\n\nTo compute the hydrophobic moment, we obtain the vector by connecting the center to the coordinate of the residue and giving it a magnitude obtained from the hydrophobic scale (in our case, this scale is obtained from17). These vectors are then added to obtain the final hydrophobic moment.\n\nThe results are outputted as the input to a graphical program TiKz (for the Edmundson wheel20 and hydrophobic moment), and Pymol scripts (for showing the peptide surface). The protein structures have been rendered using Pymol, while the figures showing the Edmundson wheel has been obtained from TiKz. The source code is written in Perl, and made available at https://github.com/sanchak/pagal and permanently available on http://dx.doi.org/10.5281/zenodo.11136.\n\n\n\nInput: αH: α helix - either PDB or fasta sequence\n\nInput: TableHS: Hydrophobic scale\n\nOutput: TikZIN: TikZ input file\n\nOutput: PymolIN: Pymol input file\n\nbegin\n\nRadius = 5 ; // Radius of Edmundson wheel\n\ninitangle = 90 ; // first residue is at 12 o’clock..\n\nloopcnt = 0 ;\n\nfinalvechydro = undefined ;\n\ncentre = (0,0);\n\nforeach Residuei in αH do\n\n/* Find X,Y coordinate on the Edmundson wheel /\n\nangle = initangle - loopcnt 100 ;\n\nx = Radius * cos(val) ;\n\ny = Radius * sin(val) ;\n\nthispoint = (x,y);\n\n\n\n/* Get Hydrophobic moment /\n\nvector = MakeVectorFrom2Points(centre,thispoint) ;\n\nhydrophobicvalue = GetHydrophobicScaleForResidue(TableHS, Residuei) ;\n\ntmpvec = normal(vector) hydrophobicvalue ;\n\nfinalvechydro = finalvechydro is not defined? tmpvec : finalvechydro + tmpvec;\n\n\n\nloopcnt++ ;\n\nend\n\nWriteTikzScript();\n\nWritePymolScript();\n\nend\n\n\nResults and discussion\n\nWe have observed an empirical structural property that applies to the residues of any AH: the distance between the Cα atoms of the ith and (i+4)th residue (denoted by D(Cαi/Cαi+4)) is (almost) equal to the distance between the carbonyl oxygens of the ith and (i+4)th residue (D(Oi/Oi+4)). We validate our hypothesis on a set of 100 high resolution, non-homologous proteins (which have 775 AHs) taken from the PISCES database (http://dunbrack.fccc.edu/PISCES.php)18. Figure 1 shows the plot of the difference between D(Cαi/Cαi+4) and D(Oi/Oi+4) for AHs specified in the PDB files (in red, mean=0.16 Å, standard deviation (sd)=0.34 Å), and for all residues separated by four residues but not part of a helix (in blue, mean=0.71 Å, sd=0.75 Å).\n\nAll 775 AHs specified in the PDB files from the 100 non-homologous high resolution structures taken from the PISCES database are in red (mean=0.16 Å, standard deviation (sd)=0.34α Å). All residues separated by four residues but not part of a helix are in blue (mean=0.71 Å, sd=0.75 Å). All AHs specified in the PDB files after correction are in green (mean=0.095 Å and sd=0.14 Å).\n\nThese results are conservative, since there are residues that are annotated as part of a helix in the PDB file which seems to be incorrect. For example, in PBD 1JET, the ninth helix spans from residues 169 to 178 - “HELIX 9 9 LYS A 169 LYS A 178 1 10”. However, the Pymol helix identification program shows part of this stretch as a random coil (Lys178 in Figure 2a). Moreover, the distance between the carbonyl oxygen (C=O) and the alpha-amino nitrogen (N-H) of the fourth residue away from the N-terminal is 7.6 Å, which makes it improbable for them to have a hydrogen bond, the primary requisite to be part of a AH. The D(Cαi/Cαi+4) and D(Oi/Oi+4) for this pair is 9 Å and 8 Å, respectively: a difference of 1 Å. Even in cases where the distance between C=O and N-H is within the 3.6 Å typically required for a hydrogen bond, (PDBid: 1ELU, 12th helix), the distances D(Cαi/Cαi+4) and D(Oi/Oi+4) for the residue pair His292-Gly296 is 6.9 Å and 3.4 Å, respectively: a difference of 3.4 Å (Figure 2b). In short, the helix annotation in the PDB database is often incorrect. Removing these problematic residues reduces the mean distance to 0.095 Å and the sd to 0.14 Å (Figure 1).\n\n(a) Lys178 in PDBid:1JET appears to be part of a random coil, but is annotated in the PDB file as a helix. (b) Gly296 in PDBid:1ELU is mis-annotated similarly.\n\nThere is variation in the D(Cαi/Cαi+4) even when considering the same pair of residues. For example, taking all pairs of Arg and Lys in the 775 AHs analyzed (Table 1), we see that the values can vary from 6.5 Å in PDBid:1H16 (helix26, pair Arg583-Lys587) to 5.8 Å in PDBid:1EYH (helix5, pair Arg72-Lys76). However, as hypothesized, D(Oi/Oi+4) is the same as D(Cαi/Cαi+4).\n\nRPair: Residue pair in the alpha helix with a hydrogen bond between carbonyl oxygen (C=O) and the alpha-amino nitrogen (N-H), Dhbond: Distance between carbonyl oxygen (C=O) and the alpha-amino nitrogen (N-H) of RPair, D(Cαi/Cαi+4): Distance between the Cα atoms of RPair, D(Oi/Oi+4): Distance between the carbonyl oxygen of RPair, δ: absolute(D(Cαi/Cαi+4) - D(Oi/Oi+4)).\n\nThe Edmundson wheel20 has been the standard way of visualizing AHs for a long time now, although there are other methods (Wenxiang diagram21) to represent AHs. The Edmundson wheel shows the alignment of residues as one looks through the helix, and gives an approximate idea of the various properties of the AH. For example, a color coding differentiation of the polar and non-polar residues gives an approximation of the hydrophobic propensity of the AH. A more mathematical representation of the hydrophobic propensity is to represent each residue with a value and a sign (direction). This results in a vector representation, called the hydrophobic moment16. We have chosen the hydrophobic scale from17 (Table 2), although any other hydrophobic scale could be also used. The color coding is as follows: all hydrophobic residues (positive values in Table 2) are colored red, while hydrophilic residues (negative values in Table 2) are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides. We now show the PAGAL representation of a few AH peptides.\n\nCecropin. A synergistic combination of two critical immune functions, pathogen surface recognition and lysis, resulted in a chimeric protein with anti-microbial properties against the Gram-negative Xylella fastidiosa12. The lytic domain is cecropin B, which attacks conserved lipid moieties and creates pores in the X. fastidiosa outer membrane13. Cecropin B consists of two AHs, joined by a short stretch of random coil. Figure 3a and b shows the Edmundson wheel and hydrophobic moment of the two AHs. It can be seen that the N-Terminal AH has a large hydrophobic moment, as well as a specific positive charge distribution. The hydrophobicity of this amphipathic AH has significant bearing on the anti-microbial properties of the peptide22. This can also be seen in a Pymol rendering of the peptide surface (Figure 4). The Pymol script for this rendering is automatically generated by PAGAL. On the other hand, the C-Terminal AH comprises mostly of hydrophobic residues. Cecropin-like peptides use the synergy of these two helices - the N-terminal attaches to charged ion on the membrane, and the hydrophobic C-terminal permeates the hydrophobic inter-membrane region (known as the ‘carpet’ model23).\n\nAll hydrophobic residues are colored in red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides. (a) N-Terminal helix of cecropin B. (b) C-Terminal helix of cecropin B. (c) KR-12 peptide fragment from cathelicidin LL-37. (d) De novo designed peptide (SP1-1) with anti-microbial activity.\n\nAll hydrophobic residues are colored in red, while hydrophilic residues are colored in blue.\n\nCathelicidin LL-37. Cathelicidin LL-37 is a critical component of the innate human immune system that protects humans against infectious diseases by targeting anionic phosphatidylglycerols in the pathogenic bacterial membranes24. Recent work has demonstrated a 12-residue peptide (KR-12) corresponding to residues 18 to 29 of LL-37 is toxic to bacterial, but not human cells14. Figure 3c shows the Edmundson wheel and hydrophobic moment of KR-12. The demarcation of the polar and non-polar residues is quite evident. The predominance of positively charged residues in the polar side of the peptide is also clearly visible.\n\nDe novo designed AMPs for plant protection. The de novo design of small AMPs that inhibit plant pathogens was the focus of a recent work15. One of the most promising candidates was a small peptide (SP1-1 - RKKRLKLLKRL, Figure 3d), which was “highly active against a broad spectrum of bacteria, but showed low hemolytic activity”15. Although the hydrophobic moment of this peptide is much smaller than that of KR-12 (Figure 3c), possibly due to the presence of Arg4 on the hydrophobic surface, the distribution of positively charged residues in this peptide is greater than for KR-12.\n\nOften, it is desirable to choose a large distribution of charged residues of a certain kind (anionic or cationic) on the hydrophilic surface. One possible method for quantifying this would be to compute a ‘charge moment’, similar to the computation of hydrophobic moments. However, such an evaluation would determine certain clearly distributions to be the same. For example, assume one semicircle of the wheel comprised only positive residues, and the other hydrophobic residues (Figure 5a). This is a slightly modified version of KR-12 from cathelicidin LL-37. If one positive residue (R5) were moved from the hydrophilic side to the hydrophobic side (I7) and replaced with a negative residue (D7) (Figure 5b), the ‘charge moment’ would remain the same, although the two conformations are clearly not the same. Note that the hydrophobic moment is also different, as expected. Thus, we resort to a simple metric to allow one to choose peptides with a large proportion of charged residue of a single kind: the ratio of the positive to the negative residues (RPNR). The two peptides mentioned above will have different RPNRs: 1 (Figure 5a) and 0.85 (Figure 5b).\n\nAll hydrophobic residues are colored in red, while the hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides. (a) Edmundson wheel of a KR-12 like peptide showing the hydrophobic moment and the ‘charge moment’. (b) Swapping one positive residue (R5) from the hydrophilic side with I7 and replacing it with a negative residue (D7), results in the same ‘charge moment’, although the characteristics of the helix has clearly changed.\n\nPAGAL generates a TikZ input file for drawing the Edmundson wheel and showing the hydrophobic moment (Supplementary File TikzInput.doc). TikZ is a package “for creating graphics programmatically” - http://www.texample.net/tikz/. PAGAL also generates a Pymol script to the peptide structure using the same color coding used in for the Edmundson wheel (Supplementary File PymolInput.doc).\n\n\nSoftware availability\n\nhttp://github.com/sanchak/pagal\n\nhttps://github.com/F1000Research/pagal/tree/v1.0\n\nhttp://dx.doi.org/10.5281/zenodo.1113625\n\nLicense: GPLv3", "appendix": "Author contributions\n\n\n\nSC wrote the computer programs. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAMD wishes to acknowledge grant support from the California Department of Food and Agriculture PD/GWSS Board. BJ acknowledges financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant.\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\nPauling L, Corey RB, Branson HR: The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci U S A. 1951; 37(4): 205–211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKabsch W, Sander C: Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983; 22(12): 2577–2637. PubMed Abstract \| Publisher Full Text\n\nJoosten RP, te Beek TA, Krieger E, et al.: A series of PDB related databases for everyday needs. Nucleic Acids Res. 2011; 39(Database issue): D411–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaupetit J, Derreumaux P, Tuffery P: PEP-FOLD: an online resource for de novo peptide structure prediction. Nucleic Acids Res. 2009; 37(Web Server issue): 498–503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaur H, Garg A, Raghava GP: PEPstr: a de novo method for tertiary structure prediction of small bioactive peptides. Protein Pept Lett. 2007; 14(7): 626–631. 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PubMed Abstract \| Publisher Full Text\n\nBrogden KA: Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol. 2005; 3(3): 238–250. PubMed Abstract \| Publisher Full Text\n\nDandekar AM, Gouran H, Ibanez AM, et al.: An engineered innate immune defense protects grapevines from Pierce disease. Proc Natl Acad Sci U S A. 2012; 109(10): 3721–3725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore AJ, Beazley WD, Bibby MC, et al.: Antimicrobial activity of cecropins. J Antimicrob Chemother. 1996; 37(6): 1077–1089. PubMed Abstract \| Publisher Full Text\n\nWang G: Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles. J Biol Chem. 2008; 283(47): 32637–32643. PubMed Abstract \| Publisher Full Text\n\nZeitler B, Herrera Diaz A, Dangel A, et al.: De-novo design of antimicrobial peptides for plant protection. PLoS One. 2013; 8(8): e71687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisenberg D, Weiss RM, Terwilliger TC: The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature. 1982; 299(5881): 371–374. PubMed Abstract \| Publisher Full Text\n\nJones MK, Anantharamaiah GM, Segrest JP: Computer programs to identify and classify amphipathic alpha helical domains. J Lipid Res. 1992; 33(2): 287–296. PubMed Abstract\n\nWang G, Dunbrack RL Jr: PISCES: a protein sequence culling server. Bioinformatics. 2003; 19(12): 1589–1591. PubMed Abstract \| Publisher Full Text\n\nEngelman DM, Steitz TA, Goldman A: Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986; 15: 321–353. PubMed Abstract \| Publisher Full Text\n\nSchiffer M, Edmundson AB: Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967; 7(2): 121–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChou KC, Zhang CT, Maggiora GM: Disposition of amphiphilic helices in heteropolar environments. Proteins. 1997; 28(1): 99–108. PubMed Abstract \| Publisher Full Text\n\nChen Y, Guarnieri MT, Vasil AI, et al.: Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob Agents Chemother. 2007; 51(4): 1398–1406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteiner H, Andreu D, Merrifield RB: Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim Biophys Acta. 1988; 939(2): 260–266. PubMed Abstract \| Publisher Full Text\n\nYang D, Chertov O, Oppenheim JJ: Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37). J Leukoc Biol. 2001; 69(5): 691–697. PubMed Abstract\n\nChakraborty S, Rao BJ, Dandekar AM: PAGAL: alpha helices structures. Zenodo. 2014. Data Source" }	[ { "id": "5988", "date": "12 Sep 2014", "name": "Ramakrishnan Nagaraj", "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\nAHs are not invariably present in ALL antimicrobial peptides. The formation of AHs in antimicrobial peptides is dependent on solvent conditions. The statement to this effect should be edited appropriately. The proposed empirical structural property of AHs is OK as it is validated on a set of high resolution non-homologous proteins from a database. Extension of the analysis to antibacterial peptides such as cecropin and LL37 may not be valid. These peptides are unstructured in water and fold into amphipathic helical structure only in media of low dielectric constant. Also, the helical structures are not rigid as observed in protein crystal structures and show considerable conformational flexibility in solution. Hence, a unique value of hydrophobic moment will not be very meaningful. Solvent effects need to be taken into account. This point (3) needs to be addressed and clarified.", "responses": [ { "c_id": "990", "date": "17 Sep 2014", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "We would like to thank you for taking the time and reviewing our paper, and for your helpful comments. We have modified our manuscript to reflect this changes, and hope that these are satisfactory." } ] }, { "id": "6102", "date": "12 Sep 2014", "name": "Guangshun Wang", "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 aimed at further define features for helices, which are key structural elements in polypeptides. Based on the traditional hydrophobic moment, they proposed the concept of charge moment. It is interesting but I am a little bit of doubtful how useful the charge moment will be. In particular, the authors found the “Swapping one positive residue (R5) from the hydrophilic side with I7 and replacing it with a negative residue (D7), results in the same ‘charge moment’” (Figure 5). In the case of helical antimicrobial peptides, such a swap may have a detrimental effect on peptide antimicrobial activity. For example, even a change of a hydrophilic residue serine on the hydrophobic face with a hydrophobic residue influenced peptide activity (Wang G et al., 2012). The authors may refine this idea and conceive the possible use of charge moment. Based on charge moment, is there any clue that interfacial charged residues of antimicrobial peptides play a larger role than non-interfacial ones in determining antimicrobial activity? Will it be possible to incorporate the observation that arginines are usually more important than lysines in determining peptide activity (see Mishra B et al., 2013)?", "responses": [ { "c_id": "989", "date": "17 Sep 2014", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "We would like to thank you for taking the time and reviewing our paper. Please find our response to your suggestions below. We have incorporated the changes in the new version.The authors aimed at further define features for helices, which are key structural elements in polypeptides. Based on the traditional hydrophobic moment, they proposed the concept of charge moment. It is interesting but I am a little bit of doubtful how useful the charge moment will be. In particular, the authors found the “Swapping one positive residue (R5) from the hydrophilic side with I7 and replacing it with a negative residue (D7), results in the same ‘charge moment’” (Figure 5). In the case of helical antimicrobial peptides, such a swap may have a detrimental effect on peptide antimicrobial activity. For example, even a change of a hydrophilic residue serine on the hydrophobic face with a hydrophobic residue influenced peptide activity (Wang G et al., 2012). The authors may refine this idea and conceive the possible use of charge moment. Based on charge moment, is there any clue that interfacial charged residues of antimicrobial peptides play a larger role than non-interfacial ones in determining antimicrobial activity? Will it be possible to incorporate the observation that arginines are usually more important than lysines in determining peptide activity (see Mishra B et al., 2013)?The relevant reference you have pointed out (Wang G et al., 2012) underlines the point are making - that the charge moment (analogous to the hydrophobic moment) is not a good metric. Thus, `we resort to a simple metric to allow one to choose peptides with a large proportion of charged residue of a single kind: the ratio of the positive to the negative residues (RPNR).'. I apologize that this point has been confusing, so I have explicitly clarified this aspect. Also, the reference helps to establish our point.Unfortunately, we would be unable to differentiate between lysines and arginines using the current methodology. However, this is a salient point that we need to discuss so that future work may address this.We hope to have addressed your concerns with these modifications." } ] } ]	1	https://f1000research.com/articles/3-206
https://f1000research.com/articles/2-286/v1	27 Dec 13	{ "type": "Research Article", "title": "Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins", "authors": [ "Sandeep Chakraborty", "Adela Rendón-Ramírez", "Bjarni Ásgeirsson", "Mouparna Dutta", "Anindya S. Ghosh", "Masataka Oda", "Ravindra Venkatramani", "Basuthkar J. Rao", "Abhaya M. Dandekar", "Félix M. Goñi", "Adela Rendón-Ramírez", "Bjarni Ásgeirsson", "Mouparna Dutta", "Anindya S. Ghosh", "Masataka Oda", "Ravindra Venkatramani", "Basuthkar J. Rao", "Abhaya M. Dandekar", "Félix M. Goñi" ], "abstract": "The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.", "keywords": [ "enzymes", "serine proteases" ], "content": "Introduction\n\nOral glucose elicits a greater insulin response than intravenous glucose infusion, a phenomenon known as the incretin effect1. This effect is mostly attributed to the intestinally derived hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP)2. These hormones have a very short half-life as they are rapidly inactivated by the ubiquitous enzyme dipeptidyl peptidase-IV (DPP4)3. The finding that the incretin effect is impaired in subjects with type 2 diabetes4 led to two major types of GLP-1 based therapies5 - intravenously or sub-cutaneously administered GLP-1 mimetics that are resistant to DPP4 (exenatide, liraglutide, etc.)6, and the orally administered gliptins that prolong the physiological actions of incretin hormones by inhibiting DPP4 (sitagliptin, vildagliptin, etc.)7–9. Due to the multifarious roles played by the DPP4 enzyme10–12, the possible side effects of these drugs (acute pancreatitis, pancreatic cancer, etc.13–15) are strongly contested by researchers who argue that current statistics are insufficient16,17 to conclusively attribute these side effects to the otherwise beneficial GLP-1 drugs18. Compound promiscuity is another phenomenon that might play a crucial role in determining the side effects of these therapies, although this aspect has rarely been pursued intensively19.\n\nPrevious work by our group has established the spatial and electrostatic congruence in cognate residue pairs of the active site in proteins with the same functionality (CLASP)20,21. CLASP analysis indicated that the phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus has spatial and electrostatic congruence with a serine protease motif22. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). The specificity of the protease activity was for a proline in the amino terminal, suggesting that PI-PLC is a prolyl peptidase, similar to the DPP4 enzyme. This finding led us to believe that the gliptins would have similar inhibitory effect on PI-PLC. In the current work, we have confirmed the inhibition of the native phospholipase activity of PI-PLC using two gliptins - vildagliptin23 (at μ-molar concentrations) and K57924 (at nano-molar concentrations).\n\nSubsequently, we used a motif derived from a DPP4 protein25, in addition to the trypsin motif used previously22, to query a comprehensive and non-redundant (50% sequence identity) list of ∼5000 human proteins with known structures using CLASP, intending to identify other proteins that might be inhibited by the gliptins. From the set of proteins with significant congruent matches with these two motifs, we identified a pancreatic lipase26 and a gastric lipase27, keeping the context of lipases, acute pancreatitis and GLP-1 based therapies in mind. Our findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies28,29, although these reports are in disagreement with other findings30,31. While it is logical and expected to find scaffolds that are congruent to trypsin and DPP4 active sites in lipases based on the current results and our previous findings22, we also show the presence of the serine catalytic triad in close proximity to the active site residues of proteins which have a completely different enzymatic mechanism (for example, in glutaminyl cyclase which is a transferase32). This corroborates the current belief that convergent evolution occurs more frequently than previously believed33. Thus, we propose a rational method to identify proteins that might have unintended and undesirable interactions with newly introduced compounds, and substantiate our claims by demonstrating the inhibition of the native phospholipase activity of PI-PLC from B. cereus using gliptins that are used in type 2 diabetes therapy.\n\n\nResults\n\nThe active sites of serine proteases differ in their specificities owing to residues other than the conserved catalytic triad. Thus, in addition to the trypsin motif used previously (Asp102, Ser195 and His57 - PDBid 1A0J)22 (Motif1), we choose another motif from a DPP4 enzyme (Asp708, Ser630 and His740 - PDBid:1N1M) (Motif2) (Table 1). Apart from the catalytic triad, we chose another non-polar residue in order to increase the specificity of the matches (Ala56 in Motif1 and Val711 in Motif2). This fourth residue is chosen as the closest residue to any one of the catalytic triad residues. Using the ability of CLASP to include stereochemically equivalent residues, this last residue could be matched by another non-polar residue - one of Gly, Ala, Val, Leu, Ile or Met. Further, it has been seen that the second (ac) and fifth (bd) (Table 1) pairwise electrostatic potential differences (EPD) are not discriminatory - thus, this pair is not used to score the EPD difference (although it is included in the distance deviation score).\n\nRmsd1 and Rmsd2 are the root mean square deviation of the scaffold with respect to Motif1 and Motif2. DPP4 - dipeptidyl peptidase-IV, PI-PLC - phosphoinositide-specific phospholipase C, PLASE - human pancreatic lipase-Related Protein 2, GPASE - human gastric lipase, QC - glutaminyl cyclase. D = Pairwise distance in Å. PD = Pairwise potential difference. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\n(a) Motif1 (Asp102, Ser195, His57, Ala56) from Trypsin (b) Motif2 (Asp708, Ser630, His740, Val711) from DPP4.\n\nDPP4 (EC 3.4.14.5), a serine protease that is expressed in many tissues (kidney, liver, lung, intestinal membranes, lymphocytes and endothelial cells), cleaves peptides with Pro or Ala residues in the second amino terminal position. Previously, we have experimentally demonstrated the existence of the serine protease domain in PI-PLC from Bacillus cereus - both by virtue of its proteolytic activity, and the inhibition of its native activity on phospholipids in the presence of serine protease inhibitors22. Furthermore, the specificity of the proteolytic activity indicated that it was a prolyl peptidase - thus, leading us to believe that DPP4 inhibitors should have a similar inhibitory effect on the PI-PLC enzyme. Table 1 shows the presence of a congruent motif in the PI-PLC protein with both Motif1 and Motif2. His32 and Asp67 are known to be a part of the active site scaffold in PI-PLC22. These proteins have completely different folds, and thus a superimposition (using both MUSTANG34 and DECAAF35) does not show any detectable similarity in their structures (Supplementary Figure 1). Figure 1 shows the active sites of these proteins, and the superimposition of these proteins based on their catalytic residues35. It can be seen that the closest nonpolar residue to the catalytic triad in trypsin and PI-PLC (Ala56 in PDBid:1A0J, Ile68 in PDBid:1PTD) is differently placed from Val711 in DPP4 (PDBid:1N1M). This is also indicated by the greater RMSD (root mean square deviation) of the scaffold in PI-PLC to Motif2 as compared to Motif1. The differences in the position of peripheral residues is the source of the diverse specificities exhibited by these proteases. Figure 2 shows the inhibition of PI-PLC using two gliptins - vildagliptin(LAF-237)23 and K57924. PI-PLC catalyzes hydrolysis of phospholipids to yield diacylglycerol and a phosphoryl alcohol. In the absence of inhibitors enzyme addition to the vesicle suspension causes an increase in turbidity due to vesicle aggregation (Figure 2a,c). Aggregation in turn occurs as a result of formation of the enzyme end-product diacylglycerol36,37. A steady-state is reached under our conditions after 6–8 min. Addition of either LAF-237 (vildagliptin) or K579 leads to an obvious inhibition of the enzyme activity. Dose-response curves for the inhibitors are shown in Figure 2 (b,d). K579 is two orders of magnitude more potent than LAF-237 as a PI-PLC inhibitor, with half-maximal inhibitory concentrations IC50 respectively of 1 μM and 100 μM.\n\n(a) Trypsin (PDBid:1A0J) (b) DPP4 (PDBid:1N1M); (c) PI-PLC (PDBid:1PTD) (d) Superimposing the active site residues using DE-CAAF35.\n\n(a,c) Time courses of enzyme activity in the presence of varying amounts of inhibitors, respectively LAF-237 and K579. The trace marked LIPOSOMES corresponds to a control in the absence of PI-PLC. (b,d) Dose-response effect of inhibitors on PI-PLC activity. Activity was computed as the extent of vesicle aggregation after 10 min enzyme activity.\n\nCurrently, the PDB database has about 25,000 human proteins. Using a identity cutoff of 50%, we chose a set of ∼5000 proteins (Supplementary Table 1) as the target proteins. Table 2 shows the best matches obtained in these ∼5000 proteins when queried by Motif1 and Motif2. Given the context of lipases, acute pancreatitis and GLP-1 based therapies, we picked two proteins - the human pancreatic lipase-related protein 2 (PDBid:2OXE)26 and a human gastric lipase (PDBid:1HLG)27 - to demonstrate the distinct possibility that these proteins might be inhibited by DPP4 inhibitors. Table 1 shows the congruence of the DPP4 motif to these proteins using Motif1 and Motif2. It is interesting to note that the gastric lipase (PDBid:1HLG) has a good match with both motifs - Leu326 in PDBid:1HLG is congruent to Ala56 in PDBid:1A0J, and Ala237 (PDBid:1HLG) is congruent to Val711 (PDBid:1N1M).\n\nSince both these proteins are lipases (hydrolases), this congruence to Motif1 and Motif2 is expected based on our previous results with PI-PLC22. However, our methodology also detects other proteins, often with a completely different enzymatic mechanism from hydrolases. A glutaminyl cyclase (PDBid:3PB432), a transferase, has a significantly congruent domain with Motif1 (lesser congruence with Motif2, as indicated by the RMSD) (Table 1). Figure 3 shows the proximity of the promiscuous scaffold to the active site of the cyclase, and also the congruence of the scaffold to Motif1.\n\n(a) The active site residues are marked in magenta. They are seen to be proximal to the identified scaffold. (b) Superimposition of Motif1 and the scaffold in glutaminyl cyclase.\n\n\nDiscussion\n\nThe controversy regarding the side effects of the dpp4 inhibitors, particularly with respect to acute pancreatitis and pancreatic cancer, continues unabated. While some researchers feel that it is not acceptable to assume that ‘absence of evidence is evidence of absence’38,39, others believe that current data are not conclusive and the ‘benefits by far outweigh the potential risks’16. Adding to the uncertainties are conflicting reports presented by different groups28–31. Notwithstanding the antagonistic views on the subject, it is unanimously accepted that current data are insufficient to establish a causal pathogenic effect of these drugs on such side effects40.\n\nVarious database studies have been undertaken in order to ascertain the effects of the GLP-1 therapies. Some studies ‘did not find an association between the use of exenatide or sitagliptin and acute pancreatitis’ with the caveat that the ‘limitations of this observational claims-based analysis cannot exclude the possibility of an increased risk’41. On the other hand, other studies have shown that the use of ‘sitagliptin or exenatide increased the odds ratio for reported pancreatitis 6-fold as compared with other therapies’14. Further, they reported that ‘pancreatic cancer was more commonly reported among patients who took sitagliptin or exenatide as compared with other therapies’14. The close relationship between chronic pancreatitis and pancreatic cancer is also a subject of intense research42. Another administrative database study of US adults with type 2 diabetes reported increased odds of hospitalization for acute pancreatitis for patients undergoing GLP-1-based therapies sitagliptin and exenatide13. Once again, such correlation of GLP-1 based therapies to acute pancreatitis is contested by other studies43.\n\nOur findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies28,29, keeping in mind that other studies contradict these reports30,31. While several studies have reported that the GLP-1 mimetics do not induce pancreatitis in rats, mouse and/or monkey44–46, these studies did not include DPP4 inhibitors, which are the compounds that might be responsible for interactions with pancreatic proteins according to our study. It is to be noted however that these mimetics may have other physiological effects and ‘the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation’47. Once again, the previous study47 has been challenged by another group who note that ‘findings previously reported in rodents may not apply to humans’48.\n\nThe orally administered gliptins differ in many aspects such as potency, excretion mechanism, target selectivity, half-life, metabolism and possible drug-drug interactions9,49,50. This difference is also highlighted in the different concentrations of vildagliptin and K579 that inhibit PI-PLC. Interestingly, the PI-PLC scaffold has a better match with the trypsin motif than with the DPP4 motif (Table 1). In order to be able to model these differences in our in silico search, it is important to be able to provide flexibility in the scoring mechanism.\n\nTo summarize, it has been noted in the case of GLP-1 based therapies that as ‘evidence of harm accumulates, but is vigorously discounted’ the ‘burden of proof now rests with those who wish to convince us of their safety’39. Surveillance programs, real-life cohort studies and case-control studies can be supplemented by rational investigations of relevant proteins based on anecdotal reports51. The methodology proposed in the current work, which specifically demonstrates the effects of the DPP4 inhibitors, also presents a rational way of determining the inadvertent interactions of newly designed compounds with proteins, and thus prevent the recurrence of drug induced diseases being detected after considerable damage has already been inflicted on humans subjected to these drugs52.\n\n\nMaterials and methods\n\nA comprehensive, non-redundant set of ∼5000 human proteins (50% identity cutoff) was obtained from the PDB database53. The CLASP package (http://www.sanchak.com/clasp) used for querying these proteins using motifs from trypsin and DPP4 is written in Perl on Ubuntu20. Hardware requirements are modest - all results here are from a simple workstation (8GB ram), and runtimes for analyzing the ∼5000 proteins was about 24 hours. Adaptive Poisson-Boltzmann Solver (APBS) and PDB2PQR packages were used to calculate the potential difference between the reactive atoms of the corresponding proteins54,55. The APBS parameters and electrostatic potential units were set as described previously in Chakraborty et al.20. All protein structures were rendered by PyMol (http://www.pymol.org/). Protein structures have been superimposed using MUSTANG34 and DECAAF35.\n\nPI-PLC was purchased from Sigma. vildagliptin(LAF-237) was obtained from Selleckchem, and K579 was obtained from Santa Cruz.\n\nVesicle preparation and characterization. The appropriate lipids were mixed in organic solution, and the solvent was evaporated to dryness under N2. Solvent traces were removed by evacuating the lipids for at least 2 hours. The lipids were then swollen in 10 mM Hepes, 150 mM NaCl, pH 7.5 buffer. Large unilamellar vesicles (LUV) were prepared from the swollen lipids by extrusion and sized by using 0.1 μm poresize Nuclepore filters, as described by Ahyayauch et al.36. LUV composition was egg phosphatidylcholine: egg phosphatidylethanolamine: cholesterol at a 2:1:1 mole ratio. The average size of LUV was measured by quasi-elastic light scattering, using a Malvern Zeta-sizer instrument. Lipid concentration, determined by phosphate analysis, was 0.3 mM in all experiments.\n\nAggregation assay. Enzyme activity was assayed measuring enzyme-induced vesicle aggregation. All assays were carried out at 39°C with continuous stirring, in 10 mM Hepes, 150 mM NaCl buffer (pH 7.5), in the presence of 0.1% BSA for optimum catalytic activity. Enzyme concentration was 0.16 U/mL, and liposomal concentration was 0.3 mM. Lipid aggregation was monitored in a Cary Varian UV-vesicle spectrometer as an increase in turbidity (absorbance at 450 nm) of the sample, as described by Villar et al.37.", "appendix": "Author contributions\n\n\n\nSC, ARR and BA performed the experiments. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFMG thanks the Spanish Ministerio de Ciencia e Innovacion for grant No. BFU 2012-36241, and the University of the Basque Country for grant No. IT 849-13. BJ and RV acknowledge financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant. BA extends gratitude to the University of Iceland Research Found for supporting the project financially. AMD wishes to acknowledge grant #12-0130-SA from California Department of Food and Agriculture CDFA PD/GWSS Board. MO was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; (grant No. 21790431).\n\n\nAcknowledgements\n\nWe thank JM Frere for critical technical inputs at various stages of the project.\n\n\nSupplementary material\n\nIt is seen that there is no structural similarity in the two proteins. (a) Using MUSTANG34. (b) Using DECAAF35.\n\n\nReferences\n\nElrick H, Stimmler L, Hlad CJ, et al.: Plasma insulin response to oral and intravenous glucose administration. J Clin Endocrinol Metab. 1964; 24(10): 1076–1082. PubMed Abstract \| Publisher Full Text\n\nBaggio LL, Drucker DJ: Biology of incretins: GLP-1 and GIP. Gastroenterology. 2007; 132(6): 2131–2157. 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PubMed Abstract \| Publisher Full Text\n\nGarg R, Chen W, Pendergrass M: Acute pancreatitis in type 2 diabetes treated with exenatide or sitagliptin: a retrospective observational pharmacy claims analysis. Diabetes Care. 2010; 33(11): 2349–2354. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJura N, Archer H, Bar-Sagi D: Chronic pancreatitis, pancreatic adenocarcinoma and the black box in-between. Cell Res. 2005; 15(1): 72–77. PubMed Abstract \| Publisher Full Text\n\nEngel SS, Round E, Golm GT, et al.: Safety and tolerability of sitagliptin in type 2 diabetes: pooled analysis of 25 clinical studies. Diabetes Ther. 2013; 4(1): 119–145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNyborg NC, Molck AM, Madsen LW, et al.: The human GLP-1 analog liraglutide and the pancreas: evidence for the absence of structural pancreatic changes in three species. Diabetes. 2012; 61(5): 1243–1249. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "3818", "date": "06 Mar 2014", "name": "Rodney Rouse", "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\nDisclaimer: I lack the protein chemistry expertise to comment on the assumptions and protein chemistry used in the computational method described in this article.The title and abstract are appropriate.The overall experimental design is simple but strong and well suited for this project. The methods were generally well described. The conclusions are not overstated and any implications are justified based on the presented data. The article is very well written.\n\nThis is a very interesting study that uses a previously defined computational method, CataLytic Active Site Prediction (CLASP), that compares structural and charge similarities of catalytic sites to identify functionally similar proteins. This methodology was used to assess the potential for adverse events based on off target effects of the inhibitors of DPP-IV. Using CLASP, the authors had previously indentified a Bacillus cerus phosphoinositide specific phospholipase-C (PI-PLC) as similar in active catalytic site to the enzyme, DPP-IV. They used laboratory techniques to verify this finding. In the present study, the authors demonstrated the ability of two separate DPP-IV inhibitors to significantly reduce the activity of this PI-PLC in the lab. Subsequent to this experimentation, the authors returned CLASP to identify catalytic sites in other proteins that might also be inhibited by DPP-IV inhibitors thereby yielding unforeseen inhibition and biological effects. As applied to the case of DPP-IV inhibitors, which are not extremely specific, the authors identify a number of other proteins that could be promiscuously impacted by DPP-IV inhibitors thereby providing mechanisms for unexpected adverse events. Although the significance of DPP-IV inhibitor related adverse events has yet to be determined, the fact that changes have been reported non-clinically and clinically are undeniable. Eventually, the benefit of these molecules may far outweigh their associated risks, but the authors provide a potential path forward for investigation of unexpected events with this class of drug. If contradictory reports persist, this path may require further illumination. The approach is theoretically similar to using structural similarities to identify off target receptor binding and consequent biological effects, an expanding approach in safety assessment and in identification of mechanisms for adverse events in the pharmaceutical lifecycle. Similarly, this method could be predictive for off target effects and suggest what those effects might be. However, whether this is a method that can be generally applicable to other molecules is beyond my ability to comment and the scope of this work. Comments/Suggestions:1) Were the inhibition experiments done in duplicate, triplicate, etc? Some slight expansion of the protocols would help with attempts to replicate.", "responses": [ { "c_id": "728", "date": "10 Mar 2014", "name": "Adela Rendón-Ramirez", "role": "Author Response", "response": "Dear Dr Rouse,We would like to thank you for taking the time and reviewing our paper. Your positive comments encourage us to further our research in this area.We concur with your statement - \"Eventually, the benefit of these molecules may far outweigh their associated risks\". And it is our endeavor to improve the accuracy and generality of our method through different compounds. We would specifically like to highlight another case of antagonist binding identified through CLASP, although in this case most alkaline phosphatases were not affected - Chakraborty et al.(2012)The data for PI-PLC inhibition using DPP4 inhibitors, as shown in Figure 2, are average values of two closely similar experiments. We will revise the manuscript to include this point when we hear from another referee.Best regards,Sandeep Chakraborty and Adela Rendón-Ramirez" } ] }, { "id": "4249", "date": "26 Mar 2014", "name": "Mark D Gorrell", "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 successful targeting of DPP4 using small molecule compounds to treat type 2 diabetes has attracted a great deal of attention towards the study of this protease. The authors applied sophisticated techniques that they have developed in order to discover that two DPP4 inhibitors, including one that is in limited clinical use, can to some extent inhibit the activity of a bacterial lipase (PI-PLC). Many lipases and esterases and hydrolases including DPP4 and related enzymes use the alpha/beta hydrolase fold and the authors show how this related protein topology can place the residues in positions that are sufficiently similar to interact with an inhibitor. The major difficulty with this paper is that it attempts to connect these data with possible clinical outcomes. No evidence for such a link is presented. Therefore, the title and much of the conclusions need to be modified so that they reflect the data without speculation. Two inhibitors of DPP4, LAF237 and K-579, were studied. K-579 is not in clinical use. LAF237 is licensed in Europe and is known to exhibit some inhibition of the DPP4-related proteases DPP8 and DPP9. The extent of inhibition of DPP8 and DPP9 by LAF237 is believed not to have physiological effects in humans. The IC50 of LAF237 on DPP9 is less than 0.01 mM. The IC50 of LAF237 on bacterial PI-PLC is 0.1mM, which is close to the lower limit of detection of inhibition of an enzyme. No mammalian homolog of PI-PLC was examined. The literature that the authors cite to suggest that DPP4 inhibition might be detrimental for human health, particularly the pancreas, is data on sitagliptin or exenatide. Exenatide is not a DPP4 inhibitor and sitagliptin is quite different to LAF237, both in protease specificity and in chemical structure. The contact points of LAF237 and sitagliptin in the catalytic site of DPP4 differ considerably. The authors present no data on sitagliptin or any other DPP4 inhibitor (other than LAF237) that in is the clinic. The images of overlaid catalytic triads of various enzymes presented in Fig 1 and Fig 3 need to be depicted in 3D in order to evaluate how close they are in 3D. Intermolecular distances should be shown on thee figures. To convince the reader that LAF237 sits into and makes contacts with enzymes other than DPP4, we need to see the compound docked into the structure of each enzyme of interest.", "responses": [ { "c_id": "1118", "date": "14 Jan 2015", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "We would like to thank you for taking the time to review this paper, and also for your insightful comments. We also apologize for the inordinate time taken to respond to your comments. A lot of this time was spent in understanding docking methods, instead of blindly applying this to the problem at hand. A by-product of this learning process was the implementation of a new method (DOCLASP) for docking molecules to proteins1. We have docked vildagliptin to the PI-PLC structure complexed with myo-inositol using DOCLASP. Based on your suggestion, we have alsodone a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date.Please find out detailed responses to your comments below.The successful targeting of DPP4 using small molecule compounds to treat type 2 diabetes has attracted a great deal of attention towards the study of this protease. The authors applied sophisticated techniques that they have developed in order to discover that two DPP4 inhibitors, including one that is in limited clinical use, can to some extent inhibit the activity of a bacterial lipase (PI-PLC). Many lipases and esterases and hydrolases including DPP4 and related enzymes use the alpha/beta hydrolase fold and the authors show how this related protein topology can place the residues in positions that are sufficiently similar to interact with an inhibitor. The major difficulty with this paper is that it attempts to connect these data with possible clinical outcomes. No evidence for such a link is presented. Therefore, the title and much of the conclusions need to be modified so that they reflect the data without speculation.We have tried to keep away from taking sides on the clinical outcomes, since that is not our forte. Also, we believe our title is innocuous in that context - it just speaks of promiscuous scaffolds. We only highlight that if (and only if) our data of PIPLC inhibition holds true for human lipases, then it might provide some arguing points for those worried about the side effects of these drugs.For example, we say ‘The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here’. If you could kindly point out specifically any speculations that is unwarranted, we will modify those. Two inhibitors of DPP4, LAF237 and K-579, were studied. K-579 is not in clinical use. LAF237 is licensed in Europe and is known to exhibit some inhibition of the DPP4-related proteases DPP8 and DPP9. The extent of inhibition of DPP8 and DPP9 by LAF237 is believed not to have physiological effects in humans.Since this study does not emphasize on the clinical relevance of the inhibitions (but on the methodology of finding such interactions), and we are not a group specializing in diabetes, we believe the choice of the inhibitors would not alter our reasoning our conclusions. The IC50 of LAF237 on DPP9 is less than 0.01 mM. The IC50 of LAF237 on bacterial PI-PLC is 0.1mM, which is close to the lower limit of detection of inhibition of an enzyme.We agree to this point. However, K-579 was inhibiting even at nanomolar concentrations. No mammalian homolog of PI-PLC was examined.We are currently evaluating that possibility. The literature that the authors cite to suggest that DPP4 inhibition might be detrimental for human health, particularly the pancreas, is data on sitagliptin or exenatide. Exenatide is not a DPP4 inhibitor and sitagliptin is quite different to LAF237, both in protease specificity and in chemical structure.We were referring to the inhibitor part of the data, but that point needs to be made explicit as you have correctly pointed out. Also, we agree that the possible difference of sitagliptin with LAF237 needs to be stated. We have modified the text to include these criticisms. Once again, we reiterate we intend not to comment on clinical outcomes or debates, but to suggest a rational methodology to act as a guide for tests that look for possible interactions. The contact points of LAF237 and sitagliptin in the catalytic site of DPP4 differ considerably. The authors present no data on sitagliptin or any other DPP4 inhibitor (other than LAF237) that in is the clinic.We have included a comprehensive study on the contact points of various inhibitors. Once again, this does not negate any of our conclusions. The images of overlaid catalytic triads of various enzymes presented in Fig 1 and Fig 3 need to be depicted in 3D in order to evaluate how close they are in 3D.The 3D images of the superimposition of these enzymes are not pleasing to the eye, since they lack structural homology. However, we have added a PyMol script in case someone wishes to do that (Superimposeproteins.p1m). The script specifies the color coding of the residues. Intermolecular distances should be shown on thee figures.Once again, we think that the intermolecular distances clutter the figure. The superimposition gives an approximate idea of the congruence. The exact values are specified in Table 1. We have modified the legend of Fig.3 to specify that. To convince the reader that LAF237 sits into and makes contacts with enzymes other than DPP4, we need to see the compound docked into the structure of each enzyme of interest.As mentioned previously, we have docked sitagliptin to PI-PLC using DOCLASP1. We have provided the Pymol script as supplementary data to help visualize the docking. There is no solved structure where LAF237 inhibits DPP4.Once again, we are thankful for the comments. We hope that we have addressed your concerns by the changes that we have made, and that the manuscript will be found suitable in the modified form.ReferencesChakraborty S. DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations [v2; ref status: awaiting peer review, http://f1000r.es/4pb] F1000Research 2014, 3:262 (doi: 10.12688/f1000research.5145.2)" } ] } ]	1	https://f1000research.com/articles/2-286
https://f1000research.com/articles/4-31/v1	29 Jan 15	{ "type": "Data Note", "title": "Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.", "authors": [ "Casey M. Bergman", "Penelope R. Haddrill", "Penelope R. Haddrill" ], "abstract": "To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center.", "keywords": [ "Drosophila melanogaster", "Wolbachia pipientis", "population genomics", "population genetics", "pool-seq", "DNA-seq" ], "content": "Introduction\n\nWhole genome shotgun sequences can now be generated easily using short-read sequencing technology for most organisms. Hundreds of resequenced genomes now exist for Drosophila melanogaster that can be used for population and genomic analysis in this model insect species (Lack et al., 2014). To contribute to the worldwide sampling of population genomic data in D. melanogaster, we have sequenced genomes of multiple isofemale lines from three populations collected on different continents reported in Verspoor & Haddrill (2011): Montpellier, France (FR, n=20), Athens, Georgia, USA (GA, n=15) and Accra, Ghana (GH, n=15). Pools of these same isofemale lines were also sequenced to be able compare results based on strain-specific sequencing to pooled sequencing. Strains sequenced here were chosen because isofemale lines exist in the Drosophila Species Stock Center and because their infection status for the Wolbachia pipientis bacterial endosymbiont had previously been determined (Verspoor & Haddrill, 2011).\n\n\nMaterials and methods\n\nIsofemale strains were selected randomly from the full population samples reported in Verspoor & Haddrill (2011). Genomic DNA for isofemale lines was prepared by snap freezing females in liquid nitrogen, then extracting DNA using a standard phenol-chloroform extraction protocol with ethanol and ammonium acetate precipitation. DNA samples were generated for each isofemale lines using 50, 25, and 25 adult females for the FR, GA and GH populations, respectively.\n\nFor pooled samples, single adult females from each isofemale line were used to construct two samples for each population. The first pooled sample contains one fly from each of the same strains that were sequenced as isofemale lines (FR_pool_20, GA_pool_15, GH_pool_15). The second pooled sample contains one fly from all isofemale lines sampled for each population reported in Verspoor & Haddrill (2011) (FR_pool_39, GA_pool_30, GH_pool_32).\n\n500 bp short-insert libraries using the Illumina Paired-End Sample Prep Kit (Part # 1005063) were constructed and 90 bp paired-end reads were generated using an Illumina HiSeq 2000 to an estimated coverage of ~50× per strain by BGI-Hong Kong. Forty-one samples were sequenced in single lanes shared typically with two other samples on a single run and 15 samples were sequenced using the same layout on two runs, generating 71 pairs of fastq files for the 56 samples. Data were generated over a total of seven sequencing runs. Raw data was filtered by BGI to remove read pairs where either read contained adapters or greater than 50% of bases with a quality value <= 5. No other trimming or filtering of the raw data was performed prior to submission using original filenames provided by BGI to the European Nucleotide Archive.\n\n\nDataset validation\n\nTo validate the quality of the raw sequence data, forward and reverse reads were analyzed using fastQC (version 0.11.2) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Forward and reverse read files for all runs had PASS status for most fastQC statistics. Per base sequence quality gave FAIL status for forward or reverse read files for all of the GA samples (which were sequenced together on one run) because of poor quality scores in the terminal 1–5 bp of the read. These poor quality termini can be easily trimmed and do not affect mappability, as the percent of reads mapped for these runs is very high (see Dataset 1).\n\nTo validate that the majority of the DNA sequenced is from the focal organism(s), untrimmed reads for each sample were mapped in paired-end mode using Bowtie (version 2.2.4) (Langmead & Salzberg, 2012) with default options to a “hologenome” reference generated by concatenating genome sequences for D. melanogaster (Genbank accession GCA_000001215.4) (Hoskins et al., 2015) and W. pipientis (Genbank accession AE017196) (Wu et al., 2004). Mapping to a hologenome was performed since many of these strains are known to be infected with Wolbachia (Verspoor & Haddrill, 2011). Unfiltered BAM files were used to estimate the proportion of reads in each sample that mapped to the expected target organisms using samtools flastat (version 0.1.19-44428cd) (Li et al., 2009). Greater than 96.8% of all reads in each run were mapped to the hologenome reference, indicating low levels of contaminating DNA in these data (Dataset 1).\n\nMapping to a hologenome also allowed us to verify if strain or sample swaps occurred in the process of producing these genome sequences by comparing predicted Wolbachia infection status with previously determined PCR-based infection status (Verspoor & Haddrill, 2011). Wolbachia infection status was predicted from genome sequences for each strain following a modified protocol from Richardson et al. (2012). Briefly, strains were predicted as \"infected\" when breadth of mapped read coverage was greater than 90% of the Wolbachia genome and mean depth of coverage was greater than one. Here, we compute breadth of coverage directly from the bedtools genomecov (version v2.22.0) (Quinlan & Hall, 2010) output rather than from a consensus sequence, as was done previously by Richardson et al. (2012). Predicted Wolbachia infection status matched experimentally determined infection status for 55/56 samples (98.2% concordance), indicating that strain or sample swaps are unlikely to have occurred during the generation of this dataset (Dataset 1). The only exception observed was for line GA08 from the Georgia population, which the WGS data indicates is infected while PCR data indicates it is uninfected. This observation can be explained by either PCR amplification failure for the GA08 stock in Verspoor & Haddrill (2011) or infection of the GA08 stock after data collection for Verspoor & Haddrill (2011). Further analysis of the Wolbachia infection status of this stock is warranted prior to use.\n\n\nData availability\n\nRaw sequence data for the 56 samples reported here can be found in the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center (https://stockcenter.ucsd.edu) under accessions 14021-0231.139, 14021-0231.140, 14021-0231.141, 14021-0231.142, 14021-0231.143, 14021-0231.144, 14021-0231.145, 14021-0231.146, 14021-0231.147, 14021-0231.148, 14021-0231.149, 14021-0231.150, 14021-0231.151, 14021-0231.152, 14021-0231.153, 14021-0231.154, 14021-0231.155, 14021-0231.156, 14021-0231.157, 14021-0231.158, 14021-0231.183, 14021-0231.184, 14021-0231.185, 14021-0231.186, 14021-0231.187, 14021-0231.188, 14021-0231.189, 14021-0231.190, 14021-0231.191, 14021-0231.192, 14021-0231.193, 14021-0231.194, 14021-0231.195, 14021-0231.196, 14021-0231.197, 14021-0231.163, 14021-0231.164, 14021-0231.165, 14021-0231.166, 14021-0231.167, 14021-0231.168, 14021-0231.170, 14021-0231.172, 14021-0231.174, 14021-0231.176, 14021-0231.177, 14021-0231.178, 14021-0231.180, 14021-0231.181 and 14021-0231.182..\n\nDescriptive statistics for validation of each run can be found in Dataset 1, DOI: 10.5256/f1000research.6090.d42636 (Bergman & Haddrill, 2014).", "appendix": "Author contributions\n\n\n\nCMB and PRH conceived the study. CMB and PRH designed the experiments. PRH conducted the experiments. CMB conducted the data analysis. CMB prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Human Frontier Science Program Young Investigator grant RGY0093/2012 to CMB and National Environmental Research Council grant NE/G013195/1 to PRH.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank BGI-Hong Kong for assistance with genome sequencing and initial data quality control analysis and Daniel Halligan for assistance with data management.\n\n\nReferences\n\nBergman CM, Haddrill PR: Dataset 1 in “Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents”. F1000Research. 2014. Data Source\n\nHoskins RA, Carlson JW, Wan KH, et al.: The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 2015. PubMed Abstract \| Publisher Full Text\n\nLack J, Cardeno C, Crepeau M, et al.: The Drosophila Genome Nexus: a population genomic resource of 623 Drosophila melanogaster genomes, including 197 genomes from a single ancestral range population. Genetics. 2015. Publisher Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4): 357–359. 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\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\nRichardson MF, Weinert LA, Welch JJ, et al.: Population genomics of the Wolbachia endosymbiont in Drosophila melanogaster. PLoS Genet. 2012; 8(12): e1003129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerspoor RL, Haddrill PR: Genetic diversity, population structure and Wolbachia infection status in a worldwide sample of Drosophila melanogaster and D. simulans populations. PLoS One. 2011; 6(10): e26318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu M, Sun LV, Vamathevan J, et al.: Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2004; 2(3): E69. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "7534", "date": "03 Feb 2015", "name": "John E. Pool", "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\nThe authors' data will add value to Drosophila population genomic resources. I see no technical flaws in the manuscript. If the authors see fit, they could a bit more context to the data. For example, they could note that a mosaic of homozygous and heterozygous regions may be expected from the isofemale line genomes. Optionally, they could also briefly put these three populations in historical context (i.e. that the species originated from sub-Saharan Africa but perhaps not western Africa specifically, that it expanded out of sub-Saharan Africa with a population bottleneck, and that North American populations are thought to have both European and African ancestry). The France and Ghana samples sequenced here may prove useful for identifying population ancestry in North American and other admixed populations. Trivial edits:Methods paragraph 1:“each isofemale lines” (delete final “s”)References - from title of Lack et al. 2015, delete second “genomes”. Update precise author information.", "responses": [] }, { "id": "7634", "date": "18 Feb 2015", "name": "Ian Dworkin", "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\nThis article primarily summaries the generation of a large set of resequenced Drosophila strains from three populations (Ghana, France and the US). Sequencing was done both individually for each isofemale strain, as well as in sequenced pools for each of three populations. While the primary goal of this research appears to be to provide the community with these additional genomic resources, the researchers were also particularly interested in examining Wolbachia infection status in the strains. Given that all raw data has been made available, it is likely that will provide an important useful resource for genomic analyses.A few minor comments:Some comparison of mapping quality for the pooled sequences (as compared to the individual isofemale strains) would have been useful.Some explanation as to why the number of individuals used for the three different sequencing pools differed would have also been helpful to understand the provenance of the data.", "responses": [] }, { "id": "7868", "date": "06 Mar 2015", "name": "Sergey Nuzhdin", "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\nThe authors have presented a succinct, but detailed description of sequence data for several populations that will certainly be useful to the Drosophila community. I see no major flaws in the manuscript. However, as a minor suggestion, it may be useful for readers if the authors update their Introduction to not only place the populations in the context of migration history, but perhaps to also briefly list the geographical areas covered by other sequence resources to clearly illustrate how their dataset adds onto the currently available resources.", "responses": [] } ]	1	https://f1000research.com/articles/4-31
https://f1000research.com/articles/4-29/v1	28 Jan 15	{ "type": "Research Article", "title": "Regulation of ryanodine receptor RyR2 by protein-protein interactions: prediction of a PKA binding site on the N-terminal domain of RyR2 and its relation to disease causing mutations", "authors": [ "Belinda Nazan Walpoth", "Burak Erman", "Belinda Nazan Walpoth" ], "abstract": "Protein-protein interactions are the key processes responsible for signaling and function in complex networks. Determining the correct binding partners and predicting the ligand binding sites in the absence of experimental data require predictive models. Hybrid models that combine quantitative atomistic calculations with statistical thermodynamics formulations are valuable tools for bioinformatics predictions. We present a hybrid prediction and analysis model for determining putative binding partners and interpreting the resulting correlations in the yet functionally uncharacterized interactions of the ryanodine RyR2 N-terminal domain. Using extensive docking calculations and libraries of hexameric peptides generated from regulator proteins of the RyR2 channel, we show that the residues 318-323 of protein kinase A, PKA, have a very high affinity for the N-terminal of RyR2. Using a coarse grained Elastic Net Model, we show that the binding site lies at the end of a pathway of evolutionarily conserved residues in RyR2. The two disease causing mutations are also on this path. The program for the prediction of the energetically responsive residues by the Elastic Net Model is freely available on request from the corresponding author.", "keywords": [ "protein-protein interactions", "protein signaling", "protein function", "ryanodine receptor RyR2", "Protein Kinase A", "ligand docking", "elastic net model", "evolutionarily conserved residues", "disease causing mutations" ], "content": "Introduction\n\nRyanodine receptors are large protein complexes consisting of approximately 5000 residues that form calcium channels that mediate the release of calcium from the sarcoplasmic reticulum, SR, to the cytosol, which is essential for muscle and cardiac rhythm and contractility. There are three forms of ryanodine receptors, RyR1, RyR2 and RyR3. RyR1 is the channel in the skeletal muscle, RyR2 is the type expressed in the heart muscle, and RyR3 is found predominantly in the brain1. The present paper focuses on RyR2. Ca++ release from the SR mediated by RyR2 is a fundamental event in cardiac muscle contraction. These receptors form a group of four homotetramers, with a large cytoplasmic assembly and a transmembrane domain called the pore region. The tridimensional structure of the full assembly is known from cryo-electron microscope studies2 with limited precision. However, the crystal structures of the first 520 amino acids of the N-terminal domain of RyR1 and the first 217 amino acids of the N-terminal domain of the wild type RyR2 and its mutated form are determined with high precision by van Petegem and collaborators3. The main mass of the receptor with dimensions of ca. 280 × 280 × 120 Å is located in the cytoplasmic region, with a stalklike transmembrane region2. The full shape of the channel and the N-terminal are shown in Figure 1.\n\nThe N-terminal region is indicated. The ribbon diagram of the first 217 amino acids of the N-terminal domain is given in the right panel.\n\nThe cytoplasmic region consists of more than 10 sub-domains that are responsible for the functioning of the receptor through binding to several modulator proteins and ligands4. The modulators include cyclic AMP and protein kinase A (PKA)4, calmodulin5, FKBP12.6 (Calstabin2)6, phosphatases 1 and 2A (PP1 and PP2A)7, sorcin8, and triadin, junctin and calsequestrin9, and several others. Among these, cyclic AMP activates PKA, which in turn phosphorylates RyR2 at SER2809 and SER2815. Despite the important role of the channel, the binding sites of the modulators on the channel are known only approximately. Calmodulin binds to residues located between the positions 3611 and 3642, FKBP12.6 binds to residues around the positions 2361–2496, PP1 around 513 and 808, PP2A around 1451 and 1768, sorcin, triadin, junctin and calsequestrin bind to the vicinity of the transmembrane domain7.\n\nFKBP12.6 binds to RyR2 with a stoichiometry of four FKBP12.6 molecules per single RyR2 channel complex. Binding of FKBP12.6 to RyR2 is required to keep the receptor closed during diastole. In addition to stabilizing individual RyR channels, FKBP12.6 is also required for coupled opening and closing between RyRs. Dissociation of FKBP12.6 from coupled RyR2 channels results in functional uncoupling of the channels leading to heart failure4. Overphosphorylation of RyR2 leads to dissociation of the regulatory protein FKBP12.6 from the channel, resulting in disease7 exhibited as arrhythmias with abnormal diastolic SR Ca++ release. Uncontrolled Ca++ release during the diastole when cytosolic Ca++ concentrations are low can cause delayed after-depolarizations (DADs) which can then lead to fatal arrhythmias. These abnormalities are linked to mutations in the RyR2, located on chromosome 1q42.1–q4310, which lead to familial polymorphic ventricular tachycardia, CPVT, and arrhythmogenic right ventricular dysplasia type 2, ARVD/C. More than 300 point mutations have been identified in RyR2, some of which are associated with the disorders observed clinically11. In this respect, the N-terminal domain of RyR2, which is known to form an allosteric structure, contains several disease-causing mutations. However, there is yet no information on the mechanisms of the mutations that lead to disease and on the role of these mutations on modulator binding.\n\nNone of the modulators discussed above, except PKA, bind to the N-terminal domain. PKA phosphorylates Ser2809 and Ser2815, and it has to anchor to nearby regions of the two serines. PKAs are known to anchor to their hosts at points other than the catalytic domains12. In this study, we generated a hexameric peptide library from the PKA and docked these on several points on the surface of the RyR2 N-terminal. Calculations showed that the hexapeptide PHE LYS GLY PRO GLY ASP from the unstructured C-terminal region of PKA binds to RyR2 with very high affinity, with a dissociation constant of 5.5 nM. For brevity, we will refer to this hexapeptide as the ‘ligand’ and represent it in single letter convention as FKGPGD.\n\nIn the last part of the paper, using a coarse grained Elastic Network Model13, we show that the binding site of the ligand lies on a path of energy responsive residues. Energy responsiveness of a residue is defined in terms of correlated fluctuations of that residue with others in the protein. In allosteric proteins, a path of highly correlated residues exists and plays crucial role in energy and signal transfer13a,14. In RyR2 we identify such a path of highly correlated residues which contains most of the evolutionarily conserved residues. The path also contains the known two disease causing mutations, A77V and R176Q.\n\n\nMaterials and methods\n\nWe used the commercial software Gold15 for docking the peptides to the surface of RyR2. The PKA chain (PDB code 2JDV) of 336 amino acids is partitioned into a library of 331 overlapping hexapeptides, such that the first peptide consists of the first six residues 1–6, the second of 2–7, and so on. Several binding sites are chosen on the surface of RyR2 as discussed below. A radius of 20 Å is used for docking. The GoldScore force field is used with rescoring on ChemScore. Flexible docking is used in the first round of calculations. Peptides with reasonable docking energies are chosen after the first run, and a more thorough and extensive docking is performed over this smaller subset. Additional calculations are made with hexapeptide libraries obtained from modulators of RyR2 that are known not to bind at the N-terminal domain as a partial check of the reliability of the method. Optimum binding is obtained for the hexapeptide FKGPGD from the residues 318–323 of 2JDV. The binding energy is obtained as -49 kJ/mol, which is significantly stronger than those of all other investigated hexapeptides. This binding energy corresponds to a dissociation constant kD = exp(ΔA/kT) of 5.5 nM.\n\nOur algorithm for the Elastic Net Model uses Cα based coarse graining which evaluates correlations between thermal fluctuations ΔR̭i and ΔR̭j in the position of residues i and j. On average, a residue has about eight to 12 neighboring residues to directly interact with. These fluctuation-based interactions are assumed harmonic as if the residues are connected by linear springs. Fluctuations in the distance between two neighboring residues induce changes in their interaction energy. Two residues are assumed neighbors in space if they are closer to each other than a given cutoff distance. This distance corresponds to the radius of the first coordination shell around a given residue, and is usually thought to be between 6.5–7.0 Å. Every pair of residues closer to each other than the cutoff distance is assumed to be connected by a linear spring. The knowledge of the tridimensional structure of the protein that has n residues allows us to write a connectivity matrix, C, where the rows and the columns identify the residue indices, from 1 to n, where the amino-end is the starting and the carboxyl-end is the terminating-end of the protein. If two residues i and j are within the cutoff distance, then Cij = 1, otherwise it is zero. Another matrix, Γij, is obtained from the connectivity matrix as\n\n\n\nWhere γ is the spring constant of the harmonic interactions. The relationship of the forces to the displacements is given by the equation ΔFi = Σj ΓijΔRj. Techniques of statistical mechanics allow us to derive several relationships between the fluctuations of residues16. The correlation between the fluctuations of residues i and j is related, for example, to the inverse of the matrix Γij as\n\n\n\nHere, the angular brackets denote the time average of the product of fluctuations of residues i and j, kB is the Boltzmann constant, T is the physiological temperature expressed in Kelvin scale, Γ-1 is the inverse matrix Γ, and its subscripts i and j acknowledge the residue indices of interest. If i = j, then Equation 2 becomes\n\n\n\nThe left hand side of Equation 3 is the mean-square fluctuations of the i’th residue which is related to experimentally available B-factors, Bi, through the equation\n\n\n\nThe mean-square fluctuations 〈(ΔRij)2〉 of the distance ΔRij between residues i and j are obtained from Equation 3 as\n\n\n\nThe derivation of Equation 5 is given in the reference17. Calculations presented therein showed that the largest few eigenvalues of Γ give information at the residue level. Smaller eigenvalues are associated with global motions. Our calculations showed that the largest five eigenvalues and the corresponding eigenvectors are satisfactory for representing fluctuations at the residue level.\n\nFluctuations of the harmonic energy between two residues are proportional to the mean square fluctuations of the distance between the two. Thus, Equation 5 is representative of energy fluctuations, and summing over all the neighbors of the residue i shows the energy response ΔUi of residue i with its surroundings:\n\n\n\nThis is a thermodynamically meaningful quantity showing the mean energy response of residue i to all fluctuations of its surroundings. These correlations extend throughout the protein, leading to specific paths along which the fluctuations propagate. Recent work shows that these paths are evolutionarily conserved14a.\n\nThe N-terminal domain of RyR2 is a signal protein of 217 amino acids. The crystal structure of the N-terminal domain of physiological RyR2 (PDB code 3IM5) and the A77V mutated crystal structure (PDB code 3IM7) have been determined by x-ray with resolutions of 2.5 and 2.2 Å, respectively, by Van Petegem and Lobo3a. The protein consists of a β-trefoil of 12 β strands held together by hydrophobic forces. A 10-residue α helix is packed against strands β4 and β5. A 3 residue 3–10 helix is present in the loop containing β3 and β4. The N-terminal contains two MIR domains, similar to the inositol 1,4,5-triphosphate receptor (IP3R), for which ligand-induced conformational changes have been studied more extensively18.\n\n\nResults and discussion\n\nThe binding free energy of FKGPGD to the surface shown in Figure 2 is obtained as -49 kJ/mol by the ChemScore potential, which corresponds to a dissociation constant of 5.5 nM. The 42% of the binding energy comes from hydrogen bonds and 39% from lipophilic interactions. The dissociation constant of 5.5 nM is at least two orders of magnitude better than the values obtained for the other hexapeptides of the library. It is therefore highly likely that PKA anchors itself on RyR2 at the position shown.\n\nResidues with which it forms hydrogen bonds are shown in yellow wire, and labeled. The two disease causing mutation residues, ALA77 and ARG176 are shown in yellow CPK.\n\nIn order to interpret the binding of the PKA on RyR2, we performed elastic net analysis of energetically responsive residues of RyR2. The residues that yield high values of the energy response defined by Equation 6 are calculated according to the scheme outlined in the Methods section. In Figure 3, the mean energy response ΔUi of residue i is presented along the ordinate as a function of residue index. The circles indicate the highest conserved residues of 3IM5, obtained from the work of Goldenberg et al. (See also the PDBSum web site22).\n\nIn Reference 20, conservation levels are ordered from 1 to 8, the latter being the highest degree of conservation. The filled circles correspond to residues with level 8. The ordinate values are in arbitrary un-normalized units.\n\nComparison of the solid curve peaks and the circles shows that there is a strong correlation between the energy responsive and conserved residues, in agreement with the recent suggestion of Lockless and Ranganathan14a. The set of conserved residues, with the highest level of conservation according to Reference 20 of the protein, all lie within the set of energetically responsive residues and are located along or in the neighborhood of the path obtained from the energetically responsive residues. On the three-dimensional structure of the protein, the peaks shown in Figure 3 constitute a path of residues that are spatial neighbors.\n\nA residue or set of residues at the surface of the protein which are energy responsive are expected to be the hotspots for binding, because these residues can exchange energy with the surroundings, and distribute the energy taken from the surroundings to the other residues of the protein. According to this conjecture, one needs to dock ligands only to the hotspots identified with the peaks in Figure 3. In our calculations, we adopted five such hotspot regions for docking. These hotspot regions are centered at: (1) VAL21, (2) VAL68, (3) ARG122, (4) SER185, and (5) ALA205. In the complex structure of the channel, some of these five surface regions may not be exposed to ligands but may be facing the other domains of the channel. However, a residue that neighbors another domain may become exposed to a ligand upon opening of the channel. We carried out the calculations for the five regions stated above, irrespective of their neighborhood.\n\nIn Figure 4, we show, in stick form, the evolutionarily highly conserved residues that lie along a path between ALA77, ARG176 and the ligand FKGPGD of PKA. The peak residues clearly form a path between the ligand and the mutation residues. The path shown in the figure contains the energetically responsive residues predicted by the GNM as may be seen from Figure 3. Using extensive docking calculations and libraries of residues obtained from regulator proteins of the RyR2 channel, we showed that residues 318–323 of PKA have a very high affinity for the N-terminal of RyR2. The location of binding is a pocket bordered by GLU171 and GLU189. GLU171 is a conserved residue and participates in calcium binding in inositol 3 receptors, IP3R. However, a ligand for RyR2 at GLU171 is not yet known. We also showed that the disease causing mutations ALA77VAL and ARG176GLN are joined by an energy interaction pathway to the ligand binding surface. Although these two mutations are responsible for arrhythmias, their exact mechanism is not known. The present model directs attention to the relationship between the residues at the binding site, the predicted path of energy responsive residues and the two disease causing mutation sites. Since binding of PKA to RyR2 results in phosphorylation of the latter, and since hyperphosphorylation leads to disease, one may indirectly conjecture that mutations in the two residues modify the binding characteristics of PKA.\n\nThe residues shown in stick form are conserved residues which are also identified by the peaks in Figure 3. The hexamer ligand is shown in ball and stick form.\n\nSuperposition of the three dimensional PDB structures of PKA and RyR2 in such a way that the residues FKGPGD of PKA are kept in the bound state gives the relative orientations of the two proteins. This is shown in Figure 5.\n\nThe sequence FKGPGD of PKA is shown in ball and stick form.\n\nUsing the Elastic Net Model, we identified the energy conduction pathway for the wild type RyR2. This path whose residues are shown in Figure 3 has several features of interest. Firstly, it contains most of the evolutionarily conserved residues. The remaining conserved residues are in the close neighborhood of the path, all making hydrogen bonds with the residues of the path. This important feature has recently been shown by Lockless and Ranganathan14a implying that evolutionary conservation is driven by energy conduction in proteins. Although no ligands for the RyR2 N-terminal have been observed until now19, the three glutamic acids, GLU171, GLU173 and GLU189 at the pocket may potentially form a binding site. This suggestion is also based on the observation that in IP3R a potential calcium binding site is formed by GLU283 and GLU285 whose location on the path coincides exactly with that of RyR2. The residue GLU173 is exposed to water and is a candidate for possible binding.\n\nThe underlying determinant of the allosteric pathway, defined as the path of energy responsive residues in the present paper, is the graph structure of the protein20. The view that proteins relay signals by energy fluctuations in response to inputs, have been recently discussed in an elegant paper by Smock and Gierasch14b. In the present study, we showed that evolutionarily conserved residues lie on the pathway of energy responsive residues. RyR2 contains two interspersed MIR domains, residues 124–178 and 164–21721. Elastic net calculations show that the residues that lie on the energy conduction pathway are closely associated with these MIR domains: the energy responsive residues either lie on the MIR domains, or they are hydrogen bonded to the residues of these domains. There is no energetically responsive residue that is not closely associated with the MIR domain. We therefore conclude that the MIR domains of RyR2 play an active role in energy transport through the protein.\n\n\nData availability\n\nData of predicted PKA binding sites on RyR223.", "appendix": "Author contributions\n\n\n\nBoth authors contributed equally to the present study.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful for the suggestions of Professor Filip van Petegem for insightful suggestions which have been incorporated into the final version of the manuscript.\n\n\nReferences\n\nKimlicka L, Van Petegem F: The structural biology of ryanodine receptors. Sci China Life Sci. 2011; 54(8): 712–724. PubMed Abstract \| Publisher Full Text\n\nHamilton SL, Serysheva II: Ryanodine receptor structure: progress and challenges. J Biol Chem. 2009; 284(7): 4047–4051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLobo PA, Van Petegem F: Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. Structure. 2009; 17(11): 1505–1514. PubMed Abstract \| Publisher Full Text\n\nLobo PA, Kimlicka L, Tung CC, et al.: The deletion of exon 3 in the cardiac ryanodine receptor is rescued by β strand switching. Structure. 2011; 19(6): 790–798. PubMed Abstract \| Publisher Full Text\n\nTung CC, Lobo PA, Kimlicka L, et al.: The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature. 2010; 468(7323): 585–588. PubMed Abstract \| Publisher Full Text\n\nWehrens XH, Marks AR: Altered function and regulation of cardiac ryanodine receptors in cardiac disease. Trends Biochem Sci. 2003; 28(12): 671–678. PubMed Abstract \| Publisher Full Text\n\nFruen BR, Bardy JM, Byrem TM, et al.: Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin. Am J Physiol Cell Physiol. 2000; 279(3): C724–C733. PubMed Abstract\n\nMarx SO, Gaburjakova J, Gaburjakova M, et al.: Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ Res. 2001; 88(11): 1151–1158. PubMed Abstract \| Publisher Full Text\n\nBrillantes AB, Ondrias K, Scott A, et al.: Stabilization of calcium-release channel (ryanodine receptor) function by Fk506-binding protein. Cell. 1994; 77(4): 513–523. PubMed Abstract \| Publisher Full Text\n\nMarx SO, Reiken S, Hisamatsu Y, et al.: PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101(4): 365–376. PubMed Abstract \| Publisher Full Text\n\nMeyers MB, Pickel VM, Sheu SS, et al.: Association of sorcin with the cardiac ryanodine receptor. J Biol Chem. 1995; 270(44): 26411–26418. PubMed Abstract \| Publisher Full Text\n\nZhang L, Kelley J, Schmeisser G, et al.: Complex formation between junction, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem. 1997; 272(37): 23389–23397. PubMed Abstract \| Publisher Full Text\n\nWang J, Prakasa K, Bomma C, et al.: Comparison of novel echocardiographic parameters of right ventricular function with ejection fraction by cardiac magnetic resonance. J Am Soc Echocardiogr. 2007; 20(9): 1058–1064. PubMed Abstract \| Publisher Full Text\n\nKimlicka L, Van Petegem F: The structural biology of ryanodine receptors. Sci China Life Sci. 2011; 54(8): 712–724. PubMed Abstract \| Publisher Full Text\n\nBetzenhauser MJ, Marks AR: Ryanodine receptor channelopathies. Pflugers Arch. 2010; 460(2): 467–480. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPriori SG, Chen SR: Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis. Circ Res. 2011; 108(7): 871–883. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas NL, Maxwell C, Mukherjee S, et al.: Ryanodine receptor mutations in arrhythmia: The continuing mystery of channel dysfunction. FEBS Lett. 2010; 584(10): 2153–2160. PubMed Abstract \| Publisher Full Text\n\nHuang LJ, Durick K, Weiner JA, et al.: Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits. J Biol Chem. 1997; 272(12): 8057–8064. PubMed Abstract \| Publisher Full Text\n\nErman B: Relationships between ligand binding sites, protein architecture and correlated paths of energy and conformational ﬂuctuations. Phys Biol. 2011; 8(5): 056003. PubMed Abstract \| Publisher Full Text\n\nHaliloglu T, Erman B: Analysis of correlations between energy and residue fluctuations in native proteins and determination of specific sites for binding. Phys Rev Lett. 2009; 102(8): 088103. PubMed Abstract \| Publisher Full Text\n\nHaliloglu T, Seyrek E, Erman B: Prediction of binding sites in receptor-ligand complexes with the Gaussian Network Model. Phys Rev Lett. 2008; 100(22): 228102. PubMed Abstract \| Publisher Full Text\n\nLockless SW, Ranganathan R: Evolutionarily conserved pathways of energetic connectivity in protein families. Science. 1999; 286(5438): 295–299. PubMed Abstract \| Publisher Full Text\n\nSmock RG, Gierasch LM: Sending signals dynamically. Science. 2009; 324(5924): 198–203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttp://www.ccdc.cam.ac.uk/products/life_sciences/gold/\n\nHaliloglu T, Erman B: Analysis of correlations between energy and residue fluctuations in native proteins and determination of specific sites for binding. Phys Rev Lett. 2009; 102(8): 088103–088106. PubMed Abstract \| Publisher Full Text\n\nYogurtcu ON, Gur M, Erman B: Statistical thermodynamics of residue fluctuations in native proteins. J Chem Phys. 2009; 130(9): 095103–13. PubMed Abstract \| Publisher Full Text\n\nHaliloglu T, Gul A, Erman B: Predicting important residues and interaction pathways in proteins using Gaussian Network Model: binding and stability of HLA proteins. PLoS Comput Biol. 2010; 6(7): e1000845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuzmen C, Erman B: Identification of ligand binding sites of proteins using the Gaussian Network Model. PLoS One. 2011; 6(1): e16474. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin CC, Baek K, Lu Z: Apo and InsP3-bound crystal structures of the ligand-binding domain of an InsP3 receptor. Nat Struct Mol Biol. 2011; 18(10): 1172–1174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuchi Z, Van Petegem F: Common allosteric mechanisms between ryanodine and inositol-1,4,5-trisphosphate receptors. Channels (Austin). 2011; 5(2): 120–123. PubMed Abstract \| Publisher Full Text\n\nvan Petegem F: Private correspondence.\n\nRader AJ, Brown SM: Correlating allostery with rigidity. Mol Biosyst. 2011; 7(2): 464–471. PubMed Abstract \| Publisher Full Text\n\nAmador FJ, Liu S, Ishiyama N, et al.: Crystal structure of type 1 ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation “hot spot” loop. Proc Natl Acad Sci U S A. 2009; 106(27): 11040–11044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPDBSUM. Reference Source\n\nErman B, Walpoth BN, Erman B: Data of predicted PKA binding sites on RyR2. figshare. 2015. Data Source" }	[ { "id": "7520", "date": "04 Feb 2015", "name": "Xiayang Qiu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors first performed computational docking studies to suggest that a hexapeptide of PKA has a high-affinity binding to the N-terminal domain of RyR2. Then, based on the elastic network model calculations, the authors argued that the putative PKA binding site on the N-terminal domain of RyR2 lies at the end of an energy conduction pathway of conserved residues. While such insights would be very important for understanding the biology of RyR2, there are a few serious points needed to be addressed prior to indexing, mostly due to the lack of experimental data to sustain the authors' claims. The author had made the assumption that the binding epitope of PKA would be a linear peptide. Why wouldn't it be a 3D structural binding epitope with contributions from residues close in space but not close in sequence? The author suggested that the hexapeptide would bind to the N-terminal domain of RyR2 with an affinity of 5.5nM, which is quite high given the relatively small and flat interface. Considering the interaction is more likely to be transient than permanent in vivo, the authors need to provide more evidences or references to either support the argument or soften the affinity claim as a theoretical prediction or hypothesis. If the hexapeptide, FKGPGD, is indeed showing such a high affinity to the N-terminal domain of RyR2 how unique is such a hexapeptide among all proteins? Would other RyR2 modular proteins share a similar sequence? Or would other proteins sharing such a hexapeptide modulate the function of RyR2? Regarding the phosphorylation sites by PKA, based on a number of references (For example, UniProt database http://www.uniprot.org/uniprot/Q92736), RyR2 is phosphorylated by PKA at two major sites, Ser2031 (Ser2030 in certain publications) and Ser2808 (Ser2809 in certain publications). On the other hand, Ser2814 (Ser2815 in certain publications) is mainly phosphorylated by CaMK2D. This would be consistent with the fact that PKA phosphorylates proteins that have the motif Arg-Arg-X-Ser exposed. In the manuscript, the authors suggested that PKA phosphorylates RyR2 at Ser2809 and Ser2815; however, Ser2815 does not seem to fit such a recognition pattern of PKA. Since experimentally PKA is known to phosphorylate RyR2 at two different sites, does such a high-affinity anchor model fit in the experimental observations? Based on this, we suggest the author re-visit the manuscript to either provide some kind of experimental evidence, or tone down some of the biological implications - focusing rather on the computation approaches and the intriguing results thereof that may or may not be true in the end.", "responses": [] }, { "id": "8042", "date": "19 Mar 2015", "name": "Ugo Bastolla", "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\nThe Ryanodine Receptor RyR2 is a key transporter of calcium in the heart, and some of its mutations are associated with fatal arrhythmias. In this paper, RyR2 is studied through docking and elastic network model (ENM) computations. The authors find that a hexa-peptide of the PKA kinase, a known regulator of RyR2, has a very high affinity for RyR2, in the nano-molecular range, and that its binding site is connected to two known disease-associated residues (A77 and R176) through a “path” of residues predicted to be “energy responsive” through ENM calculations. Furthermore, energy responsive residues are strongly conserved.I think that the paper is very interesting, although some aspects of its presentation have to be improved, in particular concerning the interesting theoretical part on energy responsiveness.I tend to agree with the other reviewer that such a large affinity between the PKA kinase and RyR2 is strange, since kinase interactions are expected to be transient. However, I think that it is unlikely that such a high affinity arises by chance. If we assume that it is correct, it may suggest a molecular mechanism for the disease, as follows. After phosphorylation, the kinase has to dissociate, which would be very difficult given the high affinity, unless a conformation change takes place upon phosphorylation and triggers the release of PKA. This mechanism may be consistent with the network of highly dynamic residues predicted by the ENM, which connects the binding site and the disease-associated site. If the mutations perturb the dynamics of RyR2, making the release of the kinase more difficult, they would result in overphosphorylation, which is the known signature of the disease. It would be interesting to investigate this hypothesis through ENM (and/or MD) computations that try to connect the phosphorylation site and the binding site of the kinase through a signaling pathway and test whether this pathway passes through the disease associated residues, as the computations presented here may suggest.Concerning the presentation, the result that energy-responsive residues are strongly conserved is very interesting. The authors relate it with the influential proposal by Lockless and Raganathan (LR) that pairs of residues energetically coupled are evolutionarily coupled as well (actually this field of correlated mutations is quite older than the LR article and it recently experienced an explosion of interest), but the LR proposal is not exactly the same as what is found here, since in this paper conservation of individual residues rather than evolutionarily correlations are observed. Thus, it would be interesting to study whether this correlation between responsiveness and conservation is general, or it is a special property of RyR2 and maybe other strongly allosteric proteins. Of course I do not require that the authors do this study in this paper, but it would be useful that they share their thoughts on this point. Since the responsiveness of a residues must be correlated with its intrinsic fluctuations (predicted B factors), it would be interesting to known whether the former or the latter correlates more strongly with conservation.Furthermore, the authors expect that responsive residues are hotspots of binding. This expectation does not seem so natural to me. Actually, I expected almost the opposite: two residues i and j that bind the same molecule must have small fluctuations of their interatomic distance <(DeltaR_ij)^2>, so that I expected that residues in a binding site must be characterized by <(DeltaR_ij)^2> smaller than generic residues. I tested this expectation some time ago, finding that it is true on the average but much weaker than I expected. Thus, the fact that responsive residues are close to binding sites can be interpreted either as a general property or as a special property of allosteric proteins in which binding and conformation changes are related. It would be interesting to discuss it.Finally, although the paper is very well written, there are some small typos in the equations: 1) In Eq.(1) the factor gamma has to appear in the second line as well; 2) Temperature has disappeared from Eq.(5) ; 3) between Eq.(5) and (6), it is said that the five largest eigenvalues are enough for representing fluctuations, I think that the authors mean “smallest eigenvalues”, and it would be useful to say that the number five holds for RyR2, other proteins may have a different number of relevant modes", "responses": [] }, { "id": "8179", "date": "01 Apr 2015", "name": "Pemra Doruker", "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\nThis is an interesting paper that utilizes alternative methodologies for protein-protein docking. Especially determination of energy responsive residues- hotspots for binding- based on elastic network model deserves attention as it has been previously assessed in terms of ligand binding by Tuzmen and Erman (2011). However, the peptide docking procedure, which has detected a six-residue long peptide ‘FKGPGD’ located on an unstructured loop, is a non-standard approach as far as to my knowledge. The authors need to discuss why they have preferred such a technique for their problem and specifically used six-residue peptides. More details on the docking procedure/steps and efficiency would also be helpful. Further validation of the resulting complex by a standard protein-protein docking algorithm would also be welcome.", "responses": [] } ]	1	https://f1000research.com/articles/4-29
https://f1000research.com/articles/4-27/v1	27 Jan 15	{ "type": "Observation Article", "title": "The curious case of the spinning neuronal mass", "authors": [ "Shiladitya Mitra" ], "abstract": "Rotating cells have been reported in past. Rotation of cells and cell clusters has been associated with migration and development. This observation reports for the first time a rotating cluster of cells isolated from the hippocampi of neonatal mouse pups. The speed of rotation in these clusters is immensely high. Further analysis of such rotating neurons can shed valuable clues on the origin of such cells, their electro-mechanical properties and their role in the development of the brain.", "keywords": [ "cell rotation", "neurons", "hippocampus", "brain development" ], "content": "Introduction\n\nRotation in cells and in cell aggregates is an interesting biophysical phenomenon. Though the exact reasons behind such rotations are still largely unknown, it has been established that such rotations are important for the process of development. Cellular rotations have been previously observed in cilia-driven rotation of Helisoma trivolis embryos1 and in cortical rotation of Xenopus eggs2. Tanner et al. (2011) identified coherent angular motion (CAMo) in breast epithelial cells which maintain it cohesively while assembling into the acini. Such a rotation was lost in malignant cancerous cells. The authors conclude that such rotational movement is essential for constructing the overall geometry of the tissue3. Collective rotations have also been observed in endothelial cells4,5 and canine kidney epithelial cells6,7 on micropatterns. Most of these rotating cells follow the yin-yang pattern of movement8.\n\nHowever isolated cells cultured in regular 2D cell culture systems have not shown full body rotation9.\n\nNeurospheres are generated by three dimensional growth of neural stem cells and progenitors when primary neurons/neuronal precursors isolated from hippocampus are allowed to grow on a low attachment vessel under proper growth conditions in a non-differentiating environment. When these primary neurons/neuronal precursors are allowed to attach and grow in a differentiating environment they differentiate into mature neurons. The procedure of generating hippocampal neurospheres as well as primary neuron cultures requires single cell suspension of neurons derived from hippocampus and their immediate plating. This piece of work shows for the first time that neurons isolated from the hippocampi of four day old neonatal pups when kept in suspension for two-three hours in media instead of immediate plating, coalesce to form neuronal mass and exhibit angular momentum resulting in spinning of the mass.\n\n\nMethods\n\nMice used for this experiment had been procured from the central Animal House facility (CCMB). They had been housed in polypropylene cages with shredded corn-cob bedding under proper care with 12 hour light and dark cycle. Four day old pups were generated by mating of male and female wild-type CD-1 (Charles River) mice. All the experiments adhered to the ethical guidelines of animal usage and were approved by the Institutional Animal Ethics Committee, CCMB (Reg. No. CPCSEA 20/1999).\n\nHippocampi were isolated from brains of three male neonatal pups of four days age. The complete protocol followed has been described elsewhere10. They were pooled and fine dissected in DMEM (Himedia) without serum and then exposed to trypsin-EDTA (Sigma) for 15min at 37°C. Trypsin inhibitor was added to stop the reaction. Cells were collected after a short spin (700 rpm for 5min at room temperature in Kubota 2800 tabletop centrifuge) and re-suspsended in NeuroCult Basal (StemCell Technologies) media (>1million/ml). The cells were passed through a 40micron column filter (Millipore) for ensuring single cell suspension. A fraction of this was used for generation of neurospheres in NeuroCult Basal Media supplemented with Mouse Proliferative Supplement (StemCell Technologies), antibiotics (Pen-Strep, Sigma), 0.02% BSA (Sigma), 0.2% Heparin (Sigma), 20ng/ul EGF (Invitrogen) and 10ng/ul βFGF (Invitrogen). The remaining was put in a 50ml falcon and incubated at 37°C for 2–3 hours. They were then plated in 4 well tissue culture dishes and in T25 flasks. In two wells of the 4 well plate, Neurobasal + B27 (Life Technologies) were added to Neurocult Basal Media in 1:1 ratio.\n\nBiological duplicates of this experiment were carried out.\n\nIn the absence of video capturing capability of Image Acquisition software Image-Pro Express, video recording was done through the eye-piece using a handheld video camera (Sony Xperia Acro S) in real time.\n\n\nObservation\n\nThe observations were recorded in three video files.\n\nDifferent sizes of rotating and spinning cluster of neurons can be visualized at different magnifications in video 1. Interestingly, not only large masses, but a cluster of four cells also exhibited such rotation/spin (Video 1e,f). The culture, since it was derived from four day old pups, resulted in a high amount of debris. Spinning and rotation of neurons can be visualized in-spite of the resistance provided by the debris around it (video 2a).\n\nThese movements observed were not because of Brownian motion as a) there was no movement of the media and the plates and b) the videos were taken after 30min-1 hour of plating. Few of these masses continued exhibiting rotation for more than 48 hours (video 3) till they settled and attached. The angle of rotation/spin was not constant and the mass also exhibited change in direction/angle (video 1b, c; video 3). Also not all such neuronal masses exhibited such movements and a majority of them were attached to the plates (video 1d). Such a rotational movement persisted even when the media was changed to NeuroCult Basal with Neurobasal supplemented with B27.\n\n\nDiscussion\n\nThe observation is the first report of rotation observed in clusters of mammalian neural cells. Cells isolated from hippocampi in the same experiment were also used for generation of neurospheres. No such rotational movement was seen there. The reason for such rotation/spin is unclear and there can be numerous possibilities.\n\nThe most likely hypothesis is that two or more migrating neuronal cells couple to each other, resulting in the generation of torque, causing angular motion. Cell migration occurs in developing organs and tissues, wound healing and cancer,11–13. Collective migration associated with cell-cell interaction has been seen in neural crest cells14 as well as in neurons of the rostal migratory stream15,16. Collective migration of cells can lead to collective rotation, as is the case of rotation of epithelial cells in the extracellular matrix17. Pernille Rorth argues that this might be because most of the cells in the cluster follow a ‘leader’ cell and, as with sheet migration, rotate in their bid to follow the leader cell18. There is also a possibility that usage of planar polarized signaling molecules such as the non-canonical Wnt-Fz pathway, could account for rotation of the epithelium18–20.\n\nNoting that even a four cell clusters exhibited such rotation (video 1e,f), it can also be argued that a single cell might be the unit responsible for such a phenotype. But considering the amount of energy required for moving a larger mass, as displayed in video 2 or 3, this is highly improbable. Hence, such a movement more likely results from the interaction of multiple cells. Since these cultures were generated from fourth day neonatal pups, there are many migratory neurons present in the hippocampus. The spin thus may be caused by the force exerted by these migratory neurons.\n\nHowever it is also plausible that such a rotation arises as a result of change in charge difference between the membrane surface and the interior of the neurons. A continuous dynamic change in the charge could result in the generation of angular momentum in a cluster of cells. However a detailed analysis of electrophysiological parameters and calcium imaging of these mass of neurons needs to be done to understand the exact reasons behind such an interesting phenotype.\n\nThe other most interesting fact about the rotating neurons was the speed at which they were rotating and that the rotation persisted for more than 48hours. Whether such rotation occurs in neurons derived from other brain regions also needs to be assessed.\n\nFurther research needs to be done to find actual reason of such rotatory movement and the origin and fate of these cells.\n\n\nData availability\n\nFigshare: Rotation of neuronal masses derived from hippocampi of four day-old mouse pups. doi: 10.6084/m9.figshare.129286621", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work was supported by grant from CSIR, India.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nI extend my thanks to Dr Satish Kumar and to the CCMB for their aid in carrying out the work.\n\n\nReferences\n\nDiefenbach TJ, Koehncke NK, Goldberg JI: Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin. J Neurobiol. 1991; 22(9): 922–34. PubMed Abstract \| Publisher Full Text\n\nGerhart J, Danilchik M, Doniach T, et al.: Cortical rotation of the Xenopus, egg: consequences for the anteroposterior pattern of embryonic dorsal development. Development. 1989; 107(Suppl): 37–51. PubMed Abstract\n\nTanner K, Mori H, Mroue R, et al.: Coherent angular motion in the establishment of multicellular architecture of glandular tissues. Proc Natl Acad Sci U S A. 2012; 109(6): 1973–1978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang S, Brangwynne CP, Parker KK, et al.: Symmetry-breaking in mammalian cell cohort migration during tissue pattern formation: role of random-walk persistence. Cell Motil Cytoskeleton. 2005; 61(4): 201–213. PubMed Abstract \| Publisher Full Text\n\nBrangwynne C, Huang S, Parker KK, et al.: Symmetry breaking in cultured mammalian cells. In Vitro Cell Dev Biol Anim. 2000; 36(9): 563–565. PubMed Abstract \| Publisher Full Text\n\nDoxzen K, Vedula SR, Leong MC, et al.: Guidance of collective cell migration by substrate geometry. Integr Biol (Camb). 2013; 5(8): 1026–1035. PubMed Abstract \| Publisher Full Text\n\nMarmaras A, Berge U, Ferrari A, et al.: A mathematical method for the 3D analysis of rotating deformable systems applied on lumen-forming MDCK cell aggregates. Cytoskeleton (Hoboken). 2010; 67(4): 224–240. PubMed Abstract \| Publisher Full Text\n\nBrangwynne C, Huang S, Parker KK, et al.: Symmetry breaking in cultured mammalian cells. In Vitro Cell Dev Biol Anim. 2000; 36(9): 563–565. PubMed Abstract \| Publisher Full Text\n\nLeong FY: Physical explanation of coupled cell-cell rotational behavior and interfacial morphology: a particle dynamics model. Biophys J. 2013; 105(10): 2301–2311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKK Pacey L, Stead S, Gleave JA, et al.: Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation. Protoc exch. 2006. Publisher Full Text\n\nFriedl P, Gilmour D: Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol. 2009; 10(7): 445–457. PubMed Abstract \| Publisher Full Text\n\nVedula SR, Ravasio A, Lim CT, et al.: Collective cell migration: a mechanistic perspective. Physiology (Bethesda). 2013; 28(6): 370–379. PubMed Abstract \| Publisher Full Text\n\nTheveneau E, Mayor R: Collective cell migration of epithelial and mesenchymal cells. Cell Mol Life Sci. 2013; 70(19): 3481–3492. PubMed Abstract \| Publisher Full Text\n\nTeddy JM, Kulesa PM: In vivo evidence for short- and long-range cell communication in cranial neural crest cells. Development. 2004; 131(24): 6141–51. PubMed Abstract \| Publisher Full Text\n\nMurase S, Horwitz AF: Directions in cell migration along the rostral migratory stream: the pathway for migration in the brain. Curr Top Dev Biol. 2004; 61: 135–52. PubMed Abstract \| Publisher Full Text\n\nNam SC, Kim Y, Dryanovski D, et al.: Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study. J Comp Neurol. 2007; 505(2): 190–208. PubMed Abstract \| Publisher Full Text\n\nFarooqui R, Fenteany G: Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J Cell Sci. 2005; 118(Pt 1): 51–63. PubMed Abstract \| Publisher Full Text\n\nRørth P: Fellow travellers: emergent properties of collective cell migration. EMBO Rep. 2012; 13(11): 984–991. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein TJ, Mlodzik M: Planar cell polarization: an emerging model points in the right direction. Annu Rev Cell Dev Biol. 2005; 21: 155–76. PubMed Abstract \| Publisher Full Text\n\nRoszko I, Sawada A, Solnica-Krezel L: Regulation of convergence and extension movements during vertebrate gastrulation by the Wnt/PCP pathway. Semin Cell Dev Biol. 2009; 20(8): 986–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitra S: Rotation of neuronal masses derived from hippocampi of four day-old mouse pups. Figshare. 2014. Data Source" }	[ { "id": "7694", "date": "17 Feb 2015", "name": "Dennis Steindler", "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 author describes spinning cells or spheres in neurosphere cultures from the mouse hippocampus. Although an interesting phenomenon, presenting a time lapse film of these structures, without any phenotyping, cell and molecular analysis of them does not allow any understanding of the nature of these structures. Furthermore, these have been described before in neurosphere cultures from the subventricular zone (see Laywell et al, PNAS, 2000; Figure 1), originating from ciliated ependymal cells that can form spheres with other ciliated cells or astrocytes. The hippocampal periventricular ciliated ependymal cells and B cell astrocytes are probably the origin of these spinning structures described here, but only characterization of them would allow this confirmation. Finally, referring to the potential physical mechanisms underlying rotation really only needs to consider the study of Sawamoto et al., Science, 2014.", "responses": [] } ]	1	https://f1000research.com/articles/4-27
https://f1000research.com/articles/3-190/v1	12 Aug 14	{ "type": "Research Note", "title": "Longevity of Atlantic Sharpnose Sharks Rhizoprionodon terraenovae and Blacknose Sharks Carcharhinus acronotus in the western North Atlantic Ocean based on tag-recapture data and direct age estimates", "authors": [ "Bryan S. Frazier", "William B. Driggers III", "Glenn F. Ulrich", "William B. Driggers III", "Glenn F. Ulrich" ], "abstract": "Longevity of Rhizoprionodon terraenovae and Carcharhinus acronotus in the western North Atlantic Ocean was examined using direct age estimates from vertebral sections and tag-recapture data. Time-at-liberty ranged from 7.7-12.1 years (mean =9.2) for R. terraenovae and 10.9-12.8 years (mean =11.9) for C. acronotus. Maximum estimated longevity was determined to be 19.8 years through tag-recapture data and 18.5 years from direct age estimates for R. terraenovae and 22.8 years through tag-recapture data and 20.5 years through direct age estimates for C. acronotus. These longevity estimates represent a large increase over previous estimates and may have significant effects on analyses that depend on longevity including lifetime fecundity, mortality rates, demographic analyses and stock assessments.", "keywords": [ "Tag-recapture data grant researchers the opportunity to synthesize a vast array of information for species they are studying. Sharks", "in particular", "are well suited for tagging studies due to their migratory behavior", "longevity", "and relatively large size", "which allows tagging at all life stages (Bonham et al.", "1949). Valuable information gained from recapture data includes greater understanding of species-specific migratory patterns", "stock structure", "spatial and temporal distribution", "site fidelity/residence times", "and life histories (Kohler & Turner", "2001)." ], "content": "Introduction\n\nTag-recapture data grant researchers the opportunity to synthesize a vast array of information for species they are studying. Sharks, in particular, are well suited for tagging studies due to their migratory behavior, longevity, and relatively large size, which allows tagging at all life stages (Bonham et al., 1949). Valuable information gained from recapture data includes greater understanding of species-specific migratory patterns, stock structure, spatial and temporal distribution, site fidelity/residence times, and life histories (Kohler & Turner, 2001).\n\nAmong life history parameters needed to best manage populations of fishes, robust longevity estimates are of paramount importance, particularly for iteroparous species, such as sharks. For example, assuming age at maturity, reproductive periodicity, and brood size are constant, the lifetime fecundity of a given species is directly linked to its lifespan. Longevity estimates for sharks are generally derived from von Bertalanffy Growth Function (VBGF) parameter estimates (i.e. growth constant); however, VBGF parameter estimates can be heavily affected by low sample sizes, incomplete sampling among size classes and/or difficulties associated with age estimation of large individuals (Goldman, 2004; Francis et al., 2007). Obtaining accurate longevity estimates can be hampered by the low probability of catching older individuals as they represent a small portion of the entire population, difficulty associated with capturing large specimens that are more capable of escaping gear than smaller conspecifics, and the reduction of older individuals in the population due to fishing pressure (Bishop et al., 2006). Additionally, age underestimation has been documented in multiple shark species including long-lived species such as the Porbeagle, Lamna nasus (Bonnaterre, 1788) (Francis et al., 2007) and the Australian School Shark, Galeorhinus galeus (L. 1758) (Kalish & Johnston, 2001), and species with intermediate life histories such as the Bonnethead, Sphyrna tiburo (L. 1758) (Frazier et al., 2014). Due to the above factors, tag-recapture records, when available, are the most reliable source of longevity data as maximum time-at-liberty for any individual within the population would represent the highest directly known longevity. Based on tag-recapture data and direct age estimates, herein we report on the longevity of Atlantic Sharpnose Rhizoprionodon terraenovae (Richardson, 1836) and Blacknose Carcharhinus acronotus (Poey, 1860) Sharks, both of which are common in the coastal waters off the southeastern United States.\n\n\nMethods\n\nSharks were captured and tagged during survey operations conducted by the South Carolina Department of Natural Resources Adult Red Drum and Coastal Shark Longline Program (SCDNR) (see Ulrich et al., 2007 for longline protocol). Collection, handling and tagging of specimens was authorized and controlled under the SCDNR scientific permit issued to employees of SCDNR.\n\nUpon capture, the fork length (FL) and sex of each shark was recorded. For those sharks deemed to be in good health, a Hallprint© nylon dart tag, labeled with unique identifying numbers, was inserted into the dorsal musculature at the base of the first dorsal fin prior to release. As tagged sharks were at liberty in the wild, recaptured specimens were reported by a number of sources, including recreational anglers, commercial fishermen and SCDNR. Estimated measurements at recapture were obtained from recreational and commercial fishermen and direct measurements were recorded from specimens recaptured by SCDNR. When possible, a sample of 8–10 vertebrae were removed from the cervical region of the vertebral column from recaptured sharks.\n\nIn the case of recaptured specimens from commercial fishermen, sharks were already deceased and vertebrae were removed. In the case of recaptured specimens from SCDNR, a IACUC protocol approved for graduate students who had previously worked with SCDNR on elasmobranch studies was followed; the vertebral column was served by serrated knife in two cervical locations.\n\nTo estimate age at recapture, vertebrae were prepared for analyses following the protocol of Frazier et al., 2014. Initial vertebral growth band counts were conducted by three unbiased readers with no knowledge of specimen length or time-at-liberty. If band counts differed among readers, sections were independently re-read until agreement was reached. Age at recapture was estimated under the assumptions: (1) the birthmark was formed prior or shortly after parturition, (2) the second band was formed 6 months later during the first winter, and (3) the third band was formed 1 year later. Therefore, we subtracted 1.5 from each total band count to calculate age in years (e.g. Loefer & Sedberry, 2003; Driggers et al., 2004).\n\nAge estimates at recapture were also determined by adding time-at-liberty to backtransformed ages at tagging. Species and sex-specific von Bertalanffy growth function parameters from Loefer & Sedberry, 2003 (R. terraenovae) and Driggers et al., 2004 (C. acronotus) were used to backtransform age at tagging using the following equation:\n\n\n\nWhere:\n\nLt = length at age t,\n\nL∞ = theoretical maximum length,\n\nk = coefficient of growth,\n\nto = theoretical age at which length equals zero.\n\nA paired t-test was used to determine if age estimates from vertebral sections were significantly different than backtransformed age estimates. Statistical results were considered significant at α < 0.05.\n\n\nResults\n\nFour R. terraenovae (three male and one female) were recaptured with times at liberty ranging from 7.7 to 12.1 years (mean ± S.D. = 9.2 ± 2.0). Length at initial tagging, and when available, length at recapture are listed in Table 1. Backtransformed age at tagging and recapture ranged from 3.8–10.3 years and from 11.8 to 19.8 years, respectively (Table 1). Vertebrae were sampled from Shark L5242 and the direct age estimate from the vertebral section was 18.5 years old. Precise measurements were taken for L5242 at tagging and recapture; over 12.1 years-at-liberty this shark grew 28 mm (2.3 mm/year). All four sharks were recaptured within 15 km of initial tagging.\n\nFork length (FL) at initial tagging, length at recapture, growth, days and years at liberty, backtransformed age at initial tagging and recapture and direct age estimates are provided.\n\n*Denotes approximate measurements as provided by recreational anglers.\n\nThree C. acronotus were recaptured (two males and one female ). Time at liberty ranged from 10.9 to 12.8 years (mean ± S.D. = 11.9 ± 1.0). Backtransformed age at tagging and recapture ranged from 4.2 to 11.9 years and 16.4 to 22.8 years, respectively (Table 1). Vertebrae were obtained from all recaptured C. acronotus. Age estimates from vertebral sections ranged from 14.5 to 20.5 years. Precise measurements were taken at capture and recapture for sharks L2910 and L1515. L2910 was at liberty for 12.0 years and grew 84 mm (7.0 mm/year). L1515 was at liberty for 12.8 years and grew 177 mm (13.8 mm/year). All recaptured C. acronotus were recovered within 15 km of initial tagging.\n\nIn recaptures with both direct and backtransformed age estimates, backtransformed age estimates were significantly larger (paired t-test, t = 4.82, P = 0.02) (Table 1). From direct age estimates, R. terraenovae observed maximum longevity increased by 9.5 years for males (previously 9+ years [Loefer & Sedberry, 2003]), and no vertebrae were available from female recaptures (Table 2). For R. terraenovae all age estimates (direct and backtransformed) were over double theoretical maximum longevities (7.1 and 6.9 years females and males respectively) from Loefer & Sedberry, 2003 (Table 2).\n\nC. acronotus observed maximum longevity from sectioned age estimates increased by 2 years for females and 8 years for males (12.5 and 10.5 years for females and males respectively [Driggers et al., 2001]). Backtransformed maximum longevity was 4.5 years older than published estimates for females and 12.3 years older for males. The backtransformed maximum longevity was below the theoretical maximum longevities (19.0 years, Driggers et al., 2004) for female C. acronotus, but above theoretical longevity (16.4 years) for males (Table 2).\n\n\nDiscussion\n\nThe backtransformed and direct age estimates documented herein greatly increase the known longevity for R. terraenovae and C. acronotus, which could significantly affect population dynamics models that include longevity as a parameter. Interestingly, the maximum directly estimated ages for both species were associated with male sharks. A review of published shark age and growth studies shows that, in most studies, that females have a greater or equal longevity than males (e.g. Carlson & Baremore, 2003; Carlson & Parsons, 1997; Carlson et al., 1999; Driggers et al., 2004; Drymon et al., 2006; Frazier et al., 2014). Therefore, we believe that we did not sample older females and suggest that the longevity estimates maximum observed longevity we observed for each species be applied to males and females.\n\nBacktransformed age estimates from recaptures were in all cases larger than direct age estimates. This could be evidence that direct ages were underestimated, however direct age estimates only differed by an average of 1.8 years (range 1.0–2.5 years). Observed differences could be due to low sample size or individual variability in growth. The rearranged VBGF gives an estimate of average age-at-length based on parameter estimates. Therefore, specimens could have been younger or older than the average estimated age at time of tagging thus accounting for the discrepancy. Conversely, if the species-specific VBGF do not adequately describe the growth of R. terraenovae and C. acronotus then the observed differences would be expected. However, the growth models utilized were from studies with robust sample sizes that included all size classes (Loefer & Sedberry, 2003; Driggers et al., 2004).\n\nThe age estimates from this study greatly increase longevity for both species; however, actual life spans could be even longer. The specimens recaptured from this study were tagged in the first few years of the SCDNR longline program (1994–1996). Given project species-specific recapture rates of less than 3% and reported tag shedding rates for nylon dart tags as high as 41% to 63% (Xiao et al., 1999), the chances of recapturing a shark at liberty for 10+ years are small. The fact that seven were encountered and all exceeded published longevity estimates lends support to this assertion. The data gathered from these recaptures also highlights the importance of continuing long term surveys and tagging efforts. While return rates of tagged sharks are notoriously low, our data demonstrate that continued tagging efforts are essential to provide the most up to date and reliable estimates of maximum longevity. Ideally, to obtain the best possible longevity estimates for a given species, future efforts should focus on tagging neonate sharks with the hope of recapturing them as they approach the end of their lifespans.", "appendix": "Author contributions\n\n\n\nBF, WD and GU were involved with longline survey operations as well as tagging and recapturing the sharks. BF and WD aged specimens. BF and WD prepared manuscript for publication, and all authors were involved in revising the draft manuscript and agree to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThese data were based on a 20 year longline survey and numerous grants have funded the survey over the years. Funding for the longline survey was provided by the Federal Aid in Sport fish Restoration Act, the Southeast Area Monitoring and Assessment Program, the Atlantic States Marine Fisheries Commission, the National Marine Fisheries Federal Assistance Program, and the South Carolina State Recreational Fisheries Advisory Committee.\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 to the many folks that have helped the longline program over the years, especially Doug Oakley, Catherine Riley, Josh Loefer, Christian Jones, Carrie Hendrix, Jonathan Richardson, Erin Levesque, Henry Davega, Rob Dunlap and Ashley Shaw. This is contribution 727 of the South Carolina Marine Resource Center.\n\n\nReferences\n\nBishop SDH, Francis MP, Duffy C, et al.: Age, growth, maturity, longevity and natural mortality of the shortfin mako shark (Isurus oxyrinchus) in New Zealand Waters. Mar Freshwater Res. 2006; 57(2): 143–154. Publisher Full Text\n\nBonham K, Sanford FB, Clegg W, et al.: Biological and vitamin A studies of dogfish (Squalus suckleye) in the State of Washington. Washington State Department of Fisheries. Biological Report. 1949; 49A: 83–114. Reference Source\n\nCarlson JK, Baremore IE: Changes in biological parameters of Atlantic sharpnose shark Rhizoprionodon terraenovae in the Gulf of Mexico: evidence for density-dependent growth and maturity? Mar Freshwater Res. 2003; 54(3): 227–234. Publisher Full Text\n\nCarlson JK, Parsons GR: Age and growth of the bonnethead shark, Sphyrna tiburo, from northwest Florida, with comments on clinal variation. Environ Biol Fish. 1997; 50(3): 331–341. Publisher Full Text\n\nCarlson JK, Cortes E, Johnson AG: Age and growth of the blacknose shark, Carcharhinus acronotus, in the eastern Gulf of Mexico. Copeia. 1999; 1999(3): 684–691. Publisher Full Text\n\nDriggers WB III, Carlson JK, Cullum BJ, et al.: Age and growth of the blacknose shark, Carcharhinus acronotus, in the western North Atlantic Ocean with comments on regional variation in growth rates. Environ Biol Fish. 2004; 71(2): 171–178. Publisher Full Text\n\nDrymon JM, Driggers WB III, Oakley D, et al.: Investigating life history difference between finetooth sharks, Carcharhinus isodon, in the northern Gulf of Mexico and the western North Atlantic Ocean. Gulf Mexico Sci. 2006; 24(1–2): 2–10. Reference Source\n\nFabens AJ: Properties and fitting of the Von Bertalanffy growth curve. Growth. 1965; 29(3): 265–89. PubMed Abstract\n\nFrancis MP, Campana SE, Jones CM: Age under-estimation in New Zealand porbeagle sharks (Lamna nasus): is there an upper limit to ages that can be determined from shark vertebrae? Mar Freshwater Res. 2007; 58(1): 10–23. Publisher Full Text\n\nFrazier BS, Driggers WB III, Adams DH, et al.: Validated age, growth and maturity of the bonnethead Sphyrna tiburo in the western North Atlantic Ocean. J Fish Biol. 2014. in press. Publisher Full Text\n\nGoldman KJ: Age and growth of elasmobranch fishes. In Elasmobranch fisheries management techniques (Musick, JA and R Bonfil editors), Asia Pacific Economic Cooperation, Singapore. 2004; 97–132. Reference Source\n\nKalish J, Johnston J: Determination of school shark age based on analysis of radiocarbon in vertebral collagen. In Use of the Bomb Radiocarbon Chronometer to Validate Fish Age (Kalish, JM Editor), Final Report FRDC Project 93/109. Fisheries Research and Development Corporation, Canberra. 2001; 116–129.\n\nKohler NE, Turner PA: Shark tagging: a review of conventional methods and studies. Environ Biol Fish. 2001; 60(1–3): 191–223. Publisher Full Text\n\nLoefer JK, Sedberry GR: Life history of the Atlantic sharpnose shark (Rhizoprionodon terraenovae) (Richardson, 1836) off the Southeastern United States. Fish Bull. 2003; 101: 75–88. Reference Source\n\nUlrich GF, Jones CM, Driggers WB II, et al.: Habitat utilization, relative abundance, and seasonality of sharks in the estuarine and nearshore waters of South Carolina. In Shark nursery grounds of the Gulf of Mexico and East Coast waters of the United States (McCandless, CT NE Kohler, and HL Pratt Jr. editors), American Fisheries Society. Symposium 50, Bethesda, Maryland. 2007; 125–139. Reference Source\n\nXiao Y, Brown LP, Walker TI, et al.: Estimation of instantaneous rates of tag shedding for school shark, Galeorhinus galeus, and gummy shark, Mustelus antarcticus, by conditional likelihood. Fish Bull. 1999; 97(1): 170–184. Reference Source" }	[ { "id": "5802", "date": "26 Aug 2014", "name": "Alastair Harry", "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 research note presents data on the long-term recapture of seven individuals of two species of coastal sharks from the Northwest Atlantic; Rhizoprionodon terranovae, and the Carcharhinus acronotus. Based on their length at tagging, all individuals appeared to be either mature or close to maturity, but were recaptured between 7.7 and 12.8 years later. As neither species was reported to be particularly long-lived these results are interesting, and provide evidence that both species live longer than currently thought. Age was also estimated and compared in four of the samples by reading sectioned vertebrae. These data contribute to growing evidence that longevity of sharks is often underestimated when based on vertebral ageing methods, and that some aspects of elasmobranch demography that have been inferred from these studies (e.g. survival) may be biased. I’m glad the authors have taken the time and effort to document these observations, as there are relatively few long-terms studies of this kind.While I think the data themselves are worthy of publication, I don’t think any of the subsequent analysis contributed hugely to the paper. Published growth curves are used to estimate the age at tagging, which is then added to the time at liberty to get a ‘backtransformed age at recapture’. Unfortunately, neither of these original growth studies presented the model variance, a detailed description of the data, or even any plots of the size at age data! Without this information, it is hard to get an idea of how variable size-at-age is for these species (and it is clearly quite variable), or the representativeness of the original sampling, etc. Personally, I think other information could be more useful, like photographs of the two oldest vertebrae sections for reference.I recommend two revisions to the research note. Firstly, some more background information is required on the tagging study itself. It is not stated how many sharks were tagged and recaptured over the duration of the whole study, nor is it clear why these seven were chosen specifically (presumably they weren't the only recaptures). Secondly, I disagree that Fabens’ theoretical method for estimating longevity is the usual method of estimating longevity. I don’t see the value with making comparisons to theoretical longevity later on in the paper and find this confusing. For example, the ‘theoretical maximum longevities’ of R. terranovae are stated to be 7.1 and 6.9 years for females and males respectively, but Loefer and Sedberry (2003) reported actual longevities of 10 and 9 years.", "responses": [ { "c_id": "1197", "date": "28 Jan 2015", "name": "Bryan Frazier", "role": "Author Response", "response": "The authors appreciate the suggested revisions offered by the reviewer. We agree that it is unfortunate that the original age and growth studies did not present individual length at age data with their published growth curves. While we felt it most appropriate to cite the peer reviewed publications, it should be noted that these data are available for Carcharhinus acronotus (SEDAR 21 DW-36, http://www.sefsc.noaa.gov/sedar/download/S21_DW_36.pdf?id=DOCUMENT) as a working paper for United States SouthEast Data, Assessment and Review C. acronotus stock assessment. Unfortunately, there are no publicly accessible documents showing variation in length-at-age for Rhizoprionodon terraenovae. The authors agree with the reviewer that images of the oldest aged vertebra for each species are appropriate, and we have added those figures. As requested, the authors have added minimal background information detailing the total tagged and recaptured of each species. The authors feel adding additional background information beyond this would not be of value to readers. The recaptures that were used for this note were chosen due to their lengthy times-at-liberty. Recaptures with shorter times-at-liberty were not used as they would be less informative given variability in length-at-age. The authors agree with the reviewer that including Faben's theoretical maximum longevities were confusing and we have removed these data from the note." } ] }, { "id": "5801", "date": "02 Sep 2014", "name": "Marcus Drymon", "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\nThis manuscript reports increased longevity for two common species of small coastal sharks (Atlantic Sharpnose Shark Rhizoprionodon terraenovae and Blacknose Shark Carcharhinus acronotus) based on tag recapture data. These findings are particularly interesting given the previously assumed longevity of these species, and are clearly relevant to population models that include longevity as a parameter for these species. This note is clearly written, and conclusions from this work are appropriately presented; moreover, these data illustrate the benefit of long-term fisheries-independent monitoring and tagging programs for long-lived species such as sharks.These data are clear, concise, and important to the continued successful management of these two species; as such, they are worthy of publication. The two tables presented are sufficient to convey the data discussed. Given the straightforward nature of these data and their appropriate interpretation by the Authors, I have no major comments. There are two typos in the first paragraph of the Discussion (detailed below) which should be addressed.\n\nIn the sentence “A review of published shark age and growth studies shows that, in most studies, that females have a greater…,” remove that. In the sentence “Therefore, we believe that we did not sample older females and suggest that the longevity estimates maximum observed longevity we observed for each species…,” revise “longevity estimates maximum observed longevity” as desired.", "responses": [ { "c_id": "1190", "date": "27 Jan 2015", "name": "Bryan Frazier", "role": "Author Response", "response": "The authors thank the reviewer for the suggested edits, all have been incorporated." } ] } ]	1	https://f1000research.com/articles/3-190

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